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Analytical and Bioanalytical Chemistry (v.403, #8)
Probing a myth: does the younger generation of scientists have it easier? by Sapna Deo; Yinan Wei; Sylvia Daunert (pp. 2065-2067).
is Associate Professor in the Department of Biochemistry and Molecular Biology at the University of Miami, Miller School of Medicine. Her research interest is in the development of novel nanobioanalytical techniques based on luminescence and quantum dots for the detection of microRNA and RNA molecules for applications in biomedicine, diagnosis, and pathogen detection. Other areas of research include the development of molecular probes for biosensing and bioimaging applications. Her research also focuses on creating luminescent nanocarriers for targeted imaging and sensing applications in biomedicine. She has appeared as an author and co-author of over 70 scientific publications, several patents, and received an NSF-CAREER Award. received her Ph.D. from Princeton University, USA in 2003 and was a GoldhaberResearch Fellow at the Brookhaven National Laboratory, USA, between 2003 and 2006. She joined the faculty at the Department of Chemistry, University of Kentucky, USA as an Assistant Professor in 2008 and received the Ralph E. Powe Junior Faculty Enhancement Award in 2010. Her group is currently working on multiple projects that range from the study of membrane protein biogenesis to the structure and function of periplasmic molecular chaperones, bacterial multidrug resistance, and the development of membrane protein based novel biomaterials and biosensors. is the Lucille P. Markey Chair of Biochemistry and Molecular Biology at the Miller School of Medicine and the Associate Director of the JT Macdonald Foundation Biomedical Nanotechnology Institute of the University of Miami. Dr. Daunert’s research interests are in bionanotechnology. Her group designs new molecular diagnostic tools and biosensors based on genetically engineered proteins and cells that find applications in the biomedical, environmental, and pharmaceutical fields. Her research also focuses on the development of targeted and responsive drug delivery systems.
Probing a myth: does the younger generation of scientists have it easier? by Sapna Deo; Yinan Wei; Sylvia Daunert (pp. 2065-2067).
is Associate Professor in the Department of Biochemistry and Molecular Biology at the University of Miami, Miller School of Medicine. Her research interest is in the development of novel nanobioanalytical techniques based on luminescence and quantum dots for the detection of microRNA and RNA molecules for applications in biomedicine, diagnosis, and pathogen detection. Other areas of research include the development of molecular probes for biosensing and bioimaging applications. Her research also focuses on creating luminescent nanocarriers for targeted imaging and sensing applications in biomedicine. She has appeared as an author and co-author of over 70 scientific publications, several patents, and received an NSF-CAREER Award. received her Ph.D. from Princeton University, USA in 2003 and was a GoldhaberResearch Fellow at the Brookhaven National Laboratory, USA, between 2003 and 2006. She joined the faculty at the Department of Chemistry, University of Kentucky, USA as an Assistant Professor in 2008 and received the Ralph E. Powe Junior Faculty Enhancement Award in 2010. Her group is currently working on multiple projects that range from the study of membrane protein biogenesis to the structure and function of periplasmic molecular chaperones, bacterial multidrug resistance, and the development of membrane protein based novel biomaterials and biosensors. is the Lucille P. Markey Chair of Biochemistry and Molecular Biology at the Miller School of Medicine and the Associate Director of the JT Macdonald Foundation Biomedical Nanotechnology Institute of the University of Miami. Dr. Daunert’s research interests are in bionanotechnology. Her group designs new molecular diagnostic tools and biosensors based on genetically engineered proteins and cells that find applications in the biomedical, environmental, and pharmaceutical fields. Her research also focuses on the development of targeted and responsive drug delivery systems.
Probing a myth: does the younger generation of scientists have it easier? by Sapna Deo; Yinan Wei; Sylvia Daunert (pp. 2065-2067).
is Associate Professor in the Department of Biochemistry and Molecular Biology at the University of Miami, Miller School of Medicine. Her research interest is in the development of novel nanobioanalytical techniques based on luminescence and quantum dots for the detection of microRNA and RNA molecules for applications in biomedicine, diagnosis, and pathogen detection. Other areas of research include the development of molecular probes for biosensing and bioimaging applications. Her research also focuses on creating luminescent nanocarriers for targeted imaging and sensing applications in biomedicine. She has appeared as an author and co-author of over 70 scientific publications, several patents, and received an NSF-CAREER Award. received her Ph.D. from Princeton University, USA in 2003 and was a GoldhaberResearch Fellow at the Brookhaven National Laboratory, USA, between 2003 and 2006. She joined the faculty at the Department of Chemistry, University of Kentucky, USA as an Assistant Professor in 2008 and received the Ralph E. Powe Junior Faculty Enhancement Award in 2010. Her group is currently working on multiple projects that range from the study of membrane protein biogenesis to the structure and function of periplasmic molecular chaperones, bacterial multidrug resistance, and the development of membrane protein based novel biomaterials and biosensors. is the Lucille P. Markey Chair of Biochemistry and Molecular Biology at the Miller School of Medicine and the Associate Director of the JT Macdonald Foundation Biomedical Nanotechnology Institute of the University of Miami. Dr. Daunert’s research interests are in bionanotechnology. Her group designs new molecular diagnostic tools and biosensors based on genetically engineered proteins and cells that find applications in the biomedical, environmental, and pharmaceutical fields. Her research also focuses on the development of targeted and responsive drug delivery systems.
Calibration of isotope amount ratios by analysis of isotope mixtures by Juris Meija (pp. 2071-2076).
is a Research Officer at the Institute for National Measurement Standards, National Research Council Canada. His research interests encompass theoretical analytical chemistry, isotope-ratio measurements, and the history of chemistry. He serves as Column Editor for the Analytical Challenge series in Analytical and Bioanalytical Chemistry and is the Secretary of the IUPAC Commission on Isotopic Abundances and Atomic Weights. The question as to what constitutes a fully calibrated isotope amount ratio measurement still remains a topic of active research. For years, the definitive calibration approach has been by means of synthetic mixtures of highly enriched isotopes with known chemical purity to give gravimetrically defined ratios. This article outlines the core concepts and assumptions of this method and illustrates the recent developments in the practical metrology of isotope amount ratio measurements.
Keywords: Metrology; Mass spectrometry; Isotope amount ratios
Calibration of isotope amount ratios by analysis of isotope mixtures by Juris Meija (pp. 2071-2076).
is a Research Officer at the Institute for National Measurement Standards, National Research Council Canada. His research interests encompass theoretical analytical chemistry, isotope-ratio measurements, and the history of chemistry. He serves as Column Editor for the Analytical Challenge series in Analytical and Bioanalytical Chemistry and is the Secretary of the IUPAC Commission on Isotopic Abundances and Atomic Weights. The question as to what constitutes a fully calibrated isotope amount ratio measurement still remains a topic of active research. For years, the definitive calibration approach has been by means of synthetic mixtures of highly enriched isotopes with known chemical purity to give gravimetrically defined ratios. This article outlines the core concepts and assumptions of this method and illustrates the recent developments in the practical metrology of isotope amount ratio measurements.
Keywords: Metrology; Mass spectrometry; Isotope amount ratios
Calibration of isotope amount ratios by analysis of isotope mixtures by Juris Meija (pp. 2071-2076).
is a Research Officer at the Institute for National Measurement Standards, National Research Council Canada. His research interests encompass theoretical analytical chemistry, isotope-ratio measurements, and the history of chemistry. He serves as Column Editor for the Analytical Challenge series in Analytical and Bioanalytical Chemistry and is the Secretary of the IUPAC Commission on Isotopic Abundances and Atomic Weights. The question as to what constitutes a fully calibrated isotope amount ratio measurement still remains a topic of active research. For years, the definitive calibration approach has been by means of synthetic mixtures of highly enriched isotopes with known chemical purity to give gravimetrically defined ratios. This article outlines the core concepts and assumptions of this method and illustrates the recent developments in the practical metrology of isotope amount ratio measurements.
Keywords: Metrology; Mass spectrometry; Isotope amount ratios
Applications of microelectromagnetic traps by Joseph R. Basore; Lane A. Baker (pp. 2077-2088).
received his Ph.D. at Texas A&M University working with Richard M. Crooks. He was then awarded a National Research Council Postdoctoral Associateship to study scanned probe microscopies with Lloyd J. Whitman at the Naval Research Laboratory in Washington, DC. He also studied nanopore membranes and single nanopore platforms as a postdoctoral associate with Charles R. Martin at the University of Florida. Among his many other awards, he was recently named the 2012 Society of Electroanaltical Chemistry Young Investigator. Lane Baker is interested in electrochemical methods for analysis and imaging. Current work in his group is focused on applications of nanopores for the development of chemical and biochemically selective membranes, sensor development and electrochemical imaging Microelectromagnetic traps (METs) have been used for almost two decades to manipulate magnetic fields. Different trap geometries have been shown to produce distinct magnetic fields and field gradients. Initially, microelectromagnetic traps were used mainly to separate and concentrate magnetic material at small scales. Recently such traps have been implemented for unique applications, for example filterless bioseparations, inductive heat generation, and biological detection. In this review, we describe recent reports in which MET geometry, current density, or external fields have been used. Descriptions of recent applications in which METs have been used to develop sensors, manipulate DNA, or block ion current are also provided. Figure Illustration of a magnetic particle trapped by the magnetic field of a microelectromagnet
Keywords: Microelectromagnetic traps; Magnetic particles; Magnetic manipulation; Microfluidics
Applications of microelectromagnetic traps by Joseph R. Basore; Lane A. Baker (pp. 2077-2088).
received his Ph.D. at Texas A&M University working with Richard M. Crooks. He was then awarded a National Research Council Postdoctoral Associateship to study scanned probe microscopies with Lloyd J. Whitman at the Naval Research Laboratory in Washington, DC. He also studied nanopore membranes and single nanopore platforms as a postdoctoral associate with Charles R. Martin at the University of Florida. Among his many other awards, he was recently named the 2012 Society of Electroanaltical Chemistry Young Investigator. Lane Baker is interested in electrochemical methods for analysis and imaging. Current work in his group is focused on applications of nanopores for the development of chemical and biochemically selective membranes, sensor development and electrochemical imaging Microelectromagnetic traps (METs) have been used for almost two decades to manipulate magnetic fields. Different trap geometries have been shown to produce distinct magnetic fields and field gradients. Initially, microelectromagnetic traps were used mainly to separate and concentrate magnetic material at small scales. Recently such traps have been implemented for unique applications, for example filterless bioseparations, inductive heat generation, and biological detection. In this review, we describe recent reports in which MET geometry, current density, or external fields have been used. Descriptions of recent applications in which METs have been used to develop sensors, manipulate DNA, or block ion current are also provided. Figure Illustration of a magnetic particle trapped by the magnetic field of a microelectromagnet
Keywords: Microelectromagnetic traps; Magnetic particles; Magnetic manipulation; Microfluidics
Applications of microelectromagnetic traps by Joseph R. Basore; Lane A. Baker (pp. 2077-2088).
received his Ph.D. at Texas A&M University working with Richard M. Crooks. He was then awarded a National Research Council Postdoctoral Associateship to study scanned probe microscopies with Lloyd J. Whitman at the Naval Research Laboratory in Washington, DC. He also studied nanopore membranes and single nanopore platforms as a postdoctoral associate with Charles R. Martin at the University of Florida. Among his many other awards, he was recently named the 2012 Society of Electroanaltical Chemistry Young Investigator. Lane Baker is interested in electrochemical methods for analysis and imaging. Current work in his group is focused on applications of nanopores for the development of chemical and biochemically selective membranes, sensor development and electrochemical imaging Microelectromagnetic traps (METs) have been used for almost two decades to manipulate magnetic fields. Different trap geometries have been shown to produce distinct magnetic fields and field gradients. Initially, microelectromagnetic traps were used mainly to separate and concentrate magnetic material at small scales. Recently such traps have been implemented for unique applications, for example filterless bioseparations, inductive heat generation, and biological detection. In this review, we describe recent reports in which MET geometry, current density, or external fields have been used. Descriptions of recent applications in which METs have been used to develop sensors, manipulate DNA, or block ion current are also provided. Figure Illustration of a magnetic particle trapped by the magnetic field of a microelectromagnet
Keywords: Microelectromagnetic traps; Magnetic particles; Magnetic manipulation; Microfluidics
Biochemical analysis with the expanded genetic lexicon by Hui-wang Ai (pp. 2089-2102).
has been an Assistant Professor of Chemistry at the University of California, Riverside since July 2011. His current research interests are developing biosensors for chemicals of environmental and biological interests, and engineering molecular tools for the interrogation of biochemical networks using light microscopy. The information used to build proteins is stored in the genetic material of every organism. In nature, ribosomes use 20 native amino acids to synthesize proteins in most circumstances. However, laboratory efforts to expand the genetic repertoire of living cells and organisms have successfully encoded more than 80 nonnative amino acids in E. coli, yeast, and other eukaryotic systems. The selectivity, fidelity, and site-specificity provided by the technology have enabled unprecedented flexibility in manipulating protein sequences and functions in cells. Various biophysical probes can be chemically conjugated or directly incorporated at specific residues in proteins, and corresponding analytical techniques can then be used to answer diverse biological questions. This review summarizes the methodology of genetic code expansion and its recent progress, and discusses the applications of commonly used analytical methods. Figure The technology to expand the genetic code of living cells and organism has provided valuable research tools for bioanalytical chemistry
Keywords: Genetic code; Nonnative amino acids; Bioorthogonal chemistry; Fluorescent probes; Biophysical probes; Photocrosslinking amino acids
Biochemical analysis with the expanded genetic lexicon by Hui-wang Ai (pp. 2089-2102).
has been an Assistant Professor of Chemistry at the University of California, Riverside since July 2011. His current research interests are developing biosensors for chemicals of environmental and biological interests, and engineering molecular tools for the interrogation of biochemical networks using light microscopy. The information used to build proteins is stored in the genetic material of every organism. In nature, ribosomes use 20 native amino acids to synthesize proteins in most circumstances. However, laboratory efforts to expand the genetic repertoire of living cells and organisms have successfully encoded more than 80 nonnative amino acids in E. coli, yeast, and other eukaryotic systems. The selectivity, fidelity, and site-specificity provided by the technology have enabled unprecedented flexibility in manipulating protein sequences and functions in cells. Various biophysical probes can be chemically conjugated or directly incorporated at specific residues in proteins, and corresponding analytical techniques can then be used to answer diverse biological questions. This review summarizes the methodology of genetic code expansion and its recent progress, and discusses the applications of commonly used analytical methods. Figure The technology to expand the genetic code of living cells and organism has provided valuable research tools for bioanalytical chemistry
Keywords: Genetic code; Nonnative amino acids; Bioorthogonal chemistry; Fluorescent probes; Biophysical probes; Photocrosslinking amino acids
Biochemical analysis with the expanded genetic lexicon by Hui-wang Ai (pp. 2089-2102).
has been an Assistant Professor of Chemistry at the University of California, Riverside since July 2011. His current research interests are developing biosensors for chemicals of environmental and biological interests, and engineering molecular tools for the interrogation of biochemical networks using light microscopy. The information used to build proteins is stored in the genetic material of every organism. In nature, ribosomes use 20 native amino acids to synthesize proteins in most circumstances. However, laboratory efforts to expand the genetic repertoire of living cells and organisms have successfully encoded more than 80 nonnative amino acids in E. coli, yeast, and other eukaryotic systems. The selectivity, fidelity, and site-specificity provided by the technology have enabled unprecedented flexibility in manipulating protein sequences and functions in cells. Various biophysical probes can be chemically conjugated or directly incorporated at specific residues in proteins, and corresponding analytical techniques can then be used to answer diverse biological questions. This review summarizes the methodology of genetic code expansion and its recent progress, and discusses the applications of commonly used analytical methods. Figure The technology to expand the genetic code of living cells and organism has provided valuable research tools for bioanalytical chemistry
Keywords: Genetic code; Nonnative amino acids; Bioorthogonal chemistry; Fluorescent probes; Biophysical probes; Photocrosslinking amino acids
Analytical approaches to determination of total choline in foods and dietary supplements by Melissa M. Phillips (pp. 2103-2112).
is a research chemist at the National Institute of Standards and Technology in Gaithersburg, MD, USA. She is involved in the certification efforts for food and dietary supplement Standard Reference Materials and is the co-coordinator of the Dietary Supplement Laboratory Quality Assurance Program. Her interests include development of new analytical methods for the determination of marker compounds and vitamins in dietary supplements and foods, as well as improving the measurement capabilities of the dietary supplement and food communities. Choline is a quaternary amine that is synthesized in the body or consumed through the diet. Choline is critical for cell membrane structure and function and in synthesis of the neurotransmitter acetylcholine. Although the human body produces this micronutrient, dietary supplementation of choline is necessary for good health. The major challenge in the analysis of choline in foods and dietary supplements is in the extraction and/or hydrolysis approach. In many products, choline is present as choline esters, which can be quantitated individually or treated with acid, base, or enzymes in order to release choline ions for analysis. A critical review of approaches based on extraction and quantitation of each choline ester as well as hydrolysis-based methods for determination of total choline in foods and dietary supplements is presented.
Keywords: Choline; Food; Dietary supplement
Analytical approaches to determination of total choline in foods and dietary supplements by Melissa M. Phillips (pp. 2103-2112).
is a research chemist at the National Institute of Standards and Technology in Gaithersburg, MD, USA. She is involved in the certification efforts for food and dietary supplement Standard Reference Materials and is the co-coordinator of the Dietary Supplement Laboratory Quality Assurance Program. Her interests include development of new analytical methods for the determination of marker compounds and vitamins in dietary supplements and foods, as well as improving the measurement capabilities of the dietary supplement and food communities. Choline is a quaternary amine that is synthesized in the body or consumed through the diet. Choline is critical for cell membrane structure and function and in synthesis of the neurotransmitter acetylcholine. Although the human body produces this micronutrient, dietary supplementation of choline is necessary for good health. The major challenge in the analysis of choline in foods and dietary supplements is in the extraction and/or hydrolysis approach. In many products, choline is present as choline esters, which can be quantitated individually or treated with acid, base, or enzymes in order to release choline ions for analysis. A critical review of approaches based on extraction and quantitation of each choline ester as well as hydrolysis-based methods for determination of total choline in foods and dietary supplements is presented.
Keywords: Choline; Food; Dietary supplement
Analytical approaches to determination of total choline in foods and dietary supplements by Melissa M. Phillips (pp. 2103-2112).
is a research chemist at the National Institute of Standards and Technology in Gaithersburg, MD, USA. She is involved in the certification efforts for food and dietary supplement Standard Reference Materials and is the co-coordinator of the Dietary Supplement Laboratory Quality Assurance Program. Her interests include development of new analytical methods for the determination of marker compounds and vitamins in dietary supplements and foods, as well as improving the measurement capabilities of the dietary supplement and food communities. Choline is a quaternary amine that is synthesized in the body or consumed through the diet. Choline is critical for cell membrane structure and function and in synthesis of the neurotransmitter acetylcholine. Although the human body produces this micronutrient, dietary supplementation of choline is necessary for good health. The major challenge in the analysis of choline in foods and dietary supplements is in the extraction and/or hydrolysis approach. In many products, choline is present as choline esters, which can be quantitated individually or treated with acid, base, or enzymes in order to release choline ions for analysis. A critical review of approaches based on extraction and quantitation of each choline ester as well as hydrolysis-based methods for determination of total choline in foods and dietary supplements is presented.
Keywords: Choline; Food; Dietary supplement
Laser ablation ICP-MS for quantitative biomedical applications by Ioana Konz; Beatriz Fernández; M. Luisa Fernández; Rosario Pereiro; Alfredo Sanz-Medel (pp. 2113-2125).
obtained her European Ph.D. in 2006 at the University of Oviedo (Spain). She was awarded the Extraordinary Doctorate Prize (Analytical Chemistry) of the University of Oviedo and the San Alberto Magno Doctoral Thesis Award. Between 2006 and 2008 she was a postdoctoral researcher in the Laboratory of Bio-inorganic and Environmental Analytical Chemistry at the IPREM in Pau (France). Since September 2008 she has performed postdoctoral research in the Analytical Spectrometry Group in the Department of Physical and Analytical Chemistry of Oviedo University. Her main scientific interests are related to optical and mass spectrometry techniques for the direct analysis of solid materials. Her current research is mainly focused on elemental/molecular analysis of nanomaterials and thin films using GD-OES and GD-MS, and the analysis of biomedical, geological, and industrial samples by LA-ICP-MS. LA-ICP-MS allows precise, relatively fast, and spatially resolved measurements of elements and isotope ratios at trace and ultratrace concentration levels with minimal sample preparation. Over the past few years this technique has undergone rapid development, and it has been increasingly applied in many different fields, including biological and medical research. The analysis of essential, toxic, and therapeutic metals, metalloids, and nonmetals in biomedical tissues is a key task in the life sciences today, and LA-ICP-MS has proven to be an excellent complement to the organic MS techniques that are much more commonly employed in the biomedical field. In order to provide an appraisal of the fast progress that is occurring in this field, this review critically describes new developments for LA-ICP-MS as well as the most important applications of LA-ICP-MS, with particular emphasis placed on the quantitative imaging of elements in biological tissues, the analysis of heteroatom-tagged proteins after their separation and purification by gel electrophoresis, and the analysis of proteins that do not naturally have ICP-MS-detectable elements in their structures, thus necessitating the use of labelling strategies. Figure New developments and most relevant applications of LA-ICP-MS are critically described in this review, with particular emphasis on quantitative strategies currently employ for analysis of proteins in biological tissues
Keywords: Laser ablation inductively coupled plasma mass spectrometry; Gel electrophoresis; Quantitative bioimaging of heteroatoms; Heteroatom-tagged protein analysis; Protein labelling
Laser ablation ICP-MS for quantitative biomedical applications by Ioana Konz; Beatriz Fernández; M. Luisa Fernández; Rosario Pereiro; Alfredo Sanz-Medel (pp. 2113-2125).
obtained her European Ph.D. in 2006 at the University of Oviedo (Spain). She was awarded the Extraordinary Doctorate Prize (Analytical Chemistry) of the University of Oviedo and the San Alberto Magno Doctoral Thesis Award. Between 2006 and 2008 she was a postdoctoral researcher in the Laboratory of Bio-inorganic and Environmental Analytical Chemistry at the IPREM in Pau (France). Since September 2008 she has performed postdoctoral research in the Analytical Spectrometry Group in the Department of Physical and Analytical Chemistry of Oviedo University. Her main scientific interests are related to optical and mass spectrometry techniques for the direct analysis of solid materials. Her current research is mainly focused on elemental/molecular analysis of nanomaterials and thin films using GD-OES and GD-MS, and the analysis of biomedical, geological, and industrial samples by LA-ICP-MS. LA-ICP-MS allows precise, relatively fast, and spatially resolved measurements of elements and isotope ratios at trace and ultratrace concentration levels with minimal sample preparation. Over the past few years this technique has undergone rapid development, and it has been increasingly applied in many different fields, including biological and medical research. The analysis of essential, toxic, and therapeutic metals, metalloids, and nonmetals in biomedical tissues is a key task in the life sciences today, and LA-ICP-MS has proven to be an excellent complement to the organic MS techniques that are much more commonly employed in the biomedical field. In order to provide an appraisal of the fast progress that is occurring in this field, this review critically describes new developments for LA-ICP-MS as well as the most important applications of LA-ICP-MS, with particular emphasis placed on the quantitative imaging of elements in biological tissues, the analysis of heteroatom-tagged proteins after their separation and purification by gel electrophoresis, and the analysis of proteins that do not naturally have ICP-MS-detectable elements in their structures, thus necessitating the use of labelling strategies. Figure New developments and most relevant applications of LA-ICP-MS are critically described in this review, with particular emphasis on quantitative strategies currently employ for analysis of proteins in biological tissues
Keywords: Laser ablation inductively coupled plasma mass spectrometry; Gel electrophoresis; Quantitative bioimaging of heteroatoms; Heteroatom-tagged protein analysis; Protein labelling
Laser ablation ICP-MS for quantitative biomedical applications by Ioana Konz; Beatriz Fernández; M. Luisa Fernández; Rosario Pereiro; Alfredo Sanz-Medel (pp. 2113-2125).
obtained her European Ph.D. in 2006 at the University of Oviedo (Spain). She was awarded the Extraordinary Doctorate Prize (Analytical Chemistry) of the University of Oviedo and the San Alberto Magno Doctoral Thesis Award. Between 2006 and 2008 she was a postdoctoral researcher in the Laboratory of Bio-inorganic and Environmental Analytical Chemistry at the IPREM in Pau (France). Since September 2008 she has performed postdoctoral research in the Analytical Spectrometry Group in the Department of Physical and Analytical Chemistry of Oviedo University. Her main scientific interests are related to optical and mass spectrometry techniques for the direct analysis of solid materials. Her current research is mainly focused on elemental/molecular analysis of nanomaterials and thin films using GD-OES and GD-MS, and the analysis of biomedical, geological, and industrial samples by LA-ICP-MS. LA-ICP-MS allows precise, relatively fast, and spatially resolved measurements of elements and isotope ratios at trace and ultratrace concentration levels with minimal sample preparation. Over the past few years this technique has undergone rapid development, and it has been increasingly applied in many different fields, including biological and medical research. The analysis of essential, toxic, and therapeutic metals, metalloids, and nonmetals in biomedical tissues is a key task in the life sciences today, and LA-ICP-MS has proven to be an excellent complement to the organic MS techniques that are much more commonly employed in the biomedical field. In order to provide an appraisal of the fast progress that is occurring in this field, this review critically describes new developments for LA-ICP-MS as well as the most important applications of LA-ICP-MS, with particular emphasis placed on the quantitative imaging of elements in biological tissues, the analysis of heteroatom-tagged proteins after their separation and purification by gel electrophoresis, and the analysis of proteins that do not naturally have ICP-MS-detectable elements in their structures, thus necessitating the use of labelling strategies. Figure New developments and most relevant applications of LA-ICP-MS are critically described in this review, with particular emphasis on quantitative strategies currently employ for analysis of proteins in biological tissues
Keywords: Laser ablation inductively coupled plasma mass spectrometry; Gel electrophoresis; Quantitative bioimaging of heteroatoms; Heteroatom-tagged protein analysis; Protein labelling
Single-molecule emulsion PCR in microfluidic droplets by Zhi Zhu; Gareth Jenkins; Wenhua Zhang; Mingxia Zhang; Zhichao Guan; Chaoyong James Yang (pp. 2127-2143).
is a Professor in the Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, China. He won a Chinese Government Award for Outstanding Students Abroad (2005) and is the recipient of American Chemical Society DAC Graduate Fellowship (2005). His current research is particularly focused on molecular engineering, molecular recognition and microfluidics. The application of microfluidic droplet PCR for single-molecule amplification and analysis has recently been extensively studied. Microfluidic droplet technology has the advantages of compartmentalizing reactions into discrete volumes, performing highly parallel reactions in monodisperse droplets, reducing cross-contamination between droplets, eliminating PCR bias and nonspecific amplification, as well as enabling fast amplification with rapid thermocycling. Here, we have reviewed the important technical breakthroughs of microfluidic droplet PCR in the past five years and their applications to single-molecule amplification and analysis, such as high-throughput screening, next generation DNA sequencing, and quantitative detection of rare mutations. Although the utilization of microfluidic droplet single-molecule PCR is still in the early stages, its great potential has already been demonstrated and will provide novel solutions to today’s biomedical engineering challenges in single-molecule amplification and analysis.
