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Analytica Chimica Acta (v.695, #1-2)
Sensitive sequence-specific molecular identification system comprising an aluminum micro-nanofluidic chip and associated real-time confocal detector by Guoliang Huang; Can Wang; Li Ma; Xu Yang; Xiaoyong Yang; Guoqing Wang (pp. 1-10).
An Aluminum micro-nanofluidic chip and a confocal optical detector were developed for sensitive real-time clinical pathogen molecular diagnostics. This is the first report of Al metal being used to build a lab-on-a-chip. The Al micro-nanofluidic chip had a surface contact angle of 93.8° and was inert to biomolecules. DNA amplification could be performed in a reaction volume as small as 39nL, and a detection limit of approximately three genomic copies for stable and specific pathogen identification was obtained. Our results indicate that the chip is promising for applications in nanobiotechnology, nanomedicine, and clinical pathogen molecular diagnostics for nanoliter droplet amplification of several genomic copies from a single bacterium.We developed a micro-nanofluidic bioreactor-detector system for isothermal DNA amplification and sensitive real-time detection of the amplified products for sequence-specific molecular identification. Aluminum (Al) chips with a range of volumes from 7.07μL to 39nL and an associated real-time confocal optical detector are described. The detector provided highly sensitive fluorescence detection and low background noise. One of the important aspects of the system was the development of a surface processing technique that afforded chips with an inert surface to improve amplification stability in micro-nanoliter reaction assays. The micro-nanofluidic system exhibited more sensitive exponential DNA amplification characteristics than a standard PCR tube amplification system with a volume of 25μL, the response time was clearly reduced at the same DNA template concentration, and the sensitivity in the number of copies of the DNA template was improved by >600-fold. Efficient amplification of nucleic acid was achieved with as few as three copies of the DNA template. This system may be useful for the development of novel lab-on-a-chip devices and shows promise for single-molecule amplification in droplet assays, with potential applications in nanobiotechnology, nanomedicine, and clinical molecular diagnostics.
Keywords: Micro-nanofluidic chip; Inert surface processing; Isothermal amplification; Confocal optical detector; Molecular diagnostics
Sensitive sequence-specific molecular identification system comprising an aluminum micro-nanofluidic chip and associated real-time confocal detector by Guoliang Huang; Can Wang; Li Ma; Xu Yang; Xiaoyong Yang; Guoqing Wang (pp. 1-10).
An Aluminum micro-nanofluidic chip and a confocal optical detector were developed for sensitive real-time clinical pathogen molecular diagnostics. This is the first report of Al metal being used to build a lab-on-a-chip. The Al micro-nanofluidic chip had a surface contact angle of 93.8° and was inert to biomolecules. DNA amplification could be performed in a reaction volume as small as 39nL, and a detection limit of approximately three genomic copies for stable and specific pathogen identification was obtained. Our results indicate that the chip is promising for applications in nanobiotechnology, nanomedicine, and clinical pathogen molecular diagnostics for nanoliter droplet amplification of several genomic copies from a single bacterium.We developed a micro-nanofluidic bioreactor-detector system for isothermal DNA amplification and sensitive real-time detection of the amplified products for sequence-specific molecular identification. Aluminum (Al) chips with a range of volumes from 7.07μL to 39nL and an associated real-time confocal optical detector are described. The detector provided highly sensitive fluorescence detection and low background noise. One of the important aspects of the system was the development of a surface processing technique that afforded chips with an inert surface to improve amplification stability in micro-nanoliter reaction assays. The micro-nanofluidic system exhibited more sensitive exponential DNA amplification characteristics than a standard PCR tube amplification system with a volume of 25μL, the response time was clearly reduced at the same DNA template concentration, and the sensitivity in the number of copies of the DNA template was improved by >600-fold. Efficient amplification of nucleic acid was achieved with as few as three copies of the DNA template. This system may be useful for the development of novel lab-on-a-chip devices and shows promise for single-molecule amplification in droplet assays, with potential applications in nanobiotechnology, nanomedicine, and clinical molecular diagnostics.
Keywords: Micro-nanofluidic chip; Inert surface processing; Isothermal amplification; Confocal optical detector; Molecular diagnostics
Determination of antioxidant additives in foodstuffs by direct measurement of gold nanoparticle formation using resonance light scattering detection by A. Andreu-Navarro; J.M. Fernández-Romero; A. Gómez-Hens (pp. 11-17).
The capability of antioxidant compounds to reduce gold(III) to gold nanoparticles has been kinetically studied in the presence of cetyltrimethylammonium bromide using stopped-flow mixing technique and resonance light scattering as detection system. This study has given rise to a simple and rapid method for the determination of several synthetic and natural antioxidants used as additives in foodstuff samples. The formation of AuNPs was monitored by measuring the initial reaction-rate of the system in about 5s, using an integration time of 0.1s. Dynamic ranges of the calibration graphs and detection limits, obtained with standard solutions of the analytes, were (μmolL−1): gallic acid (0.04–0.59, 0.01), propyl gallate (0.04–1.41, 0.01), octyl gallate (0.03–0.35, 0.08), dodecyl gallate (0.02–0.30, 0.007), butylated hydroxyanisol (0.07–0.39, 0.009), butylated hydroxytoluene (0.04–0.32, 0.01), ascorbic acid (0.11–1.72, 0.03) and sodium citrate (0.07–1.29, 0.02). The regression coefficients were higher than 0.994 in all instances. The precision of the method, expressed as RSD%, was established at two concentration levels of each analyte, with values ranging between 0.6 and 4.8%. The practical usefulness of the developed method was demonstrated by the determination of several antioxidant additives in foodstuff samples, which were extracted, appropriately diluted and assayed, obtaining recoveries between 95.4 and 99.5%. The results obtained were validated using two reference methods.
