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Use of biosensors for rapid drug residue analysis without sample deconjugation or clean-up: a possible way forward.

The drug salbutamol (SBL) is a beta-agonist that may be used illegally as an animal growth promoter. SBL is also a good example of a drug which is excreted in the form of glucuronides and sulfates. Such metabolites cause complexities in analysing for the presence of drug residues. In the majority of cases a process of deconjugation and sample clean-up is required prior to analysis. This is both time consuming and causes some loss of accuracy. In this study, the urine of calves treated with SBL orally for 3 d was collected during and after medication. Samples were assayed before and after hydrolysis by two different methods, radioimmunoassay (RIA) and a newly developed biosensor immunoassay (BIA). Some samples were also analysed by GC-MS. The results clearly showed that both screening assays (RIA and BIA) found high concentrations of SBL residues throughout the study. This was especially true in the BIA method. It was also demonstrated that urine sample analysis without the need for deconjugation or clean-up could be achieved. Results obtained by GC-MS tended to be an order of magnitude lower than the corresponding screening test results. This work showed that biosensor based veterinary drug residue testing procedures can be developed which can generate results in real time without the need for time consuming sample preparation.

Adrenergic beta-Agonists↗

Rapid determination of lactulose in milk by microdialysis and biosensors.

A simple and rapid flow system for the determination of lactulose in milk samples was developed. It is based on the hydrolysis of lactulose to galactose and fructose by the enzyme beta-galactosidase immobilised in a reactor. The amount of fructose produced was measured with an electrochemical biosensor based on the fructose dehydrogenase enzyme, K3[Fe(CN)6] as mediator and a platinum based electrochemical transducer. Parameters such as the enzyme immobilisation in the reactor and under the electrode surface, the lifetime of the beta-galactosidase reactor and of the dehydrogenase biosensor and the flow parameters were studied and optimised. Fructose was determined in the range 1 x 10(-6)-5 x 10(-3) mol l-1 with an RSD of about 2% and a detection limit of 5 x 10(-7) mol l-1. The use of a microdialysis probe as the sampling system permitted the direct measurement of lactulose in milk samples without pre-treatment in the range 1 x 10(-5)-5 x 10(-3) mol l-1. The sensitivity of the procedure allowed pasteurised, UHT and in-container sterilised milk to be distinguished.

Animals↗

Comparison of formats for the development of fiber-optic biosensors utilizing sol-gel derived materials entrapping fluorescently-labelled protein.

The development of fiber-optic biosensors requires that a biorecognition element and a fluorescent reporter group be immobilized at or near the surface of an optical element such as a planar waveguide or optical fiber. In this study, we examined a model biorecognition element-reporter group couple consisting of human serum albumin that was site-selectively labelled at Cys 34 with iodoacetoxy-nitrobenzoxadiazole (HSA-NBD). The labelled protein was encapsulated into sol-gel derived materials that were prepared either as monoliths, as beads that were formed at the distal tip of a fused silica optical fiber, or as thin films that were dipcast along the length of a glass slide or optical fiber. For fiber-based studies, the entrapped protein was excited using a helium-cadmium laser that was launched into a single optical fiber, and emission was separated from the incident radiation using a perforated mirror beam-splitter, and detected using a monochromator-photomultiplier tube assembly. Changes in fluorescence intensity were generated by denaturant-induced conformational changes in the protein or by iodide quenching. The analytical parameters of merit for the different encapsulation formats, including minimum protein loading level, response time and limit-of-detection, were examined, as were factors such as protein accessibility, leaching and photobleaching. Overall, the results indicated that both beads and films were suitable for biosensor development. In both formats, a substantial fraction of the entrapped protein remained accessible, and the entrapped protein retained a large degree of conformational flexibility. Thin films showed the most rapid response times, and provided good detection limits for a model analyte. However, the entrapment of proteins into beads at the distal tip of fibers provided better signal-to-noise and signal-to-background ratios, and required less protein for preparation. Hence, beads appear to be the most viable method for interfacing of proteins to optical fibers.

Animals↗

Novel, reagentless, amperometric biosensor for uric acid based on a chemically modified screen-printed carbon electrode coated with cellulose acetate and uricase.

