The elaboration of highly sensitive and express methods for quantitative and qualitative detection and monitoring of chemical warfare agents (CWA), organophosphate and carbamate pesticides, compounds with delayed neurotoxicity, and pathogenic microorganisms and viruses is discussed. The application of potentiometric and amperometric biosensors, automatic biosensors discriminating the neurotoxins of different classes, is performed. The information about biosensors detecting the compounds with delayed neurotoxicity through the evaluation of “neurotoxic esterase” activity in the blood is presented. The use of immunochip technology for the detection of pathogenic microorganisms and viruses is demonstrated. The enzymatic methods of destruction of organophosphorus neurotoxins are considered as the base of new defense technology

Pesticide wastes generated from livestock dipping operations containing the organophosphate (OP) insecticide coumaphos (CP) are well suited for disposal by biodegradation since they are highly concentrated (∼1 g/L), generally contained, and lack additional toxic components. In this study, a significantly enhanced efficiency of degrading CP in cattle dip waste (CDW) is reported using a dense, nongrowing cell population that functions without the addition of nutrients required for growing cell cultures. A recombinant strain of Escherichia coli containing the opd gene for organophosphate hydrolase (OPH), which is capable of active hydrolysis of OP neurotoxins including CP, was cultivated in a rich medium containing all essential nutrients. Cells were harvested and utilized in lab scale experiments in the form of either freely suspended cells or cells immobilized within a macroporous gel matrix, poly(vinyl alcohol) (PVA) cryogel. Significantly higher degradation rates were achieved with either suspended or immobilized OPH + cells compared to rates with the microbial consortium naturally present in CDW. Of the two nongrowing cell systems, the detoxification rate with immobilized cells was approximately twice that of freely suspended cells, and kinetic studies demonstrated that a higher maximum reaction rate was achieved with the immobilized cell system. A comparative study using both the CDW and pure CP substrates with free cells indicated that the CDW contained one or more factors that reduced the bioavailability of CP. The immobilized cells retained their activity over a 4‐month period of use and storage, demonstrating both sustained catalytic activity and long‐term mechanical stability.

A new selective amperometric biosensor for reagentless L-tryptophan determination has been developed using immobilized tryptophan-2-monooxygenase (TMO, EC 1.13.12.3). This enzyme-based biosensor provides a rapid-response detection system for concentrations of L-tryptophan between 25 and 1,000 microM in a batch mode system and between 100 and 50,000 microM in a flow-injection mode. The response time was 30 seconds, and the total analysis time was less than 3 minutes. The biosensor retained catalytic activity and fidelity of phenylalanine and tryptophan response for greater than 4 months with repeated usage. The biosensor selectivity to L-tryptophan was dramatically increased relative to phenylalanine when a competitive inhibitor of TMO, indole acetamide (IA), was included. The biosensor was successfully used for L-tryptophan determination in nutrition broth, giving values identical to those determined by HPLC analysis.

Modeling methods used to optimize the biocatalytic efficiency of freely suspended cells have been applied to non-growing microbial cells entrapped within a macro-porous carrier. The catalytic rate, which is dependent on cell concentration inside the biocatalyst beads, coincided with catalytic parameters for freely suspended cells. Immobilized non-growing cell systems could be optimized utilizing the characteristics of freely suspended cells without requiring extensive experimentation to define catalytic behaviour inside the biocatalyst. A dynamic diffusion-reaction model was developed and validated using experimental data for thiodiglycol degradation by Alcaligenes xylosoxidans subsp. xylosoxidans immobilized within macro-porous poly(vinyl alcohol) cryogel in a completely mixed batch bioreactor.

