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Analytical and Bioanalytical Chemistry (v.401, #7)

Stanislav S. Rubakhin and Jonathan V. Sweedler (Eds.): Mass spectrometry imaging. Principles and protocols by José Manuel Costa-Fernández (pp. 2033-2034).

Boris L. Milman: Chemical identification and its quality assurance by Angel Ríos (pp. 2035-2036).

Biomarkers by Boguslaw Buszewski; Jochen Schubert (pp. 2037-2038).

is currently Head of the Chair of Environmental Chemistry and Bioanalysis at the Faculty of Chemistry, Nicolaus Copernicus University in Torun, Poland. He serves as Vice Chair of the Committee of Analytical Chemistry of the Polish Academy of Sciences, President of the Polish Chemical Society, and is a member of the Advisory Board of the Austrian Academy of Sciences. His main scientific interests cover environmental analysis, chromatography and related techniques (HPLC, SPE, GC, CZE, adsorption, sample preparation), spectroscopy, utilization of waste and sludge, and chemometrics. Professor Dr Buszewski is also the Chairman of the Central European Group for Separation Sciences (CEGSS), President of the Societas Humboldtiana Polonorum, and member of the Steering Committee of the Division of Environmental Chemistry of the European Association for Chemical and Molecular Sciences (EuCheMS). He is also President of the European Society for Separation Science (EuSSS) and a member of the editorial advisory board of numerous international journals. a physician and chemist, is currently Professor of Anaesthesiology and Vice Director of the Department of Anaesthesiology and Intensive Care at Rostock University. His scientific interests are new non-invasive diagnostic methods in medicine, clinical breath analysis, and application of modern mass spectrometric methods for diagnostic purposes. As Vice Director of the department he is responsible for clinical anaesthesia, intensive care, emergency medicine, and pain service in a large university hospital.

The state-of-the-art determination of urinary nucleosides using chromatographic techniques “hyphenated” with advanced bioinformatic methods by Wiktoria Struck; Małgorzata Waszczuk-Jankowska; Roman Kaliszan; Michał J. Markuszewski (pp. 2039-2050).

Over the last decade metabolomics has gained increasing popularity and significance in life sciences. Together with genomics, transcriptomics and proteomics, metabolomics provides additional information on specific reactions occurring in humans, allowing us to understand some of the metabolic pathways in pathological processes. Abnormal levels of such metabolites as nucleosides in the urine of cancer patients (abnormal in relation to the levels observed in healthy volunteers) seem to be an original potential diagnostic marker of carcinogenesis. However, the expectations regarding the diagnostic value of nucleosides may only be justified once an appropriate analytical procedure has been applied for their determination. The achievement of good specificity, sensitivity and reproducibility of the analysis depends on the right choice of the phases (e.g. sample pretreatment procedure), the analytical technique and the bioinformatic approach. Improving the techniques and methods applied implies greater interest in exploration of reliable diagnostic markers. This review covers the last 11 years of determination of urinary nucleosides conducted with the use of high-performance liquid chromatography in conjunction with various types of detection, sample pretreatment methods as well as bioinformatic data processing procedures.

Keywords: Cancer markers; Bioinformatics tools; Urinary nucleosides; Chromatographic determinations; Metabolomics

MicroRNAs as biomarkers of disease onset by Maciej Ciesla; Klaudia Skrzypek; Magdalena Kozakowska; Agnieszka Loboda; Alicja Jozkowicz; Jozef Dulak (pp. 2051-2061).

MicroRNAs (miRNAs) are small, noncoding RNA molecules with the ability to posttranscriptionally regulate gene expression via targeting the 3′ untranslated region of messenger RNAs. miRNAs are critical for normal cellular functions such as the regulation of the cell cycle, differentiation, and apoptosis, and they target genes during embryonal and postnatal development, whereas their expression is unbalanced in various pathological states. Importantly, miRNAs are abundantly present in body fluids (e.g., blood), which are routinely examined in patients. These molecules circulate in free and exosome encapsulated forms, and can be efficiently detected and amplified by means of molecular biology tools such as real-time PCR. Together with relative stability, specificity, and reproducibility, they are seen as good candidates for early recognition of the onset of disease. Thus, miRNAs might be considered as biomarkers for many pathological states.

Keywords: MicroRNA; Biomarkers; Cancer; Heart diseases

Time course of ethanol and propofol exhalation after bolus injection using ion molecule reaction–mass spectrometry by Martin Grossherr; Balamurugan Varadarajan; Leif Dibbelt; Peter Schmucker; Hartmut Gehring; Andreas Hengstenberg (pp. 2063-2067).

The transit of ethanol from blood to breath gas is well characterised. It is used for intraoperative monitoring and in forensic investigations. A further substance, which can be measured in breath gas, is the phenol propofol. After a simultaneous bolus injection, the signals (time course and amplitude) of ethanol and propofol in breath gas were detected by ion molecule reaction–mass spectrometry (IMR-MS) and compared. After approval by the regional authorities, eight pigs were endotracheally intubated after a propofol-free induction with etomidate. Boluses of ethanol (16 μg/kg) and propofol (4 or 2 mg/kg) were infused alone and in combination. For both substances, breath gas concentrations were continuously measured by IMR-MS; the delay time, time to peak and amplitude were determined and compared using non-parametric statistic tests. IMR-MS allows a simultaneous continuous measurement of both substances in breath gas. Ethanol appeared (median delay time, 12 vs 29.5 s) and reached its peak concentration (median time to peak, 45.5 vs 112 s) significantly earlier than propofol. Time courses of ethanol and propofol in breath gas can be simultaneously described with IMR-MS. Differing pharmacological and physicochemical properties of the two substances can explain the earlier appearance and time to peak of ethanol in breath gas compared with propofol.

Keywords: Chemical sensors; Drug monitoring/drug screening; Gas sensors; Clinical/biomedical analysis; Mass spectrometry/ICP-MS

Determination of zearalenone and its metabolites in endometrial cancer by coupled separation techniques by Renata Gadzała-Kopciuch; Krzysztof Cendrowski; Anna Cesarz; Paweł Kiełbasa; Bogusław Buszewski (pp. 2069-2078).