Keywords: Single-molecule amplification; Emulsion PCR; Microfluidic droplets; High-throughput screening; Next generation DNA sequencing; Rare mutation detection
Single-molecule emulsion PCR in microfluidic droplets by Zhi Zhu; Gareth Jenkins; Wenhua Zhang; Mingxia Zhang; Zhichao Guan; Chaoyong James Yang (pp. 2127-2143).
is a Professor in the Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, China. He won a Chinese Government Award for Outstanding Students Abroad (2005) and is the recipient of American Chemical Society DAC Graduate Fellowship (2005). His current research is particularly focused on molecular engineering, molecular recognition and microfluidics. The application of microfluidic droplet PCR for single-molecule amplification and analysis has recently been extensively studied. Microfluidic droplet technology has the advantages of compartmentalizing reactions into discrete volumes, performing highly parallel reactions in monodisperse droplets, reducing cross-contamination between droplets, eliminating PCR bias and nonspecific amplification, as well as enabling fast amplification with rapid thermocycling. Here, we have reviewed the important technical breakthroughs of microfluidic droplet PCR in the past five years and their applications to single-molecule amplification and analysis, such as high-throughput screening, next generation DNA sequencing, and quantitative detection of rare mutations. Although the utilization of microfluidic droplet single-molecule PCR is still in the early stages, its great potential has already been demonstrated and will provide novel solutions to today’s biomedical engineering challenges in single-molecule amplification and analysis.
Keywords: Single-molecule amplification; Emulsion PCR; Microfluidic droplets; High-throughput screening; Next generation DNA sequencing; Rare mutation detection
Single-molecule emulsion PCR in microfluidic droplets by Zhi Zhu; Gareth Jenkins; Wenhua Zhang; Mingxia Zhang; Zhichao Guan; Chaoyong James Yang (pp. 2127-2143).
is a Professor in the Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, China. He won a Chinese Government Award for Outstanding Students Abroad (2005) and is the recipient of American Chemical Society DAC Graduate Fellowship (2005). His current research is particularly focused on molecular engineering, molecular recognition and microfluidics. The application of microfluidic droplet PCR for single-molecule amplification and analysis has recently been extensively studied. Microfluidic droplet technology has the advantages of compartmentalizing reactions into discrete volumes, performing highly parallel reactions in monodisperse droplets, reducing cross-contamination between droplets, eliminating PCR bias and nonspecific amplification, as well as enabling fast amplification with rapid thermocycling. Here, we have reviewed the important technical breakthroughs of microfluidic droplet PCR in the past five years and their applications to single-molecule amplification and analysis, such as high-throughput screening, next generation DNA sequencing, and quantitative detection of rare mutations. Although the utilization of microfluidic droplet single-molecule PCR is still in the early stages, its great potential has already been demonstrated and will provide novel solutions to today’s biomedical engineering challenges in single-molecule amplification and analysis.
Keywords: Single-molecule amplification; Emulsion PCR; Microfluidic droplets; High-throughput screening; Next generation DNA sequencing; Rare mutation detection
Direct analysis of biological samples using extractive electrospray ionization mass spectrometry (EESI-MS) by Haiwei Gu; Ning Xu; Huanwen Chen (pp. 2145-2153).
is a Professor of the Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation at the East China Institute of Technology (Nanchang, China). His team is developing novel ambient ionization methods and applying them to metabolomics studies for the early diagnosis of diseases. is a graduate student in the Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation at the East China Institute of Technology (Nanchang, China). Her research interests are the applications of EESI-MS. completed his doctoral thesis in Analytical Chemistry at Jilin University, China, in 2001. During 2003–2005 he was a postdoc in the group of Prof. Graham Cooks at Purdue University, USA. He was a Simon Fellow in the group of Prof. Renato Zenobi at ETH Zurich in 2006–2007. Since 2008 he has been a full professor in the East China Institute of Technology, where he founded the Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation and serves as the Laboratory Director. He received the First Class of Natural Science Award of Jiangxi Province as the Chief Contributor in 2010. His research interest is to develop new instruments and methods for directly obtaining structural information about molecules in complex samples. Mass spectrometry (MS) is one of the most widely used techniques for the analysis of biological samples. In the past decade, a novel improvement in MS was the invention of ambient ionization which stands out owing to its unique capability of direct analysis of complex samples with no or minimal pretreatment. In this review, extractive electrospray ionization (EESI), a representative ambient ionization technique, is introduced focusing on its mechanism, instrumentation, and applications in biological analysis. EESI uses a traditional ESI channel to produce primary ions which subsequently ionize neutral chemicals from the sample introduction channel through an online extraction process. When analyzing biological samples, EESI has advantages of rapid analysis, high matrix tolerance, and the ability to perform in vivo analysis. According to previous studies, EESI is able to directly analyze various chemicals in complex biological specimens in liquid, gas, and solid states. EESI can provide a sensitive and selective measurement of biological samples for both qualitative and quantitative purposes. Therefore, it is anticipated that EESI will have promising applications, especially in fields which require the fast and/or in vivo analysis of biological samples with complicated matrixes.
Keywords: Extractive electrospray ionization (EESI); Ambient mass spectrometry; Biological samples; Direct analysis
Direct analysis of biological samples using extractive electrospray ionization mass spectrometry (EESI-MS) by Haiwei Gu; Ning Xu; Huanwen Chen (pp. 2145-2153).
is a Professor of the Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation at the East China Institute of Technology (Nanchang, China). His team is developing novel ambient ionization methods and applying them to metabolomics studies for the early diagnosis of diseases. is a graduate student in the Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation at the East China Institute of Technology (Nanchang, China). Her research interests are the applications of EESI-MS. completed his doctoral thesis in Analytical Chemistry at Jilin University, China, in 2001. During 2003–2005 he was a postdoc in the group of Prof. Graham Cooks at Purdue University, USA. He was a Simon Fellow in the group of Prof. Renato Zenobi at ETH Zurich in 2006–2007. Since 2008 he has been a full professor in the East China Institute of Technology, where he founded the Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation and serves as the Laboratory Director. He received the First Class of Natural Science Award of Jiangxi Province as the Chief Contributor in 2010. His research interest is to develop new instruments and methods for directly obtaining structural information about molecules in complex samples. Mass spectrometry (MS) is one of the most widely used techniques for the analysis of biological samples. In the past decade, a novel improvement in MS was the invention of ambient ionization which stands out owing to its unique capability of direct analysis of complex samples with no or minimal pretreatment. In this review, extractive electrospray ionization (EESI), a representative ambient ionization technique, is introduced focusing on its mechanism, instrumentation, and applications in biological analysis. EESI uses a traditional ESI channel to produce primary ions which subsequently ionize neutral chemicals from the sample introduction channel through an online extraction process. When analyzing biological samples, EESI has advantages of rapid analysis, high matrix tolerance, and the ability to perform in vivo analysis. According to previous studies, EESI is able to directly analyze various chemicals in complex biological specimens in liquid, gas, and solid states. EESI can provide a sensitive and selective measurement of biological samples for both qualitative and quantitative purposes. Therefore, it is anticipated that EESI will have promising applications, especially in fields which require the fast and/or in vivo analysis of biological samples with complicated matrixes.
Keywords: Extractive electrospray ionization (EESI); Ambient mass spectrometry; Biological samples; Direct analysis
Direct analysis of biological samples using extractive electrospray ionization mass spectrometry (EESI-MS) by Haiwei Gu; Ning Xu; Huanwen Chen (pp. 2145-2153).
is a Professor of the Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation at the East China Institute of Technology (Nanchang, China). His team is developing novel ambient ionization methods and applying them to metabolomics studies for the early diagnosis of diseases. is a graduate student in the Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation at the East China Institute of Technology (Nanchang, China). Her research interests are the applications of EESI-MS. completed his doctoral thesis in Analytical Chemistry at Jilin University, China, in 2001. During 2003–2005 he was a postdoc in the group of Prof. Graham Cooks at Purdue University, USA. He was a Simon Fellow in the group of Prof. Renato Zenobi at ETH Zurich in 2006–2007. Since 2008 he has been a full professor in the East China Institute of Technology, where he founded the Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation and serves as the Laboratory Director. He received the First Class of Natural Science Award of Jiangxi Province as the Chief Contributor in 2010. His research interest is to develop new instruments and methods for directly obtaining structural information about molecules in complex samples. Mass spectrometry (MS) is one of the most widely used techniques for the analysis of biological samples. In the past decade, a novel improvement in MS was the invention of ambient ionization which stands out owing to its unique capability of direct analysis of complex samples with no or minimal pretreatment. In this review, extractive electrospray ionization (EESI), a representative ambient ionization technique, is introduced focusing on its mechanism, instrumentation, and applications in biological analysis. EESI uses a traditional ESI channel to produce primary ions which subsequently ionize neutral chemicals from the sample introduction channel through an online extraction process. When analyzing biological samples, EESI has advantages of rapid analysis, high matrix tolerance, and the ability to perform in vivo analysis. According to previous studies, EESI is able to directly analyze various chemicals in complex biological specimens in liquid, gas, and solid states. EESI can provide a sensitive and selective measurement of biological samples for both qualitative and quantitative purposes. Therefore, it is anticipated that EESI will have promising applications, especially in fields which require the fast and/or in vivo analysis of biological samples with complicated matrixes.
Keywords: Extractive electrospray ionization (EESI); Ambient mass spectrometry; Biological samples; Direct analysis
Aspects of matrix effects in applications of liquid chromatography–mass spectrometry to forensic and clinical toxicology—a review by Frank T. Peters; Daniela Remane (pp. 2155-2172).
is Head of Toxicology at the Institute of Forensic Medicine at the University Hospital Jena. He has received the Young Scientist Best Published Paper Award and the Achievement Award of The International Association of Forensic Toxicologists (TIAFT), the Young Scientist Award of the Society of Toxicological and Forensic Chemistry (GTFCh), and the Young Investigator Award of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT). Current projects of his workgroup include: studies on the metabolism of drugs and poisons by microorganisms colonizing corpses; metabolism, toxicological analysis, and prevalence of new designer drugs; and development and validation of mass spectrometry-based methods for application in forensic and clinical toxicology. In the last decade, liquid chromatography coupled to (tandem) mass spectrometry (LC–MS(−MS)) has become a versatile technique with many routine applications in clinical and forensic toxicology. However, it is well-known that ionization in LC–MS(−MS) is prone to so-called matrix effects, i.e., alteration in response due to the presence of co-eluting compounds that may increase (ion enhancement) or reduce (ion suppression) ionization of the analyte. Since the first reports on such matrix effects, numerous papers have been published on this matter and the subject has been reviewed several times. However, none of the existing reviews has specifically addressed aspects of matrix effects of particular interest and relevance to clinical and forensic toxicology, for example matrix effects in methods for multi-analyte or systematic toxicological analysis or matrix effects in (alternative) matrices almost exclusively analyzed in clinical and forensic toxicology, for example meconium, hair, oral fluid, or decomposed samples in postmortem toxicology. This review article will therefore focus on these issues, critically discussing experiments and results of matrix effects in LC–MS(−MS) applications in clinical and forensic toxicology. Moreover, it provides guidance on performance of studies on matrix effects in LC–MS(−MS) procedures in systematic toxicological analysis and postmortem toxicology.
Keywords: Liquid chromatography; Mass spectrometry; Matrix effect; Ion suppression; Ion enhancement
Aspects of matrix effects in applications of liquid chromatography–mass spectrometry to forensic and clinical toxicology—a review by Frank T. Peters; Daniela Remane (pp. 2155-2172).
is Head of Toxicology at the Institute of Forensic Medicine at the University Hospital Jena. He has received the Young Scientist Best Published Paper Award and the Achievement Award of The International Association of Forensic Toxicologists (TIAFT), the Young Scientist Award of the Society of Toxicological and Forensic Chemistry (GTFCh), and the Young Investigator Award of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT). Current projects of his workgroup include: studies on the metabolism of drugs and poisons by microorganisms colonizing corpses; metabolism, toxicological analysis, and prevalence of new designer drugs; and development and validation of mass spectrometry-based methods for application in forensic and clinical toxicology. In the last decade, liquid chromatography coupled to (tandem) mass spectrometry (LC–MS(−MS)) has become a versatile technique with many routine applications in clinical and forensic toxicology. However, it is well-known that ionization in LC–MS(−MS) is prone to so-called matrix effects, i.e., alteration in response due to the presence of co-eluting compounds that may increase (ion enhancement) or reduce (ion suppression) ionization of the analyte. Since the first reports on such matrix effects, numerous papers have been published on this matter and the subject has been reviewed several times. However, none of the existing reviews has specifically addressed aspects of matrix effects of particular interest and relevance to clinical and forensic toxicology, for example matrix effects in methods for multi-analyte or systematic toxicological analysis or matrix effects in (alternative) matrices almost exclusively analyzed in clinical and forensic toxicology, for example meconium, hair, oral fluid, or decomposed samples in postmortem toxicology. This review article will therefore focus on these issues, critically discussing experiments and results of matrix effects in LC–MS(−MS) applications in clinical and forensic toxicology. Moreover, it provides guidance on performance of studies on matrix effects in LC–MS(−MS) procedures in systematic toxicological analysis and postmortem toxicology.
Keywords: Liquid chromatography; Mass spectrometry; Matrix effect; Ion suppression; Ion enhancement
Aspects of matrix effects in applications of liquid chromatography–mass spectrometry to forensic and clinical toxicology—a review by Frank T. Peters; Daniela Remane (pp. 2155-2172).
is Head of Toxicology at the Institute of Forensic Medicine at the University Hospital Jena. He has received the Young Scientist Best Published Paper Award and the Achievement Award of The International Association of Forensic Toxicologists (TIAFT), the Young Scientist Award of the Society of Toxicological and Forensic Chemistry (GTFCh), and the Young Investigator Award of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT). Current projects of his workgroup include: studies on the metabolism of drugs and poisons by microorganisms colonizing corpses; metabolism, toxicological analysis, and prevalence of new designer drugs; and development and validation of mass spectrometry-based methods for application in forensic and clinical toxicology. In the last decade, liquid chromatography coupled to (tandem) mass spectrometry (LC–MS(−MS)) has become a versatile technique with many routine applications in clinical and forensic toxicology. However, it is well-known that ionization in LC–MS(−MS) is prone to so-called matrix effects, i.e., alteration in response due to the presence of co-eluting compounds that may increase (ion enhancement) or reduce (ion suppression) ionization of the analyte. Since the first reports on such matrix effects, numerous papers have been published on this matter and the subject has been reviewed several times. However, none of the existing reviews has specifically addressed aspects of matrix effects of particular interest and relevance to clinical and forensic toxicology, for example matrix effects in methods for multi-analyte or systematic toxicological analysis or matrix effects in (alternative) matrices almost exclusively analyzed in clinical and forensic toxicology, for example meconium, hair, oral fluid, or decomposed samples in postmortem toxicology. This review article will therefore focus on these issues, critically discussing experiments and results of matrix effects in LC–MS(−MS) applications in clinical and forensic toxicology. Moreover, it provides guidance on performance of studies on matrix effects in LC–MS(−MS) procedures in systematic toxicological analysis and postmortem toxicology.
Keywords: Liquid chromatography; Mass spectrometry; Matrix effect; Ion suppression; Ion enhancement
Protein-imprinted materials: rational design, application and challenges by Kaiguang Yang; Lihua Zhang; Zhen Liang; Yukui Zhang (pp. 2173-2183).
is an Associate Professor of Analytical Chemistry at the National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences. His research on hydrophilic molecularly imprinted nanoparticles was selected as one of the best contributions from young scientists at the 5th International Workshop on Molecular Imprinting (MIP 2008, Japan). His current research interests are focused on the preparation of novel sample-preparation and separation materials for proteomics analysis using functional nanoparticles and molecularly imprinted polymers Protein imprinting is a promising tool for generating artificial biomimetic receptors with antibody-like specific recognition sites. Recently, protein-imprinted materials, as potential antibody substitutes, have attracted much attention in many fields, for example chemical sensors, chromatographic stationary phases, and artificial enzymes, owing to their long-term storage stability, potential re-usability, resistance to harsh environment, and low cost. In this critical review, we focus our discussion on the rational preparation of protein-imprinted materials in terms of choice of template, functional monomer, crosslinker, and polymerization format. In addition, several highlighted applications of protein-imprinted materials are emphasized, not only in well-known fields but also in some unique fields, for example proteomics and tissue engineering. Finally, we propose challenges arising from the intrinsic properties of protein imprinting, for example obtaining the template, heterogeneous binding, and extrinsic competition, for example immobilized aptamers. Figure Schematic representation of fabrication and application of protein-imprinted materials
Keywords: Molecular imprinting; Protein; Antibody; Recognition sites
Protein-imprinted materials: rational design, application and challenges by Kaiguang Yang; Lihua Zhang; Zhen Liang; Yukui Zhang (pp. 2173-2183).
is an Associate Professor of Analytical Chemistry at the National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences. His research on hydrophilic molecularly imprinted nanoparticles was selected as one of the best contributions from young scientists at the 5th International Workshop on Molecular Imprinting (MIP 2008, Japan). His current research interests are focused on the preparation of novel sample-preparation and separation materials for proteomics analysis using functional nanoparticles and molecularly imprinted polymers Protein imprinting is a promising tool for generating artificial biomimetic receptors with antibody-like specific recognition sites. Recently, protein-imprinted materials, as potential antibody substitutes, have attracted much attention in many fields, for example chemical sensors, chromatographic stationary phases, and artificial enzymes, owing to their long-term storage stability, potential re-usability, resistance to harsh environment, and low cost. In this critical review, we focus our discussion on the rational preparation of protein-imprinted materials in terms of choice of template, functional monomer, crosslinker, and polymerization format. In addition, several highlighted applications of protein-imprinted materials are emphasized, not only in well-known fields but also in some unique fields, for example proteomics and tissue engineering. Finally, we propose challenges arising from the intrinsic properties of protein imprinting, for example obtaining the template, heterogeneous binding, and extrinsic competition, for example immobilized aptamers. Figure Schematic representation of fabrication and application of protein-imprinted materials
Keywords: Molecular imprinting; Protein; Antibody; Recognition sites
Protein-imprinted materials: rational design, application and challenges by Kaiguang Yang; Lihua Zhang; Zhen Liang; Yukui Zhang (pp. 2173-2183).
is an Associate Professor of Analytical Chemistry at the National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences. His research on hydrophilic molecularly imprinted nanoparticles was selected as one of the best contributions from young scientists at the 5th International Workshop on Molecular Imprinting (MIP 2008, Japan). His current research interests are focused on the preparation of novel sample-preparation and separation materials for proteomics analysis using functional nanoparticles and molecularly imprinted polymers Protein imprinting is a promising tool for generating artificial biomimetic receptors with antibody-like specific recognition sites. Recently, protein-imprinted materials, as potential antibody substitutes, have attracted much attention in many fields, for example chemical sensors, chromatographic stationary phases, and artificial enzymes, owing to their long-term storage stability, potential re-usability, resistance to harsh environment, and low cost. In this critical review, we focus our discussion on the rational preparation of protein-imprinted materials in terms of choice of template, functional monomer, crosslinker, and polymerization format. In addition, several highlighted applications of protein-imprinted materials are emphasized, not only in well-known fields but also in some unique fields, for example proteomics and tissue engineering. Finally, we propose challenges arising from the intrinsic properties of protein imprinting, for example obtaining the template, heterogeneous binding, and extrinsic competition, for example immobilized aptamers. Figure Schematic representation of fabrication and application of protein-imprinted materials
Keywords: Molecular imprinting; Protein; Antibody; Recognition sites
Microscale separation methods for enzyme kinetics assays by Tomáš Křížek; Anna Kubíčková (pp. 2185-2195).
has been a researcher at Charles University in Prague, Department of Analytical Chemistry, since 2009. His research interests are applications of capillary electrophoresis in the study of enzyme kinetics and interactions of biomolecules has been a researcher at Charles University in Prague, Department of Analytical Chemistry, since 2009. In 2007 she won the Hlavka’s Foundation prize for the young scientists. She is currently investigating HPLC methods for monitoring pharmaceutically important reactions Miniaturization continues to be one of the leading trends in analytical chemistry and one that brings advantages that can be particularly beneficial in biochemical research. Use of a miniaturized scale enables efficient analysis in a short time and requires very small amounts of samples, solvents, and reagents. This can result in a remarkable decrease in costs of enzyme kinetics studies, especially when expensive or rare enzymes and/or substrates are involved. Free zone electrophoresis is without a doubt the most common microscale separation technique for capillary and on-chip enzyme assays. Progress and applications in this field are reviewed frequently whereas other modes of separation, although successfully applied, receive only marginal interest in such publications. This review summarizes applications of less common modes of separation in capillary or chip formats, namely micellar electrokinetic chromatography, liquid chromatography, gel electrophoresis, isoelectric focusing, and isotachophoresis. Because these techniques are based on separation mechanisms different from those of free zone electrophoresis, they can be, and have been, successfully used in cases where zone electrophoresis fails. Advantages and drawbacks of these alternative separation techniques are discussed, as also are the difficulties encountered most often and solutions proposed by different research groups.