A genetically engineered strain of Escherichia coli that expresses organophosphorus hydrolase (OPH) was immobilized in a polyvinyl alcohol (PVA) cryogel to form a porous biocatalyst that successfully degrades organophosphorus (OP) neurotoxins. The impacts of both diffusion and reaction on biocatalyst efficiency were determined to enable prediction and optimization of the biocatalyst performance. The kinetic rate parameters and activation energies of pure OPH, free cell suspensions, and the immobilized cell biocatalyst were compared. Diffusion was a determining factor for paraoxon hydrolysis because of the very rapid OPH kinetics for its model substrate. Both the paraoxon diffusion through the PVA matrix and the diffusion associated with microbial transport of paraoxon were shown to impact the biocatalyst reaction. However, the enhancement in storage stability resulting from diffusional limitations provides an advantage to diffusion-limited operation. This research may serve as a guide to define the influe...

A new flow-injection amperometric biosensor based on immobilized tryptophan-2-monooxygenase (TMO) has been developed for reagentless L-tryptophan determination. Concentrations of L-tryptophan between 0·1 and 50 mM could be measured with the linear part of the calibration curve between 0·1 and 2 mM. The response time was 30 s and the total analysis time was less than 3 min. The biosensor retained activity for greater than 4 months, when operated daily at 25°C and stored at 8°C. The biosensor was characterized by a relatively high sensitivity to phenylalanine (54% that of L-tryptophan), a modest response to L-methionine (less than 6%) and virtually no response to other amino acids. However, the biosensor selectivity to L-tryptophan could be dramatically increased when indoleacetamide (IA), a competitive inhibitor of TMO, was introduced. In the presence of 10 μM IA, the biosensor response to L-phenylalanine decreased to 7·4% of the unaffected rate for L-tryptophan. In the absence of L-tryptophan and IA the biosensor could be used for L-phenylalanine de determination in the concentration range from 1 to 50 mM. The biosensor was successfully used for L-tryptophan determination in nutritionnal broth.

Alcaligenes xylosoxidans subsp. xylosoxidans (SH91) capable of biodegradation of thiodiglycol (TDG) were immobilized in poly(vinyl) alcohol (PVA) cryogels. Cryoimmobilized biocatalyst was formed as spherical granules with a diameter of 0.5 mm; the biomass concentration inside the gel matrix was as high as 10% (w/w). The immobilized cells were capable of rapid degradation of TDG in tap water or potassium phosphate buffer (100 mM, pH 8.0) containing only (NH4)2 SO4. The immobilized biocatalyst did not show any substrate inhibition up to 200 mM TDG, and retained 100% activity during three months of continuous use in a repeated-batch bioreactor.

A radically new approach for the discriminative determination of various neurotoxins has been developed. This novel biosensor combines a highly sensitive acetylcholinesterase (AChE) biosensor with immobilized organophosphate hydrolase (OPH). The value of the new concept was demonstrated by the discrimination between carbamate and organophosphorus pesticides. It was shown that the response of traditional AChE-based biosensor to mixed samples containing paraoxon and carbofuran was not simply additive, and the measured concentrations of these pesticides were very different from their real concentrations. This combined OPK/AChE system was able to improve the accuracy of the AChE-based biosensor and to uniquely distinguish paraoxon in mixed solutions containing carbofuran. The presented approach promises a new perspective for ''real world'' analyses and opens a new area of discriminative determination of various species in multicomponent solutions.

A microbial sensor for concentration measurement of phenol in aqueous solutions has been developed. Phenol-utilizing cellsPseudomonas putida GFS-8 immobilized in poly(vinyl)alcohol cryogel were used as a biological transducer. Relationships between phenol concentration in the activating medium and endogenic cell respiration have been established. Cell respiration and phenol concentration in the assay solution positively correlated at a phenol concentration range of 0.1–2.0 mg/L and were linearly dependent in the range of 0.1–1.0 mg/L. A Clark membrane electrode was the physiochemical transducer. The assay may be completed within 5 min. The cells oxidize phenol, pyrocatechol, mesityl oxide, aniline, and do not react with a number of xenobiotics, sugars, and alcohol. With the exception of aniline, most components found in waste waters from phenol production affect neither the assay process nor the ability of these cells to use phenol as exogenic respiratory substrate. The immobilized cells retained their ability to utilize phenol as an exogenic respiratory substrate for up to 1 mo.

