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The development of polyclonal and monoclonal antibodies against sulfur mustard (SM; yperite) and initial experiments towards the piezoelectric immunosensor are reported. The antibodies were produced using bovine serum albumin and thyroglobulin as carrier proteins, the hapten density was 9–11 SM molecules per one molecule of albumin. The obtained antibodies were able to bind to the piezoelectric quartz crystal sensors with immobilized SM; however, the interaction with free SM was very weak in a competitive assay format. For this reason, the sensors with immobilized antibodies were prepared and tested for affinity to SM using toluene as solvent. Specific binding of SM to polyclonal antibodies was observed in dry toluene, the monoclonal antibody 2C1.2 was able to bind SM in toluene saturated with water. The preliminary results seem promising for immunosensing of SM in organic solvents.

Francisella tularensis is a pathogenic bacterium causing the serious disease tularemia and is potentially considered as a bioweapon agent. The piezoelectric immunosensor for F. tularensis (Ft) assay was developed using the quartz crystal microbalance (QCM) with covalently immobilized Ft antigen. Samples containing the bacterium were preincubated with a highly diluted anti‐Ft IgM, and the mixture was injected to the flow cell containing the immunosensor. IgM alone as well as Ft cells alone were not giving any response, and therefore, it is assumed that the sensor responded to the IgMx–Fty complex formed during the preincubation step. The effects of pH, time of preincubation, and IgM concentration were tested. This rapid method allowed us to detect 5 · 106 Ft cells/mL. RSD of measurements was near 1%. The total time of one analysis was 35 min, and one sensor was used for more than 30 times without losing sensitivity. The antibody was a kind gift from the Defense Evaluation Research Agency, Porton D...

The electrochemical immunosensor for the detection of the herbicide acetochlor was developed. Initially, the feasibility of the immunosensor was verified using the standard ELISA technique employing the competitive assay format with immobilized acetochlor. The assay principle of the disposable immunochemical biosensor was similar. The screen-printed electrode system served as the transducer, acetochlor was covalently immobilized on the surface of the gold working electrode activated with a self-assembled monolayer of cystamine. Acetochlor was linked to this layer through S-acetylmercaptosuccinic anhydride (AMSA). A limited amount of anti-acetochlor polyclonal rabbit antibody in solution competed with the analyzed acetochlor and the secondary goat anti-rabbit antibody labeled with peroxidase was chosen for detection. The amperometric measurement of peroxidase activity was carried out using 5?aminosalicylic acid (ASA) and hydrogen peroxide as substrates. Successful regeneration of the sensing surface was achieved using pepsin at pH 2 when analyzing samples of water. However, deterioration of the sensing surface in the presence of food samples (corn, carrot, potato and milk) required adopting the disposable assay format. The detection limits of the immunosensor were 25 mg/l (drinking water), 60 mg/l (surface water) and 5 mg/l (milk), thus the sensitivity of the immunosensor assay was not sufficient for drinking water analysis. At present, the developed immunosensor allows only qualitative detection of acetochlor above the maximum residual levels in food. The potential repeated use of the immunosensor remains an important issue for future optimization.

An electrochemical immunosensor combining three monoclonal antibodies on the same measuring element produced by screen-printing was constructed for the semiquantitative group-specific detection of herbicides belonging to phenoxyalkanoic acids. The combination of different monoclonal antibodies covalently immobilized on three working electrodes and one additional electrode containing immobilized albumin provided a more reliable detection of phenoxyalkanoic acids. Peroxidase-2,4-dichlorophenoxyacetic acid conjugate was used as a tracer suitable for all the antibodies employed in a competitive immunoassay. Several herbicides were used to characterize the immunosensor. The developed immunosensor was tested on samples of surface water spiked with different herbicides and binary mixtures of herbicides. The proposed approach seems feasible for rapid screening of large series of samples for the presence of herbicides.

The piezoelectric immunosensor for the determination of acetochlor was developed. The surface of gold electrodes of piezoelectric quartz crystals was modified by self-assembled thiolayers using either 4-aminothiophenol or dithiobis(succinimidyl propionate). In the next step, the modified surface was used for the coupling of acetochlor-protein conjugates. Acetochlor was conjugated to ovalbumin using either thiopropionic acid or acetylthiosuccinimidyl anhydride (AMSA). The acetochlor-modified crystals were used for characterization of the anti-acetochlor polyclonal antibody (Ab). The kinetic rate and equilibrium constants were compared for both types of immobilization. For acetochlor immobilized through AMSA, the dissociation rate constant was 20-times lower. The possibility of using this system for the competitive determination of free acetochlor in water was further studied. The detection limit (10% decrease of relative binding of the antibody) was 0.20 mg/l). The piezoelectric crystals were used repeatedly, 100 mM formic acid served for regeneration of the sensing surface. The total time for one measurement was about 30 min including 15 min pre-incubation of antibody with sample, 10 min binding reaction and 4 min regeneration.