Amperometry in stirred solution has been used for the systematic evaluation of modified screen-printed carbon electrodes (SPCEs) with a view to developing a reagentless biosensor for uric acid. The developed system consists of a base cobalt phthalocyanine (CoPC) electrode tailored to the electrocatalytic oxidation of H2O2 by means of a cellulose acetate (CA)-uricase bilayer. Uricase was immobilized by drop-coating the enzyme onto the CA membrane covering the CoPC-SPCE. The device exploits the near-universal H2O2-generating propensity of oxidases, the permselectivity of the CA film towards H2O2 and the electrocatalytic oxidation of this product at the CoPC-SPCE. The electrochemical oxidation of the resulting Co+ species was used as the analytical signal, facilitating the application of a greatly reduced operating potential when compared with that required for direct oxidation of H2O2 at unmodified electrodes. The time required to achieve 95% of the steady-state current (t95i(ss)) was 44 s [relative standard deviation = 7.5% (n = 10)]. Amperometric calibrations were linear over the range from 13 x 10(-6) to 1 x 10(-3) mol dm-3, with the former representing the limit of detection. The CA membrane extended the linear range of the biosensor by over two orders of magnitude, when apparent Michaelis-Menten constants (Km') of immobilized and free enzymes are compared. This suggests that the process is diffusion-controlled and not governed by the kinetics of the enzyme. The precision of electrode fabrication was determined by cyclic voltammetry to be 4.9% (n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Biosensor assay of sulfadiazine and sulfamethazine residues in pork.

Biosensor-based immunochemical screening assays for the detection of sulfadiazine (SDZ) and sulfamethazine (SMT) in muscle extract from pigs were developed. Samples were extracted with aqueous buffer and then centrifuged. This simple and straightforward preparation allowed up to 40 samples to be processed and analysed in 1 d. The limits of detection for the assays were found to be 5.6 ng g-1 for SDZ and 7.4 ng g-1 for SMT. These figures were well below the European and US legal limits for sulfonamides (100 ng g-1). The precision (RSD) between runs was < 8% and the recovery was between 91 and 98%. The validation proved the assays to be accurate and the analysis of routine field samples showed good correlation with an established TLC screening procedure. No false negative or positive results were obtained with blank and spiked samples. The influence of cross-reacting metabolites on immunoassays was demonstrated by testing incurred tissue samples, collected from sulfonamide treated pigs after only a short withdrawal period. The quantitative results obtained by biosensor analysis were a combination of parent sulfonamide plus N4-acetyl metabolite while the HPLC method used for confirmatory analysis detected only the parent sulfonamide. This gave rise to some false positive results and highlighted the need to use real samples in evaluating any assay thoroughly. False negative results were not obtained.

Animals↗

Cyclodextrin-modified biosensors: comparision of cyclodextrin-linked ferrocenes as mediators in sol-gel and screen-printed formats for sensing acetylcholine.

This work reports the comparison of a sol-gel and a screen-printed biosensor format using new mediators and a sensitive thin-film (ref. 1: P. Kataky and D. Parker, Analyst, 1996, 121, 1829) to enhance the sensitivity and stability of biosensors. The new mediators were per-alkylated cyclodextrin linked ferrocenes and a control, ferrocene aminocarboxylic acid. The thin film comprised a cocktail with polyurethane, plasticiser, lipophilic anion and a perethylated beta-CD. The analyte targeted was acetylcholine using the established horseradish peroxidase-choline-oxidase-acetylcholine esterase relay. The screen-printed electrode format showed a marked decrease in oxidation potential, the magnitude of the shift depended on the structure of the mediator and the membrane covering. Although lower oxidation-potentials were observed with the sol-gel format sensors, their response was more akin to aqueous solution behaviour. Electrocatalytic currents were observed suggesting a highly efficient electron transfer process.

Journal Article↗

The study of nafion/xanthine oxidase/au colloid chemically modified biosensor and its application in the determination of hypoxanthine in myocardial cells in vivo.