Keywords: Antioxidant additives; Gold nanoparticles; Stopped-flow mixing technique; Resonance light scattering detection; Foodstuff samples
Determination of antioxidant additives in foodstuffs by direct measurement of gold nanoparticle formation using resonance light scattering detection by A. Andreu-Navarro; J.M. Fernández-Romero; A. Gómez-Hens (pp. 11-17).
The capability of antioxidant compounds to reduce gold(III) to gold nanoparticles has been kinetically studied in the presence of cetyltrimethylammonium bromide using stopped-flow mixing technique and resonance light scattering as detection system. This study has given rise to a simple and rapid method for the determination of several synthetic and natural antioxidants used as additives in foodstuff samples. The formation of AuNPs was monitored by measuring the initial reaction-rate of the system in about 5s, using an integration time of 0.1s. Dynamic ranges of the calibration graphs and detection limits, obtained with standard solutions of the analytes, were (μmolL−1): gallic acid (0.04–0.59, 0.01), propyl gallate (0.04–1.41, 0.01), octyl gallate (0.03–0.35, 0.08), dodecyl gallate (0.02–0.30, 0.007), butylated hydroxyanisol (0.07–0.39, 0.009), butylated hydroxytoluene (0.04–0.32, 0.01), ascorbic acid (0.11–1.72, 0.03) and sodium citrate (0.07–1.29, 0.02). The regression coefficients were higher than 0.994 in all instances. The precision of the method, expressed as RSD%, was established at two concentration levels of each analyte, with values ranging between 0.6 and 4.8%. The practical usefulness of the developed method was demonstrated by the determination of several antioxidant additives in foodstuff samples, which were extracted, appropriately diluted and assayed, obtaining recoveries between 95.4 and 99.5%. The results obtained were validated using two reference methods.
Keywords: Antioxidant additives; Gold nanoparticles; Stopped-flow mixing technique; Resonance light scattering detection; Foodstuff samples
Ionic liquids in solid-phase microextraction: A review by Tien D. Ho; Anthony J. Canestraro; Jared L. Anderson (pp. 18-43).
Solid-phase microextraction (SPME) has undergone a surge in popularity within the field of analytical chemistry in the past two decades since its introduction. Owing to its nature of extraction, SPME has become widely known as a quick and cost-effective sample preparation technique. Although SPME has demonstrated extraordinary versatility in sampling capabilities, the technique continues to experience a tremendous growth in innovation. Presently, increasing efforts have been directed towards the engineering of novel sorbent material in order to expand the applicability of SPME for a wider range of analytes and matrices. This review highlights the application of ionic liquids (ILs) and polymeric ionic liquids (PILs) as innovative sorbent materials for SPME. Characterized by their unique physico-chemical properties, these compounds can be structurally-designed to selectively extract target analytes based on unique molecular interactions. To examine the advantages of IL and PIL-based sorbent coatings in SPME, the field is reviewed by gathering available experimental data and exploring the sensitivity, linear calibration range, as well as detection limits for a variety of target analytes in the methods that have been developed.
Keywords: Ionic liquids; Polymeric ionic liquids; Solid-phase microextraction; Gas chromatography; Direct-immersion; Sample preparation
Ionic liquids in solid-phase microextraction: A review by Tien D. Ho; Anthony J. Canestraro; Jared L. Anderson (pp. 18-43).
Solid-phase microextraction (SPME) has undergone a surge in popularity within the field of analytical chemistry in the past two decades since its introduction. Owing to its nature of extraction, SPME has become widely known as a quick and cost-effective sample preparation technique. Although SPME has demonstrated extraordinary versatility in sampling capabilities, the technique continues to experience a tremendous growth in innovation. Presently, increasing efforts have been directed towards the engineering of novel sorbent material in order to expand the applicability of SPME for a wider range of analytes and matrices. This review highlights the application of ionic liquids (ILs) and polymeric ionic liquids (PILs) as innovative sorbent materials for SPME. Characterized by their unique physico-chemical properties, these compounds can be structurally-designed to selectively extract target analytes based on unique molecular interactions. To examine the advantages of IL and PIL-based sorbent coatings in SPME, the field is reviewed by gathering available experimental data and exploring the sensitivity, linear calibration range, as well as detection limits for a variety of target analytes in the methods that have been developed.
Keywords: Ionic liquids; Polymeric ionic liquids; Solid-phase microextraction; Gas chromatography; Direct-immersion; Sample preparation
Novel electrochemical sensor for the selective recognition of chlorogenic acid by Wilney de Jesus Rodrigues Santos; Murilo Santhiago; Inez Valeria Pagotto Yoshida; Lauro Tatsuo Kubota (pp. 44-50).
In this study, a novel sensitive molecularly imprinted electrochemical sensor was constructed for the selective detection of chlorogenic acid (CGA) by deposition of a molecularly imprinted siloxane (MIS) film, prepared by sol–gel process, onto Au bare electrode surface. Initially, a (3-mercaptopropyl)siloxane layer (MSL) was formed on the Au bare surface, followed by a siloxane layer obtained from the acid-catalyzed hydrolysis/condensation of a solution constituted by tetraethoxysilane (TEOS), phenyltriethoxysilane (PTEOS), 3-(aminopropyl)trimethoxysilane (APTMS) and CGA, as a molecular template. After the GCA extraction the MIS imprinted film was electrochemically characterized using differential pulse voltammetry (DPV). The MIS/Au sensor was tested in a solution of the CGA template and other similar molecules. This electrode displayed excellent selectivity towards CGA when compared with structurally similar molecules. Under optimized experimental conditions, the peak current response of the sensor for CGA was linear from 5.0×10−7molL−1 to 1.4×10−5molL−1, and the detection limit was 1.48×10−7molL−1. The MIS/Au sensor was successfully applied for the determination of CGA in coffee and tea samples.