The application of SPR for measurement of the concentration of human serum albumin (HSA) in urine was studied using the compact integrated SPR sensing system Spreeta. HSA was immobilized via cystamine and glutaraldehyde onto the gold sensing area and a competitive assay for HSA was developed using a limited amount of the monoclonal antibody AL-01 in solution. Measurements were carried out in the flow-through mode and the interaction between immobilized HSA and antibody was observed in real time. To obtain reproducible results, different conditions of the measurement (method of immobilization of HSA, data evaluation, concentration of antibody, regeneration procedure) were tested. The calibration curve for clinically relevant concentrations of HSA in urine samples was constructed using 300-times diluted antibody in the form of ascites fluid. The measuring range was between 0.1 and 5 mg/l of HSA, the sensing surface was successfully regenerated and suitable for more than 20 assays. The developed method was t...

The developed piezoelectric quartz crystal microbalance sensor was compared with the commercial resonant mirror-based optical biosensor IAsys. The former one was based on 10 MHz crystals and flow-through arrangement, the latter employed a stirred cuvette. The same biosensing layers were produced by immobilization of 4,4′-dichlorobiphenyl on aminosilanized surfaces through an albumin bridge. The affinity binding of three corresponding polyclonal antibodies was studied and the kinetic rate constants ka and kd were determined. The same values of constants were obtained for sheep antibody, smaller values of ka (approx. twice) were obtained for the other two lower-affinity antibodies using the piezosensor. In the additional comparison during a competitive assay of atrazine, both transducers were able to distinguish 0 and 0.1 μg/L atrazine, however, the IAsys signal was less noisy. For bioaffinity kinetic studies, the piezoelectric biosensor was found to be an economic alternative to the advanced optic...

A piezoelectric quartz crystal sensor with immobilized parathion was used for real‐time kinetic characterization of the interactions with recombinant anti‐parathion single chain Fv antibody IFRN AA01 (scFv). Parathion was linked to the sensor gold electrodes modified with a self‐assembled layer of aminothiophenol using either bovine serum albumin (BSA) or dextran as spacer molecules. The kinetic dissociation rate constant kd was 8 × 10−4 s−1 for both types of sensors, and the association rate constants ka, were 590 and 260 mol−1 1 s−1 for BSA and dextran‐linked parathion, respectively. The regeneration of BSA‐parathion coated crystals was not successful, however. Those crystals with bound scFv were used to study the formation of scFv dimers. Dextran‐parathion modified crystals were successfully regenerated using proteinase K. The affinity of scFv to dextran‐parathion was 50 X lower when compared with the affinity of the anti‐parathion monoclonal antibody (IgG, IFRN 1701) the sequence of which served for p...

Amperometric biosensors based on acetylcholinesterase or butyrylcholinesterase were used for the kinetic determination of organophosphate and carbamate pesticides. The current of the biosensor Iss was measured continuously with substrate; the addition of samples with pesticides resulted in the time decrease of the current dl/dt. The relative inhibition RI=(dl/dt)Iss was used as the signal for evaluations. For several pesticides, different calibration curves (dependencies of RI on concentration c) were obtained depending on the affinity of the individual pesticide to the cholinesterase used. This affinity was described using the bimolecular inhibition constant ki. The single calibration curve independent on the type of pesticide was obtained as the dependence of RI on the product kic, thus indicating the effects of both concentration and inhibiting properties on the response of the biosensor. The relative inhibition was used to characterise anticholinesterase toxicity of the sediments collected fr...

The detection of organophosphate and carbamate pesticides using amperometric biosensors based on immobilized cholinesterases (ChE) was investigated. If only one type of ChE was used for analysis, the sensitivities for some pesticides were not sufficient. We have prepared biosensors with immobilized acetylcholinesterase or butyrylcholinesterase and with both ChEs coimmobilized in the same enzyme layer. Both coimmobilized ChEs function simultaneously in the presence of acetylthiocholine and these biosensors exhibit high response to a wider group of pesticides compared with sensors containing only one type of ChE.