A novel hypoxanthine (Hx) microsensor was constructed. In this work, Nafion xanthine oxidase (XOD) and Au colloid were immobilized onto the surface of a Pt microelectrode. The enzyme biosensor displayed a quick and sensitive response to Hx. Under physiological conditions, a low detection limit, with high selectivity and sensitivity for Hx determination were obtained. The oxidation current [investigated using current-time (I-t) plots] was linear with Hx concentration ranging from 2.0 x 10(-7) to 2.0 x 10(-5) mol L(-1) with a calculated detection limit of 1.0 x 10(-7) mol L(-1) (S/N of 3). The biosensor should be promising for in vivo measurement of Hx without interferences and fouling. The change of Hx concentration in cardiac myocytes stimulated by L-arginine (L-Arg) and acetylcholine (Ach) was also studied.

Biosensing Techniques↗

Evaluation of interactive toxicity of chlorophenols in water and soil using lux-marked biosensors.

An assessment of the toxicity of three chlorophenol compounds (2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP) and pentachlorophenol (PCP)), individually and in combinations, was made in deionised water, soil extracts and soils using the lux-marked microbial biosensors: Escherichia coli HB101 pUCD607 and Pseudomonas fluorescens 10586r pUCD607. These biosensors responded to the bioavailable fraction of pollutants enabling a rapid and ecologically relevant toxicity test. Toxicity interaction responses of pollutant mixtures were predicted after individual compounds were assessed. Synergistic interactions were observed in the response of P. fluorescens to all combinations of chlorophenols tested, while the toxicity response of E. coli varied with the matrices tested. Soil characteristics influenced the toxicity response when compared with aqueous solutions. These results highlight the significance of interactive factors and physicochemical parameters when evaluating toxicity. To develop an understanding of pollution derived hazard assessment in soils we need to integrate a wide spectrum of parameters.

Anthelmintics↗

A glucose dehydrogenase biosensor as an additional signal amplification step in an enzyme-flow immunoassay.

Both the antibody affinity and the detectability of the label are essential in deciding the final characteristics of a heterogeneous immunoassay. This paper describes an approach to obtain a supplementary enhancement of the signal generated by using an enzyme label, e.g., by including the product of the enzymatic reaction in an additional amplification cycle during the detection step performed with an amperometric biosensor based on glucose dehydrogenase (GDH). An immunoassay format with a labelled analyte derivative that competes with the analyte present in the sample for a limited amount of antibody binding sites was employed. The beta-galactosidase label hydrolyses the substrate aminophenyl-beta-galactopyranoside, and the generated aminophenol enters then into a bioelectrocatalytic amplification cycle at the GDH biosensor. The principle was applied for determination of 4-nitrophenol, with the best minimal concentration of 1.5 microM and a midpoint of the calibration of 24 microM. The potentials and limitations of such a system are discussed.

Biosensing Techniques↗

Effects of DNA mismatches on binding affinity and kinetics of polymerase-DNA complexes as revealed by surface plasmon resonance biosensor.

In this study, surface plasmon resonance (SPR) biosensor techniques were used to obtain quantitative information on the kinetics of the DNA and polymerase I (Klenow fragment) interaction. DNA duplexes containing different base compositions at the binding site were immobilized on the SPR sensor surface via biotin-streptavidin chemistry and the subsequent binding of the polymerase was measured in real time. Various kinetic models were tested and a translocation model was shown to provide the best fit for the binding and dissociation profiles. The results revealed that the enzyme binds to DNA at both the polymerase and the exonuclease domains with different association and dissociation rates as well as affinity constants, depending on the presence of mismatches near the primer 3'-end. Introduction of unpaired bases increases the DNA binding affinity towards the exonuclease domain and promotes the translocation of DNA from the polymerase site to the exonuclease site. The results also demonstrated that SPR biosensors may be used as a sensitive technique for studying molecular recognition events such as single-base discrimination involved in protein-DNA interaction.

Animals↗

An iridium oxide reference electrode for use in microfabricated biosensors and biochips.