A new biosensor for the direct detection of organophosphorus (OP) neurotoxins has been developed utilizing cryoimmobilized, recombinant E. coli cells capable of hydrolyzing a wide spectrum of OP pesticides and chemical warfare agents. The biological transducer was provided by the enzymatic hydrolysis of OP neurotoxins by organophosphate hydrolase which generates two protons through a reaction in which P-O, P-F, P-S or P-CN bonds are cleaved, and the proton release corresponded with the quantity of organophosphate hydrolyzed. This stoichiometric relationship permitted the creation of a potentiometric biosensor for detection of OP neurotoxins and a pH-based assay was developed as a direct function of the concentration of OP neurotoxins and the immobilized biomass. In these studies utilizing paraoxon as the substrate, neurotoxin concentration was determined with two different types of measuring units containing immobilized cells: (1) a stirred batch reactor; and (2) a flow-through column minireactor. A pH glass electrode was used as the physical transducer. The linear detection range for paraoxon spanned a concentration range of 0.25-250 ppm (0.001-1.0 mM). The response times were 10 min for the batch reactors and 20 min for the flow-through systems. It was possible to use the same biocatalyst repetitively for 25 analyses with a 10 min intermediate washing of the biocatalyst required for reestablishing the starting conditions. The cryoimmobilized E. coli cells exhibited stable hydrolytic activity for over 2 months under storage in 50 mM potassiumphosphate buffer at +4 degrees C and provide the potential for the development of a stable biotransducer for detecting various OP neurotoxins.

A simple, rapid and selective biosensor for reagent-less L-tryptophan determination has been developed. Tryptophan-2-monooxygenase was immobilized on silica gel. A Clark-type membrane O-2 electrode was used as a physical transducer. A positive correlation between, signal and L-tryptophan- concentration was observed between 0.025 mM and 1.0 mM L-tryptophan. The sensitivity of the biosensor to L-phenylalanine was 30% and to L-methionine 6% relative to L-tryptophan, while there was no response to L-glycine, L-leucine, L-valine, L-isoleucine L-ornithine, L-alanine, L-proline, L-lysine and L-phenylglycine. The analysis time was 2 minutes, and the biosensor has retained activity for greater than 4 months. The biosensor was successfully used for L-tryptophan determination in the nutrition broth.

The volume productivity of the process of CO2 reduction to acetate was investigated in batch and continuous cultures of immobilized thermophilic homoacetogenic bacterium Thermoanaerobacter kivui as a function of physicochemical fermentation parameters. Cells immobilized within a polyvinyl alcohol cryogel were found to be able to completely convert an H-2/CO2 gas mixture to acetic acid. The productivity of this process increased with partial pressure of gases and also when batch fermentation was run at a controlled optimum pH. A maximum productivity of 0.53 g acetate l(-1) h(-1) achieved during continuous fermentation exceeded the productivity of the batch process by more than 30 times.

The formation of acetate from CO2/H-2 gas mixtures by the homoacetate bacterium Acetogenium kivui under various conditions was studied. The advantages and disadvantages of processes based on the use of growing, non-growing and immobilized cells are discussed. Analysis of the kinetics of acetate formation (the concentration of which reached 150-170 mmol/litre in 3 days) in growing culture has shown that the limiting factor is cell lysis. Batch experiments with non-growing cultures demonstrated that, in the absence of lysis, acetate production was constant for at least 1 month, but was considerably inhibited by traces of oxygen, analogously to growing cultures. The entrapment of A. kivui cells in poly(vinyl alcohol) cryogel beads generated a biocatalyst the activity and mechanical strength of which remained constant for at least 1 year. The immobilized cells were practically insensitive to the presence of traces of O2. The experiments with the biocatalyst were performed under batch, semi-batch and continuous conditions, the latter carried out in a flow-through bioreactor with immobilized cells. Constant acetate concentrations of 100 mmol/litre were achieved under the optimal regime, and process productivity did not change at least for 10 days.