Use of biocompatible redox-active polymers based on carbon nanotubes and modified organic matrices for development of a highly sensitive BOD biosensor.

This work investigated the use of redox-active polymers based on bovine serum albumin and chitosan, covalently bound to mediators neutral red and ferrocene and containing carbon nanotubes, for immobilization of Paracoccus yeei VKM B-3302 bacteria. The structures of produced polymers were studied by IR spectroscopy and scanning electron microscopy. Cyclic voltammetry and impedance spectroscopy found the electrochemical characteristics of the investigated systems: the heterogeneous electron transfer rate constant, the constant of the rate of interaction with P. yeei bacteria and the impedance. The systems containing carbon nanotubes and ferrocene-based redox-active polymer proved to be the most promising. Biosensors formed using the hybrid polymers had a high sensitivity with the lower boundary of 0.1 mg/dm3 of the detected BOD5 concentrations and a high correlation (R = 0.9916) with the standard BOD assay of surface water samples.

Registration of BOD using Paracoccus yeei bacteria isolated from activated sludge.

This work investigated the properties of Paracoccus yeei VKM B-3302 bacteria isolated from activated sludge and immobilized in an N-vinylpyrrolidone-modified poly(vinyl alcohol) matrix. The developed hydrogel formed a network structure to enable the entrapment of microbial cells with their viability and biocatalytic properties preserved, which ensured the technological possibility of replicating expendable biosensor receptor elements. A new ratio of the components for the synthesis selected in this work enabled producing a copolymer of an earlier undescribed chemical structure, which can be efficiently used for immobilization of highly sensitive P. yeei bacteria. A biological oxygen demand (BOD) biosensor with these bacteria and matrix was shown to possess a long-time stability exceeding that described earlier, to have a broad substrate specificity and to exceed approximately tenfold the nearest analogues by its sensitivity and the lower boundary value of 0.05 mg/dm3. The biosensor enabled assays of water samples initially attributed to pure samples (the BOD range, 0.05-5.0 mg/dm3). BOD assays of water samples from various sources showed the use of the receptor element of this composition to enable the data that closely correlated with the standard method (R 2 = 0.9990).

A mediator microbial biosensor for assaying general toxicity.

A mediator biosensor based on Paracoccus yeei bacteria for assaying the toxicity of perfumery and cosmetics samples was developed. An approach to selecting an electron-transport mediator based on the heterogeneous electron transfer constants for investigated mediators (ks) and the mediator-biomaterial interaction constants (kinteract) was proposed. Screening of nine compounds as potential mediators showed a ferrocene mediator immobilized in graphite paste to have the highest efficiency of electron transfer to the graphite-paste electrode (the heterogeneous transfer constant, 0.4 ±â€¯0.1 cm/s) and a high constant of interaction with P. yeei (0.023 ±â€¯0.001 dm3/(g·s)). A biosensor for toxicity assessment based on the ferrocene mediator and P. yeei bacteria was formed. The biosensor was tested on samples of four heavy metals (Cu2+, Zn2+, Pb2+, Cd2+) and two phenols (phenol and p-nitrophenol). Proceeding from the EC50 index, it was found that the use of the ferrocene mediator made the biosensor more sensitive to investigated toxicants than most analogues described. Toxicity determination of four perfumery and cosmetics samples by the developed biosensor showed prospects of using this system for real-time toxicity monitoring of samples.

Use of one- and two-mediator systems for developing a BOD biosensor based on the yeast Debaryomyces hansenii.

We investigated the use of one- and two-mediator systems in amperometric BOD biosensors (BOD, biochemical oxygen demand) based on the yeast Debaryomyces hansenii. Screening of nine mediators potentially capable of electron transfer - ferrocene, 1,1'-dimethylferrocene, ferrocenecarboxaldehyde, ferroceneacetonitrile, neutral red, 2,6-dichlorophenolindophenol, thionine, methylene blue and potassium ferricyanide - showed only ferrocene and neutral red to be efficient electron carriers for the eukaryotes studied. Two-mediator systems based on combinations of the investigated compounds were used to increase the efficiency of electron transfer. The developed two-mediator biosensors exceeded their one-mediator analogs by their characteristics. The most preferable two-mediator system for developing a BOD biosensor was a ferrocene-methylene blue combination that ensured a satisfactory long-time stability (43 days), selectivity, sensitivity (the lower limit of the determined BOD5 concentrations, 2.5mg О2/dm3) and speed (assay time for one sample, not greater than 10min) of BOD determination. Analysis of water samples showed that the use of a ferrocene-methylene blue two-mediator system and the yeast D. hansenii enabled registration of data that highly correlated with the results of the standard method (R=0.9913).