This study presents a selective method of isolation of zearalenone (ZON) and its metabolite, α-zearalenol (α-ZOL), in neoplastically changed human tissue by accelerated solvent and ultrasonic extractions using a mixture of acetonitrile/water (84/16% v/v) as the extraction solvent. Extraction effectiveness was determined through the selection of parameters (composition of the solvent mixture, temperature, pressure, number of cycles) with tissue contamination at the level of nanograms per gram. The produced acetonitrile/water extracts were purified, and analytes were enriched in columns packed with homemade molecularly imprinted polymers. Purified extracts were determined by liquid chromatography (LC) coupled with different detection systems (diode array detection - DAD and mass spectrometry - MS) involving the Ascentis RP-Amide as a stationary phase and gradient elution. The combination of UE-MISPE-LC (ultrasonic extraction - molecularly imprinted solid-phase extraction - liquid chromatography) produced high (R ≈ 95–98%) and repeatable (RSD < 3%) recovery values for ZON and α-ZOL.

Keywords: Zearalenone; α-Zearalenol; Molecularly imprinted polymers; Liquid chromatography; DAD/MS; Endometrial cancer

Phase-resolved real-time breath analysis during exercise by means of smart processing of PTR-MS data by Henny Schwoebel; Roland Schubert; Martin Sklorz; Sabine Kischkel; Ralf Zimmermann; Jochen K. Schubert; Wolfram Miekisch (pp. 2079-2091).

Separation of inspiratory, mixed expired and alveolar air is indispensable for reliable analysis of VOC breath biomarkers. Time resolution of direct mass spectrometers often is not sufficient to reliably resolve the phases of a breathing cycle. To realise fast on-line breath monitoring by means of direct MS utilising low-fragmentation soft ionisation, a data processing algorithm was developed to identify inspiratory and alveolar phases from MS data without any additional equipment. To test the algorithm selected breath biomarkers (acetone, isoprene, acetaldehyde and hexanal) were determined by means of quadrupole proton transfer reaction mass spectrometry (PTR-MS) in seven healthy volunteers during exercise on a stationary bicycle. The results were compared to an off-line reference method consisting of controlled alveolar breath sampling in Tedlar® bags, preconcentration by solid-phase micro extraction (SPME), separation and identification by GC-MS. Based on the data processing method, quantitative attribution of biomarkers to inspiratory, alveolar and mixed expiratory phases was possible at any time during the experiment, even under respiratory rates up to 60/min. Alveolar concentrations of the breath markers, measured by PTR-MS ranged from 130 to 2,600 ppb (acetone), 10 to 540 ppb (isoprene), 2 to 31 ppb (acetaldehyde), whereas the concentrations of hexanal were always below the limit of detection (LOD) of 3 ppb. There was good correlation between on-line PTR-MS and SPME-GC-MS measurements during phases with stable physiological parameters but results diverged during rapid changes of heart rate and minute ventilation. This clearly demonstrates the benefits of breath-resolved MS for fast on-line monitoring of exhaled VOCs. Figure Experimental setup showing bicycle ergometer and analytical pathways: Right side PTR-MS: identification of respiratory phases by means of the new algorithm. Left side: confirmation of PTR-MS data for exhaled isoprene by means of GC-MS analysis

Keywords: PTR-MS; Data processing algorithm; Stationary bicycle; Breath analysis; SPME-GC-MS

Drug detection in breath: effects of pulmonary blood flow and cardiac output on propofol exhalation by Svend Kamysek; Patricia Fuchs; Henny Schwoebel; Jan P. Roesner; Sabine Kischkel; Kathi Wolter; Christian Loeseken; Jochen K. Schubert; Wolfram Miekisch (pp. 2093-2102).

Breath analysis could offer a non-invasive means of intravenous drug monitoring if robust correlations between drug concentrations in breath and blood can be established. In this study, propofol blood and breath concentrations were determined in an animal model under varying physiological conditions. Propofol concentrations in breath were determined by means of two independently calibrated analytical methods: continuous, real-time proton transfer reaction mass spectrometry (PTR-MS) and discontinuous solid-phase micro-extraction coupled with gas chromatography mass spectrometry (SPME-GC-MS). Blood concentrations were determined by means of SPME-GC-MS. Effects of changes in pulmonary blood flow resulting in a decreased cardiac output (CO) and effects of dobutamine administration resulting in an increased CO on propofol breath concentrations and on the correlation between propofol blood and breath concentrations were investigated in seven acutely instrumented pigs. Discontinuous propofol determination in breath by means of alveolar sampling and SPME-GC-MS showed good agreement (R ² = 0.959) with continuous alveolar real-time measurement by means of PTR-MS. In all investigated animals, increasing cardiac output led to a deterioration of the relationship between breath and blood propofol concentrations (R ² = 0.783 for gas chromatography-mass spectrometry and R ² = 0.795 for PTR-MS). Decreasing pulmonary blood flow and cardiac output through banding of the pulmonary artery did not significantly affect the relationship between propofol breath and blood concentrations (R ² > 0.90). Estimation of propofol blood concentrations from exhaled alveolar concentrations seems possible by means of different analytical methods even when cardiac output is decreased. Increases in cardiac output preclude prediction of blood propofol concentration from exhaled concentrations. Figure Experimental setup for simultaneous real-time (PTR-MS) and discontinuous (SPME-GC-MS) drug determination in the breath of acutely instrumented pigs (A). In order to assess the influence of hemodynamic variables pulmonary artery blood flow was determined by means of Doppler-measurement (B).

Keywords: Breath test; Cardiac output; Propofol; Non-invasive drug monitoring; Animal model

Blood cell capture in a sawtooth dielectrophoretic microchannel by Paul V. Jones; Sarah J. R. Staton; Mark A. Hayes (pp. 2103-2111).

Biological fluids can be considered to contain information-rich mixtures of biochemicals and particles that enable clinicians to accurately diagnose a wide range of pathologies. Rapid and inexpensive analysis of blood and other bodily fluids is a topic gaining substantial attention in both science and medicine. One line of development involves microfluidic approaches that provide unique advantages over entrenched technologies, including rapid analysis times, microliter sample and reagent volumes, potentially low cost, and practical portability. The present study focuses on the isolation and concentration of human blood cells from small-volume samples of diluted whole blood. Separation of cells from the matrix of whole blood was accomplished using constant potential insulator-based gradient dielectrophoresis in a converging, sawtooth-patterned microchannel. The channel design enabled the isolation and concentration of specific cell types by exploiting variations in their characteristic physical properties. The technique can operate with isotonic buffers, allowing capture of whole cells, and reproducible capture occurred at specific locales within the channel over a global applied voltage range of 200–700 V.