Keywords: Molecularly imprinted siloxane; Sol–gel; Sensor; Chlorogenic acid recognition
Novel electrochemical sensor for the selective recognition of chlorogenic acid by Wilney de Jesus Rodrigues Santos; Murilo Santhiago; Inez Valeria Pagotto Yoshida; Lauro Tatsuo Kubota (pp. 44-50).
In this study, a novel sensitive molecularly imprinted electrochemical sensor was constructed for the selective detection of chlorogenic acid (CGA) by deposition of a molecularly imprinted siloxane (MIS) film, prepared by sol–gel process, onto Au bare electrode surface. Initially, a (3-mercaptopropyl)siloxane layer (MSL) was formed on the Au bare surface, followed by a siloxane layer obtained from the acid-catalyzed hydrolysis/condensation of a solution constituted by tetraethoxysilane (TEOS), phenyltriethoxysilane (PTEOS), 3-(aminopropyl)trimethoxysilane (APTMS) and CGA, as a molecular template. After the GCA extraction the MIS imprinted film was electrochemically characterized using differential pulse voltammetry (DPV). The MIS/Au sensor was tested in a solution of the CGA template and other similar molecules. This electrode displayed excellent selectivity towards CGA when compared with structurally similar molecules. Under optimized experimental conditions, the peak current response of the sensor for CGA was linear from 5.0×10−7molL−1 to 1.4×10−5molL−1, and the detection limit was 1.48×10−7molL−1. The MIS/Au sensor was successfully applied for the determination of CGA in coffee and tea samples.
Keywords: Molecularly imprinted siloxane; Sol–gel; Sensor; Chlorogenic acid recognition
Characterization of Hg(II) binding with different length phytochelatins using liquid chromatography and amperometric detection by Àngela Dago; Olga González-García; Cristina Ariño; José Manuel Díaz-Cruz; Miquel Esteban (pp. 51-57).
.Display Omitted► A HPLC - amperometric detection method to analyse mixtures of phytochelatins and related peptides with mercury is optimised. ► The free peptides and their complexes can be distinguished, which allows one to follow the evolution of the different Hg(II)-complexes formed.A simple and rapid methodology is optimised to analyse mixtures of different phytochelatins (PC n, n=2–5) with Hg(II) by HPLC with amperometric detection as a first step towards the analysis of extracts of plants stressed with Hg(II). The separation was achieved in a C18 column with a mobile phase of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in acetonitrile using gradient elution. Electrochemical detection with glassy carbon electrode and UV–vis detection were used in series. This methodology can clearly distinguish between the free peptides and their complexes and permits to study the evolution of the different complexes formed and predicts the possible interactions between the long chain phytochelatin complexes. ESI-MS is used as a complementary technique to find out the stoichiometries of such long chain phytochelatin complexes.
Keywords: Phytochelatins; Electrochemical detection; Liquid chromatography; Mercury complexes
Characterization of Hg(II) binding with different length phytochelatins using liquid chromatography and amperometric detection by Àngela Dago; Olga González-García; Cristina Ariño; José Manuel Díaz-Cruz; Miquel Esteban (pp. 51-57).
.Display Omitted► A HPLC - amperometric detection method to analyse mixtures of phytochelatins and related peptides with mercury is optimised. ► The free peptides and their complexes can be distinguished, which allows one to follow the evolution of the different Hg(II)-complexes formed.A simple and rapid methodology is optimised to analyse mixtures of different phytochelatins (PC n, n=2–5) with Hg(II) by HPLC with amperometric detection as a first step towards the analysis of extracts of plants stressed with Hg(II). The separation was achieved in a C18 column with a mobile phase of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in acetonitrile using gradient elution. Electrochemical detection with glassy carbon electrode and UV–vis detection were used in series. This methodology can clearly distinguish between the free peptides and their complexes and permits to study the evolution of the different complexes formed and predicts the possible interactions between the long chain phytochelatin complexes. ESI-MS is used as a complementary technique to find out the stoichiometries of such long chain phytochelatin complexes.
Keywords: Phytochelatins; Electrochemical detection; Liquid chromatography; Mercury complexes
CMK-3 nanoporous carbon as a new fiber coating for solid-phase microextraction coupled to gas chromatography–mass spectrometry by Akram Rahimi; Payman Hashemi; Alireza Badiei; Pezhman Arab; Ali Reza Ghiasvand (pp. 58-62).
CMK-3 nanoporous carbon was prepared and characterized as a highly porous fiber coating, with a highly ordered carbon framework, for solid-phase microextraction (SPME). The nanomaterial was immobilized onto platinum, stainless steel and copper metal wires for preparation of new SPME fibers. The copper-CMK-3 fiber showed superior properties and therefore was applied for extraction of some phenolic compounds in combination with GC–MS. For optimization of the extraction conditions, a simplex optimization method was used. The selected conditions were: sample volume 13ml, extraction temperature 56°C, extraction time 7min, ultrasonic time 5.5min, pH 5 and salt concentration 8.9%. The selected fiber showed some selectivity towards the polar phenolic compounds and its extraction efficiency was better than a commercial PDMS fiber. Linear calibration curves with correlation coefficients better than 0.99 and detection limits in the range from 0.002 to 0.068μgmL−1 were obtained for the fiber. No significant change was observed in the extraction efficiency of the new SPME fiber over at least 40 extractions. The fiber was successfully used for the determination of phenolic compounds in natural water samples.
Keywords: CMK-3 Nanoporous carbon; New solid-phase microextraction fiber; Phenolic compounds; Gas chromatography–mass spectrometry
CMK-3 nanoporous carbon as a new fiber coating for solid-phase microextraction coupled to gas chromatography–mass spectrometry by Akram Rahimi; Payman Hashemi; Alireza Badiei; Pezhman Arab; Ali Reza Ghiasvand (pp. 58-62).
