Facile synthesis of AgNPs@SNCDs nanocomposites as a fluorescent ‘turn on’ sensor for detection of glutathione

The present study illustrates the facile synthesis of silver nanoparticles capped with sulfur and nitrogen co‐doped carbon dots (AgNPs@SNCDs) nanocomposites and their application towards the sensitive and selective detection of glutathione (GSH) using a spectrofluorimetry method. SNCDs were synthesized using solvothermal treatment of cysteamine hydrochloride and p‐phenylenediamine. The as‐fabricated SNCDs were then utilized as capping and stabilizing agents for the preparation of AgNPs@SNCDs nanocomposites using wet chemistry. The size of AgNPs@SNCDs nanocomposites was characterized to be ~37.58 nm or even larger aggregates. Particularly, the quenched fluorescence of AgNPs@SNCDs nanocomposites could be significantly restored upon addition of GSH, and the colour of its solution changed to some extent. The fluorescence intensity ratio of AgNPs@SNCDs nanocomposites at ~450 nm and 550 nm was directly proportional to the GSH concentration within the ranges 8.35–66.83 μM and 66.83–200.5 μM, and the detection limit was 0.52 μM. Furthermore various common organic molecules had no obvious interference in the detection mode. The proposed nanosensor was successfully applied for GSH assay in actual water samples.     

Simultaneous determination of amlodipine and metoprolol in their combined dosage form using a synchronous fluorescence spectrofluorimetric method

Highly sensitive, rapid, accurate and precise synchronous fluorescence spectrofluorimetric method has been developed for simultaneous analysis of a mixture of amlodipine (AMD) and metoprolol (MET). The method relies on measuring the relative synchronous fluorescence intensity of both drugs at Δλ of 90 nm in acetate buffer solution at pH 5. The experimental parameters influencing the developed method were investigated and optimized. The method was linear over the ranges 0.2–2 μg/ml and 0.5–10 μg/ml for AMD and MET, respectively. The limits of detection were 50 ng/ml for AMD and 130 ng/ml for MET while the limits of quantitation were 150 ng/ml for AMD and 390 ng/ml for MET. The developed method was applied successfully for the determination of the two drugs in their co‐formulated tablet. The mean percent recoveries were found to be 100.51 and 99.57 for AMD and MET, respectively.     

Enantiomeric separation of malathion and malaoxon and the chiral residue analysis in food and environmental matrix

Malathion is a widely used chiral phosphorus insecticide, which has a more toxic chiral metabolite malaoxon. In this work, the enantiomers of malathion and malaoxon were separated by high-performance liquid chromatography-mass/mass (HPLC-MS/MS) with chiral columns using acetonitrile/water or methanol/water as mobile phase, and the chromatographic conditions were optimized. Based on the chiral separation, the chiral residue analysis methods for the enantiomers in soil, fruit, and vegetables were set up. Two pairs of the enantiomers were better separated on CHIRALPAK IC chiral column, and baseline simultaneous separations of malathion and malaoxon enantiomers were achieved with acetonitrile/water (40/60, v/v) as mobile phase at a flow rate of 0.5 mL/min. The elution orders were −/+ for both malathion and malaoxon measured by an optical rotation detector. The chiral residue analysis in soil, fruit, and vegetables was validated by linearity, recovery, precision, limit of detection (LOD), and limit of quantification (LOQ). The LODs and LOQs for the enantiomers of malathion were 1 μg/kg and 3–5 μg/kg and 0.08 μg/kg and 0.20–0.25 μg/kg for malaoxon enantiomers. Good linear calibration curves for each enantiomer in the matrices were obtained within the concentration range of 0.02–12 mg/L. The mean recoveries of the enantiomers of malathion and malaoxon ranged from 82.26% to 109.04%, with RSDs of 0.71–8.63%.The results confirmed that this method was capable of simultaneously determining the residue of malathion and malaoxon in food and environmental matrix on an enantiomeric level.     

