Immunoassay for rapid on-site detection of glyphosate herbicide.

Glyphosate is the most widespread herbicide and its global use is steadily increasing. Although glyphosate is considered to have low toxicity, its wide application has raised concerns about its effects on human health. The extensive use of glyphosate has risen a need of its continuous monitoring in drinking and surface waters to assure in accordance with the set standards. Within the present study, we have developed a novel assay for the on-site detection of glyphosate by combining flow-through technology with the high specificity of immunorecognition. The proposed biosensing system was based on the detection of fluorescence signal generated by the quantitative replacement of glyphosate in antigen-antibody complex with IgY-type anti-glyphosate antibodies on microbeads by synthetic 5-carboxytetramethylrhodamine (5-TAMRA) conjugated glyphosate. The working range of this assay was in low millimolar range and the time required for glyphosate detection around 0.5 h. The applicability of the immunoassay for glyphosate detection in surface water was tested and the biosensor results were validated with high-performance liquid chromatography.

Immunodetection of Streptococcus uberis pathogen in raw milk.

Streptococcus uberis is a major mastitis-causing environmental pathogen, which rapid immunodetection has not been possible due to the absence of specific anti-Str. uberis antibodies. Recently, a specific antibody against the Str. uberis adhesion molecule (SUAM) has been designed. In the present study, the specificity and affinity of this antibody towards SUAM antigenic region SAPVYLGVSTE and Str. uberis cells are characterized, using experimental and in silico bioinformatic methods. The selectivity studies and bioinformatic analyses revealed high specificity of the antibody towards Str. uberis. The Kd value of SAPVYLGVSTE/anti-Str. uberis antibody complex was 27 ±â€¯6 nM, indicating the applicability of this antibody for the detection of Str. uberis. The anti-Str. uberis antibody was used as a specific biorecognition element of a biosensor for the detection of Str. uberis bacteria in phosphate buffer and in milk and these analyses took less than 20 min. The Str. uberis biosensor was also tested in the milk of cows suffering from mastitis and the obtained results were in good agreement with the conventional identification of this pathogen by microbiological plating.

Design and production of antibodies for the detection of Streptococcus uberis.

Streptococcus uberis (S. uberis) is an important environmental pathogen causing mastitis in dairy cattle. Mastitis or mammary gland infection is the most common disease in milking cows and cause significant economic burden to farmers due to the reduction of the amount of milk and its quality. The development of rapid analytical methods for the determination of mastitis-causing pathogens in milk is of utmost importance for the early identification of the causes of mastitis and the beginning of timely treatment of cows. Combining in silico bioinformatic analysis and solid phase peptide synthesis using Fmoc chemistry, we have made two different peptides to mimic the adhesion protein of S. uberis, which is promoting the attachment of bacteria to epithelial cells. After purification with RP-HPLC, the peptides were conjugated with a larger carrier protein (KLH) and used for immunization of rabbits to produce specific antibodies. The separation of anti-S. uberis antibodies from rabbit blood antisera was carried out with affinity chromatography, using the synthetic peptides as affinity ligands. The purified antibodies showed high affinity and specificity towards S. uberis and were used for rapid bio-recognition and identification of S. uberis with an immunobiosensing system.

Determination of antibiotic residues and their interaction in milk with lactate biosensor.

Milk and dairy products are among the most important foodstuffs and the quality of raw milk is of significant importance from the point of view of human health. For rapid determination of chloramphenicol and penicillin residues in raw milk, lactate oxidase-based amperometric biosensor was used. The concentration of antibiotic residuals was determined by two characteristic reaction parameters, calculated from the biosensor transient response with the dynamic biosensor model. Both chloramphenicol and penicillin caused the decrease of the value of the kinetic parameter, but they changed the total signal change parameter in different ways. The shift of the combined total signal change parameter at the simultaneous presence of these antibiotics indicated their antagonistic effect. Due to the respiration process of bacteria in raw milk, the dynamics of the biosensor signal was different in warm and cold seasons. The respiration characteristics were added to the biosensor model as a negative linear time-depending factor. The reaction characteristic parameters, obtained with this complemented model, showed excellent alignment in different conditions and allowed to detect antibiotic residues and their interaction in raw milk.