CMK-3 nanoporous carbon was prepared and characterized as a highly porous fiber coating, with a highly ordered carbon framework, for solid-phase microextraction (SPME). The nanomaterial was immobilized onto platinum, stainless steel and copper metal wires for preparation of new SPME fibers. The copper-CMK-3 fiber showed superior properties and therefore was applied for extraction of some phenolic compounds in combination with GC–MS. For optimization of the extraction conditions, a simplex optimization method was used. The selected conditions were: sample volume 13ml, extraction temperature 56°C, extraction time 7min, ultrasonic time 5.5min, pH 5 and salt concentration 8.9%. The selected fiber showed some selectivity towards the polar phenolic compounds and its extraction efficiency was better than a commercial PDMS fiber. Linear calibration curves with correlation coefficients better than 0.99 and detection limits in the range from 0.002 to 0.068μgmL−1 were obtained for the fiber. No significant change was observed in the extraction efficiency of the new SPME fiber over at least 40 extractions. The fiber was successfully used for the determination of phenolic compounds in natural water samples.
Keywords: CMK-3 Nanoporous carbon; New solid-phase microextraction fiber; Phenolic compounds; Gas chromatography–mass spectrometry
Development and characterization of molecularly imprinted polymers for the selective enrichment of podophyllotoxin from traditional Chinese medicines by Ya Yuan; Yuzhi Wang; Meidong Huang; Ran Xu; Huan Zeng; Chan Nie; Jinhuan Kong (pp. 63-72).
In the present work, microwave heating initiated precipitation polymerization was developed to prepare podophyllotoxin (PPT) molecularly imprinted polymers (MIPs), resulting in much shorter polymerization time and better particle morphology. Prior to the polymerization, ultraviolet and FTIR spectroscopy were used to study the interactions between PPT and the functional monomers. The synthesized parameters were respectively optimized and the optimal conditions for the efficient adsorption property were template: PPT, 1mmol; functional monomer: acrylamide, 6mmol; bi-crosslinker: ethylene glycol dimethacrylate, 20mmol and divinylbenzene, 20mmol; porogen: acetonitrile, 40mL; initiator: azobisisobutyronitrile, 0.01molL−1; polymerization temperature: 60°C. FTIR spectroscopy, SEM and thermal analysis were used to characterize the MIPs. The results of the equilibrium rebinding experiments and the competitive adsorption experiments showed that these imprinted polymers exhibited good adsorption ability for the PPT. Scatchard analysis illustrated that two and one types of binding sites were generated in the MIPs and non-imprinted polymers (NIPs), respectively. Using the prepared MIPs as the solid phase extraction (SPE) sorbent, PPT was extracted selectively and efficiently from Dysosma versipellis, Sinopodophyllum hexandrum and Diphylleia sinensis. The regression equation was y=5.873×106 x+17075.659 with the correlation coefficient of 0.9994 in the concentration range of 0.005–0.4mgmL−1. After washing and eluting the SPE column with methanol and MeOH/acetic acid solution (v/v, 9:1), the limits of detection were 0.12–0.18μgmL−1 and their recoveries were in the range of 89.5–91.1% with all RSDs lower than 3.7.
Keywords: Molecularly imprinted polymer; Microwave heating; Podophyllotoxin; Traditional Chinese medicines; Solid-phase extraction
Development and characterization of molecularly imprinted polymers for the selective enrichment of podophyllotoxin from traditional Chinese medicines by Ya Yuan; Yuzhi Wang; Meidong Huang; Ran Xu; Huan Zeng; Chan Nie; Jinhuan Kong (pp. 63-72).
In the present work, microwave heating initiated precipitation polymerization was developed to prepare podophyllotoxin (PPT) molecularly imprinted polymers (MIPs), resulting in much shorter polymerization time and better particle morphology. Prior to the polymerization, ultraviolet and FTIR spectroscopy were used to study the interactions between PPT and the functional monomers. The synthesized parameters were respectively optimized and the optimal conditions for the efficient adsorption property were template: PPT, 1mmol; functional monomer: acrylamide, 6mmol; bi-crosslinker: ethylene glycol dimethacrylate, 20mmol and divinylbenzene, 20mmol; porogen: acetonitrile, 40mL; initiator: azobisisobutyronitrile, 0.01molL−1; polymerization temperature: 60°C. FTIR spectroscopy, SEM and thermal analysis were used to characterize the MIPs. The results of the equilibrium rebinding experiments and the competitive adsorption experiments showed that these imprinted polymers exhibited good adsorption ability for the PPT. Scatchard analysis illustrated that two and one types of binding sites were generated in the MIPs and non-imprinted polymers (NIPs), respectively. Using the prepared MIPs as the solid phase extraction (SPE) sorbent, PPT was extracted selectively and efficiently from Dysosma versipellis, Sinopodophyllum hexandrum and Diphylleia sinensis. The regression equation was y=5.873×106 x+17075.659 with the correlation coefficient of 0.9994 in the concentration range of 0.005–0.4mgmL−1. After washing and eluting the SPE column with methanol and MeOH/acetic acid solution (v/v, 9:1), the limits of detection were 0.12–0.18μgmL−1 and their recoveries were in the range of 89.5–91.1% with all RSDs lower than 3.7.
Keywords: Molecularly imprinted polymer; Microwave heating; Podophyllotoxin; Traditional Chinese medicines; Solid-phase extraction
Rotating disk sorbent extraction for pre-concentration of chromogenic organic compounds and direct determination by solid phase spectrophotometry by Pablo Richter; Alejandro Cañas; Carlos Muñoz; Claudio Leiva; Inés Ahumada (pp. 73-76).
