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Analytical and Bioanalytical Chemistry (v.381, #5)
Graduate student internships: developing scientists with real-world experiences
by C. K. Larive (pp. 993-995).
Trends in the development of nucleic acid biosensors for medical diagnostics by Paul A. E. Piunno; Ulrich J. Krull (pp. 1004-1011).
Some of the recent advances in the field of biosensors for nucleic acid analysis in medical diagnostic applications are highlighted. Particular attention is paid in this review to the progress made in two key areas of development: (i) enhancements achieved in device selectivity, and (ii) enhancements achieved in device sensitivity.
Keywords: Biosensors; Nucleic acids; Hybridization; Amplification; Sensitivity; Selectivity
Effects of immobilization on a FRET immunosensor for the detection of myocardial infarction by Sheila A Grant; Mary E Pierce; Darcy J Lichlyter; David A Grant (pp. 1012-1018).
A novel optical biosensor technique is being developed for the early detection of myocardial infarction by utilizing the distance-dependent chemical transduction method of fluorescence resonance energy transfer (FRET). The FRET process requires two fluorophores termed the donor and the acceptor. When in close proximity, the donor absorbs energy from the excitation source and non-radiatively transfers the energy to the acceptor, which in turn emits fluorescent energy. This distance-dependent property was utilized to detect conformational changes when antibodies combine with their respective antigens. The fluorophores were conjugated to an antibody–Protein A complex and then immobilized via silanization to the distal ends of optical fibers. Three different antibody–Protein A complexes were immobilized: generic IgG, cardiac Troponin T (cTnT), and cardiac Troponin I (cTnI). Results showed that upon the addition of the specific antigens, the antibodies underwent a conformational change, reducing the distance between the FRET fluorophores. The generic IgG responded to 233 nM antigens, whereas the cTnT biosensor had a limit of detection of 75 nM, and the cTnI biosensors had a limit of detection of 94 nM.
Keywords: Optical sensing; FRET; Myocardial infarction
Latex bead immobilisation in PDMS matrix for the detection of p53 gene point mutation and anti-HIV-1 capsid protein antibodies by Christophe A. Marquette; Agnès Degiuli; Emmanuelle Imbert-Laurenceau; Francois Mallet; Carole Chaix; Bernard Mandrand; Loïc J. Blum (pp. 1019-1024).
Two diagnostic chemiluminescent biochips were developed for either the detection of p53 gene point mutation or the serological detection of anti-HIV-1 p24 capsid protein. Both biochips were composed of 24 microarrays of latex beads spots (4×4) (150 μm in diameter, 800 μm spacing) entrapped in a poly(dimethylsiloxane) elastomer (PDMS). The latex beads, bearing oligonucleotide sequences or capsid protein, were spotted with a conventional piezoelectric spotter and subsequently transferred at the PDMS interface. The electron microscopy observation of the biochips showed how homogeneous and well distributed the spots could be. Point mutation detection on the codon 273 of the p53 gene was performed on the basis of the melting temperature difference between the perfect match sequence and the one base pair mismatch sequence. The hybridisation of a 20-mer oligonucleotide form the codon 273 including a one base pair mutation in its sequence on a biochip arrayed with non-muted and the muted complementary sequences, enabled a clear discrimination at 56°C between muted and wild sequences. Moreover, the quantitative measurement of the amount of muted sequence in a sample was possible in the range 0.4–4 pmol. Serological measurement of anti-HIV-1 p24 capsid protein on the biochip, prepared with 1-μm-diameter latex beads, enabled the detection of monoclonal antibodies in the range 1.55–775 ng mL−1. Such a range could be lowered to 0.775 ng mL−1 when using 50-nm-diameter beads, which generated a higher specific surface. The validation of the biochip for the detection of anti-HIV-1 capsid protein antibodies was performed in human sera from seropositive and seronegative patients. The positivity of the sera was easily discriminated at serum dilutions below 1:1,000.