Keywords: Dielectrophoresis; Erythrocytes; Bioparticle trapping; Separation; Blood

Physisorbed surface coatings for poly(dimethylsiloxane) and quartz microfluidic devices by M. Viefhues; S. Manchanda; T.-C. Chao; D. Anselmetti; J. Regtmeier; A. Ros (pp. 2113-2122).

Surface modifications of microfluidic devices are of essential importance for successful bioanalytical applications. Here, we investigate three different coatings for quartz and poly(dimethylsiloxane) (PDMS) surfaces. We employed a triblock copolymer with trade name F₁₀₈, poly(l-lysine)-g-poly(ethylene glycol) (PLL-PEG), as well as the hybrid coating n-dodecyl-β-d-maltoside and methyl cellulose (DDM/MC). The impact of these coatings was characterized by measuring the electroosmotic flow (EOF), contact angle, and prevention of protein adsorption. Furthermore, we investigated the influence of static coatings, i.e., the incubation with the coating agent prior to measurements, and dynamic coatings, where the coating agent was present during the measurement. We found that all coatings on PDMS as well as quartz reduced EOF, increased reproducibility of EOF, reduced protein adsorption, and improved the wettability of the surfaces. Among the coating strategies tested, the dynamic coatings with DDM/MC and F₁₀₈ demonstrated maximal reduction of EOF and protein adsorption and simultaneously best long-term stability concerning EOF. For PLL-PEG, a reversal in the EOF direction was observed. Interestingly, the static surface coating strategy with F₁₀₈ proved to be as effective to prevent protein adsorption as dynamic coating with this block copolymer. These findings will allow optimized parameter choices for coating strategies on PDMS and quartz microfluidic devices in which control of EOF and reduced biofouling are indispensable. Figure The difference between static coating (left) and dynamic surface coating (right) in a microfluidic channel is shown schematically. In the static case, the surface is incubated with the coating agent prior to a specific experiment. Dynamic coating refers to the case in which the coating agent is present in the solution during a specific experiment

Keywords: Static coating; Dynamic coating; Electroosmotic flow; Protein adsorption; PDMS; Quartz

Analysis of the flame retardant metabolites bis(1,3-dichloro-2-propyl) phosphate (BDCPP) and diphenyl phosphate (DPP) in urine using liquid chromatography–tandem mass spectrometry by E. M. Cooper; A. Covaci; A. L. N. van Nuijs; T. F. Webster; H. M. Stapleton (pp. 2123-2132).

Organophosphate triesters tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and triphenyl phosphate are widely used flame retardants (FRs) present in many products common to human environments, yet understanding of human exposure and health effects of these compounds is limited. Monitoring urinary metabolites as biomarkers of exposure can be a valuable aid for improving this understanding; however, no previously published method exists for the analysis of the primary TDCPP metabolite, bis(1,3-dichloro-2-propyl) phosphate (BDCPP), in human urine. Here, we present a method to extract the metabolites BDCPP and diphenyl phosphate (DPP) in human urine using mixed-mode anion exchange solid phase extraction and mass-labeled internal standards with analysis by atmospheric pressure chemical ionization liquid chromatography tandem mass spectrometry. The method detection limit was 8 pg mL⁻¹ urine for BDCPP and 204 pg mL⁻¹ for DPP. Recoveries of analytes spiked into urine ranged from 82 ± 10% to 91 ± 4% for BDCPP and from 72 ± 12% to 76 ± 8% for DPP. Analysis of a small number of urine samples (n = 9) randomly collected from non-occupationally exposed adults revealed the presence of both BDCPP and DPP in all samples. Non-normalized urinary concentrations ranged from 46–1,662 pg BDCPP mL⁻¹ to 287–7,443 pg DPP mL⁻¹, with geometric means of 147 pg BDCPP mL⁻¹ and 1,074 pg DPP mL⁻¹. Levels of DPP were higher than those of BDCPP in 89% of samples. The presented method is simple and sufficiently sensitive to detect these FR metabolites in humans and may be applied to future studies to increase our understanding of exposure to and potential health effects from FRs. Figure The flame retardant TDCPP is metabolized to BDCPP and detected in human urine.

Keywords: Flame retardant; Urine; Metabolite; Method

Strategy for choosing extraction procedures for NMR-based metabolomic analysis of mammalian cells by Estelle Martineau; Illa Tea; Gregory Loaëc; Patrick Giraudeau; Serge Akoka (pp. 2133-2142).

Metabolomic analysis of mammalian cells can be applied across multiple fields including medicine and toxicology. It requires the acquisition of reproducible, robust, reliable, and homogeneous biological data sets. Particular attention must be paid to the efficiency and reliability of the extraction procedure. Even though a number of recent studies have dealt with optimizing a particular protocol for specific matrices and analytical techniques, there is no universal method to allow the detection of the entire cellular metabolome. Here, we present a strategy for choosing extraction procedures from adherent mammalian cells for the global NMR analysis of the metabolome. After the quenching of cells, intracellular metabolites are extracted from the cells using one of the following solvent systems of varying polarities: perchloric acid, acetonitrile/water, methanol, methanol/water, and methanol/chloroform/water. The hydrophilic metabolite profiles are analysed using ¹H nuclear magnetic resonance (NMR) spectroscopy. We propose an original geometric representation of metabolites reflecting the efficiency of extraction methods. In the case of NMR-based analysis of mammalian cells, this methodology demonstrates that a higher portion of intracellular metabolites are extracted by using methanol or methanol/chloroform/water. The preferred method is evaluated in terms of biological variability for studying metabolic changes caused by the phenotype of four different human breast cancer cell lines, showing that the selected extraction procedure is a promising tool for metabolomic and metabonomic studies of mammalian cells. The strategy proposed in this paper to compare extraction procedures is applicable to NMR-based metabolomic studies of various systems.

Keywords: Metabolomics; NMR; Human breast cancer; Metabolite extraction; Sample preparation

Analysis of doxorubicin uptake in single human leukemia K562 cells using capillary electrophoresis coupled with laser-induced fluorescence detection by Bin Deng; Zhiming Wang; Jian Song; Yuxiu Xiao; Dan Chen; Jun Huang (pp. 2143-2152).