Generation of a mutagenized organophosphorus hydrolase for the biodegradation of the organophosphate pesticides malathion and demeton‐S

Aims: The bacterial organophosphorus hydrolase (OPH) enzyme hydrolyses and detoxifies a broad range of toxic organophosphate pesticides and warfare nerve agents by cleaving the various phosphorus‐ester bonds (P–O, P–F, P–CN, P–S); however, OPH hydrolyses these bonds with varying efficiencies. The aim of this study was to generate a variant OPH enzyme with improved hydrolytic efficiency against the poorly hydrolysed P–S class of organophosphates. Methods and Results: The gene encoding OPH was sequentially mutated at specific codons by saturation mutagenesis and screened for improved activity against the P–S substrates demeton‐S methyl and malathion. Escherichia coli lysates harbouring the variants displayed up to 177‐ and 1800‐fold improvement in specific activity against demeton‐S methyl and malathion, respectively, compared to the wild‐type lysates. The specificity constants of the purified variant proteins were improved up to 25‐fold for demeton‐S methyl and malathion compared to the wild‐type. Activity was associated with organophosphate detoxification as the hydrolysed substrate lost the ability to inhibit acetylcholinesterase. The improved hydrolytic efficiency against demeton‐S translated to the improved ability to hydrolyse the warfare agent VX. Conclusions: OPH variant enzymes were generated that displayed significantly improved ability to hydrolyse and detoxify organophosphates harbouring the P–S bond. Significance and Impact of the Study: The long‐term goal is to generate an environmentally‐friendly enzyme‐mediated bioremediation approach for the removal of toxic organophosphate compounds in the environment.     

Malathion detoxification by human hepatic carboxylesterases and its inhibition by isomalathion and other pesticides

The organophosphorothioate (OPT) pesticide malathion (MAL) in mammals is readily hydrolyzed by mammalian carboxylesterases (CE). The reaction competes with the CYP‐catalyzed formation of malaoxon (MOX), the toxic metabolite. Alterations or individual variations in CE activity may result in increased MOX formation, enhancing MAL toxicity. We have characterized the human hepatic CE activity in a panel of 18 human liver microsomes as well as the inhibitory effect of IsoMAL, a major impurity of MAL commercial formulations, parathion (PAR), chlorpyrifos (CPF), and chlorpyrifos‐oxon (CPFO). CE activity showed a low level of variation among individuals (4‐fold). The reaction consists of two different phases, differing in their affinity for MAL (K_m1 = 0.25–0.69 μM; K_m2 = 10.3–26.8 μM). The relatively low K_m1 values confirmed that human CE efficiently detoxify MAL. IsoMAL was shown to be a potent noncompetitive inhibitor of MAL detoxification (K_i = 0.6 μM), with a higher inhibitory potency than CPF and PAR (K_i = 7.5 μM and 50 μM, respectively). These two latter compounds very likely act as mixed inhibitors. CPFO showed the highest inhibitory potency toward CE‐mediated detoxification, being characterized by a K_i = 22 nM. The present results provide useful information for a better understanding of possible interactions between different OPTs and for assessing the potential cumulative risk for exposure to OPT mixtures. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:406–414, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20106     

Accurate quantitation of standard peptides used for quantitative proteomics

MS‐based proteomics has become an indispensable tool in system biology generating a need for accurate and precise quantitation of peptide standards. The presented method utilizes ultra performance LC‐MS/MS (UPLC‐MS/MS) to accurately quantify peptide standards at concentrations of 0.1–10 μM. The ability for accurate quantitation of micro‐molar concentrations has the advantages that quantitation can be performed routinely with high precision and the high sensitivity of the method minimizes the amounts required.     

Interaction of glucose‐derived carbon quantum dots with silver and gold nanoparticles and its application for the fluorescence detection of 6‐thioguanine

The interaction of glucose‐derived carbon quantum dots (CQDs) with silver (Ag) and gold (Au) nanoparticles (NPs) was explored by fluorescence spectroscopy. Both metal NPs cause an efficient quenching of CQD fluorescence, which is likely due to the energy transfer process between CQDs as donors and metal NPs as acceptors. The Stern–Volmer plots were evaluated and corresponding quenching constants were found to be 1.9 × 10¹⁰ and 2.2 × 10⁸ M^?1 for AgNPs and AuNPs, respectively. The analytical applicability of these systems was demonstrated for turn‐on fluorescence detection of the anti‐cancer drug, 6‐thioguanine. Because the CQD–AgNP system had much higher sensitivity than the CQD–AuNP system, we used it as a selective fluorescence probe in a turn‐on assay of 6‐thioguanine. Under optimum conditions, the calibration graph was linear from 0.03 to 1.0 μM with a detection limit of 0.01 μM. The developed method was applied to the analysis of human plasma samples with satisfactory results.     