A novel and very simple microextraction approach for pre-concentration and direct solid phase spectrophotometric measurement has been developed for the determination of chromogenic analytes. The model analyte to assess this approach was the chromophore malachite green (MG). The analyte was extracted from water samples onto a small rotating disk made of Teflon containing a sorbent phase of polydimethylsiloxane (PDMS) on one of its surfaces. We refer to the extraction procedure as rotating disk sorptive extraction (RDSE). After extraction, the sorbent phase with the concentrated analyte was separated from the Teflon disk and used directly for MG determination by solid phase spectrophotometry at 624nm, without the necessity of a desorption step.Chemical and extraction variables such as concentration of sodium sulfate, pH, disk rotational velocity, extraction time, and temperature were studied in order to establish the best conditions for extraction. Under optimum conditions, the extraction of MG was carried out in 18min and 90min, for sample volumes of 100mL or 1000mL, respectively. The detection limit, based on three times the standard deviation of the blank phase (3 σb), was 1.4μgL−1, and the repeatability, expressed as relative standard deviation (RSD), for 20μgL−1 MG was 8.1%. This study also applied the method to real samples, obtaining quantitative recovery (mean recovery of 99.3%).The PDMS phases could be reused after desorbing the MG into methanol for 3h. Replacement of the PDMS film onto the disk is very easy and low cost.
Keywords: Rotating disk sorptive extraction; Water samples; Polydimethylsiloxane; Malachite green; Chromophores
Rotating disk sorbent extraction for pre-concentration of chromogenic organic compounds and direct determination by solid phase spectrophotometry by Pablo Richter; Alejandro Cañas; Carlos Muñoz; Claudio Leiva; Inés Ahumada (pp. 73-76).
A novel and very simple microextraction approach for pre-concentration and direct solid phase spectrophotometric measurement has been developed for the determination of chromogenic analytes. The model analyte to assess this approach was the chromophore malachite green (MG). The analyte was extracted from water samples onto a small rotating disk made of Teflon containing a sorbent phase of polydimethylsiloxane (PDMS) on one of its surfaces. We refer to the extraction procedure as rotating disk sorptive extraction (RDSE). After extraction, the sorbent phase with the concentrated analyte was separated from the Teflon disk and used directly for MG determination by solid phase spectrophotometry at 624nm, without the necessity of a desorption step.Chemical and extraction variables such as concentration of sodium sulfate, pH, disk rotational velocity, extraction time, and temperature were studied in order to establish the best conditions for extraction. Under optimum conditions, the extraction of MG was carried out in 18min and 90min, for sample volumes of 100mL or 1000mL, respectively. The detection limit, based on three times the standard deviation of the blank phase (3 σb), was 1.4μgL−1, and the repeatability, expressed as relative standard deviation (RSD), for 20μgL−1 MG was 8.1%. This study also applied the method to real samples, obtaining quantitative recovery (mean recovery of 99.3%).The PDMS phases could be reused after desorbing the MG into methanol for 3h. Replacement of the PDMS film onto the disk is very easy and low cost.
Keywords: Rotating disk sorptive extraction; Water samples; Polydimethylsiloxane; Malachite green; Chromophores
Identification and quantification of glucosamine in rabbit cartilage and correlation with plasma levels by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry by Elisabetta Pastorini; Stefania Vecchiotti; Carolina Colliva; Stefano Persiani; Roberto Rotini; Giulia Roatti; Lorenzo Zaccarelli; Lucio Claudio Rovati; Aldo Roda (pp. 77-83).
Display Omitted► Optimization of an HPLC-ESI-MS/MS method for glucosamine in rabbit cartilage. ► Application of the method to an in-vivo study. ► Glucosamine presence in cartilage in physiological condition. ► Significant increase of cartilage glucosamine concentration after dosing. ► Good correlation between cartilage glucosamine levels and plasma concentrations.A new HPLC–ESI-MS/MS method for the determination of glucosamine (2-amino-2-deoxy-d-glucose) in rabbit cartilage was developed and optimized. Glucosamine was extracted from cartilage by cryogenic grinding followed by protein precipitation with trichloroacetic acid. The HPLC separation was achieved with a polymer-based amino column using a mobile phase composed of 10mM ammonium acetate (pH 7.5)–acetonitrile (20:80%, v/v) at 0.3mLmin−1 flow rate.d-[1-13C]Glucosamine was used as internal standard. Selective detection was performed by tandem mass spectrometry with electrospray source, operating in positive ionization mode and in multiple reaction monitoring acquisition ( m/ z 180→72 and 181→73 for glucosamine and internal standard, respectively). Limit of quantification was 0.045ng injected, corresponding to 0.25μgg−1 in cartilage. Linearity was obtained up to 20μgg−1 ( R2>0.991). Precision values (%R.S.D.) were <10%. Accuracy (% bias) ranged from −6.0% to 12%. Mean recoveries obtained at 3 concentration levels were higher than 81% (%R.S.D.≤8%). The method was applied to measure glucosamine levels in rabbit cartilage and plasma after single oral administration of glucosamine sulfate at a dose of 98mgkg−1 ( n=6). Glucosamine was present in cartilage in physiological condition before the treatment. After dosing, mean concentration of cartilage glucosamine significantly increased from 461 to 1040ngg−1. Cartilage glucosamine levels resulted to be well correlated with plasma concentrations, which therefore are useful to predict the target cartilage concentration and its pharmacological activity.
Keywords: Crystalline glucosamine sulfate; Glucosamine; High performance liquid chromatography-electrospray ionization-tandem mass spectrometry; Knee osteoarthritis; Cartilage; Rabbit
Identification and quantification of glucosamine in rabbit cartilage and correlation with plasma levels by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry by Elisabetta Pastorini; Stefania Vecchiotti; Carolina Colliva; Stefano Persiani; Roberto Rotini; Giulia Roatti; Lorenzo Zaccarelli; Lucio Claudio Rovati; Aldo Roda (pp. 77-83).