The doxorubicin (DOX) uptake in single human leukemia K562 cells with changes in both drug dosage and exposure period was studied using capillary electrophoresis (CE) coupled with laser-induced fluorescence (LIF) detection. The cells were treated with DOX at different concentrations (1, 3, 10, 20, 30, and 50 μM) and for different exposure times (1, 3, 5, 24, and 48 h). At least 20 cells were analyzed for each DOX-treated cell population. A marked heterogeneity in DOX uptake among single cells was observed, because the relative standard deviation of the uptake of DOX by single cells ranged from 24.0% to 61.1% within each cell population. The cell-to-cell heterogeneity in DOX uptake first decreased and then became constant with increasing drug concentration, but it did not exhibit regular variation with increasing exposure time. The mean DOX uptake was a linear function of drug concentration (r ≥ 0.9667). In terms of the correlation with exposure time, the mean DOX uptake reached its maximum at 3 h for the cell populations treated with 1–10 μM DOX, while it kept increasing during 48 h exposure of cell populations to 20–50 μM DOX. Because it eliminates DOX fluorescence quenching and sample loss, the CE-LIF method directly detects the true DOX uptake by single cells, and thus presents accurate information on both the cell-to-cell heterogeneity in DOX uptake and the patterns of DOX uptake in K562 cells as functions of drug concentration and exposure time. Figure CE-LIF analysis of DOX uptake in single human leukemia K562 cells. a DOX uptakes of single K562 cells treated with 1 μM DOX for 3 h; The RSD% for the DOX uptake of single K562 cells as functions of b the extracellular DOX concentration and c the exposure time; Mean DOX uptake of single K562 cells as functions of d the extracellular DOX concentration and e the exposure time

Keywords: Doxorubicin; Single-cell analysis; Capillary electrophoresis; Cellular heterogeneity; Drug uptake; Human leukemia K562 cells

Chemometric evaluation of nine alcohol biomarkers in a large population of clinically-classified subjects: pre-eminence of ethyl glucuronide concentration in hair for confirmatory classification by Valentina Pirro; Valeria Valente; Paolo Oliveri; Angela De Bernardis; Alberto Salomone; Marco Vincenti (pp. 2153-2164).

An important goal of forensic and clinical toxicology is to identify biological markers of ethanol consumption that allow an objective diagnosis of chronic alcohol misuse. Blood and head hair samples were collected from 175 subjects—objectively classified as non-drinkers (N = 65), social drinkers (N = 51) and active heavy drinkers (N = 59)—and analyzed to determine eight traditional indirect biomarkers of ethanol consumption [aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (γ-GT), alkaline phosphatase (ALP), mean corpuscular volume (MCV), carbohydrate-deficient transferrin (CDT), and cholesterol and triglycerides in blood] and one direct biomarker [ethyl glucuronide (EtG) in head hair]. The experimental values obtained from these determinations were submitted to statistical evaluations. In particular, Kruskal–Wallis, Mann–Whitney and ROC curve analyses, together with principal component analysis (PCA), allowed the diagnostic performances of the various biomarkers to be evaluated and compared consistently. From these evaluations, it was possible to deduce that EtG measured in head hair is the only biomarker that can conclusively discriminate active heavy drinkers from social and non-drinkers, using a cut-off value of 30 pg/mg. In contrast, a few indirect biomarkers such as ALP, cholesterol, and triglycerides showed extremely low diagnostic abilities and may convey misleading information. AST and ALT proved to be highly correlated and exhibited quite low sensitivity and specificity. Consequently, either of these parameters can be discarded without compromising the classification efficiency. Among the indirect biomarkers, γ-GT provided the highest diagnostic accuracy, while CDT and MCV yielded high specificity but low sensitivity. It was therefore concluded that EtG in head hair is the only biomarker capable of supporting a confirmatory diagnosis of chronic alcohol abuse in both forensic and clinical practice, while it was found that γ-GT, CDT, MCV, and AST—whether used alone or in combination—do not allow the conclusive classification of subjects according to ethanol consumption. However, a diagnostic strategy combining these four parameters could be formulated in order to create a multivariate model capable of screening suspected active heavy drinkers. Figure PCA of 175 patients clinically classified as non-drinkers (green dots, N = 65), social drinkers (yellow dots, N = 51) and active heavy drinkers (red dots, N = 59). Score and loading (six parameters) bi-plot of PC1 and PC2.

Keywords: Ethyl glucuronide; Hair; Alcohol biomarker; Chemometric; PCA; ROC curve

S-glutathionyl quantification in the attomole range using glutaredoxin-3-catalyzed cysteine derivatization and capillary gel electrophoresis with laser-induced fluorescence detection by Cheng Zhang; Cynthia Rodriguez; Magdalena L. Circu; Tak Yee Aw; June Feng (pp. 2165-2175).

S-glutathionylation (Pr–SSG) is a specific post-translational modification of cysteine residues by the addition of glutathione. S-Glutathionylated proteins induced by oxidative or nitrosative stress play an essential role in understanding the pathogenesis of the aging and age-related disorder, such as Alzheimer’s disease (AD). The purpose of this research is to develop a novel and ultrasensitive method to accurately and rapidly quantify the Pr–SSG by using capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). The derivatization method is based on the specific reduction of protein-bound S-glutathionylation with glutaredoxin (Grx) and labeling with thiol-reactive fluorescent dye (Dylight 488 maleimide). The experiments were performed by coupling the derivatization method with CGE-LIF to study electrophoretic profiling in in vitro oxidative stress model–S-glutathionylated bovine serum albumin (BSA-SSG), oxidant-induced human colon adenocarcinoma (HT-29) cells, brain tissues, and whole blood samples from an AD transgenic (Tg) mouse model. The results showed almost an eightfold increase in S-glutathionyl abundance when subjecting HT-29 cells in an oxidant environment, resulting in Pr–SSG at 232 ± 10.64 (average ±SD; n = 3) nmol/mg. In the AD–Tg mouse model, an initial quantitative measurement demonstrated the extent of protein S-glutathionylation in three brain regions (hippocampus, cerebellum, and cerebrum), ranging from 1 to 10 nmol/mg. Additionally, we described our developed method to potentially serve as a highly desirable diagnostic tool for monitoring S-glutathionylated protein profile in minuscule amount of whole blood. The whole blood samples for S-glutathionyl expression of 5-month-old AD–Tg mice are quantified as 16.3 μmol/L (=7.2 nmol/mg protein). Altogether, this is a fast, easy, and accurate method, reaching the lowest limit of Pr–SSG detection at 1.8 attomole (amol) level, reported to date. Figure Quantitative electrophoretic profiling of MQ-mediated glutathionylation in HT-29 cells and Pr–SSG of brain tissues and blood in AD–Tg/WT mice by using CGE-LIF

Keywords: S–glutathionylation; AD–Tg mice; Pr–SSG; Grx; Thiol quantification; CGE-LIF

A rapid analytical method based on microwave-assisted extraction for the determination of drugs of abuse in vitreous humor by Purificación Fernández; Santiago Seoane; Cristina Vázquez; Ana M. Bermejo; Antonia M. Carro; Rosa A. Lorenzo (pp. 2177-2186).