Targeted On‐line SPE‐LC‐MS/MS Assay for the Quantitation of 12 Apolipoproteins from Human Blood

Laborious sample pretreatment of biological samples represents the most limiting factor for the translation of targeted proteomics assays from research to clinical routine. An optimized method for the simultaneous quantitation of 12 major apolipoproteins (apos) combining on‐line SPE and fast LC‐MS/MS analysis in 6.5 min total run time was developed, reducing the manual sample pretreatment time of 3 μL serum or plasma by 60%. Within‐run and between‐day imprecisions below 10 and 15% (n = 10) and high recovery rates (94–131%) were obtained applying the high‐throughput setup. High‐quality porcine trypsin was used, which outperformed cost‐effective bovine trypsin regarding digestion efficiency. Comparisons with immunoassays and another LC‐MS/MS assay demonstrated good correlation (Pearson's R: 0.81–0.98). Further, requirements on sample quality concerning sampling, processing, and long‐term storage up to 1 year were investigated revealing significant influences of the applied sampling material and coagulant on quantitation results. Apo profiles of 1339 subjects of the LIFE‐Adult‐Study were associated with lifestyle and physiological parameters as well as establish parameters of lipid metabolism (e.g., triglycerides, cholesterol). Besides gender effects, most significant impact was seen regarding lipid‐lowering medication. In conclusion, this novel highly standardized, high‐throughput targeted proteomics assay utilizes a fast, simultaneous analysis of 12 apos from least sample amounts.     

Indirect electrochemiluminescence detection of lysine and histidine separated by capillary electrophoresis based on charge displacement

Indirect electrochemiluminescence (ECL) detection was applied for the analysis of lysine (Lys) and histidine (His) separated by capillary electrophoresis (CE). With the most effective electrophoretic buffer system, which contained 15 mM phosphate buffer (pH = 5.8) and 0.5 mM Tripropylamine (TPA), fast separation of the two basic amino acids could be performed within 7 min. The linear ranges were 10–35 μM, 35–150 μM for Lys; and 5–35 μM, 35–150 μM for His. The detection limits (S/N = 3) were 0.3 μM for Lys and 1.0 μM for His, respectively. The proposed method was also successfully used for the determination of Lys in the oral pharmaceutical formulations. Copyright © 2012 John Wiley & Sons, Ltd.     

In vitro screening of Fe2+‐chelating effect by a Fenton's reaction–luminol chemiluminescence system

In vitro screening of a Fe²⁺‐chelating effect using a Fenton's reaction–luminol chemiluminescence (CL) system is described. The luminescence between the reactive oxygen species generated by the Fenton's reaction and luminol was decreased on capturing Fe²⁺ using a chelator. The proposed method can prevent the consumption of expensive seed compounds (drug discovery candidates) owing to the high sensitivity of CL detection. Therefore, the assay could be performed using small volumes of sample solution (150 μL) at micromolar concentrations. After optimization of the screening conditions, the efficacies of conventional chelators such as ethylenediaminetetraacetic acid (EDTA), diethylentriaminepentaacetic acid (DETAPAC), deferoxamine, deferiprone and 1,10‐phenanthroline were examined. EC₅₀ values for these compounds (except 1,10‐phenanthroline) were in the range 3.20 ± 0.87 to 9.57 ± 0.64 μM (n = 3). Rapid measurement of the Fe²⁺‐chelating effect with an assay run time of a few minutes could be achieved using the proposed method. In addition, the specificity of the method was discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Enantiopurity Determination of the Enantiomers of the Triple Reuptake Inhibitor Indatraline

The present study describes the development of two approaches for the determination of the enantiopurity of both enantiomers of indatraline. Initially, a method was developed using different chiral solvating agents (CSAs) for diastereomeric discrimination regarding signal separation in ¹H nuclear magnetic resonance (NMR) spectroscopy, revealing MTPA as a promising choice for the differentiation of the indatraline enantiomers. This CSA was also tested for its ideal molar ratio, temperature, and solvent. Optimized conditions could be achieved that made determination of enantiopurity for (1R,3S)‐indatraline up to 98.9% enantiomeric excess (ee) possible. To quantify even higher enantiopurities, a high‐performance liquid chromatography (HPLC) method based on a modified β‐cyclodextrine phase was established. The influence of buffer type, concentration, pH value, percentage and kind of organic modifier, temperature, injection volume as well as sample solvent on chromatographic parameters was investigated. Afterwards, the reliability of the established HPLC method was demonstrated by validation according to the ICH guideline Q2(R1) regarding specificity, accuracy, precision, linearity, and quantitation limit. The developed method proved to be strictly linear within a concentration range of 1.25–1000 μM for the (1R,3S)‐enantiomer and 1.25‐750 μM for its mirror image that enables a reliable determination of enantiopurities up to 99.75% ee for the (1R,3S)‐enantiomer and up to 99.67% ee for the (1S,3R)‐enantiomer. Chirality 25:923–933, 2013. © 2013 Wiley Periodicals, Inc.     