Keywords: Enzyme assays; Micellar electrokinetic chromatography; Capillary gel electrophoresis; Capillary isoelectric focusing; Capillary liquid chromatography; Chip
Microscale separation methods for enzyme kinetics assays by Tomáš Křížek; Anna Kubíčková (pp. 2185-2195).
has been a researcher at Charles University in Prague, Department of Analytical Chemistry, since 2009. His research interests are applications of capillary electrophoresis in the study of enzyme kinetics and interactions of biomolecules has been a researcher at Charles University in Prague, Department of Analytical Chemistry, since 2009. In 2007 she won the Hlavka’s Foundation prize for the young scientists. She is currently investigating HPLC methods for monitoring pharmaceutically important reactions Miniaturization continues to be one of the leading trends in analytical chemistry and one that brings advantages that can be particularly beneficial in biochemical research. Use of a miniaturized scale enables efficient analysis in a short time and requires very small amounts of samples, solvents, and reagents. This can result in a remarkable decrease in costs of enzyme kinetics studies, especially when expensive or rare enzymes and/or substrates are involved. Free zone electrophoresis is without a doubt the most common microscale separation technique for capillary and on-chip enzyme assays. Progress and applications in this field are reviewed frequently whereas other modes of separation, although successfully applied, receive only marginal interest in such publications. This review summarizes applications of less common modes of separation in capillary or chip formats, namely micellar electrokinetic chromatography, liquid chromatography, gel electrophoresis, isoelectric focusing, and isotachophoresis. Because these techniques are based on separation mechanisms different from those of free zone electrophoresis, they can be, and have been, successfully used in cases where zone electrophoresis fails. Advantages and drawbacks of these alternative separation techniques are discussed, as also are the difficulties encountered most often and solutions proposed by different research groups.
Keywords: Enzyme assays; Micellar electrokinetic chromatography; Capillary gel electrophoresis; Capillary isoelectric focusing; Capillary liquid chromatography; Chip
Microscale separation methods for enzyme kinetics assays by Tomáš Křížek; Anna Kubíčková (pp. 2185-2195).
has been a researcher at Charles University in Prague, Department of Analytical Chemistry, since 2009. His research interests are applications of capillary electrophoresis in the study of enzyme kinetics and interactions of biomolecules has been a researcher at Charles University in Prague, Department of Analytical Chemistry, since 2009. In 2007 she won the Hlavka’s Foundation prize for the young scientists. She is currently investigating HPLC methods for monitoring pharmaceutically important reactions Miniaturization continues to be one of the leading trends in analytical chemistry and one that brings advantages that can be particularly beneficial in biochemical research. Use of a miniaturized scale enables efficient analysis in a short time and requires very small amounts of samples, solvents, and reagents. This can result in a remarkable decrease in costs of enzyme kinetics studies, especially when expensive or rare enzymes and/or substrates are involved. Free zone electrophoresis is without a doubt the most common microscale separation technique for capillary and on-chip enzyme assays. Progress and applications in this field are reviewed frequently whereas other modes of separation, although successfully applied, receive only marginal interest in such publications. This review summarizes applications of less common modes of separation in capillary or chip formats, namely micellar electrokinetic chromatography, liquid chromatography, gel electrophoresis, isoelectric focusing, and isotachophoresis. Because these techniques are based on separation mechanisms different from those of free zone electrophoresis, they can be, and have been, successfully used in cases where zone electrophoresis fails. Advantages and drawbacks of these alternative separation techniques are discussed, as also are the difficulties encountered most often and solutions proposed by different research groups.
Keywords: Enzyme assays; Micellar electrokinetic chromatography; Capillary gel electrophoresis; Capillary isoelectric focusing; Capillary liquid chromatography; Chip
Pump-probe optical microscopy for imaging nonfluorescent chromophores by Lu Wei; Wei Min (pp. 2197-2202).
is an Assistant Professor in the Department of Chemistry at Columbia University. He is also a member of the Kavli Institute for Brain Science at Columbia University. The Min group is dedicated to developing novel and powerful optical bioimaging techniques and applying them to visualize and understand the dynamic behavior of biomolecules and their delicate interactions in living cells and organisms. Many chromophores absorb light intensely but have undetectable fluorescence. Hence microscopy techniques other than fluorescence are highly desirable for imaging these chromophores inside live cells, tissues, and organisms. The recently developed pump-probe optical microscopy techniques provide fluorescence-free contrast mechanisms by employing several fundamental light–molecule interactions including excited state absorption, stimulated emission, ground state depletion, and the photothermal effect. By using the pump pulse to excite molecules and the subsequent probe pulse to interrogate the created transient states on a laser scanning microscope, pump-probe microscopy offers imaging capability with high sensitivity and specificity toward nonfluorescent chromophores. Single-molecule sensitivity has even been demonstrated. Here we review and summarize the underlying principles of this emerging class of molecular imaging techniques. Figure Apparatus used for pump-probe microscopy
Keywords: Pump-probe microscopy; Label-free imaging; Excited state absorption; Stimulated emission; Ground state depletion; Nonfluorescent chromophore
Pump-probe optical microscopy for imaging nonfluorescent chromophores by Lu Wei; Wei Min (pp. 2197-2202).
is an Assistant Professor in the Department of Chemistry at Columbia University. He is also a member of the Kavli Institute for Brain Science at Columbia University. The Min group is dedicated to developing novel and powerful optical bioimaging techniques and applying them to visualize and understand the dynamic behavior of biomolecules and their delicate interactions in living cells and organisms. Many chromophores absorb light intensely but have undetectable fluorescence. Hence microscopy techniques other than fluorescence are highly desirable for imaging these chromophores inside live cells, tissues, and organisms. The recently developed pump-probe optical microscopy techniques provide fluorescence-free contrast mechanisms by employing several fundamental light–molecule interactions including excited state absorption, stimulated emission, ground state depletion, and the photothermal effect. By using the pump pulse to excite molecules and the subsequent probe pulse to interrogate the created transient states on a laser scanning microscope, pump-probe microscopy offers imaging capability with high sensitivity and specificity toward nonfluorescent chromophores. Single-molecule sensitivity has even been demonstrated. Here we review and summarize the underlying principles of this emerging class of molecular imaging techniques. Figure Apparatus used for pump-probe microscopy
Keywords: Pump-probe microscopy; Label-free imaging; Excited state absorption; Stimulated emission; Ground state depletion; Nonfluorescent chromophore
Pump-probe optical microscopy for imaging nonfluorescent chromophores by Lu Wei; Wei Min (pp. 2197-2202).
is an Assistant Professor in the Department of Chemistry at Columbia University. He is also a member of the Kavli Institute for Brain Science at Columbia University. The Min group is dedicated to developing novel and powerful optical bioimaging techniques and applying them to visualize and understand the dynamic behavior of biomolecules and their delicate interactions in living cells and organisms. Many chromophores absorb light intensely but have undetectable fluorescence. Hence microscopy techniques other than fluorescence are highly desirable for imaging these chromophores inside live cells, tissues, and organisms. The recently developed pump-probe optical microscopy techniques provide fluorescence-free contrast mechanisms by employing several fundamental light–molecule interactions including excited state absorption, stimulated emission, ground state depletion, and the photothermal effect. By using the pump pulse to excite molecules and the subsequent probe pulse to interrogate the created transient states on a laser scanning microscope, pump-probe microscopy offers imaging capability with high sensitivity and specificity toward nonfluorescent chromophores. Single-molecule sensitivity has even been demonstrated. Here we review and summarize the underlying principles of this emerging class of molecular imaging techniques. Figure Apparatus used for pump-probe microscopy
Keywords: Pump-probe microscopy; Label-free imaging; Excited state absorption; Stimulated emission; Ground state depletion; Nonfluorescent chromophore
Conformational analyses of peptides and proteins by vibrational Raman optical activity by Shigeki Yamamoto (pp. 2203-2212).
has been a post-doctoral fellow at Kwansei Gakuin University since November 2011. His current research interests are the development of analytical methods for solution structures of proteins from Raman optical activity spectra and quantum mechanical calculations. Recent developments of vibrational Raman optical activity (ROA) spectroscopy enabled the detailed analyses of the backbone and side chain conformations of peptides and proteins in solution phases. ROA can be used as a powerful analytical technique for determining not only the structures of conformers, but also their populations even for systems in fast conformational equilibria where NMR spectroscopy is difficult to be applied. ROA enabled the monitoring of the secondary structures of denatured or unfolded proteins, such as an amyloid fibril and its prefibril intermediates.
Keywords: Raman optical activity; ROA; Peptide conformation; Solution structure; Amyloid fibril
Conformational analyses of peptides and proteins by vibrational Raman optical activity by Shigeki Yamamoto (pp. 2203-2212).
has been a post-doctoral fellow at Kwansei Gakuin University since November 2011. His current research interests are the development of analytical methods for solution structures of proteins from Raman optical activity spectra and quantum mechanical calculations. Recent developments of vibrational Raman optical activity (ROA) spectroscopy enabled the detailed analyses of the backbone and side chain conformations of peptides and proteins in solution phases. ROA can be used as a powerful analytical technique for determining not only the structures of conformers, but also their populations even for systems in fast conformational equilibria where NMR spectroscopy is difficult to be applied. ROA enabled the monitoring of the secondary structures of denatured or unfolded proteins, such as an amyloid fibril and its prefibril intermediates.
Keywords: Raman optical activity; ROA; Peptide conformation; Solution structure; Amyloid fibril
Conformational analyses of peptides and proteins by vibrational Raman optical activity by Shigeki Yamamoto (pp. 2203-2212).
has been a post-doctoral fellow at Kwansei Gakuin University since November 2011. His current research interests are the development of analytical methods for solution structures of proteins from Raman optical activity spectra and quantum mechanical calculations. Recent developments of vibrational Raman optical activity (ROA) spectroscopy enabled the detailed analyses of the backbone and side chain conformations of peptides and proteins in solution phases. ROA can be used as a powerful analytical technique for determining not only the structures of conformers, but also their populations even for systems in fast conformational equilibria where NMR spectroscopy is difficult to be applied. ROA enabled the monitoring of the secondary structures of denatured or unfolded proteins, such as an amyloid fibril and its prefibril intermediates.
Keywords: Raman optical activity; ROA; Peptide conformation; Solution structure; Amyloid fibril
Extraction and stirring integrated techniques: examples and recent advances by Rafael Lucena (pp. 2213-2223).
has been Associate Professor in the Analytical Chemistry department of the University of Cordoba (Spain) since 2010. His main research interests are the development of new microextraction techniques and the evaluation of ionic liquids and carbon nanostructures in this context. Apart from this main topic, he also works on the development of mid-IR sensors and their applications in industrial process control. He has co-authored approximately 40 scientific articles and three book chapters on these topics. Extraction techniques, which focus on selectivity and sensitivity enhancement by isolation and preconcentration of target analytes, are essential in many analytical methods. Because many extraction techniques occur under diffusion-controlled conditions, stirring of the sample solution is required to accelerate the extraction by favoring diffusion of the analytes from the bulk solution to the extractant phase. This stirring may be performed by use of an external device or by integrating extraction and stirring in the same device. This review focuses on the latter techniques, which are promising methods for sample treatment. First, stir-bar-sorptive extraction, the most widely used method, is considered, paying special attention to the development of new coatings. Finally, a general overview of novel integrated techniques in both solid-phase and liquid-phase microextraction is presented; their main characteristics and marked trends are reported. Figure
Keywords: Stir-bar-sorptive extraction; New coatings; Rotating disk-sorptive extraction; Stir-rod-sorptive extraction; Stir-membrane extraction; Liquid-phase microextraction
Extraction and stirring integrated techniques: examples and recent advances by Rafael Lucena (pp. 2213-2223).
has been Associate Professor in the Analytical Chemistry department of the University of Cordoba (Spain) since 2010. His main research interests are the development of new microextraction techniques and the evaluation of ionic liquids and carbon nanostructures in this context. Apart from this main topic, he also works on the development of mid-IR sensors and their applications in industrial process control. He has co-authored approximately 40 scientific articles and three book chapters on these topics. Extraction techniques, which focus on selectivity and sensitivity enhancement by isolation and preconcentration of target analytes, are essential in many analytical methods. Because many extraction techniques occur under diffusion-controlled conditions, stirring of the sample solution is required to accelerate the extraction by favoring diffusion of the analytes from the bulk solution to the extractant phase. This stirring may be performed by use of an external device or by integrating extraction and stirring in the same device. This review focuses on the latter techniques, which are promising methods for sample treatment. First, stir-bar-sorptive extraction, the most widely used method, is considered, paying special attention to the development of new coatings. Finally, a general overview of novel integrated techniques in both solid-phase and liquid-phase microextraction is presented; their main characteristics and marked trends are reported. Figure
Keywords: Stir-bar-sorptive extraction; New coatings; Rotating disk-sorptive extraction; Stir-rod-sorptive extraction; Stir-membrane extraction; Liquid-phase microextraction
Extraction and stirring integrated techniques: examples and recent advances by Rafael Lucena (pp. 2213-2223).
has been Associate Professor in the Analytical Chemistry department of the University of Cordoba (Spain) since 2010. His main research interests are the development of new microextraction techniques and the evaluation of ionic liquids and carbon nanostructures in this context. Apart from this main topic, he also works on the development of mid-IR sensors and their applications in industrial process control. He has co-authored approximately 40 scientific articles and three book chapters on these topics. Extraction techniques, which focus on selectivity and sensitivity enhancement by isolation and preconcentration of target analytes, are essential in many analytical methods. Because many extraction techniques occur under diffusion-controlled conditions, stirring of the sample solution is required to accelerate the extraction by favoring diffusion of the analytes from the bulk solution to the extractant phase. This stirring may be performed by use of an external device or by integrating extraction and stirring in the same device. This review focuses on the latter techniques, which are promising methods for sample treatment. First, stir-bar-sorptive extraction, the most widely used method, is considered, paying special attention to the development of new coatings. Finally, a general overview of novel integrated techniques in both solid-phase and liquid-phase microextraction is presented; their main characteristics and marked trends are reported. Figure
Keywords: Stir-bar-sorptive extraction; New coatings; Rotating disk-sorptive extraction; Stir-rod-sorptive extraction; Stir-membrane extraction; Liquid-phase microextraction
Advances and trends in the design, analysis, and characterization of polymer–protein conjugates for “PEGylaided” bioprocesses by José González-Valdez; Marco Rito-Palomares; Jorge Benavides (pp. 2225-2235).
has been Assistant Professor of the Biotechnology and Food Engineering Department at Tecnológico de Monterrey (ITESM, Campus Monterrey) since January 2007. His current research interests are developing bioprocesses for production and purification of high-value biomolecules, optimizing methods for protein analysis, and exploring new application for PEGylated proteins In addition to their use as therapeutics and because of their enhanced properties, PEGylated proteins have potential application in fields such as bioprocessing. However, the use of PEGylated conjugates to improve the performance of bioprocess has not been widely explored. This limited additional industrial use of PEG–protein conjugates can be attributed to the fact that PEGylation reactions, separation of the products, and final characterization of the structure and activity of the resulting species are not trivial tasks. The development of bioprocessing operations based on PEGylated proteins relies heavily in the use of analytical tools that must sometimes be adapted from the strategies used in pharmaceutical conjugate development. For instance, to evaluate conjugate performance in bioprocessing operations, both chromatographic and non-chromatographic steps must be used to separate and quantify the resulting reaction species. Characterization of the conjugates by mass spectrometry, circular dichroism, and specific activity assays, among other adapted techniques, is then required to evaluate the feasibility of using the conjugates in any operation. Correct selection of the technical and analytical methods in each of the steps from design of the PEGylation reaction to its final engineering application will ensure success in implementing a “PEGylaided” process. In this context, the objective of this review is to describe technological and analytical trends in developing successful applications of PEGylated conjugates in bioprocesses and to describe potential fields in which these proteins can be exploited.
Keywords: PEGylation; PEG–protein conjugates; Bioprocessing
Advances and trends in the design, analysis, and characterization of polymer–protein conjugates for “PEGylaided” bioprocesses by José González-Valdez; Marco Rito-Palomares; Jorge Benavides (pp. 2225-2235).
has been Assistant Professor of the Biotechnology and Food Engineering Department at Tecnológico de Monterrey (ITESM, Campus Monterrey) since January 2007. His current research interests are developing bioprocesses for production and purification of high-value biomolecules, optimizing methods for protein analysis, and exploring new application for PEGylated proteins In addition to their use as therapeutics and because of their enhanced properties, PEGylated proteins have potential application in fields such as bioprocessing. However, the use of PEGylated conjugates to improve the performance of bioprocess has not been widely explored. This limited additional industrial use of PEG–protein conjugates can be attributed to the fact that PEGylation reactions, separation of the products, and final characterization of the structure and activity of the resulting species are not trivial tasks. The development of bioprocessing operations based on PEGylated proteins relies heavily in the use of analytical tools that must sometimes be adapted from the strategies used in pharmaceutical conjugate development. For instance, to evaluate conjugate performance in bioprocessing operations, both chromatographic and non-chromatographic steps must be used to separate and quantify the resulting reaction species. Characterization of the conjugates by mass spectrometry, circular dichroism, and specific activity assays, among other adapted techniques, is then required to evaluate the feasibility of using the conjugates in any operation. Correct selection of the technical and analytical methods in each of the steps from design of the PEGylation reaction to its final engineering application will ensure success in implementing a “PEGylaided” process. In this context, the objective of this review is to describe technological and analytical trends in developing successful applications of PEGylated conjugates in bioprocesses and to describe potential fields in which these proteins can be exploited.
Keywords: PEGylation; PEG–protein conjugates; Bioprocessing
Advances and trends in the design, analysis, and characterization of polymer–protein conjugates for “PEGylaided” bioprocesses by José González-Valdez; Marco Rito-Palomares; Jorge Benavides (pp. 2225-2235).
has been Assistant Professor of the Biotechnology and Food Engineering Department at Tecnológico de Monterrey (ITESM, Campus Monterrey) since January 2007. His current research interests are developing bioprocesses for production and purification of high-value biomolecules, optimizing methods for protein analysis, and exploring new application for PEGylated proteins In addition to their use as therapeutics and because of their enhanced properties, PEGylated proteins have potential application in fields such as bioprocessing. However, the use of PEGylated conjugates to improve the performance of bioprocess has not been widely explored. This limited additional industrial use of PEG–protein conjugates can be attributed to the fact that PEGylation reactions, separation of the products, and final characterization of the structure and activity of the resulting species are not trivial tasks. The development of bioprocessing operations based on PEGylated proteins relies heavily in the use of analytical tools that must sometimes be adapted from the strategies used in pharmaceutical conjugate development. For instance, to evaluate conjugate performance in bioprocessing operations, both chromatographic and non-chromatographic steps must be used to separate and quantify the resulting reaction species. Characterization of the conjugates by mass spectrometry, circular dichroism, and specific activity assays, among other adapted techniques, is then required to evaluate the feasibility of using the conjugates in any operation. Correct selection of the technical and analytical methods in each of the steps from design of the PEGylation reaction to its final engineering application will ensure success in implementing a “PEGylaided” process. In this context, the objective of this review is to describe technological and analytical trends in developing successful applications of PEGylated conjugates in bioprocesses and to describe potential fields in which these proteins can be exploited.
Keywords: PEGylation; PEG–protein conjugates; Bioprocessing
Biological responses related to agonistic, antagonistic and synergistic interactions of chemical species by T. García-Barrera; J. L. Gómez-Ariza; M. González-Fernández; F. Moreno; M. A. García-Sevillano; V. Gómez-Jacinto (pp. 2237-2253).
has been an associate professor of analytical chemistry at the University of Huelva (Spain) since 2009. Her research interests focus on environmental studies, such as the speciation of Hg/Se and As/Se, and metallomics. Her expertise includes liquid and gas chromatography, atomic spectroscopy, mass spectrometry and sample treatment. She has been honoured with the Andalusian Government’s award for young researchers “X Premio Tercer Milenio”, as well as the “Extraordinary Ph.D. Prize” from the University of Huelva, and prizes from the Illustrious Official College of Chemists of Seville, the Association of Chemical and Basic Industries of Huelva, the Regional Andalusian Group of the Spanish Society of Analytical Chemistry, and the Academy of Sciences, Arts and Letters of Huelva. The fact that the essential or toxic character of elements is species specific has encouraged the development of analytical strategies for chemical speciation over the last twenty years; indeed, there are now a great number of them that provide very good performance. However, biological systems are exposed to a complex environment in which species of elements can interact in a synergistic/antagonistic fashion. Thus, the metabolism of trace elements cannot be considered in isolation. On the other hand, biological systems are dynamic, so it is necessary to study the trafficking of species of elements between organs, tissues or cell compartments in order to decipher the biochemical processes of the interactions in which they are involved. Although the application of liquid chromatography–inductively coupled plasma-based “metallomics” methods in combination with organic mass spectrometry can provide much-needed insight, new analytical strategies are required to really understand the role of species of elements in biological systems and the mechanisms of their interactions. In the present paper, the interactions of the most widely studied elements in this context (Se, Hg and As) are discussed, as well as other important interactions between different elements. Figure Interactions of chemical species in biology
Keywords: Speciation; Metals; Agonists; Synergist; Antagonist; Trafficking; Multispeciation; Mass spectrometry; ICP-MS
Biological responses related to agonistic, antagonistic and synergistic interactions of chemical species by T. García-Barrera; J. L. Gómez-Ariza; M. González-Fernández; F. Moreno; M. A. García-Sevillano; V. Gómez-Jacinto (pp. 2237-2253).
has been an associate professor of analytical chemistry at the University of Huelva (Spain) since 2009. Her research interests focus on environmental studies, such as the speciation of Hg/Se and As/Se, and metallomics. Her expertise includes liquid and gas chromatography, atomic spectroscopy, mass spectrometry and sample treatment. She has been honoured with the Andalusian Government’s award for young researchers “X Premio Tercer Milenio”, as well as the “Extraordinary Ph.D. Prize” from the University of Huelva, and prizes from the Illustrious Official College of Chemists of Seville, the Association of Chemical and Basic Industries of Huelva, the Regional Andalusian Group of the Spanish Society of Analytical Chemistry, and the Academy of Sciences, Arts and Letters of Huelva. The fact that the essential or toxic character of elements is species specific has encouraged the development of analytical strategies for chemical speciation over the last twenty years; indeed, there are now a great number of them that provide very good performance. However, biological systems are exposed to a complex environment in which species of elements can interact in a synergistic/antagonistic fashion. Thus, the metabolism of trace elements cannot be considered in isolation. On the other hand, biological systems are dynamic, so it is necessary to study the trafficking of species of elements between organs, tissues or cell compartments in order to decipher the biochemical processes of the interactions in which they are involved. Although the application of liquid chromatography–inductively coupled plasma-based “metallomics” methods in combination with organic mass spectrometry can provide much-needed insight, new analytical strategies are required to really understand the role of species of elements in biological systems and the mechanisms of their interactions. In the present paper, the interactions of the most widely studied elements in this context (Se, Hg and As) are discussed, as well as other important interactions between different elements. Figure Interactions of chemical species in biology
Keywords: Speciation; Metals; Agonists; Synergist; Antagonist; Trafficking; Multispeciation; Mass spectrometry; ICP-MS
Biological responses related to agonistic, antagonistic and synergistic interactions of chemical species by T. García-Barrera; J. L. Gómez-Ariza; M. González-Fernández; F. Moreno; M. A. García-Sevillano; V. Gómez-Jacinto (pp. 2237-2253).
has been an associate professor of analytical chemistry at the University of Huelva (Spain) since 2009. Her research interests focus on environmental studies, such as the speciation of Hg/Se and As/Se, and metallomics. Her expertise includes liquid and gas chromatography, atomic spectroscopy, mass spectrometry and sample treatment. She has been honoured with the Andalusian Government’s award for young researchers “X Premio Tercer Milenio”, as well as the “Extraordinary Ph.D. Prize” from the University of Huelva, and prizes from the Illustrious Official College of Chemists of Seville, the Association of Chemical and Basic Industries of Huelva, the Regional Andalusian Group of the Spanish Society of Analytical Chemistry, and the Academy of Sciences, Arts and Letters of Huelva. The fact that the essential or toxic character of elements is species specific has encouraged the development of analytical strategies for chemical speciation over the last twenty years; indeed, there are now a great number of them that provide very good performance. However, biological systems are exposed to a complex environment in which species of elements can interact in a synergistic/antagonistic fashion. Thus, the metabolism of trace elements cannot be considered in isolation. On the other hand, biological systems are dynamic, so it is necessary to study the trafficking of species of elements between organs, tissues or cell compartments in order to decipher the biochemical processes of the interactions in which they are involved. Although the application of liquid chromatography–inductively coupled plasma-based “metallomics” methods in combination with organic mass spectrometry can provide much-needed insight, new analytical strategies are required to really understand the role of species of elements in biological systems and the mechanisms of their interactions. In the present paper, the interactions of the most widely studied elements in this context (Se, Hg and As) are discussed, as well as other important interactions between different elements. Figure Interactions of chemical species in biology
Keywords: Speciation; Metals; Agonists; Synergist; Antagonist; Trafficking; Multispeciation; Mass spectrometry; ICP-MS
Dual labeling of biomolecules using MeCAT and DOTA derivatives: application to quantitative proteomics by Ahmed H. El-Khatib; Diego Esteban-Fernández; Michael W. Linscheid (pp. 2255-2267).