Robust and simple validated analytical methods are required in postmortem toxicology to confirm immunoassay screening analysis of drugs of abuse. In this work, microwave-assisted extraction (MAE) was evaluated as an alternative method for extraction of target compounds such as cocaine, benzoylecgonine, cocaethylene, morphine, codeine, 6-monoacetylmorphine, methadone, and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine from vitreous humor. The MAE procedure parameters, namely, extraction temperature, time, and solvent volume, were optimized using a central composite design and applying desirability functions. The optimal conditions for extraction were 80 °C, 8 min, and 15 mL of dichloromethane solvent. The MAE–high-performance liquid chromatography–diode-array detection method was validated, showing its capability for the detection of concentrations in the range from 33 to 76 ng mL⁻¹ and recoveries in the range from 87 to 99.3% for all drugs. The MAE-based method was tested for 15 vitreous humor samples from forensic cases and its performance was compared with that of a solid-phase extraction method previously developed by our group. In general, better recovery and precision were achieved with the use of the MAE-based procedure.

Keywords: Cocaine; Opiates; Microwave-assisted extraction; Experimental design; Vitreous humor; High-performance liquid chromatography

Quantification of total and free concentrations of R- and S-warfarin in human plasma by ultrafiltration and LC-MS/MS by Berit Packert Jensen; Paul Ken Leong Chin; Evan James Begg (pp. 2187-2193).

A sensitive LC-MS/MS assay for quantification of total and free concentrations of R- and S-warfarin in plasma was required to support clinical studies on warfarin enantiomers. Several ultrafiltration devices were evaluated for separation of free warfarin from plasma proteins. The highest precision and lowest non-specific binding was obtained for Centrifree ultrafiltration devices. R- and S-warfarin were extracted from plasma (total) and ultrafiltrate (free) by liquid–liquid extraction with methyl tert-butyl ether using d₆-warfarin as internal standard. Mean extraction recovery was 68 ± 4%. The enantiomers were separated on a Chirobiotic V column with isocratic elution using 40% methanol and 0.03% acetic acid in water. Negative mode electrospray ionisation was used for MS/MS detection, monitoring the ion transition m/z 307/161. Calibration curves (quadratic, weighted 1/x) were fitted over the range of 20–2,000 ng/ml (r ² ≥ 0.995) in plasma and 0.5–20 ng/ml (r ² ≥ 0.998) in ultrafiltrate. The lower limit of quantification for R- and S-warfarin was 0.5 ng/ml in ultrafiltrate. Intra- and interday precision (% RSD) and bias were within 10% in all cases, and matrix effects were negligible. The assay was applied successfully to analysis of samples from clinical studies. Figure LC-MS/MS chromatogram of free R- and S-warfarin in human ultrafiltrate

Keywords: Warfarin; Enantiomer; Free; Unbound; Ultrafiltration; Plasma

Highly specific, sensitive and rapid enzyme immunoassays for the measurement of acetaminophen in serum by Ramadan A. Abuknesha; Michael Paleodimos; Fiona Jeganathan (pp. 2195-2204).

Acetaminophen antibodies were purified using affinity chromatography and labelled with horseradish peroxidase (HRP). The antibody–HRP conjugate and a new acetaminophen derivative were used in the construction of two immunoassay methods facilitating the direct quantitative measurement of acetaminophen in serum: a 96-well microtitre plate and coated-tube ELISAs. A minimum detection limit of 0.2 μg mL⁻¹ and a dynamic range of 0.2 to 10 μg mL⁻¹ in serum were achieved using the 96-well microtitre plate ELISA. The tube assay was optimised for the measurement of the clinically critical acetaminophen concentration of 50 to 250 μg mL⁻¹ of serum. The quantitative and specific tests could be completed within less than an hour. Common drugs including aspirin showed less than 0.1% cross-reactivity.

Keywords: Acetaminophen; ELISA

Quantitative determination of isoniazid in biological samples by cation-selective exhaustive injection–sweeping–micellar electrokinetic chromatography by I-Lin Tsai; Hsiang-Yin Liu; Ping-Hung Kuo; Jann-Yuan Wang; Li-Jiuan Shen; Ching-Hua Kuo (pp. 2205-2214).

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis, infects approximately one third of the current world population. Isoniazid is one of the most frequently used first-line anti-TB drugs. In this study, we developed a sensitive cation-selective exhaustive injection–sweeping–micellar electrokinetic chromatography method (CSEI-Sweep-MEKC) for analyzing isoniazid in human plasma. Parameters including acetonitrile (ACN) percentage in the separation buffer; the injection time, and concentration of the high-conductivity buffer; sodium dodecyl sulfate (SDS) concentration; phosphate concentration in the sample matrix; and the sample injection time were all optimized to obtain the best analytical performance. The optimal background electrolyte comprised 50 mM phosphate buffer, 100 mM SDS, and 15% ACN. Non-micelle background electrolyte, containing 75 mM phosphate buffer and 15% ACN, was first injected into the capillary, followed by a short plug of 200 mM phosphate (high-conductivity buffer). Run-to-run repeatability (n = 3) and intermediate precision (n = 3) of peak area ratios were found to be lower than 8.7% and 11.4% RSD, respectively. The accuracy of the method was within 98.1–106.9%. The limit of detection of isoniazod in human plasma was 9 ng mL⁻¹. Compared with conventional MEKC, the enhancement factor of the CSEI-Sweep-MEKC method was 85 in plasma samples. The developed method was successfully used to determine isoniazid concentration in patient plasma. The results demonstrated that CSEI-Sweep-MEKC has the potential to analyze isoniazid in human plasma for therapeutic drug monitoring and clinical research.

Keywords: Isoniazid; Human plasma; Cation-selective exhaustive injection; Sweeping

Determination of dextromethorphan in human plasma using pipette tip solid-phase extraction and gas chromatography–mass spectrometry by Chika Hasegawa; Takeshi Kumazawa; Seisaku Uchigasaki; Xiao-Pen Lee; Keizo Sato; Masaru Terada; Kunihiko Kurosaki (pp. 2215-2223).