[Competition between redox mediator and oxygen in the microbial fuel cell].

The maximal rates and effective constants of 2,6-dichlorphenolindophenol and oxygen reduction by bacterim Gluconobacter oxydans in bacterial fuel cells under different conditions were evaluated. In an open-circuit mode, the rate of 2,6-dichlorphenolindophenol reduction coupled with ethanol oxidation under oxygen and nirogen atmospheres were 1.0 and 1.1 µM s­1 g­1, respectively. In closed-circuit mode, these values were 0.4 and 0.44 µM s­1 g­1, respectively. The initial rate of mediator reduction with the use of membrane fractions of bacteria in oxygen and nitrogen atmospheres in open-circuit mode were 6.3 and 6.9 µM s­1 g­1, whereas these values in closed-circuit mode comprised 2.2 and 2.4 µM s­1 g­1, respectively. The oxygen reduction rates in the presence and absence of 2,6-dichlorphenolindophenol were 0.31 and 0.32 µM s­1 g­1, respectively. The data obtained in this work demonstrated independent electron transfer from bacterial redox centers to the mediator and the absence of competition between the redox mediator and oxygen. The results can make it possible to reduce costs of microbial fuel cells based on activity of acetic acid bacteria G. oxydans.

A yeast co-culture-based biosensor for determination of waste water contamination levels.

Artificial microbial co-cultures were formed to develop the receptor element of a biosensor for assessment of biological oxygen demand (BOD). The co-cultures possessed broad substrate specificities and enabled assays of water and fermentation products within a broad BOD range (2.4-80 mg/dm(3)) with a high correlation to the standard method (R = 0.9988). The use of the co-cultures of the yeasts Pichia angusta, Arxula adeninivorans and Debaryomyces hansenii immobilized in N-vinylpyrrolidone-modified poly(vinyl alcohol) enabled developing a BOD biosensor possessing the characteristics not inferior to those in the known biosensors. The results are indicative of a potential of using these co-cultures as the receptor element base in prototype models of instruments for broad application.

[Biosensors and biofuel cells: research focused on practical application (review)].

Studies on the production of microbial and enzyme biosensor analyzers and microbial biofuel cells for potential practical application are discussed.

[Bioanode for a microbial fuel cell based on Gluconobacter oxydans inummobilized into a polymer matrix].

Acetic acid bacteria Gluconobacter oxydans subsp. industrius RKM V-1280 were immobilized into a synthetic matrix based on polyvinyl alcohol modified with N-vinylpyrrolidone and used as biocatalysts for the development ofbioanodes for microbial fuel cells. The immobilization method did not significantly affect bacterial substrate specificity. Bioanodes based on immobilized bacteria functioned stably for 7 days. The maximum voltage (fuel cell signal) was reached when 100-130 µM of an electron transport mediator, 2,6-dichlorophenolindophenol, was added into the anode compartment. The fuel cell signals reached a maximum at a glucose concentration higher than 6 mM. The power output of the laboratory model of a fuel cell based on the developed bioanode reached 7 mW/m2 with the use of fermentation industry wastes as fuel.

BOD biosensor based on the yeast Debaryomyces hansenii immobilized in poly(vinyl alcohol) modified by N-vinylpyrrolidone.

An amperometric biosensor for assessing the biochemical oxygen demand (BOD) was formed by immobilizing Debaryomyces hansenii VKM Y-2482 yeast cells in poly(vinyl alcohol) modified by N-vinylpyrrolidone. Modification provided for a high sensitivity and stability of the bioreceptor. A high oxidative activity of the receptor element and the absence of any toxic effect of assayed compounds were shown for 34 substrates (alcohols, carbohydrates, carboxylic acids, amino acids, nitrophenols and surfactants) that may occur in wastewaters. Estimates of the measurement range and region of the linear dependence of signals on the BOD level, pH and temperature sensitivities, dependences of signals on concentrations of salts, stability, Michaelis kinetic constants and assay rates were obtained. The BOD values determined by the biosensor in assayed wastewater samples were shown to have a high correlation with those obtained by the standard dilution method.