The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Vibrio parahaemolyticus

Aims: The current study was aimed to develop a loop‐mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay for rapid and specific detection of Vibrio parahaemolyticus. Methods and Results: Biotinylated LAMP amplicons were produced by a set of four designed primers that recognized specifically the V. parahaemolyticus thermolabile haemolysin (tlh) gene followed by hybridization with an FITC‐labelled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP–LFD method accurately identified 28 isolates of V. parahaemolyticus but did not detect 24 non‐parahaemolyticus Vibrio isolates and 35 non‐Vibrio bacterial isolates. The sensitivity of LAMP–LFD for V. parahaemolyticus detection in pure cultures was 120 CFU ml^?1. In the case of spiked shrimp samples without enrichment, the detection limit for V. parahaemolyticus was 1·8 × 10³ CFU g^?1 or equivalent to 3 CFU per reaction while that of conventional PCR was 30 CFU per reaction. Conclusions: The established LAMP–LFD assay targeting tlh gene was specific, rapid and sensitive for identification of V. parahaemolyticus. Significance and Impact of the Study: The developed LAMP–LFD assay provided a valuable tool for detection of V. parahaemolyticus and can be used effectively for identification of V. parahaemolyticus in contaminated food sample.     

Detection and enumeration of Dekkera anomala in beer, cola, and cider using real-time PCR

Aims: In this article, a quantitative real‐time PCR assay for detection and enumeration of the spoilage yeast Dekkera anomala in beer, cola, apple cider, and brewing wort is presented as an improvement upon existing detection methods, which are very time‐consuming and not always accurate. Methods and Results: Primers were designed to exclude other organisms common in these beverages, and the assay was linear over 6 log units of cell concentrations. The addition of large amounts of non‐target yeast DNA did not affect the efficiency of this assay. A standard curve of known DNA was established by plotting the C_t values obtained from the QPCR against the log of plate counts on yeast peptone dextrose medium and unknowns showed exceptional correlation when tested against this standard curve. The assay was found to detect D. anomala at levels of 10–14 CFU ml^?1 in either cola or beer and at levels of 9·4–25·0 CFU ml^?1 in apple cider. The assay was also used to follow the growth of D. anomala in brewing wort. Conclusions: The results indicate that real‐time PCR is an effective tool for rapid, accurate detection and quantitation of D. anomala in beer, cola and apple cider. Significance and Impact of the Study: This method gives a faster and more efficient technique to screen beer, cola, and cider samples and reduce spoilage by D. anomala. Faster screening may allow for significant reduction in economic loss because of reduced spoilage.     

A novel colorimetric assay of β‐D‐glucans in basidiomycete strains by alcian blue dye in a 96‐well microtiter plate

Basidiomycete strains synthesize several types of β‐d ‐glucans, which play a major role in the medicinal properties of mushrooms. Therefore, the specific quantification of these β‐d ‐glucans in mushroom strains is of great biochemical importance. Because published assay methods for these β‐d ‐glucans present some disadvantages, a novel colorimetric assay method for β‐d ‐glucan with alcian blue dye was developed. The complex formation was detected by following the decrease in absorbance in the range of 620 nm and by hypsochromic shift from 620 to 606 nm (～14 nm) in UV‐Vis spectrophotometer. Analysis of variance was used for optimization of the slope of the calibration curve by using the assay mixture containing 0.017% (w/v) alcian blue in 2% (v/v) acetic acid at pH 3.0. The high‐throughput colorimetric assay method on microtiter plates was used for quantification of β‐d ‐glucans in the range of 0–0.8 μg, with a slope of 44.15 × 10^?2 and a limit of detection of 0.017 μg/well. Recovery experiments were carried out by using a sample of Hericium erinaceus, which exhibited a recovery of 95.8% for β‐1,3‐d ‐glucan. The present assay method exhibited a 10‐fold higher sensitivity and a 59‐fold lower limit of detection compared with the published method with congo red. β‐d ‐glucans of several mushrooms strains were isolated from fruiting bodies and mycelia, and they were quantified by this assay method. This assay method is fast, specific, simple, and it can be used to quantify β‐d ‐glucans from other biological sources. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1526–1535, 2015     