Display Omitted► Optimization of an HPLC-ESI-MS/MS method for glucosamine in rabbit cartilage. ► Application of the method to an in-vivo study. ► Glucosamine presence in cartilage in physiological condition. ► Significant increase of cartilage glucosamine concentration after dosing. ► Good correlation between cartilage glucosamine levels and plasma concentrations.A new HPLC–ESI-MS/MS method for the determination of glucosamine (2-amino-2-deoxy-d-glucose) in rabbit cartilage was developed and optimized. Glucosamine was extracted from cartilage by cryogenic grinding followed by protein precipitation with trichloroacetic acid. The HPLC separation was achieved with a polymer-based amino column using a mobile phase composed of 10mM ammonium acetate (pH 7.5)–acetonitrile (20:80%, v/v) at 0.3mLmin−1 flow rate.d-[1-13C]Glucosamine was used as internal standard. Selective detection was performed by tandem mass spectrometry with electrospray source, operating in positive ionization mode and in multiple reaction monitoring acquisition ( m/ z 180→72 and 181→73 for glucosamine and internal standard, respectively). Limit of quantification was 0.045ng injected, corresponding to 0.25μgg−1 in cartilage. Linearity was obtained up to 20μgg−1 ( R2>0.991). Precision values (%R.S.D.) were <10%. Accuracy (% bias) ranged from −6.0% to 12%. Mean recoveries obtained at 3 concentration levels were higher than 81% (%R.S.D.≤8%). The method was applied to measure glucosamine levels in rabbit cartilage and plasma after single oral administration of glucosamine sulfate at a dose of 98mgkg−1 ( n=6). Glucosamine was present in cartilage in physiological condition before the treatment. After dosing, mean concentration of cartilage glucosamine significantly increased from 461 to 1040ngg−1. Cartilage glucosamine levels resulted to be well correlated with plasma concentrations, which therefore are useful to predict the target cartilage concentration and its pharmacological activity.
Keywords: Crystalline glucosamine sulfate; Glucosamine; High performance liquid chromatography-electrospray ionization-tandem mass spectrometry; Knee osteoarthritis; Cartilage; Rabbit
A highly sensitive and rapid organophosphate biosensor based on enhancement of CdS–decorated graphene nanocomposite by Kun Wang; Qian Liu; Lina Dai; Jiajia Yan; Chang Ju; Baijing Qiu; Xiangyang Wu (pp. 84-88).
Display Omitted► Based on acetylcholinesterase immobilized on CdS–decorated graphene (CdS–G) nanocomposite, a rapid and sensitive organophosphates amperometric biosensor was constructed. ► The as-prepared biosensor showed high affinity to acetylthiocholine with a Michaelis–Menten constant value of 0.24mM. ► A rapid inhibition time was obtained due to the integration of the CdS–G nanocomposite. ► The resulting biosensor exhibited a reliable linear relationship between the inhibition and log[carbaryl] from 2ngmL−1 up to 2μgmL−1.This work reports a rapid and sensitive organophosphates (OPs) amperometric biosensor based on acetylcholinesterase (AChE) immobilized on CdS–decorated graphene (CdS–G) nanocomposite. The as-prepared biosensor shows high affinity to acetylthiocholine (ATCl) with a Michaelis–Menten constant ( Km) value of 0.24mM. A rapid inhibition time (2min) is obtained due to the integration of the CdS–G nanocomposite. Based on the inhibition of OPs on the enzymatic activity of the immobilized AChE, and used carbaryl as the model compound, the resulting biosensor exhibits excellent performance for OPs detection including good reproducibility, acceptable stability, and a reliable linear relationship between the inhibition and log[carbaryl] from 2ngmL−1 up to 2μgmL−1 with a detection limit of 0.7ngmL−1, which provides a new promising tool for analysis of enzyme inhibitors.
Keywords: Acetylcholinesterase; Organophosphorus pesticides; Graphene nanocomposites; Inhibition; Biosensor
A highly sensitive and rapid organophosphate biosensor based on enhancement of CdS–decorated graphene nanocomposite by Kun Wang; Qian Liu; Lina Dai; Jiajia Yan; Chang Ju; Baijing Qiu; Xiangyang Wu (pp. 84-88).
Display Omitted► Based on acetylcholinesterase immobilized on CdS–decorated graphene (CdS–G) nanocomposite, a rapid and sensitive organophosphates amperometric biosensor was constructed. ► The as-prepared biosensor showed high affinity to acetylthiocholine with a Michaelis–Menten constant value of 0.24mM. ► A rapid inhibition time was obtained due to the integration of the CdS–G nanocomposite. ► The resulting biosensor exhibited a reliable linear relationship between the inhibition and log[carbaryl] from 2ngmL−1 up to 2μgmL−1.This work reports a rapid and sensitive organophosphates (OPs) amperometric biosensor based on acetylcholinesterase (AChE) immobilized on CdS–decorated graphene (CdS–G) nanocomposite. The as-prepared biosensor shows high affinity to acetylthiocholine (ATCl) with a Michaelis–Menten constant ( Km) value of 0.24mM. A rapid inhibition time (2min) is obtained due to the integration of the CdS–G nanocomposite. Based on the inhibition of OPs on the enzymatic activity of the immobilized AChE, and used carbaryl as the model compound, the resulting biosensor exhibits excellent performance for OPs detection including good reproducibility, acceptable stability, and a reliable linear relationship between the inhibition and log[carbaryl] from 2ngmL−1 up to 2μgmL−1 with a detection limit of 0.7ngmL−1, which provides a new promising tool for analysis of enzyme inhibitors.