Dextromethorphan was extracted from human plasma samples (100 μL) using MonoTip C₁₈ tips, which are packed with C₁₈-bonded monolithic silica gel that is attached to the inside of the tip. The samples, which contained dextromethorphan and trimeprazine as an internal standard (IS), were mixed with 200 μL of distilled water and 50 μL of 1 mol/L glycine–sodium hydroxide buffer (pH 10). The mixture was extracted to the C₁₈ phase of the tip by 20 sequential aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C₁₈ phase were then eluted with methanol by five sequential aspirating/dispensing cycles. The eluate was injected directly into a gas chromatograph and detected by a mass spectrometer with selected ion monitoring in positive electron ionization mode. An Equity-5 fused silica capillary column (30 m × 0.32 mm i.d., film thickness 0.5 μm) gave adequate separation of the dextromethorphan, IS, and impurities. The recoveries of dextromethorphan and the IS spiked into plasma were >87.4%. The regression equation for dextromethorphan showed excellent linearity from 2.5 to 320 ng/mL of plasma, and the limit of detection was 1.25 ng/mL of plasma. The intraday and interday coefficients of variation were less than 10.5% and 14.7%, respectively. The accuracy ranged from 91.9% to 107%. The validated method was successfully used to quantify the plasma concentration of dextromethorphan in a human subject after oral administration of the drug.

Keywords: Dextromethorphan; Solid-phase extraction; Pipette tip; Gas chromatography; Mass spectrometry

Discrimination of bacteria from Jamaican bauxite soils using laser-induced breakdown spectroscopy by Dawn E. Lewis; Jorge Martinez; Charlemagne A. Akpovo; Lewis Johnson; Ashvini Chauhan; Maurice D. Edington (pp. 2225-2236).

Soil bacteria are sensitive to ecological change and can be assessed to gauge anthropogenic influences and ecosystem health. In recent years, there has been a significant increase in the focus on new technologies that can be applied to the evaluation of soil quality. Laser-induced breakdown spectroscopy (LIBS) is a promising technique that has been used for the investigation and characterization of explosives, solids, liquids, gases, biological and environmental samples. In this study, bacteria from un-mined and a chronosequence of reclaimed bauxite soils were isolated on Luria–Bertani agar media. Polymerase chain reaction amplification of the bacterial 16S rDNA, sequencing, and phylogenetic analysis were applied to each isolated soil bacteria from the sample sites resulting in the identification and classification of the organisms. Femtosecond LIBS performed on the isolated bacteria showed atomic and ionic emission lines in the spectrum containing inorganic elements such as sodium (Na), magnesium (Mg), potassium (K), zinc (Zn), and calcium (Ca). Principal component analysis and partial least squares regression analysis were performed on the acquired bacterial spectra demonstrating that LIBS has the potential to differentiate and discriminate among bacteria in the un-mined and reclaimed chronosequence of bauxite soils.

Keywords: Bacteria; Bauxite soils; Laser-induced breakdown spectroscopy; Polymerase chain reaction; Chemometric analysis

Arsenopyrite and pyrite bioleaching: evidence from XPS, XRD and ICP techniques by Marzia Fantauzzi; Cristina Licheri; Davide Atzei; Giovanni Loi; Bernhard Elsener; Giovanni Rossi; Antonella Rossi (pp. 2237-2248).

In this work, a multi-technical bulk and surface analytical approach was used to investigate the bioleaching of a pyrite and arsenopyrite flotation concentrate with a mixed microflora mainly consisting of Acidithiobacillus ferrooxidans. X-ray diffraction, X-ray photoelectron spectroscopy (XPS) and X-ray-induced Auger electron spectroscopy mineral surfaces investigations, along with inductively coupled plasma-atomic emission spectroscopy and carbon, hydrogen, nitrogen and sulphur determination (CHNS) analyses, were carried out prior and after bioleaching. The flotation concentrate was a mixture of pyrite (FeS₂) and arsenopyrite (FeAsS); after bioleaching, 95% of the initial content of pyrite and 85% of arsenopyrite were dissolved. The chemical state of the main elements (Fe, As and S) at the surface of the bioreactor feed particles and of the residue after bioleaching was investigated by X-ray photoelectron and X-ray excited Auger electron spectroscopy. After bioleaching, no signals of iron, arsenic and sulphur originating from pyrite and arsenopyrite were detected, confirming a strong oxidation and the dissolution of the particles. On the surfaces of the mineral residue particles, elemental sulphur as reaction intermediate of the leaching process and precipitated secondary phases (Fe–OOH and jarosite), together with adsorbed arsenates, was detected. Evidence of microbial cells adhesion at mineral surfaces was also produced: carbon and nitrogen were revealed by CHNS, and nitrogen was also detected on the bioleached surfaces by XPS. This was attributed to the deposition, on the mineral surfaces, of the remnants of a bio-film consisting of an extra-cellular polymer layer that had favoured the bacterial action.

Keywords: X-ray photoelectron spectroscopy (XPS); Surface analysis; Pyrite; Arsenopyrite; Acidithiobacillus ferrooxidans ; Biohydrometallurgy; Bio-oxidation; Bioleaching; Biomining; Extra-cellular polymeric substance (EPS)

Development of a monoclonal antibody-based, congener-specific and solvent-tolerable direct enzyme-linked immunosorbent assay for the detection of 2,2′,4,4′-tetrabromodiphenyl ether in environmental samples by Jia Wang; Hao Li; Weilin L. Shelver; Zhanhui Wang; Qing X. Li; Ji Li; Ting Xu (pp. 2249-2258).

A sensitive direct enzyme-linked immunosorbent assay (ELISA) for the specific detection of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) in environmental samples was developed. A hapten mimicking BDE-47 was synthesized by introducing a butyric acid spacer into 5-hydroxy-BDE-47 and coupled to keyhole limpet hemocyanin to form an immunogen for the production of monoclonal antibodies (Mabs) against BDE-47. The most sensitive direct ELISA was formatted with a Mab, designated as 4F2, in combination with 5-(2,4-dibromophenoxy)pentanoic acid peroxidase as a tracer. The inhibition half-maximum concentrations and limit of detection of BDE-47 in phosphate buffered saline with 25% DMSO were 1.4 ± 0.05 and 0.1 ng mL⁻¹, respectively. Cross-reactivity values of the ELISA with the tested BDE congeners and metabolites were ≤5.8%. This assay was used to determine BDE-47 in soil, sediment and house dust samples after ultrasonic extraction, simple cleanup and concentration steps. The average recoveries, repeatabilities (intraday extractions and analyses), and intra-laboratory reproducibilities (interday extractions and analyses) were in a range of 92–126%, 8–19% and 9–25%, respectively. Applied to 44 real samples, the results of this assay displayed a statistically significant correlation with those of a gas chromatography–mass spectrometry method (R ² = 0.79-0.85), indicating this ELISA is a suitable tool for environmental analyses of BDE-47. Figure Direct competitive ELISA

Keywords: 2,2′,4,4′-Tetrabromodiphenyl ether; Polybrominated diphenyl ethers; Flame retardant; Enzyme-linked immunosorbent assay; Monoclonal antibody

Preparation and characterization of a molecularly imprinted polymer by grafting on silica supports: a selective sorbent for patulin toxin by Dayun Zhao; Jingfu Jia; Xuelei Yu; Xiangjun Sun (pp. 2259-2273).