is a post-doctoral researcher at the Humboldt Universitaet zu Berlin (Department of Chemistry, Prof. Dr. Michael W. Linscheid group). He has worked in several prestigious research groups related with speciation analysis and was part of the Spanish National Reference Laboratory for heavy metals in food and feed. Nowadays, his scientific interest includes the combined used of elemental and molecular mass spectrometry for metallomics studies and quantification of biomolecules. In this study, single and dual labeling of primary amino and thiol groups of target peptides is presented as a proof of concept. The proposed method allows flexible, independent and sequential labeling of the mentioned residues using lanthanides introduced via DOTA-complexes (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The efficiency of the method was optimized using cysteine-containing standard peptides and then applied to bovine serum albumin (BSA) and human serum albumin (HSA) to demonstrate qualitative and quantitative aspects of this strategy. For amino labeling, cysteinyl peptides were immobilized on Sepharose-6B resin and labeled with DOTA-NHS ester followed by metallation with lanthanides. Thiol labeling was carried out using lanthanide-containing metal-coded affinity tags (MeCAT) after elution of peptides from the resin. Complete dual labeling of the standard peptides was demonstrated by liquid chromatography electrospray ionization mass spectrometry, whereas more than 80 % of the detected peptides of BSA and HSA were completely dual-labeled. Parallel detection by LC coupled to inductively coupled plasma mass spectrometry (ICP-MS) delivered reliable quantitative information. Thus, the versatile lanthanide choice in both labeling steps allowed estimating primary amino and thiol stoichiometries for the studied samples using different lanthanides. On the other hand, enhancement of ICP-MS signal was achieved as expected when all positions were labeled with the same lanthanide. Finally, linear calibrations of the signal for most of the labeled peptides by standard additions of digested BSA showed a suitable behaviour for quantitative applications and demonstrated the pre-concentration capability of the employed resin. Figure
Keywords: Peptide/protein quantification; Amino and thiol labeling; DOTA-NHS; MeCAT; ICP-MS; On-resin labeling
Dual labeling of biomolecules using MeCAT and DOTA derivatives: application to quantitative proteomics by Ahmed H. El-Khatib; Diego Esteban-Fernández; Michael W. Linscheid (pp. 2255-2267).
is a post-doctoral researcher at the Humboldt Universitaet zu Berlin (Department of Chemistry, Prof. Dr. Michael W. Linscheid group). He has worked in several prestigious research groups related with speciation analysis and was part of the Spanish National Reference Laboratory for heavy metals in food and feed. Nowadays, his scientific interest includes the combined used of elemental and molecular mass spectrometry for metallomics studies and quantification of biomolecules. In this study, single and dual labeling of primary amino and thiol groups of target peptides is presented as a proof of concept. The proposed method allows flexible, independent and sequential labeling of the mentioned residues using lanthanides introduced via DOTA-complexes (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The efficiency of the method was optimized using cysteine-containing standard peptides and then applied to bovine serum albumin (BSA) and human serum albumin (HSA) to demonstrate qualitative and quantitative aspects of this strategy. For amino labeling, cysteinyl peptides were immobilized on Sepharose-6B resin and labeled with DOTA-NHS ester followed by metallation with lanthanides. Thiol labeling was carried out using lanthanide-containing metal-coded affinity tags (MeCAT) after elution of peptides from the resin. Complete dual labeling of the standard peptides was demonstrated by liquid chromatography electrospray ionization mass spectrometry, whereas more than 80 % of the detected peptides of BSA and HSA were completely dual-labeled. Parallel detection by LC coupled to inductively coupled plasma mass spectrometry (ICP-MS) delivered reliable quantitative information. Thus, the versatile lanthanide choice in both labeling steps allowed estimating primary amino and thiol stoichiometries for the studied samples using different lanthanides. On the other hand, enhancement of ICP-MS signal was achieved as expected when all positions were labeled with the same lanthanide. Finally, linear calibrations of the signal for most of the labeled peptides by standard additions of digested BSA showed a suitable behaviour for quantitative applications and demonstrated the pre-concentration capability of the employed resin. Figure
Keywords: Peptide/protein quantification; Amino and thiol labeling; DOTA-NHS; MeCAT; ICP-MS; On-resin labeling
Dual labeling of biomolecules using MeCAT and DOTA derivatives: application to quantitative proteomics by Ahmed H. El-Khatib; Diego Esteban-Fernández; Michael W. Linscheid (pp. 2255-2267).
is a post-doctoral researcher at the Humboldt Universitaet zu Berlin (Department of Chemistry, Prof. Dr. Michael W. Linscheid group). He has worked in several prestigious research groups related with speciation analysis and was part of the Spanish National Reference Laboratory for heavy metals in food and feed. Nowadays, his scientific interest includes the combined used of elemental and molecular mass spectrometry for metallomics studies and quantification of biomolecules. In this study, single and dual labeling of primary amino and thiol groups of target peptides is presented as a proof of concept. The proposed method allows flexible, independent and sequential labeling of the mentioned residues using lanthanides introduced via DOTA-complexes (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The efficiency of the method was optimized using cysteine-containing standard peptides and then applied to bovine serum albumin (BSA) and human serum albumin (HSA) to demonstrate qualitative and quantitative aspects of this strategy. For amino labeling, cysteinyl peptides were immobilized on Sepharose-6B resin and labeled with DOTA-NHS ester followed by metallation with lanthanides. Thiol labeling was carried out using lanthanide-containing metal-coded affinity tags (MeCAT) after elution of peptides from the resin. Complete dual labeling of the standard peptides was demonstrated by liquid chromatography electrospray ionization mass spectrometry, whereas more than 80 % of the detected peptides of BSA and HSA were completely dual-labeled. Parallel detection by LC coupled to inductively coupled plasma mass spectrometry (ICP-MS) delivered reliable quantitative information. Thus, the versatile lanthanide choice in both labeling steps allowed estimating primary amino and thiol stoichiometries for the studied samples using different lanthanides. On the other hand, enhancement of ICP-MS signal was achieved as expected when all positions were labeled with the same lanthanide. Finally, linear calibrations of the signal for most of the labeled peptides by standard additions of digested BSA showed a suitable behaviour for quantitative applications and demonstrated the pre-concentration capability of the employed resin. Figure
Keywords: Peptide/protein quantification; Amino and thiol labeling; DOTA-NHS; MeCAT; ICP-MS; On-resin labeling
Facile identification of photocleavable reactive metabolites and oxidative stress biomarkers in proteins via mass spectrometry by Jolene K. Diedrich; Ryan R. Julian (pp. 2269-2277).
has been at the University of California at Riverside since 2005 and is presently an Associate Professor in the Department of Chemistry. He was recipient of the NSF Career Award in 2008 and the ASMS Research Award in 2006. The Julian group focuses on the development of new techniques utilizing mass spectrometry to study protein structure and folding, post-translational modifications, and radical directed protein sequencing. Described herein is a method which combines bond selective fragmentation by photodissociation with online liquid chromatographic separation and mass spectrometric analysis. Photoexcitation of proteins or peptides with 266-nm light does not normally yield abundant fragmentation; however, incorporation of a suitable carbon–sulfur or carbon–halogen bond that is proximal to a chromophore allows access to direct dissociation pathways, resulting in homolytic cleavage of these bonds. Radicals generated through this process can cause further dissociation of the peptide backbone, which is useful for site specifically identifying the point of modification. Two specific applications of this technique for peptide analysis in model systems are presented: (1) identification of reactive metabolites which covalently modify cysteine residues, and (2) characterization of halogenated tyrosine residues which are biomarkers related to oxidative stress. In both cases, these naturally occurring post translational modifications create photocleavable bonds which can be fragmented by 266-nm light. The selectivity offered by photodissociation allows facile identification of the peptides of interest even in complex mixtures, and subsequent selective radical directed backbone fragmentation pinpoints the site of modification. This combination greatly simplifies data analysis and provides more confident assignments. Fig Photocleavable metabolites are easily identified by mass specific losses.
Keywords: Thyroglobulin; Iodine; Quinone; Radical; Myeloperoxidase; Proteomics
Facile identification of photocleavable reactive metabolites and oxidative stress biomarkers in proteins via mass spectrometry by Jolene K. Diedrich; Ryan R. Julian (pp. 2269-2277).
has been at the University of California at Riverside since 2005 and is presently an Associate Professor in the Department of Chemistry. He was recipient of the NSF Career Award in 2008 and the ASMS Research Award in 2006. The Julian group focuses on the development of new techniques utilizing mass spectrometry to study protein structure and folding, post-translational modifications, and radical directed protein sequencing. Described herein is a method which combines bond selective fragmentation by photodissociation with online liquid chromatographic separation and mass spectrometric analysis. Photoexcitation of proteins or peptides with 266-nm light does not normally yield abundant fragmentation; however, incorporation of a suitable carbon–sulfur or carbon–halogen bond that is proximal to a chromophore allows access to direct dissociation pathways, resulting in homolytic cleavage of these bonds. Radicals generated through this process can cause further dissociation of the peptide backbone, which is useful for site specifically identifying the point of modification. Two specific applications of this technique for peptide analysis in model systems are presented: (1) identification of reactive metabolites which covalently modify cysteine residues, and (2) characterization of halogenated tyrosine residues which are biomarkers related to oxidative stress. In both cases, these naturally occurring post translational modifications create photocleavable bonds which can be fragmented by 266-nm light. The selectivity offered by photodissociation allows facile identification of the peptides of interest even in complex mixtures, and subsequent selective radical directed backbone fragmentation pinpoints the site of modification. This combination greatly simplifies data analysis and provides more confident assignments. Fig Photocleavable metabolites are easily identified by mass specific losses.
Keywords: Thyroglobulin; Iodine; Quinone; Radical; Myeloperoxidase; Proteomics
Facile identification of photocleavable reactive metabolites and oxidative stress biomarkers in proteins via mass spectrometry by Jolene K. Diedrich; Ryan R. Julian (pp. 2269-2277).
has been at the University of California at Riverside since 2005 and is presently an Associate Professor in the Department of Chemistry. He was recipient of the NSF Career Award in 2008 and the ASMS Research Award in 2006. The Julian group focuses on the development of new techniques utilizing mass spectrometry to study protein structure and folding, post-translational modifications, and radical directed protein sequencing. Described herein is a method which combines bond selective fragmentation by photodissociation with online liquid chromatographic separation and mass spectrometric analysis. Photoexcitation of proteins or peptides with 266-nm light does not normally yield abundant fragmentation; however, incorporation of a suitable carbon–sulfur or carbon–halogen bond that is proximal to a chromophore allows access to direct dissociation pathways, resulting in homolytic cleavage of these bonds. Radicals generated through this process can cause further dissociation of the peptide backbone, which is useful for site specifically identifying the point of modification. Two specific applications of this technique for peptide analysis in model systems are presented: (1) identification of reactive metabolites which covalently modify cysteine residues, and (2) characterization of halogenated tyrosine residues which are biomarkers related to oxidative stress. In both cases, these naturally occurring post translational modifications create photocleavable bonds which can be fragmented by 266-nm light. The selectivity offered by photodissociation allows facile identification of the peptides of interest even in complex mixtures, and subsequent selective radical directed backbone fragmentation pinpoints the site of modification. This combination greatly simplifies data analysis and provides more confident assignments. Fig Photocleavable metabolites are easily identified by mass specific losses.
Keywords: Thyroglobulin; Iodine; Quinone; Radical; Myeloperoxidase; Proteomics
LC-MS/MS biopharmaceutical glycoanalysis: identification of desirable reference material characteristics by John E. Schiel; Jennifer Au; Hua-Jun He; Karen W. Phinney (pp. 2279-2289).
has been a Research Chemist at the National Institute of Standards and Technology since July 2009. He was the recipient of the Bioanalysis Young Investigator Award and an ACS Division of Analytical Chemistry Fellowship. John’s research is directed toward improving the reliability of mass spectrometry-based glycoprotein characterization through reference material development. Glycosylation, the enzymatic addition of carbohydrates to a protein, is one of the most abundant post-translational modifications found in nature. There is variability in the number, location, and identity of glycans attached. As a result, a glycoprotein consists of a number of glycoforms with different combinations of glycans, potentially resulting in different stability, toxicity, and activity. This is especially important in the biopharmaceutical industry where product consistency and safety are vital. Glycoprotein analysis involves numerous mass spectrometry based techniques, each of which provides various aspects of characterization. The current paper describes two commonly used analytical techniques for glycoprotein characterization. In one experiment, nonspecific proteolysis is combined with a two-tiered mass spectrometry approach (MALDI-TOF and LC-MS/MS) to gain glycosylation site and glycan identity. In a second approach, glycans were enzymatically released, labeled with a fluorescent dye, and analyzed using LC-Fluorescence-MS/MS to give glycan identification and relative quantification. The type and degree of information yielded by each method is assessed in an effort to identify desired reference material characteristics for improving biopharmaceutical glycoanalysis.
Keywords: Biopharmaceutical; Glycoprotein; Glycan; Mass spectrometry; Standards; Reference material
LC-MS/MS biopharmaceutical glycoanalysis: identification of desirable reference material characteristics by John E. Schiel; Jennifer Au; Hua-Jun He; Karen W. Phinney (pp. 2279-2289).
has been a Research Chemist at the National Institute of Standards and Technology since July 2009. He was the recipient of the Bioanalysis Young Investigator Award and an ACS Division of Analytical Chemistry Fellowship. John’s research is directed toward improving the reliability of mass spectrometry-based glycoprotein characterization through reference material development. Glycosylation, the enzymatic addition of carbohydrates to a protein, is one of the most abundant post-translational modifications found in nature. There is variability in the number, location, and identity of glycans attached. As a result, a glycoprotein consists of a number of glycoforms with different combinations of glycans, potentially resulting in different stability, toxicity, and activity. This is especially important in the biopharmaceutical industry where product consistency and safety are vital. Glycoprotein analysis involves numerous mass spectrometry based techniques, each of which provides various aspects of characterization. The current paper describes two commonly used analytical techniques for glycoprotein characterization. In one experiment, nonspecific proteolysis is combined with a two-tiered mass spectrometry approach (MALDI-TOF and LC-MS/MS) to gain glycosylation site and glycan identity. In a second approach, glycans were enzymatically released, labeled with a fluorescent dye, and analyzed using LC-Fluorescence-MS/MS to give glycan identification and relative quantification. The type and degree of information yielded by each method is assessed in an effort to identify desired reference material characteristics for improving biopharmaceutical glycoanalysis.
Keywords: Biopharmaceutical; Glycoprotein; Glycan; Mass spectrometry; Standards; Reference material
LC-MS/MS biopharmaceutical glycoanalysis: identification of desirable reference material characteristics by John E. Schiel; Jennifer Au; Hua-Jun He; Karen W. Phinney (pp. 2279-2289).
has been a Research Chemist at the National Institute of Standards and Technology since July 2009. He was the recipient of the Bioanalysis Young Investigator Award and an ACS Division of Analytical Chemistry Fellowship. John’s research is directed toward improving the reliability of mass spectrometry-based glycoprotein characterization through reference material development. Glycosylation, the enzymatic addition of carbohydrates to a protein, is one of the most abundant post-translational modifications found in nature. There is variability in the number, location, and identity of glycans attached. As a result, a glycoprotein consists of a number of glycoforms with different combinations of glycans, potentially resulting in different stability, toxicity, and activity. This is especially important in the biopharmaceutical industry where product consistency and safety are vital. Glycoprotein analysis involves numerous mass spectrometry based techniques, each of which provides various aspects of characterization. The current paper describes two commonly used analytical techniques for glycoprotein characterization. In one experiment, nonspecific proteolysis is combined with a two-tiered mass spectrometry approach (MALDI-TOF and LC-MS/MS) to gain glycosylation site and glycan identity. In a second approach, glycans were enzymatically released, labeled with a fluorescent dye, and analyzed using LC-Fluorescence-MS/MS to give glycan identification and relative quantification. The type and degree of information yielded by each method is assessed in an effort to identify desired reference material characteristics for improving biopharmaceutical glycoanalysis.
Keywords: Biopharmaceutical; Glycoprotein; Glycan; Mass spectrometry; Standards; Reference material
Analysis of carbon and nitrogen co-metabolism in yeast by ultrahigh-resolution mass spectrometry applying 13C- and 15N-labeled substrates simultaneously by Lars M. Blank; Rahul R. Desphande; Andreas Schmid; Heiko Hayen (pp. 2291-2305).
is Professor in the Department of Food Chemistry, University of Wuppertal in Germany. He has received the Award for Younger Chemists from the Division Analytical Chemistry of the German Chemical Society (2007). His current research interests include the development of analytical methods for the determination of metabolites, lipids, and organic pollutants with particular emphasis on LC/MS and novel ionization techniques. Alternative metabolic pathways inside a cell can be deduced using stable isotopically labeled substrates. One prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of single-element isotopes, mainly 13C. Here, we demonstrate the application of direct infusion nanospray, ultrahigh-resolution Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) for metabolic studies using differently labeled elemental isotopes simultaneously—i.e., 13C and 15N—in amino acids of a total protein hydrolysate. The optimized strategy for the analysis of metabolism by a hybrid linear ion trap-FTICR-MS comprises the collection of multiple adjacent selected ion monitoring scans. By limiting both the width of the mass range and the number of ions entering the ICR cell with automated gain control, sensitive measurements of isotopologue distribution were possible without compromising mass accuracy and isotope intensity mapping. The required mass-resolving power of more than 60,000 is only achievable on a routine basis by FTICR and Orbitrap mass spectrometers. Evaluation of the method was carried out by comparison of the experimental data to the natural isotope abundances of selected amino acids and by comparison to GC/MS results obtained from a labeling experiment with 13C-labeled glucose. The developed method was used to shed light on the complexity of the yeast Saccharomyces cerevisiae carbon–nitrogen co-metabolism by administering both 13C-labeled glucose and 15N-labeled alanine. The results indicate that not only glutamate but also alanine acts as an amino donor during alanine and valine synthesis. Metabolic studies using FTICR-MS can exploit new possibilities by the use of multiple-labeled elemental isotopes. Figure Analysis of carbon and nitrogen co-metabolism in yeast by nanospray-FTICR-MS determination of 13C and 15N incorporation into proteinogenic amino acids
Keywords: 13C- and 15N-labeling experiment; Mass spectrometry; Yeast; Saccharomyces cerevisiae ; Amino acid metabolism
Analysis of carbon and nitrogen co-metabolism in yeast by ultrahigh-resolution mass spectrometry applying 13C- and 15N-labeled substrates simultaneously by Lars M. Blank; Rahul R. Desphande; Andreas Schmid; Heiko Hayen (pp. 2291-2305).
is Professor in the Department of Food Chemistry, University of Wuppertal in Germany. He has received the Award for Younger Chemists from the Division Analytical Chemistry of the German Chemical Society (2007). His current research interests include the development of analytical methods for the determination of metabolites, lipids, and organic pollutants with particular emphasis on LC/MS and novel ionization techniques. Alternative metabolic pathways inside a cell can be deduced using stable isotopically labeled substrates. One prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of single-element isotopes, mainly 13C. Here, we demonstrate the application of direct infusion nanospray, ultrahigh-resolution Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) for metabolic studies using differently labeled elemental isotopes simultaneously—i.e., 13C and 15N—in amino acids of a total protein hydrolysate. The optimized strategy for the analysis of metabolism by a hybrid linear ion trap-FTICR-MS comprises the collection of multiple adjacent selected ion monitoring scans. By limiting both the width of the mass range and the number of ions entering the ICR cell with automated gain control, sensitive measurements of isotopologue distribution were possible without compromising mass accuracy and isotope intensity mapping. The required mass-resolving power of more than 60,000 is only achievable on a routine basis by FTICR and Orbitrap mass spectrometers. Evaluation of the method was carried out by comparison of the experimental data to the natural isotope abundances of selected amino acids and by comparison to GC/MS results obtained from a labeling experiment with 13C-labeled glucose. The developed method was used to shed light on the complexity of the yeast Saccharomyces cerevisiae carbon–nitrogen co-metabolism by administering both 13C-labeled glucose and 15N-labeled alanine. The results indicate that not only glutamate but also alanine acts as an amino donor during alanine and valine synthesis. Metabolic studies using FTICR-MS can exploit new possibilities by the use of multiple-labeled elemental isotopes. Figure Analysis of carbon and nitrogen co-metabolism in yeast by nanospray-FTICR-MS determination of 13C and 15N incorporation into proteinogenic amino acids
Keywords: 13C- and 15N-labeling experiment; Mass spectrometry; Yeast; Saccharomyces cerevisiae ; Amino acid metabolism
Analysis of carbon and nitrogen co-metabolism in yeast by ultrahigh-resolution mass spectrometry applying 13C- and 15N-labeled substrates simultaneously by Lars M. Blank; Rahul R. Desphande; Andreas Schmid; Heiko Hayen (pp. 2291-2305).
is Professor in the Department of Food Chemistry, University of Wuppertal in Germany. He has received the Award for Younger Chemists from the Division Analytical Chemistry of the German Chemical Society (2007). His current research interests include the development of analytical methods for the determination of metabolites, lipids, and organic pollutants with particular emphasis on LC/MS and novel ionization techniques. Alternative metabolic pathways inside a cell can be deduced using stable isotopically labeled substrates. One prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of single-element isotopes, mainly 13C. Here, we demonstrate the application of direct infusion nanospray, ultrahigh-resolution Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) for metabolic studies using differently labeled elemental isotopes simultaneously—i.e., 13C and 15N—in amino acids of a total protein hydrolysate. The optimized strategy for the analysis of metabolism by a hybrid linear ion trap-FTICR-MS comprises the collection of multiple adjacent selected ion monitoring scans. By limiting both the width of the mass range and the number of ions entering the ICR cell with automated gain control, sensitive measurements of isotopologue distribution were possible without compromising mass accuracy and isotope intensity mapping. The required mass-resolving power of more than 60,000 is only achievable on a routine basis by FTICR and Orbitrap mass spectrometers. Evaluation of the method was carried out by comparison of the experimental data to the natural isotope abundances of selected amino acids and by comparison to GC/MS results obtained from a labeling experiment with 13C-labeled glucose. The developed method was used to shed light on the complexity of the yeast Saccharomyces cerevisiae carbon–nitrogen co-metabolism by administering both 13C-labeled glucose and 15N-labeled alanine. The results indicate that not only glutamate but also alanine acts as an amino donor during alanine and valine synthesis. Metabolic studies using FTICR-MS can exploit new possibilities by the use of multiple-labeled elemental isotopes. Figure Analysis of carbon and nitrogen co-metabolism in yeast by nanospray-FTICR-MS determination of 13C and 15N incorporation into proteinogenic amino acids
Keywords: 13C- and 15N-labeling experiment; Mass spectrometry; Yeast; Saccharomyces cerevisiae ; Amino acid metabolism
Direct analysis in real time mass spectrometry combined with single-drop liquid–liquid–liquid microextraction for the rapid analysis of multiple phytohormones in fruit juice by Yu Bai; Jialing Zhang; Yu Bai; Huwei Liu (pp. 2307-2314).
is an Associate Professor of Analytical Chemistry at Peking University. She obtained her Ph.D. in chemistry from the Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, in 2004. She was awarded a Canadian Institutes of Health Research (CIHR) postdoctoral fellowship in 2007. She also won the first prize in the Science and Technology Progress Award of Jilin Province in 2006. Her major research interests focus on the application of nanomaterials to the separation and analysis of biological samples, and the establishment of sensitive methods for the analysis and screening of bioactive molecules based on mass spectrometry. A rapid, simple, and efficient method for the fast determination of multiple phytohormones was developed in this work, based on single-drop liquid–liquid–liquid microextraction (SD-LLLME) combined with direct analysis in real-time mass spectrometry (DART-MS). Six phytohormones—indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), and gibberellin A3 (GA3)—were analyzed simultaneously using this method, and the conditions employed for DART-MS and SD-LLLME were optimized systematically. Satisfactory results were obtained in terms of linearity (R 2 values for all phytohormones were 0.991–0.996), sensitivity (limits of detection were 0.65–72 ng/mL), and repeatability (RSD values were 6.9–14%). In addition, the proposed method was applied to determine the endogenous phytohormones in three kinds of fruit juice. Different concentrations of phytohormones were detected with satisfactory recoveries, and the whole analytical procedure took no more than 30 min. Therefore, this combination of SD-LLLME and DART-MS was shown to be a suitable and effective approach for the fast analysis of targets present at trace level concentrations in complex matrices.