Effect of spermidine and its metabolites on the activity of pea seedlings diamine oxidase and the problems of biosensing of biogenic amines with this enzyme.

Spermidine is one of the several biogenic amines, produced during the microbial decarboxylation of proteins. Individual biogenic amines in the formed mixtures are frequently analyzed with oxygen sensor based biosensors, as their content serves as a good biomarker for the determination of food quality. In these biosensors, diamine oxidase from pea seedlings (PSAO), catalyzing the oxidation of various biogenic amines by dissolved oxygen is commonly used for the bio-recognition of amines. However, in the presence of spermidine and/or its metabolite 1,3-diaminopropane, the activity of PSAO and the sensitivity of PSAO-based biosensors decrease due to inhibition. The inhibition constant of soluble spermidine, acting as an inhibiting substrate toward PSAO, was found to be (40±15) mM in freshly prepared solution and (0.28±0.05) mM in solution, incubated 30 days at room temperature. The inhibition constant of 1,3-diaminopropane, acting as a competitive inhibitor, was (0.43±0.12) mM as determined through the oxidation reaction of cadaverine. The metabolic half-life of soluble spermidine was 7 days at room temperature and 186 days at 4 °C. The kinetic measurements were carried out with an oxygen sensor; the composition of the solution of degraded spermidine was analyzed with MS.

Analyzing the biosensor signal in flows: studies with glucose optrodes.

Responses of enzymatic bio-optrodes in flow regime were studied and an original model was proposed with the aim of establishing a reliable method for a quick determination of biosensor signal parameters, applicable for biosensor calibration. A dual-optrode glucose biosensor, comprising of a glucose bio-optrode and a reference oxygen optrode, both placed into identical flow channels, was developed and used as a model system. The signal parameters of this biosensor at different substrate concentrations were not dependent on the speed of the probe flow and could be determined from the initial part of the biosensor transient phase signal, providing a valuable tool for rapid analysis. In addition, the model helped to design the biosensor system with reduced impact of enzyme inactivation to the system stability (20% decrease of the enzyme activity lead to only a 1% decrease of the slope of the calibration curve) and hence significantly prolong the effective lifetime of bio-optrodes.

Dynamic model of amperometric biosensors. Characterisation of glucose biosensor output.

An integrated model for the characterisation of the output signal course of oxidase-bound amperometric biosensors is presented and evaluated in the case of glucose biosensors. This model integrates two earlier proposed models, the model of oxygen transducer-based biosensors, allowing the prediction of steady state parameters from the transient response and the dynamic signal lag model, characterising the electrochemical diffusion-limited sensors. The integrated model allows the characterisation of the whole biosensor signal output, originating from the output curve itself with errors less than 3% and no need to determine the system's geometrical, diffusion and partition parameters.

Calibration of glucose biosensors by using pre-steady state kinetic data.

A new method for biosensor calibration and data processing, allowing the prediction of steady state parameters from the analysis of transient response curves (Rinken et al., 1996. Analytical Letters 29, 859), has been evaluated in the case of an oxygen sensor based two-substrate enzyme electrode for glucose determination. The electrochemical glucose biosensor was prepared by covering the surface of oxygen sensor with glucose oxidase (EC 1.1.3.4) immobilized in nylon mesh. This decreased the oxygen flow to the sensor in the presence of glucose and resulted in time-dependent decrease of the biosensor signal. Except the lag period of the response in the beginning of the assay, the oxygen consumption by the immobilized enzyme was described by an exponential function: [formula: see text] The parameter C, which corresponded to the steady-state output of the biosensor, was found to be the most suitable for glucose determination. The non-linear fitting for data of over 1000 independent experiments to the equation above always revealed correlation coefficients greater than 0.97. The calculation of the steady state parameter from the transient phase data makes the analysis fast and precise, especially for sensors with thick membranes, being convenient to use in the case of enzyme electrodes. The theoretical essence of the parameter C also gives valuable information for the optimal design of biosensors.