A new molecularly imprinted polymer (MIP) has been prepared on silica beads using the radical “grafting from” polymerization method for selective extraction of minor contaminant mycotoxin of patulin (PTL). After the introduction of amino groups onto the silica surface with 3-aminopropyltriethoxysilane, azo initiator onto the silica surface was achieved by the reaction of surface amino groups with 4,4′-azobis(4-cyanopentanoic acid). The scale-up synthesis of MIP was then carried out in the presence of 6-hydroxynicotinic acid as template substitute, functional, and cross-linking monomers. The prepared sorbent was characterized using FT-IR spectroscopy, scanning electron microscopy, elemental analysis, and the adsorption–desorption selectivity, and the capacity characteristic of the polymer was investigated by a conventional batch adsorption test and Scatchard plot analysis. The results indicated that coated polymers had specific adsorption to PTL as compared with its co-occurring 5-hydroxymethyl-2-furaldehyde (hydroxymethylfurfural (HMF)), at the same bulk concentration for solution of PTL and HMF, the maximum absorbance in the solid-phase extraction (SPE) method to PTL were 93.97% or 0.654 μg/mg while to HMF they were 76.89% or 0.496 μg/mg. Scatchard analysis revealed that two classes of binding sites were formed in PTL-MIP with dissociation constants of 3.2 × 10⁻² and 5.0 × 10⁻³ mg/mL and the affinity binding sites of 8.029 and 1.364 mg/g, respectively. The recoveries of PTL were more than 90% for the developed MISPE and around 75% for the traditional liquid–liquid extraction in spiked apple juice samples. It was concluded that the method is suitable for the scale-up synthesis of PTL-MIP grafted on silica, and the polymer can be effectively applied as SPE coupled with high-performance liquid chromatography (HPLC) for the determination of PTL in apple juice or other related products. Figure HPLC chromatograms of loading, washing, and eluting fractions of PTL and HMF from the MIP cartridge. Test samples in each chromatogram from top to bottom: mixed standard of HMF and PTL (10 mg/L), residue solution through the cartridge, first elution and second elution washed with 1 mL of 1% (w/v) HAc aqueous solution.

Keywords: Molecular imprinted polymers (MIPs); Patulin; Mimic; Grafting; Silica

Covalent imprinted polymer for selective and rapid enrichment of ractopamine by a noncovalent approach by Yi-Wei Tang; Guo-Zhen Fang; Shuo Wang; Jia-Le Li (pp. 2275-2282).

A novel molecularly imprinted polymer (MIP) for the separation and concentration of ractopamine (RAC) was prepared by a covalent imprinting approach and the template was removed successfully by hydrolysis, so that four carboxylic acid groups were left in the cavities and could specifically rebind RAC through noncovalent interaction: hydrogen bonding. The conditions for synthesis of the MIP were optimized during the polymerization process, and a molar ratio of template–functional monomer complexes to cross-linker of 1:3 was confirmed. The adsorption capacity of the MIP was 4.1-fold that of the nonimprinted polymer, and the adsorption reaction reached equilibrium after 25 min at 50 mg L^-1 concentration. The results of the competitive adsorption test showed that the MIPs had specific recognition ability for the analyte RAC. In addition, the important factors affecting the efficiency of the method which was developed using the MIPs as a solid-phase sorbent for separation and determination of RAC combined with high-performance liquid chromatography with fluorescence detection were optimized. Under the optimum experimental conditions, the linear range of the calibration curve in the method was 0.05-5 μg L^-1 (R ² = 0.98) and the limit of detection (signal-to-noise ratio of 3) was 0.01 μg L^-1. The proposed method was applied to determination of RAC in spiked feedstuffs and urine samples, with recoveries ranging from 74.17 to 114.46% and relative standard deviation (n = 3) below 4.55 in all cases. Figure The molecularly imprinted polymer obtained by a covalent imprinting method. MMPEMPM (4-((3S)-3-(N-(2-(methacryloyloxy)-2-(4-(methacryloyloxy)phenyl)ethyl)methacrylamido)butyl)phenyl methacrylate), EGDMA ethylene glycol dimethacrylate, AIBN azobis(isobutryonitrile), red oxygen, gray carbon, white hydrogen, blue nitrogen

Keywords: Covalent imprinted polymer; Ractopamine; Solid-phase extraction

Characterisation of non-viable whole barley, wheat and sorghum grains using near-infrared hyperspectral data and chemometrics by Cushla M. McGoverin; Paulina Engelbrecht; Paul Geladi; Marena Manley (pp. 2283-2289).

Undesired germination of cereal grains diminishes process utility and economic return. Pre-germination, the term used to describe untimely germination, leads to reduced viability of a grain sample. Accurate and rapid identification of non-viable grain is necessary to reduce losses associated with pre-germination. Viability of barley, wheat and sorghum grains was investigated with near-infrared hyperspectral imaging. Principal component analyses applied to cleaned hyperspectral images were able to differentiate between viable and non-viable classes in principal component (PC) five for barley and sorghum and in PC6 for wheat. An OH stretching and deformation combination mode (1,920–1,940 nm) featured in the loading line plots of these PCs; this water-based vibrational mode was a major contributor to the viable/non-viable differentiation. Viable and non-viable classes for partial least squares-discriminant analysis (PLS-DA) were assigned from PC scores that correlated with incubation time. The PLS-DA predictions of the viable proportion correlated well with the viable proportion observed using the tetrazolium test. Partial least squares regression analysis could not be used as a source of contrast in the hyperspectral images due to sampling issues. Figure PCA score image for barley kernels

Keywords: Barley; Near-infrared hyperspectral imaging; Pre-germination; Sorghum; Viability; Wheat

Novel surface molecularly imprinted sol–gel polymer applied to the online solid phase extraction of methyl-3-quinoxaline-2-carboxylic acid and quinoxaline-2-carboxylic acid from pork muscle by Zhen-Juan Duan; Li-Peng Fan; Guo-Zhen Fang; Jiang-Hua Yi; Shuo Wang (pp. 2291-2299).