Keywords: Biochip; Chemiluminescence; HIV-1; Immunochip; Latex beads; p53; p24; PDMS; Point mutation
Multianalyte bioanalytical devices: scientific potential and business requirements
by Andreas Brecht (pp. 1025-1026).
157-nm Laser ablation of polymeric layers for fabrication of biomolecule microarrays by Antonios M. Douvas; Panagiota S. Petrou; Sotirios E. Kakabakos; Konstantinos Misiakos; Panagiotis Argitis; Evagelia Sarantopoulou; Zoe Kollia; Alkiviadis C. Cefalas (pp. 1027-1032).
A new methodology for protein microarray fabrication is proposed based on the ablation of polymer film using laser at 157 nm (F2). The polymer has been selected among others with the criterion of negligible protein adsorption. Improved results have been obtained by pretreatment of the polymer surface with an inert protein. The use of 157-nm laser radiation allowed very good depth control during the polymeric layer ablation process. In addition the importance of laser ablation at 157 nm is based on the fact that irradiated surfaces indicate limited chemical change due to the fact that laser ablation at 157 nm is only photochemical, thus avoiding excessive surface heating and damage. Results of protein microarray fabrication are presented to illustrate the viability of the proposed method.
Keywords: Biomolecule micropatterning; Protein microarrays; 157-nm Laser; Polymer ablation; Protein adsorption; Blocking
Towards a fast-responding, label-free electrochemical DNA biosensor by M. Mir; I. Katakis (pp. 1033-1035).
DNA sensors and sensor arrays (biochips) have become an important tool in molecular biology and biotechnology in recent years. For low-throughput, easy-to-use devices it is desirable that they be of low cost, reagentless, and label-free. Displacement sensors with electrochemical detection offer these advantages, and therefore the development of such a detection principle is show in this work. An HRP-labeled oligonucleotide was sub-optimally pre-hybridized with a capture probe and was displaced upon introduction of the fully complementary probe target, producing a decrease in signal that was proportional to the sample concentration. This detection scheme has been demonstrated colorimetrically and electrochemically, obtaining a total signal displacement of 55% only 5 min after introduction of the sample.
Keywords: Label free; DNA electrochemical biosensor; Diffusional mediator; Displacement
A new label-free amperometric immunosenor for rubella vaccine by Lingyan Zhang; Ruo Yuan; Xiaoqing Huang; Yaqin Chai; Dianping Tang; Shurui Cao (pp. 1036-1040).
A novel label-free amperometric immunosensor for the detection of rubella vaccine was developed by immobilizing anti-rubella serum on bilayer nano-Au/polymerized o-phenylenediamine film with electrodeposited Prussian Blue (PB) as an electrode transfer mediator on the platinum electrode. The redox reactions of PB as a probe on the platinum surface were blocked due to the binding of the antibody to the antigen, which was investigated by cyclic voltammetry. Therefore, the interaction of the antibody with various concentrations of antigen could be detected by measurements of amperometric response in PBS, and the amperometric response on the surface of the modified electrode was inversely proportional to the concentration of rubella vaccine in the sample. The immunosensor showed a specific response to rubella vaccine in the range 8.1×10−8–8.0×10−6 lgCCID50/ml (cell culture infectious dose) and a detection limit of 4.010−8 lgCCID50/ml at a signal-to-noise ratio of 3. To summarize, the present work provides a low-cost, fast response time, highly sensitive and easy-to-prepare method for the determination of antigen in biological products.
Keywords: Label-free; Amperometric; Rubella vaccine; Immunosensor; Nano-Au
Lichen-based biosensor for the determination of benzene and 2-chlorophenol: microcalorimetric and amperometric investigations by Marta Letizia Antonelli; Luigi Campanella; Patrizia Ercole (pp. 1041-1048).