Keywords: Acetylcholinesterase; Organophosphorus pesticides; Graphene nanocomposites; Inhibition; Biosensor
Chitosan coated carbon fiber microelectrode for selective in vivo detection of neurotransmitters in live zebrafish embryos by Rıfat Emrah Özel; Kenneth N. Wallace; Silvana Andreescu (pp. 89-95).
Chitosan coated fiber electrodes are sensitive to serotonin detection while rejecting physiological levels of ascorbic acid interferences.We report the development of a chitosan modified carbon fiber microelectrode for in vivo detection of serotonin. We find that chitosan has the ability to reject physiological levels of ascorbic acid interferences and facilitate selective and sensitive detection of in vivo levels of serotonin, a common catecholamine neurotransmitter. Presence of chitosan on the microelectrode surface was investigated using scanning electron microscopy (SEM) and cyclic voltammetry (CV). The electrode was characterized using differential pulse voltammetry (DPV). A detection limit of 1.6nM serotonin with a sensitivity of 5.12nA/μM, a linear range from 2 to 100nM and a reproducibility of 6.5% for n=6 electrodes were obtained. Chitosan modified microelectrodes selectively measure serotonin in presence of physiological levels of ascorbic acid. In vivo measurements were performed to measure concentration of serotonin in the live embryonic zebrafish intestine. The sensor quantifies in vivo intestinal levels of serotonin while successfully rejecting ascorbic acid interferences. We demonstrate that chitosan can be used as an effective coating to reject ascorbic acid interferences at carbon fiber microelectrodes, as an alternative to Nafion, and that chitosan modified microelectrodes are reliable tools for in vivo monitoring of changes in neurotransmitter levels.
Keywords: Carbon fiber microelectrode; Chitosan; Ascorbic acid; Interferences; In vivo detection of neurotransmitters; Zebrafish; Intestine
Chitosan coated carbon fiber microelectrode for selective in vivo detection of neurotransmitters in live zebrafish embryos by Rıfat Emrah Özel; Kenneth N. Wallace; Silvana Andreescu (pp. 89-95).
Chitosan coated fiber electrodes are sensitive to serotonin detection while rejecting physiological levels of ascorbic acid interferences.We report the development of a chitosan modified carbon fiber microelectrode for in vivo detection of serotonin. We find that chitosan has the ability to reject physiological levels of ascorbic acid interferences and facilitate selective and sensitive detection of in vivo levels of serotonin, a common catecholamine neurotransmitter. Presence of chitosan on the microelectrode surface was investigated using scanning electron microscopy (SEM) and cyclic voltammetry (CV). The electrode was characterized using differential pulse voltammetry (DPV). A detection limit of 1.6nM serotonin with a sensitivity of 5.12nA/μM, a linear range from 2 to 100nM and a reproducibility of 6.5% for n=6 electrodes were obtained. Chitosan modified microelectrodes selectively measure serotonin in presence of physiological levels of ascorbic acid. In vivo measurements were performed to measure concentration of serotonin in the live embryonic zebrafish intestine. The sensor quantifies in vivo intestinal levels of serotonin while successfully rejecting ascorbic acid interferences. We demonstrate that chitosan can be used as an effective coating to reject ascorbic acid interferences at carbon fiber microelectrodes, as an alternative to Nafion, and that chitosan modified microelectrodes are reliable tools for in vivo monitoring of changes in neurotransmitter levels.
Keywords: Carbon fiber microelectrode; Chitosan; Ascorbic acid; Interferences; In vivo detection of neurotransmitters; Zebrafish; Intestine
Gold nanoparticle–fluorophore complex for conditionally fluorescing signal mediator by Jianting Wang; Samuel Achilefu; Michael Nantz; Kyung A. Kang (pp. 96-104).
Fluorescent contrast agents with high specificity and sensitivity are valuable for accurate disease detection and diagnosis. Spherical gold nanoparticles (GNPs) can be smartly utilized for developing highly effective agents. The strong electromagnetic (plasmon) field on their surface can be very effective in influencing the electrons of fluorophores and, thus, manipulating the fluorescence output (i.e., either quenching or enhancement). Fluorescence quenching can be used for negative sensing, or for conditional de-quenching to increase the specificity. Fluorescence enhancement allows sensing to be more sensitive. The level of fluorescence alteration depends on the GNP size, the excitation and emission wavelengths and quantum yield of the fluorophore, and the distance between the GNP and the fluorophore. To understand the mechanisms of the fluorescence change by GNP, we have theoretically analyzed the parameters involved in the fluorescence alteration for commonly used fluorophores, with an emphasis on quenching.The results showed that the fluorescence of fluorophores with the excitation (Ex) and emission (Ex) wavelengths close to the GNP resonance peak tended to be significantly quenched by GNPs. For those fluorophores emitting fluorescence in red or near infrared, to achieve quenching, the distance between GNP and the fluorophore was required to be very short. In general, a shorter distance resulted in more quenching. Bigger GNPs require a shorter distance to achieve the same level of quenching. The fluorescence of a fluorophore with a lower quantum yield (especially the one with emission in far-red or near-infrared) is more difficult to be quenched by GNPs (requires very short distance). Instead, it can be enhanced.Based on the theoretical study, we have developed a near-infrared contrast agent, i.e., Cypate conjugated GNP via a short peptide spacer. Normally the fluorescence of Cypate was quenched. The spacer has a motif of a substrate for urokinase type plasminogen activator (uPA; cancer-secreting enzyme). This contrast agent emits fluorescence only in the presence of uPA, where the uPA cleaves the spacer. This design can be used in characterization of the cancer type and also in diagnosing other diseases with signature enzymes.