A new molecularly imprinted polymer (MIP), selective for major metabolites of quinoxaline-1,4-dioxides was firstly prepared by combining surface molecular imprinting technique with the sol–gel process. Methyl-3-quinoxaline-2-carboxylic acid (MQCA) was used as template, 3-aminopropyltriethoxysilane as functional monomer, and tetraethoxysilicane as cross-linker. The MIP was characterized by Fourier transform infrared and evaluated through static adsorption experiments. The results indicated that MIP had high adsorption capacity, fast binding kinetics for MQCA, and the polymer showed a high degree of cross-reactivity for quinoxaline-2-carboxylic acid (QCA). The MIP was then applied as a selective sorbent in an online solid phase extraction (SPE) coupled with high-performance liquid chromatography (HPLC). For a 50-mL sample solution, enrichment factors of 1,349 and 1,046 for QCA and MQCA, respectively, and limits of detection (S/N = 3) of 0.8 and 2 ng L⁻¹ for QCA and MQCA, respectively, were obtained (corresponding to 0.02 and 0.04 ng g⁻¹ in solid samples for final 100 mL of sample solutions of 5 g of pork). The sample preparation protocol was simplified and only included one step extraction with acetonitrile (MeCN) after the release of target analytes through acidic hydrolysis without further sample cleanup. The new MIP-SPE-HPLC method was successfully applied to the quantification of trace QCA and MQCA in pork muscle with good recoveries ranging from 67% to 80% and RSD below 8%. Figure A new molecularly imprinted polymer (MIP), selective for methyl-3-quinoxaline-2-carboxylic acid (MQCA) and quinoxaline-2-carboxylic acid (QCA), was prepared by combining surface molecular imprinting technique with the sol–gel process. The MIP could be applied as a selective sorbent of an online solid phase extraction (SPE) coupled with high-performance liquid chromatography (HPLC)

Keywords: Quinoxaline-2-carboxylic acid; Methyl-3-quinoxaline-2-carboxylic acid; Molecularly imprinted polymer; Sol–gel; Online solid phase extraction; Pork

A clinical trial for therapeutic drug monitoring using microchip-based fluorescence polarization immunoassay by Tomoya Tachi; Tetsunari Hase; Yukihiro Okamoto; Noritada Kaji; Takeshi Arima; Hiroyuki Matsumoto; Masashi Kondo; Manabu Tokeshi; Yoshinori Hasegawa; Yoshinobu Baba (pp. 2301-2305).

Microchip analysis is a promising method for therapeutic drug monitoring. This led us to evaluate a microchip-based fluorescence polarization immunoassay (FPIA) system for point-of-care testing on patients being treated with theophylline. The sera were collected from 20 patients being treated with theophylline. Fluorescence polarization was measured on the microchip and theophylline concentrations in serum were obtained. Regression analysis of the correlations was done between the results given by the microchip-based FPIA and the conventional cloned enzyme donor immunoassay (CEDIA), and between the results given by the microchip-based FPIA and the conventional particle-enhanced turbidimetric inhibition immunoassay (PETINIA). We successfully carried out a quantitative analysis of theophylline in serum at values near its therapeutic range in 65 s. The results obtained by the microchip-based FPIA correlated well with CEDIA and PETINIA results; the correlation coefficients (R ²) were 0.986 and 0.989, respectively. The FPIA system is a simple and rapid method for point-of-care testing of drugs in serum, and its accuracy is the same as the conventional CEDIA and PETINIA. It is essential to use real samples from patients and to confirm good correlations with conventional methods for a study on the realization of microchip.

Keywords: Therapeutic drug monitoring; Microchip-based fluorescence polarization immunoassay; Theophylline

Spectrophotometric quantification of lactose in solution with a peroxidase-based enzymatic cascade reaction system by Sara Fornera; Kenjiro Yazawa; Peter Walde (pp. 2307-2310).

A spectrophotometric assay was developed for the quantification of lactose in aqueous solution via a one-pot enzymatic cascade reaction at 25 °C and pH 7.2. Lactose (0.2–1.8 mM), E. coli β-galactosidase (β-Gal), Aspergillus niger glucose oxidase (GOD), horseradish peroxidase (HRP) and o-phenylenediamine (OPD) were incubated, and the increase in absorbance at 417 nm (A ₄₁₇) due to the formation of DAP (2,3-diaminophenazine), the dimeric oxidation product of OPD, was followed. The increase in A ₄₁₇ was found to depend linearly on the initial lactose concentration via three consecutive but simultaneously occurring enzymatic reaction steps catalyzed by β-Gal, GOD, and HRP. No pre-incubation of lactose with β-Gal is needed with this simple lactose assay.

Keywords: Lactose; β-Galactosidase; Glucose oxidase; Horseradish peroxidase; o-Phenylenediamine; Enzymatic cascade reaction

Chitosan-coated polystyrene microplate for covalent immobilization of enzyme by Yaodong Zhang; Li Li; Caihong Yu; Tingting Hei (pp. 2311-2317).

Microplates made of polystyrene have been widely used for immunoassays. Protein molecules that have been immobilized on a hydrophobic polystyrene microplate by passive adsorption lose their activity and suffer considerable denaturation. A new chitosan-coated microplate suitable for the covalent immobilization of enzymes has been developed. The primary amino groups of chitosan were exploited for this covalent coupling of proteins. The optical transmittance of the chitosan-coated microplate, at wavelengths of 400–800 nm, was estimated to be suitable for its application in chromogenic reaction-based bioassays. The immobilization efficiency of the chitosan-coated microplate was demonstrated to be far superior to that of a conventional microplate when tested using acetylcholinesterase (AChE) and β-glucosidase as model biomolecules, and the chitosan-coated microplate may thus have potential applications in biosensing and bioreactor systems. Figure Comparison of the optical transmittances of chitosan-coated microplates in the wavelength range monitored in chromogenic reactions employed for immunoassays with the transmittance of a conventional polystyrene microplate

Keywords: Bioassays; Enzymes; Biosensor; Covalent immobilization; Chitosan

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Analytical and Bioanalytical Chemistry (v.401, #7)

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Analytical and Bioanalytical Chemistry (v.401, #7)