Keywords: Direct analysis in real time (DART); Single drop liquid–liquid–liquid microextraction; Phytohormone; Fruit juice; Mass spectrometry
Direct analysis in real time mass spectrometry combined with single-drop liquid–liquid–liquid microextraction for the rapid analysis of multiple phytohormones in fruit juice by Yu Bai; Jialing Zhang; Yu Bai; Huwei Liu (pp. 2307-2314).
is an Associate Professor of Analytical Chemistry at Peking University. She obtained her Ph.D. in chemistry from the Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, in 2004. She was awarded a Canadian Institutes of Health Research (CIHR) postdoctoral fellowship in 2007. She also won the first prize in the Science and Technology Progress Award of Jilin Province in 2006. Her major research interests focus on the application of nanomaterials to the separation and analysis of biological samples, and the establishment of sensitive methods for the analysis and screening of bioactive molecules based on mass spectrometry. A rapid, simple, and efficient method for the fast determination of multiple phytohormones was developed in this work, based on single-drop liquid–liquid–liquid microextraction (SD-LLLME) combined with direct analysis in real-time mass spectrometry (DART-MS). Six phytohormones—indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), and gibberellin A3 (GA3)—were analyzed simultaneously using this method, and the conditions employed for DART-MS and SD-LLLME were optimized systematically. Satisfactory results were obtained in terms of linearity (R 2 values for all phytohormones were 0.991–0.996), sensitivity (limits of detection were 0.65–72 ng/mL), and repeatability (RSD values were 6.9–14%). In addition, the proposed method was applied to determine the endogenous phytohormones in three kinds of fruit juice. Different concentrations of phytohormones were detected with satisfactory recoveries, and the whole analytical procedure took no more than 30 min. Therefore, this combination of SD-LLLME and DART-MS was shown to be a suitable and effective approach for the fast analysis of targets present at trace level concentrations in complex matrices.
Keywords: Direct analysis in real time (DART); Single drop liquid–liquid–liquid microextraction; Phytohormone; Fruit juice; Mass spectrometry
Direct analysis in real time mass spectrometry combined with single-drop liquid–liquid–liquid microextraction for the rapid analysis of multiple phytohormones in fruit juice by Yu Bai; Jialing Zhang; Yu Bai; Huwei Liu (pp. 2307-2314).
is an Associate Professor of Analytical Chemistry at Peking University. She obtained her Ph.D. in chemistry from the Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, in 2004. She was awarded a Canadian Institutes of Health Research (CIHR) postdoctoral fellowship in 2007. She also won the first prize in the Science and Technology Progress Award of Jilin Province in 2006. Her major research interests focus on the application of nanomaterials to the separation and analysis of biological samples, and the establishment of sensitive methods for the analysis and screening of bioactive molecules based on mass spectrometry. A rapid, simple, and efficient method for the fast determination of multiple phytohormones was developed in this work, based on single-drop liquid–liquid–liquid microextraction (SD-LLLME) combined with direct analysis in real-time mass spectrometry (DART-MS). Six phytohormones—indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), and gibberellin A3 (GA3)—were analyzed simultaneously using this method, and the conditions employed for DART-MS and SD-LLLME were optimized systematically. Satisfactory results were obtained in terms of linearity (R 2 values for all phytohormones were 0.991–0.996), sensitivity (limits of detection were 0.65–72 ng/mL), and repeatability (RSD values were 6.9–14%). In addition, the proposed method was applied to determine the endogenous phytohormones in three kinds of fruit juice. Different concentrations of phytohormones were detected with satisfactory recoveries, and the whole analytical procedure took no more than 30 min. Therefore, this combination of SD-LLLME and DART-MS was shown to be a suitable and effective approach for the fast analysis of targets present at trace level concentrations in complex matrices.
Keywords: Direct analysis in real time (DART); Single drop liquid–liquid–liquid microextraction; Phytohormone; Fruit juice; Mass spectrometry
Analysis of colorectal adenocarcinoma tissue by desorption electrospray ionization mass spectrometric imaging by Stefanie Gerbig; Ottmar Golf; Julia Balog; Julia Denes; Zsolt Baranyai; Attila Zarand; Erzsebet Raso; Jozsef Timar; Zoltan Takats (pp. 2315-2325).
is currently a Reader in Medical Mass Spectrometry at the Imperial College London, Department of Surgery and Cancer. He received an ERC starting grant in 2008 and won the Mattauch-Herzog prize from the German Society for Mass Spectrometry, as a Research Fellow at Justus Liebig University Giessen, Institute of Inorganic and Analytical Chemistry (2008–2012). His research is focused on the development of novel mass spectrometry-based techniques capable of in vivo, in situ, and automated characterization of biological samples. Negative ion desorption electrospray ionization (DESI) was used for the analysis of an ex vivo tissue sample set comprising primary colorectal adenocarcinoma samples and colorectal adenocarcinoma liver metastasis samples. Frozen sections (12 μm thick) were analyzed by means of DESI imaging mass spectrometry (IMS) with spatial resolution of 100 μm using a computer-controlled DESI imaging stage mounted on a high resolution Orbitrap mass spectrometer. DESI-IMS data were found to predominantly feature complex lipids, including phosphatidyl-inositols, phophatidyl-ethanolamines, phosphatidyl-serines, phosphatidyl-ethanolamine plasmalogens, phosphatidic acids, phosphatidyl-glycerols, ceramides, sphingolipids, and sulfatides among others. Molecular constituents were identified based on their exact mass and MS/MS fragmentation spectra. An identified set of molecules was found to be in good agreement with previously reported DESI imaging data. Different histological tissue types were found to yield characteristic mass spectrometric data in each individual section. Histological features were identified by comparison to hematoxylin–eosin stained neighboring sections. Ions specific to certain histological tissue types (connective tissue, smooth muscle, healthy mucosa, healthy liver parenchyma, and adenocarcinoma) were identified by semi-automated screening of data. While each section featured a number of tissue-specific species, no potential global biomarker was found in the full sample set for any of the tissue types. As an alternative approach, data were analyzed by principal component analysis (PCA) and linear discriminant analysis (LDA) which resulted in efficient separation of data points based on their histological types. A pixel-by-pixel tissue identification method was developed, featuring the PCA/LDA analysis of authentic data set, and localization of unknowns in the resulting 60D, histologically assigned LDA space. Novel approach was found to yield results which are in 95% agreement with the results of classical histology. KRAS mutation status was determined for each sample by standard molecular biology methods and a similar PCA/LDA approach was developed to assess the feasibility of the determination of this important parameter using solely DESI imaging data. Results showed that the mutant and wild-type samples fully separated. DESI-MS and molecular biology results were in agreement in 90% of the cases. Figure Mass spectrometric analysis of phospholipid distribution in colorectal adenocarcinoma using DESI-MS and multivariate statistical methods.
Keywords: Desorption electrospray ionization; Mass spectrometry imaging; Phospholipid profile; Multivariate statistical analysis; Colorectal adenocarcinoma; KRAS mutation
Analysis of colorectal adenocarcinoma tissue by desorption electrospray ionization mass spectrometric imaging by Stefanie Gerbig; Ottmar Golf; Julia Balog; Julia Denes; Zsolt Baranyai; Attila Zarand; Erzsebet Raso; Jozsef Timar; Zoltan Takats (pp. 2315-2325).
is currently a Reader in Medical Mass Spectrometry at the Imperial College London, Department of Surgery and Cancer. He received an ERC starting grant in 2008 and won the Mattauch-Herzog prize from the German Society for Mass Spectrometry, as a Research Fellow at Justus Liebig University Giessen, Institute of Inorganic and Analytical Chemistry (2008–2012). His research is focused on the development of novel mass spectrometry-based techniques capable of in vivo, in situ, and automated characterization of biological samples. Negative ion desorption electrospray ionization (DESI) was used for the analysis of an ex vivo tissue sample set comprising primary colorectal adenocarcinoma samples and colorectal adenocarcinoma liver metastasis samples. Frozen sections (12 μm thick) were analyzed by means of DESI imaging mass spectrometry (IMS) with spatial resolution of 100 μm using a computer-controlled DESI imaging stage mounted on a high resolution Orbitrap mass spectrometer. DESI-IMS data were found to predominantly feature complex lipids, including phosphatidyl-inositols, phophatidyl-ethanolamines, phosphatidyl-serines, phosphatidyl-ethanolamine plasmalogens, phosphatidic acids, phosphatidyl-glycerols, ceramides, sphingolipids, and sulfatides among others. Molecular constituents were identified based on their exact mass and MS/MS fragmentation spectra. An identified set of molecules was found to be in good agreement with previously reported DESI imaging data. Different histological tissue types were found to yield characteristic mass spectrometric data in each individual section. Histological features were identified by comparison to hematoxylin–eosin stained neighboring sections. Ions specific to certain histological tissue types (connective tissue, smooth muscle, healthy mucosa, healthy liver parenchyma, and adenocarcinoma) were identified by semi-automated screening of data. While each section featured a number of tissue-specific species, no potential global biomarker was found in the full sample set for any of the tissue types. As an alternative approach, data were analyzed by principal component analysis (PCA) and linear discriminant analysis (LDA) which resulted in efficient separation of data points based on their histological types. A pixel-by-pixel tissue identification method was developed, featuring the PCA/LDA analysis of authentic data set, and localization of unknowns in the resulting 60D, histologically assigned LDA space. Novel approach was found to yield results which are in 95% agreement with the results of classical histology. KRAS mutation status was determined for each sample by standard molecular biology methods and a similar PCA/LDA approach was developed to assess the feasibility of the determination of this important parameter using solely DESI imaging data. Results showed that the mutant and wild-type samples fully separated. DESI-MS and molecular biology results were in agreement in 90% of the cases. Figure Mass spectrometric analysis of phospholipid distribution in colorectal adenocarcinoma using DESI-MS and multivariate statistical methods.
Keywords: Desorption electrospray ionization; Mass spectrometry imaging; Phospholipid profile; Multivariate statistical analysis; Colorectal adenocarcinoma; KRAS mutation
Analysis of colorectal adenocarcinoma tissue by desorption electrospray ionization mass spectrometric imaging by Stefanie Gerbig; Ottmar Golf; Julia Balog; Julia Denes; Zsolt Baranyai; Attila Zarand; Erzsebet Raso; Jozsef Timar; Zoltan Takats (pp. 2315-2325).
is currently a Reader in Medical Mass Spectrometry at the Imperial College London, Department of Surgery and Cancer. He received an ERC starting grant in 2008 and won the Mattauch-Herzog prize from the German Society for Mass Spectrometry, as a Research Fellow at Justus Liebig University Giessen, Institute of Inorganic and Analytical Chemistry (2008–2012). His research is focused on the development of novel mass spectrometry-based techniques capable of in vivo, in situ, and automated characterization of biological samples. Negative ion desorption electrospray ionization (DESI) was used for the analysis of an ex vivo tissue sample set comprising primary colorectal adenocarcinoma samples and colorectal adenocarcinoma liver metastasis samples. Frozen sections (12 μm thick) were analyzed by means of DESI imaging mass spectrometry (IMS) with spatial resolution of 100 μm using a computer-controlled DESI imaging stage mounted on a high resolution Orbitrap mass spectrometer. DESI-IMS data were found to predominantly feature complex lipids, including phosphatidyl-inositols, phophatidyl-ethanolamines, phosphatidyl-serines, phosphatidyl-ethanolamine plasmalogens, phosphatidic acids, phosphatidyl-glycerols, ceramides, sphingolipids, and sulfatides among others. Molecular constituents were identified based on their exact mass and MS/MS fragmentation spectra. An identified set of molecules was found to be in good agreement with previously reported DESI imaging data. Different histological tissue types were found to yield characteristic mass spectrometric data in each individual section. Histological features were identified by comparison to hematoxylin–eosin stained neighboring sections. Ions specific to certain histological tissue types (connective tissue, smooth muscle, healthy mucosa, healthy liver parenchyma, and adenocarcinoma) were identified by semi-automated screening of data. While each section featured a number of tissue-specific species, no potential global biomarker was found in the full sample set for any of the tissue types. As an alternative approach, data were analyzed by principal component analysis (PCA) and linear discriminant analysis (LDA) which resulted in efficient separation of data points based on their histological types. A pixel-by-pixel tissue identification method was developed, featuring the PCA/LDA analysis of authentic data set, and localization of unknowns in the resulting 60D, histologically assigned LDA space. Novel approach was found to yield results which are in 95% agreement with the results of classical histology. KRAS mutation status was determined for each sample by standard molecular biology methods and a similar PCA/LDA approach was developed to assess the feasibility of the determination of this important parameter using solely DESI imaging data. Results showed that the mutant and wild-type samples fully separated. DESI-MS and molecular biology results were in agreement in 90% of the cases. Figure Mass spectrometric analysis of phospholipid distribution in colorectal adenocarcinoma using DESI-MS and multivariate statistical methods.
Keywords: Desorption electrospray ionization; Mass spectrometry imaging; Phospholipid profile; Multivariate statistical analysis; Colorectal adenocarcinoma; KRAS mutation
Elucidating the role of select cytoplasmic proteins in altering diffusion of integrin receptors by Suzanne Sander; Neha Arora; Emily A. Smith (pp. 2327-2337).
is currently an Assistant Professor of Chemistry at Iowa State University and a Faculty Scientist at U.S. Department of Energy, The Ames Laboratory. She is the recipient of a National Science Foundation CAREER award in 2009. Her research interests are fluorescence and Raman instrument development for the analysis of nanomaterials, plant and animal tissue; and elucidating the molecular events that lead to changes in the organization of the cell membrane. Cytoplasmic proteins that affect integrin diffusion in the cell membrane are identified using a combination of fluorescence recovery after photobleaching (FRAP) and RNA interference. Integrin receptors are essential for many cellular events, and alterations in lateral diffusion are one mechanism for modulating their function. In cells expressing native cytoplasmic protein concentrations and spread on a slide containing integrin extracellular ligand, 45 ± 2% of the integrin is mobile with a time-dependent 5.2 ± 0.9 × 10−9 cm2/s diffusion coefficient at 1 s. The time exponent is 0.90 ± 0.07, indicating integrin diffusion moderately slows at longer times. The role of a specific cytoplasmic protein in altering integrin diffusion is revealed through changes in the FRAP curve after reducing the cytoplasmic protein’s expression. Decreased expression of cytoplasmic proteins rhea, focal adhesion kinase (FAK), or steamer duck decreases the integrin mobile fraction. For rhea and FAK, there is a concomitant shift to Brownian (i.e., time-independent) diffusion at reduced concentrations of these proteins. In contrast, when the expression of actin 42A, dreadlocks, paxillin, integrin-linked kinase (ILK), or vinculin is reduced, integrin diffusion generally becomes more constrained with an increase in the integrin mobile fraction. This same change in integrin diffusion is measured in the absence of integrin extracellular ligand. The results indicate breaking the extracellular ligand–integrin–cytoskeletal linkage alters integrin diffusion properties, and, in most cases, there is no correlation between integrin and lipid diffusion properties. Figure Schematic of a cell membrane depicting the interaction of integrin receptors with cytoplasmic proteins. Fluorescence recovery after photobleaching was used to measure diffusion properties of integrins before (black curve) and after (blue curve) reducing the expression of specific cytoplasmic proteins via RNA interference
Keywords: Fluorescence recovery after photobleaching (FRAP); RNA interference (RNAi); Cell membrane; αPS2CβPS integrin; Lateral diffusion; Cytoskeleton
Elucidating the role of select cytoplasmic proteins in altering diffusion of integrin receptors by Suzanne Sander; Neha Arora; Emily A. Smith (pp. 2327-2337).
is currently an Assistant Professor of Chemistry at Iowa State University and a Faculty Scientist at U.S. Department of Energy, The Ames Laboratory. She is the recipient of a National Science Foundation CAREER award in 2009. Her research interests are fluorescence and Raman instrument development for the analysis of nanomaterials, plant and animal tissue; and elucidating the molecular events that lead to changes in the organization of the cell membrane. Cytoplasmic proteins that affect integrin diffusion in the cell membrane are identified using a combination of fluorescence recovery after photobleaching (FRAP) and RNA interference. Integrin receptors are essential for many cellular events, and alterations in lateral diffusion are one mechanism for modulating their function. In cells expressing native cytoplasmic protein concentrations and spread on a slide containing integrin extracellular ligand, 45 ± 2% of the integrin is mobile with a time-dependent 5.2 ± 0.9 × 10−9 cm2/s diffusion coefficient at 1 s. The time exponent is 0.90 ± 0.07, indicating integrin diffusion moderately slows at longer times. The role of a specific cytoplasmic protein in altering integrin diffusion is revealed through changes in the FRAP curve after reducing the cytoplasmic protein’s expression. Decreased expression of cytoplasmic proteins rhea, focal adhesion kinase (FAK), or steamer duck decreases the integrin mobile fraction. For rhea and FAK, there is a concomitant shift to Brownian (i.e., time-independent) diffusion at reduced concentrations of these proteins. In contrast, when the expression of actin 42A, dreadlocks, paxillin, integrin-linked kinase (ILK), or vinculin is reduced, integrin diffusion generally becomes more constrained with an increase in the integrin mobile fraction. This same change in integrin diffusion is measured in the absence of integrin extracellular ligand. The results indicate breaking the extracellular ligand–integrin–cytoskeletal linkage alters integrin diffusion properties, and, in most cases, there is no correlation between integrin and lipid diffusion properties. Figure Schematic of a cell membrane depicting the interaction of integrin receptors with cytoplasmic proteins. Fluorescence recovery after photobleaching was used to measure diffusion properties of integrins before (black curve) and after (blue curve) reducing the expression of specific cytoplasmic proteins via RNA interference
Keywords: Fluorescence recovery after photobleaching (FRAP); RNA interference (RNAi); Cell membrane; αPS2CβPS integrin; Lateral diffusion; Cytoskeleton
Elucidating the role of select cytoplasmic proteins in altering diffusion of integrin receptors by Suzanne Sander; Neha Arora; Emily A. Smith (pp. 2327-2337).
is currently an Assistant Professor of Chemistry at Iowa State University and a Faculty Scientist at U.S. Department of Energy, The Ames Laboratory. She is the recipient of a National Science Foundation CAREER award in 2009. Her research interests are fluorescence and Raman instrument development for the analysis of nanomaterials, plant and animal tissue; and elucidating the molecular events that lead to changes in the organization of the cell membrane. Cytoplasmic proteins that affect integrin diffusion in the cell membrane are identified using a combination of fluorescence recovery after photobleaching (FRAP) and RNA interference. Integrin receptors are essential for many cellular events, and alterations in lateral diffusion are one mechanism for modulating their function. In cells expressing native cytoplasmic protein concentrations and spread on a slide containing integrin extracellular ligand, 45 ± 2% of the integrin is mobile with a time-dependent 5.2 ± 0.9 × 10−9 cm2/s diffusion coefficient at 1 s. The time exponent is 0.90 ± 0.07, indicating integrin diffusion moderately slows at longer times. The role of a specific cytoplasmic protein in altering integrin diffusion is revealed through changes in the FRAP curve after reducing the cytoplasmic protein’s expression. Decreased expression of cytoplasmic proteins rhea, focal adhesion kinase (FAK), or steamer duck decreases the integrin mobile fraction. For rhea and FAK, there is a concomitant shift to Brownian (i.e., time-independent) diffusion at reduced concentrations of these proteins. In contrast, when the expression of actin 42A, dreadlocks, paxillin, integrin-linked kinase (ILK), or vinculin is reduced, integrin diffusion generally becomes more constrained with an increase in the integrin mobile fraction. This same change in integrin diffusion is measured in the absence of integrin extracellular ligand. The results indicate breaking the extracellular ligand–integrin–cytoskeletal linkage alters integrin diffusion properties, and, in most cases, there is no correlation between integrin and lipid diffusion properties. Figure Schematic of a cell membrane depicting the interaction of integrin receptors with cytoplasmic proteins. Fluorescence recovery after photobleaching was used to measure diffusion properties of integrins before (black curve) and after (blue curve) reducing the expression of specific cytoplasmic proteins via RNA interference
Keywords: Fluorescence recovery after photobleaching (FRAP); RNA interference (RNAi); Cell membrane; αPS2CβPS integrin; Lateral diffusion; Cytoskeleton
Fluorescence recovery after photobleaching reveals the biochemistry of nucleocytoplasmic exchange by Ranieri Bizzarri; Francesco Cardarelli; Michela Serresi; Fabio Beltram (pp. 2339-2351).
received his PhD in Chemistry from Scuola Normale Superiore (SNS), Pisa, Italy. He is currently researcher at the Biophysics Institute of the Italian National Research Council. He was a visiting scientist at Cornell University (USA), CNRS—Université de Paris XII (France), and Twente University (the Netherlands). His scientific interests mainly concern the development of fluorescent probes and related imaging techniques to report on biochemical processes in living cells. Fluorescence recovery after photobleaching (FRAP) can help unveil subtle dynamical and biochemical properties of intracellular components. A peculiar aspect of this method is that it is based on the change of optical properties only, whereas dynamics and biochemistry of the molecules of interest are not perturbed. This makes FRAP particularly suitable for the study of protein translocation, e.g., between nucleus and cytoplasm. Here we present a comprehensive theoretical treatment of FRAP applied to protein nucleocytoplasmic translocation by passive diffusion and/or energy-driven processes across the nuclear envelope. Our mathematical model is validated by experimental FRAP studies with functionalized fluorescent protein chimeras. Using this approach we demonstrate that molecular crowding at the nuclear pore does not hamper passive diffusion and calculate the dimension of the nuclear pore size (5.33 nm). Additionally, our FRAP analysis reveals the biochemical parameters (maximum translocation rate and dissociation constant of the transport complex in cytoplasm) associated with the active import of a prototypical nuclear localization sequence (NLS of SV40) and related mutants. We demonstrate that transportin binding and active import into the nucleus are independent processes that can be separately modulated. The present results are discussed in light of their potential to help in engineering sequences for intracellular targeted delivery of sensors and/or therapeutic compounds. Finally, the limits of validity of our mathematical model are addressed. Figure Nucleocytoplasmic translocation. Left: Pictorial description of translocation processes across the nuclear pore: passive diffusion (up) and active transport (down). Right: time-lapse images of Fluorescence Recovery After Photobleaching (FRAP) technique applied to monitor the dynamic nucleocytoplasmic exchange of fluorescent molecules
Keywords: Fluorescence/Luminescence; Kinetics; Optical sensors; Biological samples; Bioanalytical methods
Fluorescence recovery after photobleaching reveals the biochemistry of nucleocytoplasmic exchange by Ranieri Bizzarri; Francesco Cardarelli; Michela Serresi; Fabio Beltram (pp. 2339-2351).