Keywords: Fluorescence quenching; Fluorescence manipulation; Gold nanoparticle; Molecular imaging; Contrast agent; Enzyme triggered detection
Gold nanoparticle–fluorophore complex for conditionally fluorescing signal mediator by Jianting Wang; Samuel Achilefu; Michael Nantz; Kyung A. Kang (pp. 96-104).
Fluorescent contrast agents with high specificity and sensitivity are valuable for accurate disease detection and diagnosis. Spherical gold nanoparticles (GNPs) can be smartly utilized for developing highly effective agents. The strong electromagnetic (plasmon) field on their surface can be very effective in influencing the electrons of fluorophores and, thus, manipulating the fluorescence output (i.e., either quenching or enhancement). Fluorescence quenching can be used for negative sensing, or for conditional de-quenching to increase the specificity. Fluorescence enhancement allows sensing to be more sensitive. The level of fluorescence alteration depends on the GNP size, the excitation and emission wavelengths and quantum yield of the fluorophore, and the distance between the GNP and the fluorophore. To understand the mechanisms of the fluorescence change by GNP, we have theoretically analyzed the parameters involved in the fluorescence alteration for commonly used fluorophores, with an emphasis on quenching.The results showed that the fluorescence of fluorophores with the excitation (Ex) and emission (Ex) wavelengths close to the GNP resonance peak tended to be significantly quenched by GNPs. For those fluorophores emitting fluorescence in red or near infrared, to achieve quenching, the distance between GNP and the fluorophore was required to be very short. In general, a shorter distance resulted in more quenching. Bigger GNPs require a shorter distance to achieve the same level of quenching. The fluorescence of a fluorophore with a lower quantum yield (especially the one with emission in far-red or near-infrared) is more difficult to be quenched by GNPs (requires very short distance). Instead, it can be enhanced.Based on the theoretical study, we have developed a near-infrared contrast agent, i.e., Cypate conjugated GNP via a short peptide spacer. Normally the fluorescence of Cypate was quenched. The spacer has a motif of a substrate for urokinase type plasminogen activator (uPA; cancer-secreting enzyme). This contrast agent emits fluorescence only in the presence of uPA, where the uPA cleaves the spacer. This design can be used in characterization of the cancer type and also in diagnosing other diseases with signature enzymes.
Keywords: Fluorescence quenching; Fluorescence manipulation; Gold nanoparticle; Molecular imaging; Contrast agent; Enzyme triggered detection
Development of an on-column affinity smart polymer gel glucose sensor by Chongdee Thammakhet; Panote Thavarungkul; Proespichaya Kanatharana (pp. 105-112).
► Phenylboronic acid as a chemoselective ligand in an affinity smart polymer gel. ► Affinity smart polymer gel coated inside a short capillary tube. ► Optical density change of the gel depends on the concentration of glucose. ► On-column detection coupled with a flow injection system. ► Possibly automated glucose detection.An on-column affinity smart polymer gel glucose sensor was developed as a non-enzymatic glucose sensor. A copolymer of 3-acrylamidophenylboronic acid and acrylamide, the so called “smart polymer”, was synthesized in situ in a 5cm long capillary tube with a detection window to provide the on-column detection. The optical density of this semitransparent affinity smart polymer gel, coated inside the tube, decreased with increasing glucose concentration and was detected using a UV–vis detector at 500nm. The capillary tube was incorporated into a flow injection system. Under optimum conditions, a linear dynamic range of 0.5–16.0mM with a limit of detection of 0.5mM (S/N≥3) was obtained. A single coated affinity smart polymer gel had good stability for up to 250 consecutive injections with relative standard deviation of less than 5%. The analysis time for each injection was 6min. Ten glucose samples prepared in distilled water were analyzed by the developed method and the results compared well with those obtained from the conventional dinitrosalicylic acid (DNS) method ( P>0.05). Real urine samples with known glucose levels were analyzed and the developed sensor provided comparable results to those from the normal strip test technique. Acceptable percentage recoveries, ranging from 88±2% to 103±4% from the spiked urine sample, were obtained.
Keywords: Non-enzymatic glucose sensor; On-column detection; Phenylboronic acid; Affinity smart polymer gel
Development of an on-column affinity smart polymer gel glucose sensor by Chongdee Thammakhet; Panote Thavarungkul; Proespichaya Kanatharana (pp. 105-112).
► Phenylboronic acid as a chemoselective ligand in an affinity smart polymer gel. ► Affinity smart polymer gel coated inside a short capillary tube. ► Optical density change of the gel depends on the concentration of glucose. ► On-column detection coupled with a flow injection system. ► Possibly automated glucose detection.An on-column affinity smart polymer gel glucose sensor was developed as a non-enzymatic glucose sensor. A copolymer of 3-acrylamidophenylboronic acid and acrylamide, the so called “smart polymer”, was synthesized in situ in a 5cm long capillary tube with a detection window to provide the on-column detection. The optical density of this semitransparent affinity smart polymer gel, coated inside the tube, decreased with increasing glucose concentration and was detected using a UV–vis detector at 500nm. The capillary tube was incorporated into a flow injection system. Under optimum conditions, a linear dynamic range of 0.5–16.0mM with a limit of detection of 0.5mM (S/N≥3) was obtained. A single coated affinity smart polymer gel had good stability for up to 250 consecutive injections with relative standard deviation of less than 5%. The analysis time for each injection was 6min. Ten glucose samples prepared in distilled water were analyzed by the developed method and the results compared well with those obtained from the conventional dinitrosalicylic acid (DNS) method ( P>0.05). Real urine samples with known glucose levels were analyzed and the developed sensor provided comparable results to those from the normal strip test technique. Acceptable percentage recoveries, ranging from 88±2% to 103±4% from the spiked urine sample, were obtained.
Keywords: Non-enzymatic glucose sensor; On-column detection; Phenylboronic acid; Affinity smart polymer gel