Preliminary microcalorimetric studies have been performed to analyse the response of a whole epiphytic lichen tissue (Evernia prunastri) to 2-chlorophenol (2Cl-ϕ), a pollutant of oil mill waste-water, in order to evaluate whether the tissue might be used to assess the toxic characteristics of polluted waters. The obtained results (lichen viability expressed in hours, enthalpy variations for the 2Cl-ϕ/lichen interactions) were used to create a lichen-based biosensor that uses an amperometric oxygen electrode (a Clark electrode) as a transducer. The lichen catalyses aromatic ring cleavage (via pyrocatechase enzymes present in the lichen), and transforms aromatic substances like 2Cl-ϕ into muconic acid (C6H6O4). Following a full electroanalytical characterisation, the performance of the proposed lichen biosensor was compared to that of a biosensor based on Pseudomonas putida cells, which was originally constructed to monitor benzene in different matrices (water, air, petrol and oil) and was tested in our laboratory previously.
Keywords: LichenEvernia prunastriBenzene; 2-Chlorophenol; Amperometric biosensor; Microcalorimetry
A novel amperometric sensor and chromatographic detector for determination of parathion by Chunya Li; Changfa Wang; Yong Ma; Wei Bao; Shengshui Hu (pp. 1049-1055).
A novel amperometric sensor and chromatographic detector for determination of parathion has been fabricated from a multi-wall carbon nano-tube (MWCNT)/Nafion film-modified glassy-carbon electrode (GCE). The electrochemical response to parathion at the MWCNT/Nafion film electrode was investigated by cyclic voltammetry and linear sweep voltammetry. The redox current of parathion at the MWCNT/Nafion film electrode was significantly higher than that at the bare GCE, the MWCNT-modified GCE, and the Nafion-modified GCE. The results indicated that the MWCNT/Nafion film had an efficient electrocatalytic effect on the electrochemical response to parathion. The peak current was proportional to the concentration of parathion in the range 5.0×10−9–2.0×10−5 mol L−1. The detection limit was 1.0×10−9 mol L−1 (after 120 s accumulation). In high-performance liquid chromatography with electrochemical detection (HPLC–ED) a stable and sensitive current response was obtained for parathion at the MWCNT/Nafion film electrode. The linear range for parathion was over four orders of magnitude and the detection limit was 6.0×10−9 mol L−1. Application of the method for determination of parathion in rice was satisfactory.
Keywords: Carbon nano-tube; Nafion; Sensor; Parathion; High-performance liquid chromatography
Application of an SPR-based receptor assay for the determination of biologically active recombinant bone morphogenetic protein-2 by Janine Wendler; Luis Felipe Vallejo; Ursula Rinas; Ursula Bilitewski (pp. 1056-1064).
Human bone morphogenetic protein-2 (hBMP-2) is a member of the human transforming growth factor-β superfamily. Biologically active bone morphogenetic protein-2 (BMP-2) is a dimeric protein that binds in the first step of the signal transduction cascade to specific receptors on the cell-surface. This specific interaction of the dimeric protein with the extracellular ligand-binding domain (ECD) of the receptor was used to develop a receptor-based assay based on an optical biosensor system (Biacore 2000, Biacore AB, Uppsala, Sweden). The ECD of the BMP-receptor type IA, tagged with the Fc part of IgG (BMPR-IA-Fc), was immobilised on the surface of a dextran-protein A-coated sensor chip. Calibration curves were obtained with purified and biologically active recombinant hBMP-2 (rhBMP-2) that showed a linear range from approximately 5 to 250 nM rhBMP-2. Moreover, this assay was used to quantitatively follow the generation of biologically active protein during the renaturation from unfolded and reduced monomers to biologically active dimers. A refolding mixture containing renatured dimeric rhBMP-2 and not correctly folded monomers, was used as the sample solution without any further pre-treatment. It was proven that only the biologically active dimers were recognised by the immobilised receptor, so the generation of biologically active rhBMP-2 during the renaturation process could be monitored directly and rapidly. Furthermore, the results from the optical sensor obtained during the renaturation process showed a good correlation with the data obtained by non-reducing SDS-PAGE analysis carried out at the end of the renaturation process. These data show that the disulphide-bonded dimer corresponds to the biologically active protein capable of binding the BMP-receptor type IA.