received his PhD in Chemistry from Scuola Normale Superiore (SNS), Pisa, Italy. He is currently researcher at the Biophysics Institute of the Italian National Research Council. He was a visiting scientist at Cornell University (USA), CNRS—Université de Paris XII (France), and Twente University (the Netherlands). His scientific interests mainly concern the development of fluorescent probes and related imaging techniques to report on biochemical processes in living cells. Fluorescence recovery after photobleaching (FRAP) can help unveil subtle dynamical and biochemical properties of intracellular components. A peculiar aspect of this method is that it is based on the change of optical properties only, whereas dynamics and biochemistry of the molecules of interest are not perturbed. This makes FRAP particularly suitable for the study of protein translocation, e.g., between nucleus and cytoplasm. Here we present a comprehensive theoretical treatment of FRAP applied to protein nucleocytoplasmic translocation by passive diffusion and/or energy-driven processes across the nuclear envelope. Our mathematical model is validated by experimental FRAP studies with functionalized fluorescent protein chimeras. Using this approach we demonstrate that molecular crowding at the nuclear pore does not hamper passive diffusion and calculate the dimension of the nuclear pore size (5.33 nm). Additionally, our FRAP analysis reveals the biochemical parameters (maximum translocation rate and dissociation constant of the transport complex in cytoplasm) associated with the active import of a prototypical nuclear localization sequence (NLS of SV40) and related mutants. We demonstrate that transportin binding and active import into the nucleus are independent processes that can be separately modulated. The present results are discussed in light of their potential to help in engineering sequences for intracellular targeted delivery of sensors and/or therapeutic compounds. Finally, the limits of validity of our mathematical model are addressed. Figure Nucleocytoplasmic translocation. Left: Pictorial description of translocation processes across the nuclear pore: passive diffusion (up) and active transport (down). Right: time-lapse images of Fluorescence Recovery After Photobleaching (FRAP) technique applied to monitor the dynamic nucleocytoplasmic exchange of fluorescent molecules
Keywords: Fluorescence/Luminescence; Kinetics; Optical sensors; Biological samples; Bioanalytical methods
Fluorescence recovery after photobleaching reveals the biochemistry of nucleocytoplasmic exchange by Ranieri Bizzarri; Francesco Cardarelli; Michela Serresi; Fabio Beltram (pp. 2339-2351).
received his PhD in Chemistry from Scuola Normale Superiore (SNS), Pisa, Italy. He is currently researcher at the Biophysics Institute of the Italian National Research Council. He was a visiting scientist at Cornell University (USA), CNRS—Université de Paris XII (France), and Twente University (the Netherlands). His scientific interests mainly concern the development of fluorescent probes and related imaging techniques to report on biochemical processes in living cells. Fluorescence recovery after photobleaching (FRAP) can help unveil subtle dynamical and biochemical properties of intracellular components. A peculiar aspect of this method is that it is based on the change of optical properties only, whereas dynamics and biochemistry of the molecules of interest are not perturbed. This makes FRAP particularly suitable for the study of protein translocation, e.g., between nucleus and cytoplasm. Here we present a comprehensive theoretical treatment of FRAP applied to protein nucleocytoplasmic translocation by passive diffusion and/or energy-driven processes across the nuclear envelope. Our mathematical model is validated by experimental FRAP studies with functionalized fluorescent protein chimeras. Using this approach we demonstrate that molecular crowding at the nuclear pore does not hamper passive diffusion and calculate the dimension of the nuclear pore size (5.33 nm). Additionally, our FRAP analysis reveals the biochemical parameters (maximum translocation rate and dissociation constant of the transport complex in cytoplasm) associated with the active import of a prototypical nuclear localization sequence (NLS of SV40) and related mutants. We demonstrate that transportin binding and active import into the nucleus are independent processes that can be separately modulated. The present results are discussed in light of their potential to help in engineering sequences for intracellular targeted delivery of sensors and/or therapeutic compounds. Finally, the limits of validity of our mathematical model are addressed. Figure Nucleocytoplasmic translocation. Left: Pictorial description of translocation processes across the nuclear pore: passive diffusion (up) and active transport (down). Right: time-lapse images of Fluorescence Recovery After Photobleaching (FRAP) technique applied to monitor the dynamic nucleocytoplasmic exchange of fluorescent molecules
Keywords: Fluorescence/Luminescence; Kinetics; Optical sensors; Biological samples; Bioanalytical methods
Chemiluminescence detection flow cells for flow injection analysis and high-performance liquid chromatography by Jessica M. Terry; Stephan Mohr; Peter R. Fielden; Nick J. Goddard; Neil W. Barnett; Don C. Olson; Duane K. Wolcott; Paul S. Francis (pp. 2353-2360).
is a Senior Lecturer in Chemistry at Deakin University (Australia) and currently holds an Australian Research Council Future Fellowship to study the chemistry, spectroscopy and analytical applications of chemiluminescence and electrochemiluminescence reactions. For his research in these areas, he has received a Victoria Young Tall Poppy Science Award (Australian Institute of Policy and Science), the Robert Cattrall Medal (Royal Australian Chemical Institute) and the Gordon F. Kirkbright Award (Association of British Spectroscopists). We have examined a range of new and previously described flow cells for chemiluminescence detection. The reactions of acidic potassium permanganate with morphine and amoxicillin were used as model systems representing the many fast chemiluminescence reactions between oxidising agents and organic analytes, and the preliminary partial reduction of the reagent was exploited to further increase the rates of reaction. The comparison was then extended to high-performance liquid chromatography separations of α- and β-adrenergic agonists, with permanganate chemiluminescence detection. Flow cells constructed by machining novel channel designs into white polymer materials (sealed with transparent films or plates) have enabled improvements in mixing efficiency and overall transmission of light to the photodetector. Fig Chemiluminescence from the reaction of morphine and permanganate in a serpentine flow cell.
Keywords: Chemiluminescence; Flow cells; Acidic potassium permanganate; High-performance liquid chromatography
Chemiluminescence detection flow cells for flow injection analysis and high-performance liquid chromatography by Jessica M. Terry; Stephan Mohr; Peter R. Fielden; Nick J. Goddard; Neil W. Barnett; Don C. Olson; Duane K. Wolcott; Paul S. Francis (pp. 2353-2360).
is a Senior Lecturer in Chemistry at Deakin University (Australia) and currently holds an Australian Research Council Future Fellowship to study the chemistry, spectroscopy and analytical applications of chemiluminescence and electrochemiluminescence reactions. For his research in these areas, he has received a Victoria Young Tall Poppy Science Award (Australian Institute of Policy and Science), the Robert Cattrall Medal (Royal Australian Chemical Institute) and the Gordon F. Kirkbright Award (Association of British Spectroscopists). We have examined a range of new and previously described flow cells for chemiluminescence detection. The reactions of acidic potassium permanganate with morphine and amoxicillin were used as model systems representing the many fast chemiluminescence reactions between oxidising agents and organic analytes, and the preliminary partial reduction of the reagent was exploited to further increase the rates of reaction. The comparison was then extended to high-performance liquid chromatography separations of α- and β-adrenergic agonists, with permanganate chemiluminescence detection. Flow cells constructed by machining novel channel designs into white polymer materials (sealed with transparent films or plates) have enabled improvements in mixing efficiency and overall transmission of light to the photodetector. Fig Chemiluminescence from the reaction of morphine and permanganate in a serpentine flow cell.
Keywords: Chemiluminescence; Flow cells; Acidic potassium permanganate; High-performance liquid chromatography
Chemiluminescence detection flow cells for flow injection analysis and high-performance liquid chromatography by Jessica M. Terry; Stephan Mohr; Peter R. Fielden; Nick J. Goddard; Neil W. Barnett; Don C. Olson; Duane K. Wolcott; Paul S. Francis (pp. 2353-2360).
is a Senior Lecturer in Chemistry at Deakin University (Australia) and currently holds an Australian Research Council Future Fellowship to study the chemistry, spectroscopy and analytical applications of chemiluminescence and electrochemiluminescence reactions. For his research in these areas, he has received a Victoria Young Tall Poppy Science Award (Australian Institute of Policy and Science), the Robert Cattrall Medal (Royal Australian Chemical Institute) and the Gordon F. Kirkbright Award (Association of British Spectroscopists). We have examined a range of new and previously described flow cells for chemiluminescence detection. The reactions of acidic potassium permanganate with morphine and amoxicillin were used as model systems representing the many fast chemiluminescence reactions between oxidising agents and organic analytes, and the preliminary partial reduction of the reagent was exploited to further increase the rates of reaction. The comparison was then extended to high-performance liquid chromatography separations of α- and β-adrenergic agonists, with permanganate chemiluminescence detection. Flow cells constructed by machining novel channel designs into white polymer materials (sealed with transparent films or plates) have enabled improvements in mixing efficiency and overall transmission of light to the photodetector. Fig Chemiluminescence from the reaction of morphine and permanganate in a serpentine flow cell.
Keywords: Chemiluminescence; Flow cells; Acidic potassium permanganate; High-performance liquid chromatography
Binding of the ionic liquid cation 1-alkyl-3-methylimidazolium to p-tetranitrocalix[4]arene probed by fluorescent indicator displacement by Shubha Pandey; Maroof Ali; Ganesh Kamath; Siddharth Pandey; Sheila N. Baker; Gary A. Baker (pp. 2361-2366).
is an assistant professor of Chemistry at the University of Missouri–Columbia. He has won a number of awards and fellowships including the Presidential Early Career Award for Scientists and Engineers (PECASE), the George W. Thorn Award, the Eugene P. Wigner Fellowship, the Frederick Reines Postdoctoral Fellowship, and the American Chemical Society Division of Analytical Chemistry Graduate Fellowship. His current research interests include expedient and eco-friendly routes toward tailored, “exotic,” or morphologically distinct nanomaterials, advanced functional nanotechnology to address the energy challenge, approaches for effective chemical analysis in resource-poor settings, and all manner of ionic liquid research. Acridine orange (AO) was used as a fluorescent probe molecule to study the encapsulation of an alkylimidazolium cation from a water-soluble ionic liquid (IL) within two cavitand species, p-tetranitrocalix[4]arene (1) and calix[4]resorcinarene (2), both in alkaline aqueous media. The addition of IL to the preformed [1⋅AO] adduct resulted in significantly increased fluorescence due to the expulsion of AO from the inclusion complex to the aqueous phase by competitive recognition of the 1-alkyl-3-methylimidazolium cation ([C n mim]+, n = 4 and 6) by 1. Conversely, the fluorescence signal dropped upon the addition of IL to the [2⋅AO] host–guest complex due to unfavorable binding between [C n mim]+ and 2. The formation of these postulated adducts is corroborated using ab initio calculations, which also provide evidence for the location of [bmim]+ at the lower external rim of [2⋅AO], providing an explanation for the observed luminescence quenching in the latter case. These results point to a number of different paths for exploration, ranging from the fluorescence monitoring of IL contamination in groundwater to the “daisy chaining” of macrocyles toward supramolecular ionic networks. They also broadly encourage the exploration of ILs in host–guest-based optical and mass spectrometric sensory systems. Figure The acridine orange (AO) adduct with the p-tetranitrocalix[4]arene cavitand (1) displays fluorescence turn-on when challenged with the 1-butyl-3-methylimidazolium ([bmim]+) cation in aqueous media due to expulsion of AO from the inclusion complex to the aqueous phase by competitive recognition of [bmim]+.
Keywords: Ionic liquids; Calixarene; Fluorescence; Cavitand; Host–guest complex
Binding of the ionic liquid cation 1-alkyl-3-methylimidazolium to p-tetranitrocalix[4]arene probed by fluorescent indicator displacement by Shubha Pandey; Maroof Ali; Ganesh Kamath; Siddharth Pandey; Sheila N. Baker; Gary A. Baker (pp. 2361-2366).
is an assistant professor of Chemistry at the University of Missouri–Columbia. He has won a number of awards and fellowships including the Presidential Early Career Award for Scientists and Engineers (PECASE), the George W. Thorn Award, the Eugene P. Wigner Fellowship, the Frederick Reines Postdoctoral Fellowship, and the American Chemical Society Division of Analytical Chemistry Graduate Fellowship. His current research interests include expedient and eco-friendly routes toward tailored, “exotic,” or morphologically distinct nanomaterials, advanced functional nanotechnology to address the energy challenge, approaches for effective chemical analysis in resource-poor settings, and all manner of ionic liquid research. Acridine orange (AO) was used as a fluorescent probe molecule to study the encapsulation of an alkylimidazolium cation from a water-soluble ionic liquid (IL) within two cavitand species, p-tetranitrocalix[4]arene (1) and calix[4]resorcinarene (2), both in alkaline aqueous media. The addition of IL to the preformed [1⋅AO] adduct resulted in significantly increased fluorescence due to the expulsion of AO from the inclusion complex to the aqueous phase by competitive recognition of the 1-alkyl-3-methylimidazolium cation ([C n mim]+, n = 4 and 6) by 1. Conversely, the fluorescence signal dropped upon the addition of IL to the [2⋅AO] host–guest complex due to unfavorable binding between [C n mim]+ and 2. The formation of these postulated adducts is corroborated using ab initio calculations, which also provide evidence for the location of [bmim]+ at the lower external rim of [2⋅AO], providing an explanation for the observed luminescence quenching in the latter case. These results point to a number of different paths for exploration, ranging from the fluorescence monitoring of IL contamination in groundwater to the “daisy chaining” of macrocyles toward supramolecular ionic networks. They also broadly encourage the exploration of ILs in host–guest-based optical and mass spectrometric sensory systems. Figure The acridine orange (AO) adduct with the p-tetranitrocalix[4]arene cavitand (1) displays fluorescence turn-on when challenged with the 1-butyl-3-methylimidazolium ([bmim]+) cation in aqueous media due to expulsion of AO from the inclusion complex to the aqueous phase by competitive recognition of [bmim]+.
Keywords: Ionic liquids; Calixarene; Fluorescence; Cavitand; Host–guest complex
Binding of the ionic liquid cation 1-alkyl-3-methylimidazolium to p-tetranitrocalix[4]arene probed by fluorescent indicator displacement by Shubha Pandey; Maroof Ali; Ganesh Kamath; Siddharth Pandey; Sheila N. Baker; Gary A. Baker (pp. 2361-2366).
is an assistant professor of Chemistry at the University of Missouri–Columbia. He has won a number of awards and fellowships including the Presidential Early Career Award for Scientists and Engineers (PECASE), the George W. Thorn Award, the Eugene P. Wigner Fellowship, the Frederick Reines Postdoctoral Fellowship, and the American Chemical Society Division of Analytical Chemistry Graduate Fellowship. His current research interests include expedient and eco-friendly routes toward tailored, “exotic,” or morphologically distinct nanomaterials, advanced functional nanotechnology to address the energy challenge, approaches for effective chemical analysis in resource-poor settings, and all manner of ionic liquid research. Acridine orange (AO) was used as a fluorescent probe molecule to study the encapsulation of an alkylimidazolium cation from a water-soluble ionic liquid (IL) within two cavitand species, p-tetranitrocalix[4]arene (1) and calix[4]resorcinarene (2), both in alkaline aqueous media. The addition of IL to the preformed [1⋅AO] adduct resulted in significantly increased fluorescence due to the expulsion of AO from the inclusion complex to the aqueous phase by competitive recognition of the 1-alkyl-3-methylimidazolium cation ([C n mim]+, n = 4 and 6) by 1. Conversely, the fluorescence signal dropped upon the addition of IL to the [2⋅AO] host–guest complex due to unfavorable binding between [C n mim]+ and 2. The formation of these postulated adducts is corroborated using ab initio calculations, which also provide evidence for the location of [bmim]+ at the lower external rim of [2⋅AO], providing an explanation for the observed luminescence quenching in the latter case. These results point to a number of different paths for exploration, ranging from the fluorescence monitoring of IL contamination in groundwater to the “daisy chaining” of macrocyles toward supramolecular ionic networks. They also broadly encourage the exploration of ILs in host–guest-based optical and mass spectrometric sensory systems. Figure The acridine orange (AO) adduct with the p-tetranitrocalix[4]arene cavitand (1) displays fluorescence turn-on when challenged with the 1-butyl-3-methylimidazolium ([bmim]+) cation in aqueous media due to expulsion of AO from the inclusion complex to the aqueous phase by competitive recognition of [bmim]+.
Keywords: Ionic liquids; Calixarene; Fluorescence; Cavitand; Host–guest complex
Biologically driven neural platform invoking parallel electrophoretic separation and urinary metabolite screening by Tessa Page; Huong Thi Huynh Nguyen; Lindsey Hilts; Lorena Ramos; Grady Hanrahan (pp. 2367-2375).
is the John Stauffer Endowed Professor of Analytical Chemistry and Co-Director of the Hugh and Hazel Darling Center for Applied Scientific Computing (CASC) at California Lutheran University (CLU). His research interests span separation science, chemometrics, computational neural networks, environmental analysis, and the use of swarm intelligence metaheuristics to model complex chemical and biologicaI systems. He has written or co-written numerous peer-reviewed technical papers and is the author of five books detailing the use of modern chemometric methods and computational modeling techniques. This work reveals a computational framework for parallel electrophoretic separation of complex biological macromolecules and model urinary metabolites. More specifically, the implementation of a particle swarm optimization (PSO) algorithm on a neural network platform for multiparameter optimization of multiplexed 24-capillary electrophoresis technology with UV detection is highlighted. Two experimental systems were examined: (1) separation of purified rabbit metallothioneins and (2) separation of model toluene urinary metabolites and selected organic acids. Results proved superior to the use of neural networks employing standard back propagation when examining training error, fitting response, and predictive abilities. Simulation runs were obtained as a result of metaheuristic examination of the global search space with experimental responses in good agreement with predicted values. Full separation of selected analytes was realized after employing optimal model conditions. This framework provides guidance for the application of metaheuristic computational tools to aid in future studies involving parallel chemical separation and screening. Adaptable pseudo-code is provided to enable users of varied software packages and modeling framework to implement the PSO algorithm for their desired use. Figure Nature-inspired metaheuristic algorithms are showcasing themselves as attractive analytical tools for scalable and dynamical optimization of modern separation technology
Keywords: Multiplexed capillary electrophoresis; Neural networks; Swarm intelligence; Urinary metabolites; Biomarkers
Biologically driven neural platform invoking parallel electrophoretic separation and urinary metabolite screening by Tessa Page; Huong Thi Huynh Nguyen; Lindsey Hilts; Lorena Ramos; Grady Hanrahan (pp. 2367-2375).
is the John Stauffer Endowed Professor of Analytical Chemistry and Co-Director of the Hugh and Hazel Darling Center for Applied Scientific Computing (CASC) at California Lutheran University (CLU). His research interests span separation science, chemometrics, computational neural networks, environmental analysis, and the use of swarm intelligence metaheuristics to model complex chemical and biologicaI systems. He has written or co-written numerous peer-reviewed technical papers and is the author of five books detailing the use of modern chemometric methods and computational modeling techniques. This work reveals a computational framework for parallel electrophoretic separation of complex biological macromolecules and model urinary metabolites. More specifically, the implementation of a particle swarm optimization (PSO) algorithm on a neural network platform for multiparameter optimization of multiplexed 24-capillary electrophoresis technology with UV detection is highlighted. Two experimental systems were examined: (1) separation of purified rabbit metallothioneins and (2) separation of model toluene urinary metabolites and selected organic acids. Results proved superior to the use of neural networks employing standard back propagation when examining training error, fitting response, and predictive abilities. Simulation runs were obtained as a result of metaheuristic examination of the global search space with experimental responses in good agreement with predicted values. Full separation of selected analytes was realized after employing optimal model conditions. This framework provides guidance for the application of metaheuristic computational tools to aid in future studies involving parallel chemical separation and screening. Adaptable pseudo-code is provided to enable users of varied software packages and modeling framework to implement the PSO algorithm for their desired use. Figure Nature-inspired metaheuristic algorithms are showcasing themselves as attractive analytical tools for scalable and dynamical optimization of modern separation technology
Keywords: Multiplexed capillary electrophoresis; Neural networks; Swarm intelligence; Urinary metabolites; Biomarkers
Biologically driven neural platform invoking parallel electrophoretic separation and urinary metabolite screening by Tessa Page; Huong Thi Huynh Nguyen; Lindsey Hilts; Lorena Ramos; Grady Hanrahan (pp. 2367-2375).
is the John Stauffer Endowed Professor of Analytical Chemistry and Co-Director of the Hugh and Hazel Darling Center for Applied Scientific Computing (CASC) at California Lutheran University (CLU). His research interests span separation science, chemometrics, computational neural networks, environmental analysis, and the use of swarm intelligence metaheuristics to model complex chemical and biologicaI systems. He has written or co-written numerous peer-reviewed technical papers and is the author of five books detailing the use of modern chemometric methods and computational modeling techniques. This work reveals a computational framework for parallel electrophoretic separation of complex biological macromolecules and model urinary metabolites. More specifically, the implementation of a particle swarm optimization (PSO) algorithm on a neural network platform for multiparameter optimization of multiplexed 24-capillary electrophoresis technology with UV detection is highlighted. Two experimental systems were examined: (1) separation of purified rabbit metallothioneins and (2) separation of model toluene urinary metabolites and selected organic acids. Results proved superior to the use of neural networks employing standard back propagation when examining training error, fitting response, and predictive abilities. Simulation runs were obtained as a result of metaheuristic examination of the global search space with experimental responses in good agreement with predicted values. Full separation of selected analytes was realized after employing optimal model conditions. This framework provides guidance for the application of metaheuristic computational tools to aid in future studies involving parallel chemical separation and screening. Adaptable pseudo-code is provided to enable users of varied software packages and modeling framework to implement the PSO algorithm for their desired use. Figure Nature-inspired metaheuristic algorithms are showcasing themselves as attractive analytical tools for scalable and dynamical optimization of modern separation technology
Keywords: Multiplexed capillary electrophoresis; Neural networks; Swarm intelligence; Urinary metabolites; Biomarkers
Microchip electrophoresis with amperometric detection for the study of the generation of nitric oxide by NONOate salts by Dulan B. Gunasekara; Matthew K. Hulvey; Susan M. Lunte; José Alberto Fracassi da Silva (pp. 2377-2384).
graduated in Chemistry from the Sao Paulo State University in 1996, where he also received his Ph.D. in Analytical Chemistry in 2001. This was followed by a postdoctoral position at the Laboratory of Integrated Systems, in Polytechnic School in the University of Sao Paulo. In 2004, he obtained a position at State University of Campinas, in Campinas, São Paulo, Brazil. In 2010, he was a visiting scholar at The Ralph Adams Institute for Bioanalytical Chemistry, University of Kansas (USA). His main research interests are focused on bioanalytical methods and instrumentation for capillary and microchip electrophoresis. Microchip electrophoresis (ME) with electrochemical detection was used to monitor nitric oxide (NO) production from diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) and 1-(hydroxyl-NNO-azoxy)-l-proline disodium salt (PROLI/NO). NO was generated through acid hydrolysis of these NONOate salts. The products of acid hydrolysis were introduced into a 5-cm separation channel using gated injection. The separation was accomplished using reverse polarity and a background electrolyte consisting of 10 mM boric acid and 2 mM tetradecyltrimethylammonium bromide, pH 11. Electrochemical detection was performed using an isolated potentiostat in an in-channel configuration. Potentials applied to the working electrode, typically higher than +1.0 V vs. Ag/AgCl, allowed the direct detection of nitrite, NO, DEA/NO, and PROLI/NO. Baseline resolution was achieved for the separation of PROLI/NO and NO while resolution between DEA/NO and NO was poor (1.0 ± 0.2). Nitrite was present in all samples tested. Figure Microchip electrophoresis with electrochemical detection was used to detect nitric oxide generated from NONOate salts
Keywords: Nitric oxide; Microchip electrophoresis; Reactive nitrogen species; Amperometric detection; NONOates
Microchip electrophoresis with amperometric detection for the study of the generation of nitric oxide by NONOate salts by Dulan B. Gunasekara; Matthew K. Hulvey; Susan M. Lunte; José Alberto Fracassi da Silva (pp. 2377-2384).
graduated in Chemistry from the Sao Paulo State University in 1996, where he also received his Ph.D. in Analytical Chemistry in 2001. This was followed by a postdoctoral position at the Laboratory of Integrated Systems, in Polytechnic School in the University of Sao Paulo. In 2004, he obtained a position at State University of Campinas, in Campinas, São Paulo, Brazil. In 2010, he was a visiting scholar at The Ralph Adams Institute for Bioanalytical Chemistry, University of Kansas (USA). His main research interests are focused on bioanalytical methods and instrumentation for capillary and microchip electrophoresis. Microchip electrophoresis (ME) with electrochemical detection was used to monitor nitric oxide (NO) production from diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) and 1-(hydroxyl-NNO-azoxy)-l-proline disodium salt (PROLI/NO). NO was generated through acid hydrolysis of these NONOate salts. The products of acid hydrolysis were introduced into a 5-cm separation channel using gated injection. The separation was accomplished using reverse polarity and a background electrolyte consisting of 10 mM boric acid and 2 mM tetradecyltrimethylammonium bromide, pH 11. Electrochemical detection was performed using an isolated potentiostat in an in-channel configuration. Potentials applied to the working electrode, typically higher than +1.0 V vs. Ag/AgCl, allowed the direct detection of nitrite, NO, DEA/NO, and PROLI/NO. Baseline resolution was achieved for the separation of PROLI/NO and NO while resolution between DEA/NO and NO was poor (1.0 ± 0.2). Nitrite was present in all samples tested. Figure Microchip electrophoresis with electrochemical detection was used to detect nitric oxide generated from NONOate salts
Keywords: Nitric oxide; Microchip electrophoresis; Reactive nitrogen species; Amperometric detection; NONOates
Microchip electrophoresis with amperometric detection for the study of the generation of nitric oxide by NONOate salts by Dulan B. Gunasekara; Matthew K. Hulvey; Susan M. Lunte; José Alberto Fracassi da Silva (pp. 2377-2384).