In this paper we argue for the use of iridium oxide (IrO(x)) electrodes as quasi-reference electrodes in microfabricated biosensors and biochips that operate in buffered solutions. The simple microfabrication of these electrodes consists of a one-step electrodeposition of IrO(x) onto a microfabricated platinum (Pt) electrode. The IrO(x) electrode potential was found to vary less than 20 mV over 9 days after stabilization for 1 day in a phosphate-buffered saline (PBS) solution; this behavior of the electrode potential was found to be easily reproduced. Moreover, the electrode potential was found to vary by less than 15 mV in the initial hour of its use; this behavior of the electrode potential was also found to be reproducible. The performance of a microfabricated glucose sensor employing an IrO(x) reference electrode is characterized in this paper in order to evaluate the usefulness of this new IrO(x) electrode as a quasi-reference electrode. The glucose sensor consists of a recessed microfabricated Pt electrode array, an electrodeposited IrO(x) film, an inner layer composed of an electropolymerized poly(m-phenylenediamine)/glucose oxidase (PMPD/GOx) film, and an outer or protective layer composed of Teflon and polyurethane (PU) films. The response of this sensor was found to be equivalent to the response of the same sensor employing a commercial Ag/AgCl reference electrode. These results show that a microfabricated IrO(x) electrode can be used as a quasi-reference electrode in microfabricated biosensors and biochips operating in buffered solutions.

Biosensing Techniques↗

Novel fluorescent biosensor for pathogenic toxins using cyclic polypeptide conjugates.

This work describes a two-step conjugate synthesis of a new fluorescent analog of microcystin-LR and its subsequent utilization for the development of an optical biosensor for cyanobacteria toxins. The biosensor concept is based on the competitive binding between the native microcystin and its fluorescent analog at immobilized alkaline phosphatase enzymes.

Bacterial Toxins↗

Surface properties and electromagnetic excitation of a piezoelectric gallium phosphate biosensor.

The surface properties of GaPO4 have been studied by secondary ion mass spectrometry, X-ray photoelectron spectroscopy and electromagnetic acoustic wave excitation in order to explore the potential of this relatively new piezoelectric material as a biosensor. The X-ray photoelectron spectrum of the substrate shows a Ga-rich surface (Ga:P = 1.4), while the negative secondary ion mass spectrum is similar to that of other phosphates, with PO3- and PO2- being the main fragments derived from the substrate. Surface analysis reveals that the linker protein for biotinylated moieties, neutravidin, is both readily chemisorbed to bare gallium phosphate at pH 7.5 and attached to p-hydroxy benzaldehyde-treated devices, establishing the possibility to exploit the surface chemistry of the phosphate for the fabrication of an electrode-free acoustic wave biosensor. Preliminary results regarding the detection of the adsorption of neutravidin with an electromagnetic field-excited GaPO4 device incorporated in a FIA configuration showed comparable results with those obtained with a quartz-sensor equivalent. The frequency shift for the adsorbed protein layer at the device fundamental frequency was 200 Hz and the noise was routinely around 13 Hz. The possibility to use the electrodeless acoustic GaPO4 device at higher harmonics in the liquid phase has also been confirmed.

Biosensing Techniques↗

A smartphone-integrated plasmonic biosensor for amplification-free detection of African swine fever virus.

African Swine Fever Virus (ASFV) poses a catastrophic threat to global swine production, with recent outbreaks across Europe, Asia, and the Caribbean, significantly elevating the biosecurity risk to the United States' billion-dollar pork industry. Current diagnostic gold standards are laboratory-dependent and introduce critical delays in outbreak response. To address this gap, a plasmonic biosensor based on functionalized gold nanoparticles (GNPs) was developed for the rapid, amplification-free detection of ASFV. GNPs were surface-functionalized with 11-mercaptoundecanoic acid (MUDA) and combined in situ with ASFV-specific oligonucleotide probes targeting a conserved region of the p72 (B646L) gene. The detection mechanism relies on acid-induced aggregation: hybridization of target ASFV DNA to the probe generates a rigid duplex that shields the nanoparticles from acid-induced destabilization, maintaining a ruby-red color, whereas in the absence of target DNA the GNPs aggregate, producing a visible red-to-blue color shift. The optimized plasmonic biosensor demonstrated 100% analytical specificity, with no cross-reactivity against a panel of 19 non-target bacterial genomic DNA samples representative of the swine environment. Detection limits determined by the IUPAC 3&#x3c3; criterion were 285 copies per reaction for Probe 1 and 402 copies per reaction for Probe 2, within the same order of magnitude as the qPCR reference assay run on the same dilution series (approximately 312 copies per reaction) under the experimental conditions used here. A smartphone-based Bio-Analytics App employing an RGB color-conversion algorithm served as a quantitative reader, yielding signal-to-noise ratios (S/N) that strongly correlated with benchtop spectrophotometric readings (A520/A620 ratio, R2 = 0.96) and achieved diagnostic concordance with qPCR binary calls. This platform offers a robust and low-cost (&#x223c;$2 per test), amplification-free approach to ASFV screening with potential for point-of-need deployment, subject to future validation in clinical specimens.