Keywords: Surface plasmon resonance (SPR); Human recombinant bone morphogenetic protein-2; Receptor sensor; Biological activity; Receptor-ligand interaction; Renaturation
Interference of non-specific peroxidases in the fluorescence detection of superoxide radical by hydroethidine oxidation: a new assay for H2O2 by Nikolaos Patsoukis; Ioannis Papapostolou; Christos D. Georgiou (pp. 1065-1072).
The present study shows that hydroethidine (HE), used for in-vivo qualitative fluorescent detection of superoxide anion, can be also oxidized by H2O2 via non-specific peroxidase (horseradish peroxidase and myeloperoxidase) catalysis, forming fluorescent oxidation products. These products give broad excitation/emission peaks (490–495/580–600 nm) near the excitation/emission peaks (475/580 nm) of the HE-superoxide oxidation product, and this may pose serious interference problems to the fluorescent detection of the superoxide radical. The study suggests cautionary use of the HE-superoxide anion assay mainly for detection of reactive oxygen species. A byproduct of this study was the development of a simple and sensitive HE-horseradish peroxidase assay for the in-vitro quantification of H2O2 in biological tissues with a sensitivity of 1 μmol L−1.
Keywords: Horseradish peroxidase; Myeloperoxidase; Superoxide radical
Overlapping voltammetric peaks—an analytical procedure for simultaneous determination of trace metals. Application to food and environmental matrices by Clinio Locatelli (pp. 1073-1081).
Voltammetric methods are very suitable, versatile and rapid techniques for simultaneous determination of metals in complex matrices. The present work, determination of Cu(II), Sn(II), Sb(III), Tl(I), and Pb(II) by square-wave anodic-stripping voltammetry and Cr(VI) by square-wave adsorptive-stripping voltammetry, is an interesting example of the possibility of simultaneous determination of each single element in food and environmental samples, even in the presence of reciprocal interference. Dibasic ammonium citrate, pH 6.3 or 8.2, was employed as supporting electrolyte. The voltammetric measurements were carried out using a stationary hanging mercury drop electrode as working electrode and a platinum electrode and an Ag|AgCl|KClsat electrode as auxiliary and reference electrodes, respectively. The analytical procedure was verified by analysis of standard reference materials—wholemeal BCR-CRM 189, wheat flour NIST-SRM 1567a, rice flour NIST-SRM 1568a, estuarine sediment BCR-CRM 277, river sediment BCR-CRM 320, and Montana soil with moderately elevated traces NIST-SRM 2711. Precision and accuracy, expressed as relative standard deviation and relative error, respectively, were generally below 6% whereas limits of detection for each element were below 0.069 μg g−1. In the presence of reciprocal interference the standard addition method considerably improved the resolution of the voltammetric technique, even for very high element concentration ratios. After being set up on the standard reference materials the analytical procedure was transferred and applied to commercial samples of meal and soil samples taken from sites devoted to agricultural practice. A critical comparison with graphite furnace atomic-absorption spectroscopy is also discussed.
Keywords: Interference; Trace metals; Meal; Soil; Voltammetry; Spectroscopy
Speciation analysis of calcium, iron, and zinc in casein phosphopeptide fractions from toddler milk-based formula by anion exchange and reversed-phase high-performance liquid chromatography–mass spectrometry/flame atomic-absorption spectroscopy by Esther Miquel; Amparo Alegría; Reyes Barberá; Rosaura Farré (pp. 1082-1088).