Quantification of Lawsonia intracellularis in porcine faeces by real‐time PCR

Aim: To develop and to validate a method for the quantification of Lawsonia intracellularis in porcine faeces by real‐time PCR. Methods and Results: A real‐time PCR including a calibrator based on plasmid DNA for quantification by means of ΔΔCt method was evaluated. The parameters specificity, detection limit, quantification limit, linearity, range, repeatability, precision and recovery were validated. The detection limit of the agent was 1 copy per reaction, and quantification was reliable between 10¹ and 10⁷ copies per μl reaction volume. The linearity calculated by logistic regression revealed a slope of ?3·329 reflecting an efficiency of 99·7% for the assay. Moreover, it was shown that storage of samples and repetition of tests including DNA isolation by same or other investigators did not influence the outcome. Conclusion: The quantification method described herein revealed consistent results for the quantitation of L. intracellularis in porcine faeces samples. Significance and Impact of the Study: In contrast to common PCR in combination with gel electrophoresis, this validated quantification method based on real‐time PCR enhances a reliable quantification and is even more sensitive.     

Micellar‐based spectrofluorimetric method for the selective determination of ledipasvir in the presence of sofosbuvir: application to dosage forms and human plasma

A fast, low‐cost, sensitive, and selective spectrofluorimetric method for the determination of ledipasvir was developed and validated. The method is based on an enhancement in the native fluorescence intensity of ledipasvir by 500% of its original value by the formation of hydrogen bonds between the cited drug and Tween‐20 in the micellar system (pH = 5.0). All fluorescence measurements were carried out at 425 nm and 340 nm for emission and excitation wavelengths, respectively. A linear relationship between the concentration of ledipasvir and the observed fluorescence intensity was achieved in the range of 0.1–2.0 μg ml^?1 with 0.028, 0.084 μg ml^?1, for detection and quantitation limits, respectively. The acquired selectivity and sensitivity using the proposed method facilitate the analysis of ledipasvir in spiked human plasma with sufficient percentage recovery (95.36–99.30%). The proposed method was developed and validated according to International Council for Harmonisation (ICH) guidelines. Moreover, the cited drug was successfully determined in its pharmaceutical dosage form using the proposed method. In addition, the validity of the proposed results was statistically confirmed using Student's t‐test, variance ratio F‐test, and interval hypothesis test.     

Sublethal effects of malathion on boll weevil (Coleoptera: Curculionidae) fecundity when maintained on cotton squares or artificial diet

Mated 3‐day‐old female boll weevils, Anthonomus grandis grandis Boheman, reared from field‐infested cotton (Gossypium hirsutum L.) squares were topically treated with an estimated LD₅₀ of malathion (2 μg) to assess its effects on fecundity, oviposition, and body fat condition. Two different food sources, cotton squares and artificial diet, were assessed in malathion‐treated and nontreated (control) weevils. The LD₅₀ caused ～50% mortality in the square‐fed malathion treatment, but the artificial diet‐fed malathion‐treated weevils were less susceptible. LD₅₀ survivors fed on the squares produced ≥ 9 times more chorionated eggs in the ovaries and oviposited ≥ 19‐fold more than survivors fed artificial diet, regardless of the malathion treatment. Boll weevils that survived a 2 μg LD₅₀ malathion and also fed squares were ～4.5‐fold leaner than diet‐fed weevils. Our findings demonstrate that non‐resistant boll weevils surviving a sublethal dose of malathion will reproduce without any delay or significant loss in fecundity, and the food source for which boll weevils are maintained when conducting these assays will directly affect the results. The significance of these findings and how they are related to the final stages of eradicating the boll weevil from the US are discussed.     