Journal Article↗

An evaluation of hybridization kinetics in biosensors using a single-fractal analysis.

The diffusion-limited hybridization kinetics of analyte in solution to receptor immobilized on a biosensor or immunosensor surface is analysed within a fractal framework. The data may be analysed by a single-fractal analysis. This was indicated by the regression analysis provided by Sigmaplot [Sigmaplot Users Manual (1993) Jandel Scientific, San Rafael, CA]. It is of interest to note that the binding-rate coefficient and the fractal dimension both exhibit changes in the same and in the opposite directions for the single example presented in each case. The binding-rate coefficient(s) expressions developed as a function of the analyte (DNA) concentration in solution and the fractal dimension are of particular value, since they provide a means to better control biosensor or immunosensor performance and provide physical insights into the hybridization process.

Base Sequence↗

Electrochemical studies on polysorbate-20 (Tween 20)-entrapped haemoglobin and its application in a hydrogen peroxide biosensor.

Haemoglobin (Hb) was entrapped in polysorbate 20 and then modified on a pyrolytic graphite electrode. Electrochemical studies revealed that a pair of stable and well-defined redox peaks attributed to the direct redox reaction of Hb could be observed in a phosphate buffer solution (pH 6.0). The anodic and cathodic peaks were located at -236 and -316 mV (versus a saturated calomel reference electrode) separately. The formal potential, E0', was linearly varied with pH in the range from 3.0 to 10.0 with a slope of -48.0 mV.pH-1. Moreover, the protein was capable of catalysing the reduction of H2O2. Accordingly, an unmediated biosensor for H2O2 was prepared with a linear range from 8.0x10(-7) to 1.0x10(-3) M. This biosensor exhibited good stability, sensitivity and reproducibility.

Biosensing Techniques↗

Effects of mediators on the laccase biosensor response in paracetamol detection.

An enzyme electrode suitable for paracetamol detection was developed by immobilizing laccase on a dissolved-oxygen probe surface. The immobilization procedure was achieved by means of gelatin, which was then cross-linked with glutaraldehyde. The measurement was based on the detection of oxygen consumption in relation to analyte oxidation. The optimum experimental conditions for the biosensor were investigated and the system was calibrated for paracetamol. Also the effects of three different mediators, namely HBT (1-hydroxybenzotriazole), VLA [violuric acid (5-isonitrosobarbituric acid)] and TEMPO (2,2',6,6'-tetramethylpiperidine-N-oxyl radical) were tested for the biosensor's response. As a result, it was observed that HBT has a remarkable effect on the signal by providing more oxygen consumption during the enzymatic reaction. A linear relationship between sensor responses and analyte concentrations was obtained over the concentration range 2.0-15.0 microM, whereas, in the presence of the mediator HBT, this range became 0.5-3.0 microM.

Acetaminophen↗

Rapid antibody biosensor assays for environmental analysis.

Traditionally, biosensor development has focused on molecules with a defined metabolic role that can be exploited by enzyme-based systems. Antibodies have the ability to move beyond this range of analytes, and are particularly useful in detecting small, hapten molecules. Electrochemically based biosensor developments have been less fruitful in this regard, as enzyme labelling is required, and such assays require the separation from bound and unbound species. These separations and the removal of background signals result in the increased complexity of the assay format, making it unsuitable for rapid sensor analysis. We have developed an electrochemical sensor based on antibodies that does not require the separation of bound and unbound molecules in a competition immunoassay format. This removes the need for several washing and separation steps as is normally employed in this type of assay. This allows single-step immunoassays to be performed using this system, and also allows for the real-time monitoring of antibody-antigen interactions. We have shown that such assays are possible in both batch and flow-injection formats and we are currently developing an assay for the pesticide atrazine. Tentative results show that analysis with this system is possible in the p.p.m. to p.p.b. range.

Aniline Compounds↗