Casein phosphopeptides (CPP) are phosphorylated casein-derived peptides that can be released by in-vitro or in-vivo enzymatic hydrolysis of αs1-casein, αs2-casein, and β-casein (CN). Many of these peptides contain a highly polar acidic sequence of three phosphoseryl groups followed by two glutamic acid residues. These domains are binding sites for minerals such as calcium, iron, and zinc and play an important role in mineral bioavailability. The aim of this study was speciation analysis of calcium, iron, and zinc in CPP fractions from the soluble fraction of a toddler milk-based formula. Methods for CPP separation by anion-exchange high-performance liquid chromatography (AE-HPLC) were combined with CPP identification by reversed-phase high performance liquid chromatography–electrospray ionization mass spectrometry and determination of the calcium, iron, zinc, and phosphorus content of the fractions obtained by AE-HPLC. Calcium and phosphorus were detected in all the analyzed AE-HPLC fractions. Calcium and zinc could be bound to CPP derived from αs1-CN and αs2-CN in fraction 3. Iron could be bound to CPP in fraction 4 in which β-CN(15-34)4P was present with the cluster sequence S(P)S(P)S(P)EE. The results obtained prove the different distribution of calcium, iron, and zinc in heterogeneous CPP fractions.
Keywords: Casein phosphopeptides; Speciation; Minerals; High-performance liquid chromatography; Mass spectrometry.
Photometric detection of cyclodextrins in liquid chromatography by using iodine generated electrochemically in-situ by Toshiya Hiroi; Jiye Jin; Toyohide Takeuchi (pp. 1089-1094).
A method has been developed for photometric detection of cyclodextrins (CD) in liquid chromatography using iodine (I2) generated electrochemically in-situ. Iodide ion in the mobile phase was electrochemically oxidized to I2 which was subsequently reacted with I−, in an electrochemical flow cell, forming I3−. The absorbance of I3− was found to be greatly enhanced when CD were present in the mobile phase. The absorbance enhancement was caused by the change in the mole fraction of I3−, because of the inclusion reaction of I3− with CD. On the basis of this phenomenon, CD were detected by means of a photodiode-array UV–visible detector positioned downstream of the electrochemical flow cell. The signals were found to be linearly dependent on CD concentration. Because the formation constants of I3− with CD decrease in the order α-CD>β-CD>γ-CD, α-CD was most detectable by the method. Detection limits were 1.0 μmol L−1 for α-CD, 65 μmol L−1 for monoG1-β-CD, 100 μmol L−1 for β-CD, and 200 μmol L−1 for γ-CD.
Keywords: Cyclodextrins; Iodine; Inclusion reaction; Electrochemical flow cell; Cyclic voltammetry; Liquid chromatography
Determination of thiol by high-performance liquid chromatography and fluorescence detection with 5-methyl-(2-(m-iodoacetylaminophenyl)benzoxazole by Shu-Cai Liang; Hong Wang; Zhi-Min Zhang; Hua-Shan Zhang (pp. 1095-1100).
A new high-performance liquid chromatographic (HPLC) method for measuring low molecular weight (LMW) thiol-containing compounds, including cysteine (CysH), glutathione (GSH), N-acetylcysteine (Nac), penicillamine (PA), and 2-mercaptoethanol (2-ME), has been developed by using 5-methyl-(2-(m-iodoacetylaminophenyl)benzoxazole (MIPBO) as fluorescence-labeling reagent. The derivatization and separation conditions have been investigated in detail. Detection limits ranging from 3.5 to 15.0 fmol were achieved for the thiols investigated in a 16 min separation with detection wavelengths 310 and 375 nm for the excitation and emission, respectively. The utility of the proposed method has been validated by measuring CysH in human urine samples.
Keywords: High-performance liquid chromatography (HPLC); Fluorescence detection; 5-Methyl-(2-(m-iodoacetylaminophenyl)benzoxazole (MIPBO); Thiol
A test strip for chloride analysis in environmental water
by L. F. Capitán-Vallvey; E. Arroyo Guerrero; C. Berenguer Merelo; M. D. Fernández Ramos (pp. 1101-1101).