A combination of ultrasonic-assisted extraction with RRLC-QQQ method for the determination of artemisinin in the Chinese herb Artemisia annua L

Introduction – Artemisinin, the primary active ingredient of the Chinese herb Artemisia annua L., is known to have considerable anti‐malaria properties. However, rapid, sensitive and selective method for the determination of artemisinin in it is not currently available. Objective – To develop and validate an efficient method for extraction and analysis of artemisinin from the plant samples of Artemisia annua L. by rapid resolution liquid chromatography triple quadrupole mass spectrometry (RRLC‐QQQ). Methodology – Following ultrasound‐assisted extraction (USE), RRLC‐QQQ was utilised to separate and determine artemisinin from the plant sample of Artemisia annua L. The LC separation, QQQ‐MS detection and multiple reaction monitoring (MRM) mode were optimised, and the method validation concluding selectivity, calibration, accuracy and precision, and recovery were also evaluated. Results – LC separation was performed with an isocratic elution of 20% of methanol–water (10 mmol/L ammonium acetate, pH 4.0) on a C₁₈ column. The triple quadrupole MS detection was carried out under MRM mode of precursor ion [M + H]⁺ → fragment ions m/z 265.1 and m/z 247.2. The limits of detection and quantitation of artemisinin were 0.20 and 0.75 ng/mL, respectively. The intra‐ and inter‐day precisions did not exceed 3.71%, and the deviation of the intra‐ and inter‐day mean values did not exceed ±7.50. The average recoveries for artemisinin ranged from 92.45 to 103.8% with an RSD from 2.47 to 2.79%. Conclusion – The developed RRLC‐QQQ assay is an efficient method for separation and determination of artemisinin from the plant samples of Artemisia annua L. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous determination of reduced and oxidized glutathione in peripheral blood mononuclear cells by liquid chromatography–electrospray mass spectrometry

We developed a sensitive and specific liquid chromatography–electrospray mass spectrometric (HPLC–ESI-MS) assay for the simultaneous determination of reduced and oxidized glutathione (GSH and GSSG) in peripheral blood mononuclear cells (PBMC). Following derivatization with N-ethylmaleimide to prevent GSH auto-oxidation, addition of thiosalicylic acid as internal standard, and protein precipitation with cold acetonitrile, the samples were injected into a diol column, eluted with acetonitrile–1% aqueous acetic acid (25:75) and detected by the ESI-MS system. The optimized method exhibited a good detection limit for both analytes (0.01 and 0.05 μM for GSH and GSSG, respectively). Good linearity was reached in the 0.01–20 μM range for GSH and 0.05–20 μM for GSSG. The mean recoveries of GSH and GSSG were 98.5–100.6% and 105.8–111.5%, respectively. The run-to-run repeatability for retention time and peak area was RSD% 0.06 and 1.75 for GSH and 0.18 and 2.50 for GSSG. The optimized method was applied to GSH and GSSG assay in PBMC analyzing 20 healthy individuals.     

Application of Exchangeable Biochemical Reactors with Oxidase‐Catalase‐Co‐immobilizates and Immobilized Microorganisms in a Microfluidic Chip‐Calorimeter

Several methods for the quantitative detection of different compounds, e.g., L‐amino acids, sugars or alcohols in liquid media were developed by application of an automatic measuring unit including a fluid chip‐calorimeter FCC‐21. For this purpose, enzymes were immobilized covalently on the inner and outer surface of CPG (controlled porous glass)‐spherules with an outer diameter of 100 μm and filled into a micro flow‐through reaction chamber (V_R = 20 μL). The design of the measuring cell allows for easy insertion into the calorimeter device of a stored series of comfortably pre‐fabricated measuring cells. These cells can be filled with different enzyme immobilizates. Different oxidases were used and co‐immobilized with catalase for the improvement of the detection sensitivity. A signal amplification could be achieved up to a factor of 3.5 with this configuration. β‐D‐glucose, ethanol and L‐lysine could be detected in a range of 0.25–1.75 mM using glucose oxidase, alcohol oxidase and lysine oxidase. The group of oxidases in combination with the enzymatic catalysis of the intermediate H₂O₂ allows the quantitative detection of a large number of analytes. A good measurement and storage stability could be achieved for several weeks by this immobilization method. In addition to enzyme‐based detection reactions, it was shown that living microorganisms can be immobilized in the reaction chamber. Thus, the system can be used as a whole‐cell biosensor. The quantitative detection of phenol in the range of 10–100 μM could be performed using the actinomycete Rhodococcus sp. immobilized on glass beads by means of embedding into polymers.