graduated in Chemistry from the Sao Paulo State University in 1996, where he also received his Ph.D. in Analytical Chemistry in 2001. This was followed by a postdoctoral position at the Laboratory of Integrated Systems, in Polytechnic School in the University of Sao Paulo. In 2004, he obtained a position at State University of Campinas, in Campinas, São Paulo, Brazil. In 2010, he was a visiting scholar at The Ralph Adams Institute for Bioanalytical Chemistry, University of Kansas (USA). His main research interests are focused on bioanalytical methods and instrumentation for capillary and microchip electrophoresis. Microchip electrophoresis (ME) with electrochemical detection was used to monitor nitric oxide (NO) production from diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) and 1-(hydroxyl-NNO-azoxy)-l-proline disodium salt (PROLI/NO). NO was generated through acid hydrolysis of these NONOate salts. The products of acid hydrolysis were introduced into a 5-cm separation channel using gated injection. The separation was accomplished using reverse polarity and a background electrolyte consisting of 10 mM boric acid and 2 mM tetradecyltrimethylammonium bromide, pH 11. Electrochemical detection was performed using an isolated potentiostat in an in-channel configuration. Potentials applied to the working electrode, typically higher than +1.0 V vs. Ag/AgCl, allowed the direct detection of nitrite, NO, DEA/NO, and PROLI/NO. Baseline resolution was achieved for the separation of PROLI/NO and NO while resolution between DEA/NO and NO was poor (1.0 ± 0.2). Nitrite was present in all samples tested. Figure Microchip electrophoresis with electrochemical detection was used to detect nitric oxide generated from NONOate salts
Keywords: Nitric oxide; Microchip electrophoresis; Reactive nitrogen species; Amperometric detection; NONOates
Reaction of β-blockers and β-agonist pharmaceuticals with aqueous chlorine. Investigation of kinetics and by-products by liquid chromatography quadrupole time-of-flight mass spectrometry by José Benito Quintana; Rosario Rodil; Rafael Cela (pp. 2385-2395).
was a postdoc at the Technical University of Berlin (2004–2006) and later a research associate (“Isidro Parga Pondal” program) at the University of A Coruña (2006–2008). He was awarded a “Ramón y Cajal” research fellowship in the area of chemistry, and since 2008, he has held this position at the University of Santiago de Compostela. His current research interests are emerging pollutants in water, with particular emphasis on drugs of abuse; environmental transformation processes, e.g. transformation due to chlorination; LC-MS and GC-MS, including high-resolution analyzers; and the development of novel extraction techniques. is currently a researcher of Analytical Chemistry at the University of Santiago de Compostela (Spain) supported by the “Ramon y Cajal” program. She received a Ph.D. degree from Santiago de Compostela University (Spain) in 2005 and then she had two postdoctoral appointments at the Helmholtz Centre of Environmental Research-UFZ Leipzig (Germany; 2005–2006) and at the University of A Coruña (Spain; 2006–2009). Rosario Rodil’s research interests include the development of analytical methodology for the determination of emerging organic pollutants, the study of the presence and distribution of emerging pollutants in the marine environment and transformation of pollutants during water treatment processes. The degradation of two β-blockers (atenolol and propranolol) and one β-receptor agonist (salbutamol) during water chlorination was investigated by liquid chromatography–mass spectrometry (LC-MS). An accurate-mass quadrupole time-of-flight system (QTOF) was used to follow the time course of the pharmaceuticals and also used in the identification of the by-products. The degradation kinetics of these drugs was investigated at different concentrations of chlorine, bromide and sample pH by means of a Box–Behnken experimental design. Depending on these factors, dissipation half-lives varied in the ranges 68–145 h for atenolol, 1.3–33 min for salbutamol and 42–8362 min for propranolol. Normally, an increase in chlorine dosage and pH resulted in faster degradation of these pharmaceuticals. Moreover, the presence of bromide in water samples also resulted in a faster transformation of atenolol at low chlorine doses. The use of an accurate-mass high-resolution LC-QTOF-MS system permitted the identification of a total of 14 by-products. The transformation pathway of β-blockers/agonists consisted mainly of halogenations, hydroxylations and dealkylations. Also, many of these by-products are stable, depending on the chlorination operational parameters employed.
Keywords: Pharmaceuticals; Chlorination; By-products; Liquid chromatography–mass spectrometry (LC-MS); Time-of-flight (TOF)
Reaction of β-blockers and β-agonist pharmaceuticals with aqueous chlorine. Investigation of kinetics and by-products by liquid chromatography quadrupole time-of-flight mass spectrometry by José Benito Quintana; Rosario Rodil; Rafael Cela (pp. 2385-2395).
was a postdoc at the Technical University of Berlin (2004–2006) and later a research associate (“Isidro Parga Pondal” program) at the University of A Coruña (2006–2008). He was awarded a “Ramón y Cajal” research fellowship in the area of chemistry, and since 2008, he has held this position at the University of Santiago de Compostela. His current research interests are emerging pollutants in water, with particular emphasis on drugs of abuse; environmental transformation processes, e.g. transformation due to chlorination; LC-MS and GC-MS, including high-resolution analyzers; and the development of novel extraction techniques. is currently a researcher of Analytical Chemistry at the University of Santiago de Compostela (Spain) supported by the “Ramon y Cajal” program. She received a Ph.D. degree from Santiago de Compostela University (Spain) in 2005 and then she had two postdoctoral appointments at the Helmholtz Centre of Environmental Research-UFZ Leipzig (Germany; 2005–2006) and at the University of A Coruña (Spain; 2006–2009). Rosario Rodil’s research interests include the development of analytical methodology for the determination of emerging organic pollutants, the study of the presence and distribution of emerging pollutants in the marine environment and transformation of pollutants during water treatment processes. The degradation of two β-blockers (atenolol and propranolol) and one β-receptor agonist (salbutamol) during water chlorination was investigated by liquid chromatography–mass spectrometry (LC-MS). An accurate-mass quadrupole time-of-flight system (QTOF) was used to follow the time course of the pharmaceuticals and also used in the identification of the by-products. The degradation kinetics of these drugs was investigated at different concentrations of chlorine, bromide and sample pH by means of a Box–Behnken experimental design. Depending on these factors, dissipation half-lives varied in the ranges 68–145 h for atenolol, 1.3–33 min for salbutamol and 42–8362 min for propranolol. Normally, an increase in chlorine dosage and pH resulted in faster degradation of these pharmaceuticals. Moreover, the presence of bromide in water samples also resulted in a faster transformation of atenolol at low chlorine doses. The use of an accurate-mass high-resolution LC-QTOF-MS system permitted the identification of a total of 14 by-products. The transformation pathway of β-blockers/agonists consisted mainly of halogenations, hydroxylations and dealkylations. Also, many of these by-products are stable, depending on the chlorination operational parameters employed.
Keywords: Pharmaceuticals; Chlorination; By-products; Liquid chromatography–mass spectrometry (LC-MS); Time-of-flight (TOF)
Reaction of β-blockers and β-agonist pharmaceuticals with aqueous chlorine. Investigation of kinetics and by-products by liquid chromatography quadrupole time-of-flight mass spectrometry by José Benito Quintana; Rosario Rodil; Rafael Cela (pp. 2385-2395).
was a postdoc at the Technical University of Berlin (2004–2006) and later a research associate (“Isidro Parga Pondal” program) at the University of A Coruña (2006–2008). He was awarded a “Ramón y Cajal” research fellowship in the area of chemistry, and since 2008, he has held this position at the University of Santiago de Compostela. His current research interests are emerging pollutants in water, with particular emphasis on drugs of abuse; environmental transformation processes, e.g. transformation due to chlorination; LC-MS and GC-MS, including high-resolution analyzers; and the development of novel extraction techniques. is currently a researcher of Analytical Chemistry at the University of Santiago de Compostela (Spain) supported by the “Ramon y Cajal” program. She received a Ph.D. degree from Santiago de Compostela University (Spain) in 2005 and then she had two postdoctoral appointments at the Helmholtz Centre of Environmental Research-UFZ Leipzig (Germany; 2005–2006) and at the University of A Coruña (Spain; 2006–2009). Rosario Rodil’s research interests include the development of analytical methodology for the determination of emerging organic pollutants, the study of the presence and distribution of emerging pollutants in the marine environment and transformation of pollutants during water treatment processes. The degradation of two β-blockers (atenolol and propranolol) and one β-receptor agonist (salbutamol) during water chlorination was investigated by liquid chromatography–mass spectrometry (LC-MS). An accurate-mass quadrupole time-of-flight system (QTOF) was used to follow the time course of the pharmaceuticals and also used in the identification of the by-products. The degradation kinetics of these drugs was investigated at different concentrations of chlorine, bromide and sample pH by means of a Box–Behnken experimental design. Depending on these factors, dissipation half-lives varied in the ranges 68–145 h for atenolol, 1.3–33 min for salbutamol and 42–8362 min for propranolol. Normally, an increase in chlorine dosage and pH resulted in faster degradation of these pharmaceuticals. Moreover, the presence of bromide in water samples also resulted in a faster transformation of atenolol at low chlorine doses. The use of an accurate-mass high-resolution LC-QTOF-MS system permitted the identification of a total of 14 by-products. The transformation pathway of β-blockers/agonists consisted mainly of halogenations, hydroxylations and dealkylations. Also, many of these by-products are stable, depending on the chlorination operational parameters employed.
Keywords: Pharmaceuticals; Chlorination; By-products; Liquid chromatography–mass spectrometry (LC-MS); Time-of-flight (TOF)
A method to determine the kinetics of multiple proteins in human infants with respiratory distress syndrome by Michael S. Bereman; Daniela M. Tomazela; Hillary S. Heins; Manuela Simonato; Paola E. Cogo; Aaron Hamvas; Bruce W. Patterson; F. Sessions Cole; Michael J. MacCoss (pp. 2397-2402).
is currently in the MacCoss Laboratory in the department of Genome Sciences at the University of Washington. He was awarded a NIH Genome training fellowship in 2010 to study the genetic regulators of surfactant metabolism that lead to neonatal respiratory distress syndrome. His work focuses on the development of mass spectrometric methods to investigate protein turnover using both in vivo and in vitro models. His goal is to discover protein kinetic characteristics that are associated with disease acuity and progression—ultimately allowing the elucidation of therapeutic targets. We report a method to measure in vivo turnover of four proteins from sequential tracheal aspirates obtained from human newborn infants with respiratory distress syndrome using targeted proteomics. We detected enrichment for all targeted proteins approximately 3 h from the start of infusion of [5,5,5-2H3] leucine, secretion times that varied from 1.2 to 2.5 h, and half lives that ranged between 10 and 21 h. Complement factor B, a component of the alternative pathway of complement activation, had an approximately twofold-longer half-life than the other three proteins. In addition, the kinetics of mature and carboxy-terminal tryptic peptides from the same protein (surfactant protein B) were not statistically different (p = 0.49).
Keywords: Protein turnover; Respiratory distress syndrome; Selected reaction monitoring; SRM; Protein kinetics; Protein metabolism
A method to determine the kinetics of multiple proteins in human infants with respiratory distress syndrome by Michael S. Bereman; Daniela M. Tomazela; Hillary S. Heins; Manuela Simonato; Paola E. Cogo; Aaron Hamvas; Bruce W. Patterson; F. Sessions Cole; Michael J. MacCoss (pp. 2397-2402).
is currently in the MacCoss Laboratory in the department of Genome Sciences at the University of Washington. He was awarded a NIH Genome training fellowship in 2010 to study the genetic regulators of surfactant metabolism that lead to neonatal respiratory distress syndrome. His work focuses on the development of mass spectrometric methods to investigate protein turnover using both in vivo and in vitro models. His goal is to discover protein kinetic characteristics that are associated with disease acuity and progression—ultimately allowing the elucidation of therapeutic targets. We report a method to measure in vivo turnover of four proteins from sequential tracheal aspirates obtained from human newborn infants with respiratory distress syndrome using targeted proteomics. We detected enrichment for all targeted proteins approximately 3 h from the start of infusion of [5,5,5-2H3] leucine, secretion times that varied from 1.2 to 2.5 h, and half lives that ranged between 10 and 21 h. Complement factor B, a component of the alternative pathway of complement activation, had an approximately twofold-longer half-life than the other three proteins. In addition, the kinetics of mature and carboxy-terminal tryptic peptides from the same protein (surfactant protein B) were not statistically different (p = 0.49).
Keywords: Protein turnover; Respiratory distress syndrome; Selected reaction monitoring; SRM; Protein kinetics; Protein metabolism
A method to determine the kinetics of multiple proteins in human infants with respiratory distress syndrome by Michael S. Bereman; Daniela M. Tomazela; Hillary S. Heins; Manuela Simonato; Paola E. Cogo; Aaron Hamvas; Bruce W. Patterson; F. Sessions Cole; Michael J. MacCoss (pp. 2397-2402).
is currently in the MacCoss Laboratory in the department of Genome Sciences at the University of Washington. He was awarded a NIH Genome training fellowship in 2010 to study the genetic regulators of surfactant metabolism that lead to neonatal respiratory distress syndrome. His work focuses on the development of mass spectrometric methods to investigate protein turnover using both in vivo and in vitro models. His goal is to discover protein kinetic characteristics that are associated with disease acuity and progression—ultimately allowing the elucidation of therapeutic targets. We report a method to measure in vivo turnover of four proteins from sequential tracheal aspirates obtained from human newborn infants with respiratory distress syndrome using targeted proteomics. We detected enrichment for all targeted proteins approximately 3 h from the start of infusion of [5,5,5-2H3] leucine, secretion times that varied from 1.2 to 2.5 h, and half lives that ranged between 10 and 21 h. Complement factor B, a component of the alternative pathway of complement activation, had an approximately twofold-longer half-life than the other three proteins. In addition, the kinetics of mature and carboxy-terminal tryptic peptides from the same protein (surfactant protein B) were not statistically different (p = 0.49).
Keywords: Protein turnover; Respiratory distress syndrome; Selected reaction monitoring; SRM; Protein kinetics; Protein metabolism
Accumulation and efflux of polychlorinated biphenyls in Escherichia coli by Shen Geng; Jun Fang; Kendrick B. Turner; Sylvia Daunert; Yinan Wei (pp. 2403-2409).
has been an Assistant Professor of Chemistry at the University of Kentucky since August 2008. She has won the 2010 Ralph E. Powe Junior Faculty Enhancement Award. Her current research interests include the understanding of the biogenesis process of membrane proteins, structural and functional characterization of multidrug transporters, and development of biosensors. Polychlorinated biphenyls (PCBs) are environmental pollutants that have been associated with numerous adverse health effects in human and animals. Hydroxylated PCBs (HPCBs) are the product of the oxidative metabolism of PCBs. The presence of hydroxyl groups in HPCBs makes these compounds more hydrophilic than the parent PCBs. One of the best approaches to break down and remove these contaminants is bioremediation; an environmentally friendly process that uses microorganisms to degrade hazardous chemicals into non-toxic ones. In this study, we investigated the cellular accumulation and toxicity of selected PCBs and HPCBs in Gram-negative bacteria, using Escherichia coli as a model organism. We found that none of the five PCBs tested were toxic to E. coli, presumably due to their limited bioavailability. Nevertheless, different HPCBs tested showed different levels of toxicity. Furthermore, we demonstrated that the primary multidrug efflux system in E. coli, AcrAB–TolC, facilitated the efflux of HPCBs out of the cell. Since AcrAB–TolC is constitutively expressed in E. coli and is conserved in all sequenced Gram-negative bacterial genomes, our results suggest that the efflux activities of multidrug resistant pumps may affect the accumulation and degradation of PCBs in Gram-negative bacteria. Figure The major multidrug efflux pump AcrAB-TolC in Gram-negative bacteria actively transports hydroxylated PCBs out of cells.
Keywords: Hydroxylated polychlorinated biphenyl; Toxicity; Multidrug efflux pump; Whole cell sensing
Accumulation and efflux of polychlorinated biphenyls in Escherichia coli by Shen Geng; Jun Fang; Kendrick B. Turner; Sylvia Daunert; Yinan Wei (pp. 2403-2409).
has been an Assistant Professor of Chemistry at the University of Kentucky since August 2008. She has won the 2010 Ralph E. Powe Junior Faculty Enhancement Award. Her current research interests include the understanding of the biogenesis process of membrane proteins, structural and functional characterization of multidrug transporters, and development of biosensors. Polychlorinated biphenyls (PCBs) are environmental pollutants that have been associated with numerous adverse health effects in human and animals. Hydroxylated PCBs (HPCBs) are the product of the oxidative metabolism of PCBs. The presence of hydroxyl groups in HPCBs makes these compounds more hydrophilic than the parent PCBs. One of the best approaches to break down and remove these contaminants is bioremediation; an environmentally friendly process that uses microorganisms to degrade hazardous chemicals into non-toxic ones. In this study, we investigated the cellular accumulation and toxicity of selected PCBs and HPCBs in Gram-negative bacteria, using Escherichia coli as a model organism. We found that none of the five PCBs tested were toxic to E. coli, presumably due to their limited bioavailability. Nevertheless, different HPCBs tested showed different levels of toxicity. Furthermore, we demonstrated that the primary multidrug efflux system in E. coli, AcrAB–TolC, facilitated the efflux of HPCBs out of the cell. Since AcrAB–TolC is constitutively expressed in E. coli and is conserved in all sequenced Gram-negative bacterial genomes, our results suggest that the efflux activities of multidrug resistant pumps may affect the accumulation and degradation of PCBs in Gram-negative bacteria. Figure The major multidrug efflux pump AcrAB-TolC in Gram-negative bacteria actively transports hydroxylated PCBs out of cells.
Keywords: Hydroxylated polychlorinated biphenyl; Toxicity; Multidrug efflux pump; Whole cell sensing
Accumulation and efflux of polychlorinated biphenyls in Escherichia coli by Shen Geng; Jun Fang; Kendrick B. Turner; Sylvia Daunert; Yinan Wei (pp. 2403-2409).
has been an Assistant Professor of Chemistry at the University of Kentucky since August 2008. She has won the 2010 Ralph E. Powe Junior Faculty Enhancement Award. Her current research interests include the understanding of the biogenesis process of membrane proteins, structural and functional characterization of multidrug transporters, and development of biosensors. Polychlorinated biphenyls (PCBs) are environmental pollutants that have been associated with numerous adverse health effects in human and animals. Hydroxylated PCBs (HPCBs) are the product of the oxidative metabolism of PCBs. The presence of hydroxyl groups in HPCBs makes these compounds more hydrophilic than the parent PCBs. One of the best approaches to break down and remove these contaminants is bioremediation; an environmentally friendly process that uses microorganisms to degrade hazardous chemicals into non-toxic ones. In this study, we investigated the cellular accumulation and toxicity of selected PCBs and HPCBs in Gram-negative bacteria, using Escherichia coli as a model organism. We found that none of the five PCBs tested were toxic to E. coli, presumably due to their limited bioavailability. Nevertheless, different HPCBs tested showed different levels of toxicity. Furthermore, we demonstrated that the primary multidrug efflux system in E. coli, AcrAB–TolC, facilitated the efflux of HPCBs out of the cell. Since AcrAB–TolC is constitutively expressed in E. coli and is conserved in all sequenced Gram-negative bacterial genomes, our results suggest that the efflux activities of multidrug resistant pumps may affect the accumulation and degradation of PCBs in Gram-negative bacteria. Figure The major multidrug efflux pump AcrAB-TolC in Gram-negative bacteria actively transports hydroxylated PCBs out of cells.
Keywords: Hydroxylated polychlorinated biphenyl; Toxicity; Multidrug efflux pump; Whole cell sensing
Solid-phase microextraction of 2,4,6-trinitrotoluene using a molecularly imprinted-based fiber by Federica Bianchi; Marco Giannetto; G. Mori; G. D’Agostino; M. Careri; A. Mangia (pp. 2411-2418).
obtained her PhD in Chemical Science in 2002 at the University of Parma. She is researcher at the Department of General and Inorganic Chemistry, Analytical Chemistry, Physical Chemistry of the University of Parma. Her research interests mainly focus on the development of novel materials for sample treatment and on the development of MS-based analytical methods. graduated in 1966 in Chemistry at the University of Messina. In 2000, he achieved a PhD in Chemical Sciences at the University of Parma where he has been a researcher at the Department of General and In organic Chemistry, Analytical Chemistry, Physical Chemistry, since 2001. His scientific activity is focused on the realization and characterization of new chemical sensors and biosensors based on innovative recognition materials and with different transduction mechanisms. A molecularly imprinted polymer with trinitrotoluene as the template molecule was synthesized and used as the novel coating for solid-phase microextraction of the nitroaromatic explosive 2,4,6-trinitrotoluene for its selective determination. The fiber was characterized in terms of coating thickness, morphology, intra- and inter-batch repeatability and extraction efficiency. An average thickness of 50 ± 4 μm with a uniform distribution of the coating was obtained. Good performances of the developed procedure in term of both intra-batch and inter-batch repeatability with relative standard deviations <8% were obtained. Finally, detection and quantitation limits in the low nanogram per kilogram levels were achieved proving the superior extraction capability of the developed coating, obtaining gas chromatography-mass spectrometry responses about two times higher than those achieved using commercial devices. Figure Preparation of a MIP-based fiber for the solid-phase microextraction of 2,4,6-trinitrotoluene
Keywords: Solid-phase microextraction; Molecularly imprinted polymer; 2,4,6-Trinitrotoluene; Coating; Explosives
Solid-phase microextraction of 2,4,6-trinitrotoluene using a molecularly imprinted-based fiber by Federica Bianchi; Marco Giannetto; G. Mori; G. D’Agostino; M. Careri; A. Mangia (pp. 2411-2418).
obtained her PhD in Chemical Science in 2002 at the University of Parma. She is researcher at the Department of General and Inorganic Chemistry, Analytical Chemistry, Physical Chemistry of the University of Parma. Her research interests mainly focus on the development of novel materials for sample treatment and on the development of MS-based analytical methods. graduated in 1966 in Chemistry at the University of Messina. In 2000, he achieved a PhD in Chemical Sciences at the University of Parma where he has been a researcher at the Department of General and In organic Chemistry, Analytical Chemistry, Physical Chemistry, since 2001. His scientific activity is focused on the realization and characterization of new chemical sensors and biosensors based on innovative recognition materials and with different transduction mechanisms. A molecularly imprinted polymer with trinitrotoluene as the template molecule was synthesized and used as the novel coating for solid-phase microextraction of the nitroaromatic explosive 2,4,6-trinitrotoluene for its selective determination. The fiber was characterized in terms of coating thickness, morphology, intra- and inter-batch repeatability and extraction efficiency. An average thickness of 50 ± 4 μm with a uniform distribution of the coating was obtained. Good performances of the developed procedure in term of both intra-batch and inter-batch repeatability with relative standard deviations <8% were obtained. Finally, detection and quantitation limits in the low nanogram per kilogram levels were achieved proving the superior extraction capability of the developed coating, obtaining gas chromatography-mass spectrometry responses about two times higher than those achieved using commercial devices. Figure Preparation of a MIP-based fiber for the solid-phase microextraction of 2,4,6-trinitrotoluene
Keywords: Solid-phase microextraction; Molecularly imprinted polymer; 2,4,6-Trinitrotoluene; Coating; Explosives
Solid-phase microextraction of 2,4,6-trinitrotoluene using a molecularly imprinted-based fiber by Federica Bianchi; Marco Giannetto; G. Mori; G. D’Agostino; M. Careri; A. Mangia (pp. 2411-2418).
obtained her PhD in Chemical Science in 2002 at the University of Parma. She is researcher at the Department of General and Inorganic Chemistry, Analytical Chemistry, Physical Chemistry of the University of Parma. Her research interests mainly focus on the development of novel materials for sample treatment and on the development of MS-based analytical methods. graduated in 1966 in Chemistry at the University of Messina. In 2000, he achieved a PhD in Chemical Sciences at the University of Parma where he has been a researcher at the Department of General and In organic Chemistry, Analytical Chemistry, Physical Chemistry, since 2001. His scientific activity is focused on the realization and characterization of new chemical sensors and biosensors based on innovative recognition materials and with different transduction mechanisms. A molecularly imprinted polymer with trinitrotoluene as the template molecule was synthesized and used as the novel coating for solid-phase microextraction of the nitroaromatic explosive 2,4,6-trinitrotoluene for its selective determination. The fiber was characterized in terms of coating thickness, morphology, intra- and inter-batch repeatability and extraction efficiency. An average thickness of 50 ± 4 μm with a uniform distribution of the coating was obtained. Good performances of the developed procedure in term of both intra-batch and inter-batch repeatability with relative standard deviations <8% were obtained. Finally, detection and quantitation limits in the low nanogram per kilogram levels were achieved proving the superior extraction capability of the developed coating, obtaining gas chromatography-mass spectrometry responses about two times higher than those achieved using commercial devices. Figure Preparation of a MIP-based fiber for the solid-phase microextraction of 2,4,6-trinitrotoluene
Keywords: Solid-phase microextraction; Molecularly imprinted polymer; 2,4,6-Trinitrotoluene; Coating; Explosives
Ultra-trace determination of mercury in river waters after online UV digestion of humic matter by Kerstin Leopold; Anja Zierhut; Jessica Huber (pp. 2419-2428).
is an Associate Professor of Analytical Chemistry at the University of Ulm at the Institute of Analytical and Bioanalytical Chemistry. Her group is developing new methodologies for trace metal analysis in environmental biological and medical samples. Thereby, nanoparticles play an important role, either as a tool to enhance analytical procedures, or as target analyte. A fully automated online ultraviolet (UV) digestion method for subsequent mercury (Hg) quantification in humic matter containing river waters is reported. The new developed flow injection analysis system (FIAS) consists basically of a UV lamp, a meander-form quartz glass reaction tube for online irradiation of the sample, and a nano-gold collector for preconcentration of dissolved mercury species. The FIAS is coupled to an atomic fluorescence spectrometer (AFS) for Hg detection. The optimized procedure allows accurate mercury quantification in water samples with up to 15 mg C L−1 as dissolved organic carbon by addition of only 1% (v/v) of hydrogen peroxide solution and online UV irradiation for 6 min. Addition of strong oxidants and any other reagents is avoided due to the use of the catalytic active nano-gold collector. Here, preconcentration of Hg species, release of mercury as Hg0, and AFS measurement are performed without addition of any reagents. Hence, the proposed approach offers significant advantages over existing methods. Analytical figures of merit showed the good performance of the developed method: The limit of quantification was found to be as low as 0.14 ng Hg L−1. The linear working range is from 0.1 to 200 ng Hg L−1 and relative standard deviation is <6.0% (n = 9). The system was successfully validated by comparison of the mercury concentrations found in model and real water samples obtained by the reference method EPA 1631 and the proposed method. Furthermore, application to six real river waters confirmed the feasibility of the proposed approach. Figure Ultra-trace determination of mercury in river waters after online UV digestion of humic matter
Keywords: Mercury trace analysis; Natural waters; Humic matter; Online digestion; H2O2-assisted UV digestion; Preconcentration; Nano-gold collector; Flow injection analysis
Ultra-trace determination of mercury in river waters after online UV digestion of humic matter by Kerstin Leopold; Anja Zierhut; Jessica Huber (pp. 2419-2428).
is an Associate Professor of Analytical Chemistry at the University of Ulm at the Institute of Analytical and Bioanalytical Chemistry. Her group is developing new methodologies for trace metal analysis in environmental biological and medical samples. Thereby, nanoparticles play an important role, either as a tool to enhance analytical procedures, or as target analyte. A fully automated online ultraviolet (UV) digestion method for subsequent mercury (Hg) quantification in humic matter containing river waters is reported. The new developed flow injection analysis system (FIAS) consists basically of a UV lamp, a meander-form quartz glass reaction tube for online irradiation of the sample, and a nano-gold collector for preconcentration of dissolved mercury species. The FIAS is coupled to an atomic fluorescence spectrometer (AFS) for Hg detection. The optimized procedure allows accurate mercury quantification in water samples with up to 15 mg C L−1 as dissolved organic carbon by addition of only 1% (v/v) of hydrogen peroxide solution and online UV irradiation for 6 min. Addition of strong oxidants and any other reagents is avoided due to the use of the catalytic active nano-gold collector. Here, preconcentration of Hg species, release of mercury as Hg0, and AFS measurement are performed without addition of any reagents. Hence, the proposed approach offers significant advantages over existing methods. Analytical figures of merit showed the good performance of the developed method: The limit of quantification was found to be as low as 0.14 ng Hg L−1. The linear working range is from 0.1 to 200 ng Hg L−1 and relative standard deviation is <6.0% (n = 9). The system was successfully validated by comparison of the mercury concentrations found in model and real water samples obtained by the reference method EPA 1631 and the proposed method. Furthermore, application to six real river waters confirmed the feasibility of the proposed approach. Figure Ultra-trace determination of mercury in river waters after online UV digestion of humic matter
Keywords: Mercury trace analysis; Natural waters; Humic matter; Online digestion; H2O2-assisted UV digestion; Preconcentration; Nano-gold collector; Flow injection analysis
Ultra-trace determination of mercury in river waters after online UV digestion of humic matter by Kerstin Leopold; Anja Zierhut; Jessica Huber (pp. 2419-2428).
is an Associate Professor of Analytical Chemistry at the University of Ulm at the Institute of Analytical and Bioanalytical Chemistry. Her group is developing new methodologies for trace metal analysis in environmental biological and medical samples. Thereby, nanoparticles play an important role, either as a tool to enhance analytical procedures, or as target analyte. A fully automated online ultraviolet (UV) digestion method for subsequent mercury (Hg) quantification in humic matter containing river waters is reported. The new developed flow injection analysis system (FIAS) consists basically of a UV lamp, a meander-form quartz glass reaction tube for online irradiation of the sample, and a nano-gold collector for preconcentration of dissolved mercury species. The FIAS is coupled to an atomic fluorescence spectrometer (AFS) for Hg detection. The optimized procedure allows accurate mercury quantification in water samples with up to 15 mg C L−1 as dissolved organic carbon by addition of only 1% (v/v) of hydrogen peroxide solution and online UV irradiation for 6 min. Addition of strong oxidants and any other reagents is avoided due to the use of the catalytic active nano-gold collector. Here, preconcentration of Hg species, release of mercury as Hg0, and AFS measurement are performed without addition of any reagents. Hence, the proposed approach offers significant advantages over existing methods. Analytical figures of merit showed the good performance of the developed method: The limit of quantification was found to be as low as 0.14 ng Hg L−1. The linear working range is from 0.1 to 200 ng Hg L−1 and relative standard deviation is <6.0% (n = 9). The system was successfully validated by comparison of the mercury concentrations found in model and real water samples obtained by the reference method EPA 1631 and the proposed method. Furthermore, application to six real river waters confirmed the feasibility of the proposed approach. Figure Ultra-trace determination of mercury in river waters after online UV digestion of humic matter
Keywords: Mercury trace analysis; Natural waters; Humic matter; Online digestion; H2O2-assisted UV digestion; Preconcentration; Nano-gold collector; Flow injection analysis
Fingerprinting of red wine by headspace solid-phase dynamic extraction of volatile constituents by Jens Laaks; Thomas Letzel; Torsten C. Schmidt; Maik A. Jochmann (pp. 2429-2436).
has been an academic counsellor in the group of Torsten Schmidt at the University of Duisburg-Essen since October 2009. His current research interests are developing methods for compound-specific isotope analysis and microextraction techniques. Headspace solid-phase dynamic extraction (HS-SPDE) was investigated for its applicability in quality control analysis of wine volatiles using gas chromatography–mass spectrometry. In total, 196 German red wines were analysed and 22 flavour-relevant alcohols and esters were quantified. The method detection limits were between 0.1 and 9.3 μg L-1, allowing the dilution of the samples to decrease matrix and competition effects. Quantification resulted in a concentration range from about 1 μg L-1 linalool up to 380 mg L-1 2-methyl-1-propanol. The measurement uncertainty budget was determined for all compounds in a “top-down” approach and was between 2.5 and 7.9 %, with an average of 5.5 %. A surveillance of the extraction performance of the HS-SPDE devices showed constant results for up to 400 extractions using one extraction needle tip. A chromatogram library for quality and authenticity control of wine samples was created using commercially available chromatogram comparison software.
Keywords: Solid-phase dynamic extraction; Wine authenticity; Flavour compounds
Fingerprinting of red wine by headspace solid-phase dynamic extraction of volatile constituents by Jens Laaks; Thomas Letzel; Torsten C. Schmidt; Maik A. Jochmann (pp. 2429-2436).
has been an academic counsellor in the group of Torsten Schmidt at the University of Duisburg-Essen since October 2009. His current research interests are developing methods for compound-specific isotope analysis and microextraction techniques. Headspace solid-phase dynamic extraction (HS-SPDE) was investigated for its applicability in quality control analysis of wine volatiles using gas chromatography–mass spectrometry. In total, 196 German red wines were analysed and 22 flavour-relevant alcohols and esters were quantified. The method detection limits were between 0.1 and 9.3 μg L-1, allowing the dilution of the samples to decrease matrix and competition effects. Quantification resulted in a concentration range from about 1 μg L-1 linalool up to 380 mg L-1 2-methyl-1-propanol. The measurement uncertainty budget was determined for all compounds in a “top-down” approach and was between 2.5 and 7.9 %, with an average of 5.5 %. A surveillance of the extraction performance of the HS-SPDE devices showed constant results for up to 400 extractions using one extraction needle tip. A chromatogram library for quality and authenticity control of wine samples was created using commercially available chromatogram comparison software.
Keywords: Solid-phase dynamic extraction; Wine authenticity; Flavour compounds
Fingerprinting of red wine by headspace solid-phase dynamic extraction of volatile constituents by Jens Laaks; Thomas Letzel; Torsten C. Schmidt; Maik A. Jochmann (pp. 2429-2436).
has been an academic counsellor in the group of Torsten Schmidt at the University of Duisburg-Essen since October 2009. His current research interests are developing methods for compound-specific isotope analysis and microextraction techniques. Headspace solid-phase dynamic extraction (HS-SPDE) was investigated for its applicability in quality control analysis of wine volatiles using gas chromatography–mass spectrometry. In total, 196 German red wines were analysed and 22 flavour-relevant alcohols and esters were quantified. The method detection limits were between 0.1 and 9.3 μg L-1, allowing the dilution of the samples to decrease matrix and competition effects. Quantification resulted in a concentration range from about 1 μg L-1 linalool up to 380 mg L-1 2-methyl-1-propanol. The measurement uncertainty budget was determined for all compounds in a “top-down” approach and was between 2.5 and 7.9 %, with an average of 5.5 %. A surveillance of the extraction performance of the HS-SPDE devices showed constant results for up to 400 extractions using one extraction needle tip. A chromatogram library for quality and authenticity control of wine samples was created using commercially available chromatogram comparison software.
Keywords: Solid-phase dynamic extraction; Wine authenticity; Flavour compounds
Pulsed rf-GD-TOFMS for depth profile analysis of ultrathin layers using the analyte prepeak region by J. Pisonero; R. Valledor; A. Licciardello; C. Quirós; J. I. Martín; A. Sanz-Medel; N. Bordel (pp. 2437-2448).
is “Ramón y Cajal” Researcher at the Laser and Plasma Spectroscopy Group ( www.unioviedo.es/gelp ) in the Department of Physics, at University of Oviedo, Spain.He was awarded the 2009 International Masao Horiba Award and, very recently, the 2011 National Prize to Novel Researchers in Experimental Physics, given by the Royal Spanish Society of Physics. His main interest is related to fundamental studies and applications in direct solid analysis using glow discharge spectroscopies and laser ablation techniques. Direct solid analysis of ultrathin layers is investigated using pulsed radiofrequency (rf) glow discharge (GD) time-of-flight mass spectrometry (TOFMS). In particular, previous studies have always integrated the detected ion signals in the afterglow region of the rf-GD pulse, which is known to be the most sensitive one. Nevertheless, the analytical capabilities of other pulse time regions have not been evaluated in detail. Therefore, in this work, we investigate the analyte prepeak region, which is the pulse region where the analyte ions peak after the initial sputtering process of each GD pulse, aiming at obtaining improved depth profile analysis with high depth resolution and with minimum polyatomic spectral interferences. To perform these studies, challenging ultrathin Si–Co bilayers deposited on a Si substrate were investigated. The thickness of the external Si layer was 30 nm for all the samples, whilst the internal Co layer thicknesses were 30, 10, 5, 2 and 1 nm, respectively. It should be remarked that the top layer and the substrate have the same matrix composition (Si > 99.99%). Therefore, the selected samples are suitable to evaluate the response of the Si ion signal in the presence of an ultrathin Co layer as well as the possible oxygen contaminations or its reactions. Additionally, these samples have been evaluated using time-of-flight secondary ion mass spectrometry, and the results compare well to those obtained by our pulsed rf-GD time-of-flight mass spectrometry results.
Keywords: Glow discharge; Mass spectrometry; Depth profile analysis; Nanometer layers; Analyte prepeak; Afterglow; SIMS
Pulsed rf-GD-TOFMS for depth profile analysis of ultrathin layers using the analyte prepeak region by J. Pisonero; R. Valledor; A. Licciardello; C. Quirós; J. I. Martín; A. Sanz-Medel; N. Bordel (pp. 2437-2448).
is “Ramón y Cajal” Researcher at the Laser and Plasma Spectroscopy Group ( www.unioviedo.es/gelp ) in the Department of Physics, at University of Oviedo, Spain.He was awarded the 2009 International Masao Horiba Award and, very recently, the 2011 National Prize to Novel Researchers in Experimental Physics, given by the Royal Spanish Society of Physics. His main interest is related to fundamental studies and applications in direct solid analysis using glow discharge spectroscopies and laser ablation techniques. Direct solid analysis of ultrathin layers is investigated using pulsed radiofrequency (rf) glow discharge (GD) time-of-flight mass spectrometry (TOFMS). In particular, previous studies have always integrated the detected ion signals in the afterglow region of the rf-GD pulse, which is known to be the most sensitive one. Nevertheless, the analytical capabilities of other pulse time regions have not been evaluated in detail. Therefore, in this work, we investigate the analyte prepeak region, which is the pulse region where the analyte ions peak after the initial sputtering process of each GD pulse, aiming at obtaining improved depth profile analysis with high depth resolution and with minimum polyatomic spectral interferences. To perform these studies, challenging ultrathin Si–Co bilayers deposited on a Si substrate were investigated. The thickness of the external Si layer was 30 nm for all the samples, whilst the internal Co layer thicknesses were 30, 10, 5, 2 and 1 nm, respectively. It should be remarked that the top layer and the substrate have the same matrix composition (Si > 99.99%). Therefore, the selected samples are suitable to evaluate the response of the Si ion signal in the presence of an ultrathin Co layer as well as the possible oxygen contaminations or its reactions. Additionally, these samples have been evaluated using time-of-flight secondary ion mass spectrometry, and the results compare well to those obtained by our pulsed rf-GD time-of-flight mass spectrometry results.
Keywords: Glow discharge; Mass spectrometry; Depth profile analysis; Nanometer layers; Analyte prepeak; Afterglow; SIMS
Pulsed rf-GD-TOFMS for depth profile analysis of ultrathin layers using the analyte prepeak region by J. Pisonero; R. Valledor; A. Licciardello; C. Quirós; J. I. Martín; A. Sanz-Medel; N. Bordel (pp. 2437-2448).
is “Ramón y Cajal” Researcher at the Laser and Plasma Spectroscopy Group ( www.unioviedo.es/gelp ) in the Department of Physics, at University of Oviedo, Spain.He was awarded the 2009 International Masao Horiba Award and, very recently, the 2011 National Prize to Novel Researchers in Experimental Physics, given by the Royal Spanish Society of Physics. His main interest is related to fundamental studies and applications in direct solid analysis using glow discharge spectroscopies and laser ablation techniques. Direct solid analysis of ultrathin layers is investigated using pulsed radiofrequency (rf) glow discharge (GD) time-of-flight mass spectrometry (TOFMS). In particular, previous studies have always integrated the detected ion signals in the afterglow region of the rf-GD pulse, which is known to be the most sensitive one. Nevertheless, the analytical capabilities of other pulse time regions have not been evaluated in detail. Therefore, in this work, we investigate the analyte prepeak region, which is the pulse region where the analyte ions peak after the initial sputtering process of each GD pulse, aiming at obtaining improved depth profile analysis with high depth resolution and with minimum polyatomic spectral interferences. To perform these studies, challenging ultrathin Si–Co bilayers deposited on a Si substrate were investigated. The thickness of the external Si layer was 30 nm for all the samples, whilst the internal Co layer thicknesses were 30, 10, 5, 2 and 1 nm, respectively. It should be remarked that the top layer and the substrate have the same matrix composition (Si > 99.99%). Therefore, the selected samples are suitable to evaluate the response of the Si ion signal in the presence of an ultrathin Co layer as well as the possible oxygen contaminations or its reactions. Additionally, these samples have been evaluated using time-of-flight secondary ion mass spectrometry, and the results compare well to those obtained by our pulsed rf-GD time-of-flight mass spectrometry results.
Keywords: Glow discharge; Mass spectrometry; Depth profile analysis; Nanometer layers; Analyte prepeak; Afterglow; SIMS
Microchip-based immunoassays with application of silicon dioxide nanoparticle film by Yun Li; Qin-Shu Kang; Guo-Ping Sun; Li-Jin Su; Zhen-Hua Zheng; Zhen-Feng Zhang; Han-Zhong Wang; Zhi-Ke He; Wei-Hua Huang (pp. 2449-2457).
is currently Professor at the College of Chemistry and Molecular Sciences, Wuhan University. His research interest focuses on bioanalysis using microelectrode electrochemistry and microfluidic-based systems. Highly sensitive detection of proteins offers the possibility of early and rapid diagnosis of various diseases. Microchip-based immunoassay integrates the benefits from both immunoassays (high specificity of target sample) and microfluidics (fast analysis and low sample consumption). However, direct capture of proteins on bare microchannel surface suffers from low sensitivity due to the low capacity of microsystem. In this study, we demonstrated a microchip-based heterogeneous immunoassay using functionalized SiO2 nanoparticles which were covalently assembled on the surface of microchannels via a liquid-phase deposition technique. The formation of covalent bonds between SiO2 nanoparticles and polydimethylsiloxane substrate offered sufficient stability of the microfluidic surface, and furthermore, substantially enhanced the protein capturing capability, mainly due to the increased surface-area-to-volume ratio. IgG antigen and FITC-labeled anti-IgG antibody conjugates were adopted to compare protein-enrichment effect, and the fluorescence signals were increased by ∼75-fold after introduction of functionalized SiO2 nanoparticles film. Finally, a proof-of-concept experiment was performed by highly efficient capture and detection of inactivated H1N1 influenza virus using a microfluidic chip comprising highly ordered SiO2 nanoparticles coated micropillars array. The detection limit of H1N1 virus antigen was 0.5 ng mL−1, with a linear range from 20 to 1,000 ng mL−1 and mean coefficient of variance of 4.71 %. Figure SiO2 nanoparticles were covalently assembled on the surface of microfluidic channels via a liquid-phase deposition technique, and inactivated H1N1 influenza viruses were immunocaptured and detected with a microfluidic chip comprising highly ordered SiO2 nanoparticles coated micropillars array.
Keywords: Microfluidic chip; Liquid-phase deposition (LPD); SiO2 nanoparticles; Immunoassays; H1N1 influenza virus
Microchip-based immunoassays with application of silicon dioxide nanoparticle film by Yun Li; Qin-Shu Kang; Guo-Ping Sun; Li-Jin Su; Zhen-Hua Zheng; Zhen-Feng Zhang; Han-Zhong Wang; Zhi-Ke He; Wei-Hua Huang (pp. 2449-2457).
is currently Professor at the College of Chemistry and Molecular Sciences, Wuhan University. His research interest focuses on bioanalysis using microelectrode electrochemistry and microfluidic-based systems. Highly sensitive detection of proteins offers the possibility of early and rapid diagnosis of various diseases. Microchip-based immunoassay integrates the benefits from both immunoassays (high specificity of target sample) and microfluidics (fast analysis and low sample consumption). However, direct capture of proteins on bare microchannel surface suffers from low sensitivity due to the low capacity of microsystem. In this study, we demonstrated a microchip-based heterogeneous immunoassay using functionalized SiO2 nanoparticles which were covalently assembled on the surface of microchannels via a liquid-phase deposition technique. The formation of covalent bonds between SiO2 nanoparticles and polydimethylsiloxane substrate offered sufficient stability of the microfluidic surface, and furthermore, substantially enhanced the protein capturing capability, mainly due to the increased surface-area-to-volume ratio. IgG antigen and FITC-labeled anti-IgG antibody conjugates were adopted to compare protein-enrichment effect, and the fluorescence signals were increased by ∼75-fold after introduction of functionalized SiO2 nanoparticles film. Finally, a proof-of-concept experiment was performed by highly efficient capture and detection of inactivated H1N1 influenza virus using a microfluidic chip comprising highly ordered SiO2 nanoparticles coated micropillars array. The detection limit of H1N1 virus antigen was 0.5 ng mL−1, with a linear range from 20 to 1,000 ng mL−1 and mean coefficient of variance of 4.71 %. Figure SiO2 nanoparticles were covalently assembled on the surface of microfluidic channels via a liquid-phase deposition technique, and inactivated H1N1 influenza viruses were immunocaptured and detected with a microfluidic chip comprising highly ordered SiO2 nanoparticles coated micropillars array.
Keywords: Microfluidic chip; Liquid-phase deposition (LPD); SiO2 nanoparticles; Immunoassays; H1N1 influenza virus
Microchip-based immunoassays with application of silicon dioxide nanoparticle film by Yun Li; Qin-Shu Kang; Guo-Ping Sun; Li-Jin Su; Zhen-Hua Zheng; Zhen-Feng Zhang; Han-Zhong Wang; Zhi-Ke He; Wei-Hua Huang (pp. 2449-2457).
is currently Professor at the College of Chemistry and Molecular Sciences, Wuhan University. His research interest focuses on bioanalysis using microelectrode electrochemistry and microfluidic-based systems. Highly sensitive detection of proteins offers the possibility of early and rapid diagnosis of various diseases. Microchip-based immunoassay integrates the benefits from both immunoassays (high specificity of target sample) and microfluidics (fast analysis and low sample consumption). However, direct capture of proteins on bare microchannel surface suffers from low sensitivity due to the low capacity of microsystem. In this study, we demonstrated a microchip-based heterogeneous immunoassay using functionalized SiO2 nanoparticles which were covalently assembled on the surface of microchannels via a liquid-phase deposition technique. The formation of covalent bonds between SiO2 nanoparticles and polydimethylsiloxane substrate offered sufficient stability of the microfluidic surface, and furthermore, substantially enhanced the protein capturing capability, mainly due to the increased surface-area-to-volume ratio. IgG antigen and FITC-labeled anti-IgG antibody conjugates were adopted to compare protein-enrichment effect, and the fluorescence signals were increased by ∼75-fold after introduction of functionalized SiO2 nanoparticles film. Finally, a proof-of-concept experiment was performed by highly efficient capture and detection of inactivated H1N1 influenza virus using a microfluidic chip comprising highly ordered SiO2 nanoparticles coated micropillars array. The detection limit of H1N1 virus antigen was 0.5 ng mL−1, with a linear range from 20 to 1,000 ng mL−1 and mean coefficient of variance of 4.71 %. Figure SiO2 nanoparticles were covalently assembled on the surface of microfluidic channels via a liquid-phase deposition technique, and inactivated H1N1 influenza viruses were immunocaptured and detected with a microfluidic chip comprising highly ordered SiO2 nanoparticles coated micropillars array.
Keywords: Microfluidic chip; Liquid-phase deposition (LPD); SiO2 nanoparticles; Immunoassays; H1N1 influenza virus