[Aerobic methylobacteria as the basis for a biosensor for dichloromethane detection].

Cells of dichloromethane (DChM) bacteria-destructors were immobilized by sorption on different types of membranes, which were fixed on the measuring surface of a pH-sensitive field transistor. The presence of DChM in the medium (0.6-8.8 mM) led to a change in the transistor's output signal, which was determined by the appearance of H+ ions in the medium due to DChM utilization by methylobateria. Among four strains of methylobacteria--Methylobacterium dichloromethanicum DM4, Methylobacterium extorquens DM 17, Methylopila helvetica DM6, and Ancylobacter dichloromethanicus DM 16--the highest and most stable activity toward DChM degradation was observed in the strain M. dichloromethanicum DM4. Among 11 types of membranes for cell immobilization, Millipore nitrocellulose membranes and chromatographic fiber paper GF/A, which allow one to obtain stable biosensor signals for 2 weeks without a bioreceptor change, were chosen as optimal carriers.

[Receptor elements for biosensors in two ways of methylotrophic yeast immobilization].

Receptor elements for biosensors based on Hansenula polymorpha NCYC 495 In yeast cells for ethanol assay were developed using two ways of cell immobilization, i.e., physical adsorption on a glass fiber membrane and covalent binding on a modified nitrocellulose membrane. The linear diapason of ethanol assays for a biosensor based on yeast cells adsorbed on glass fiber was 0.05-1.18; for a biosensor based on yeasts immobilized on a nitrocellulose membrane, 0.2-1.53 mM. Receptor elements based on sorbed cells possessed 2.5 times higher long-term stability. The time response was 1.5 times less for cells immobilized using DEAE-dextran and benzochinone. The results of ethyl alcohol assays using biosensors based on cells immobilized via adsorption and covalent binding, as well as using the standard areometric method, had high correlation coefficients (0.998 and 0.997, respectively, for the two ways of immobilization). The results indicate the possibility to consider the described models of receptor elements for biosensors as prototypes for experimental samples for practical use.

[Degradation of the EDTA and EDTA complexes with metals by immobilized cells of Chelativorans oligotrophicus LPM-4 bacteria].

Enzymatic oxidative degradation of EDTA and EDTA complexes with metals has been investigated using immobilized cells of Chelativorans oligotrophicus LPM-4. A polarographic method, which makes it possible to register oxygen consumption by cells, has been used. For the first time, it has been indicated that the Cd-EDTA and Ni-EDTA complexes undergo degradation by the bacteria under study.

[Microbial biosensors for detection of biological oxygen demand (a review)].

The review briefs recent advances in application of biosensors for determining biological oxygen demand (BOD) in water. Special attention is focused on the principles of operation of microbial BOD sensors; the information about biorecognition elements in such systems and the methods used for immobilization of biological components in film biosensors is summarized. Characteristics of some BOD sensor models are considered in detail.

[Nanobiotechnology and biosensor research].

Nanobiotechnology is defined as an interdisciplinary field of science that studies the application of fine-sized biological objects (of nanoscale, 1-100 nm) to design the devices and systems of the same size that utilize for new purposes the unusual, known, or previously unknown effects. Analysis demonstrates that the final goals, approaches, solution methods, and applications of nanostructures and biological sensors have much in common. This brief review attempts to systematize a number of the available data and pick out an organic connection of the new research direction with the field of biosensor technology, which have reached the level of sustainable development.

[Use of NMR spectroscopy in studies of sorbitol and glucose transformation by Gluconobacter oxydans].

NMR spectroscopy was applied for studying the products of glucose and sorbitol oxidation by cells of Gluconobacter oxydans. An analysis of 1H NMR spectra showed that the transformation of glucose results in the formation of diketogluconic acid, and sorbitol is oxidized to sorbose. In the 32P NMR spectra, only a signal of inorganic phosphate was detected, which accumulated in the medium as a result of cell lysis.

[Microbial, enzymatic, and immune biosensors for ecological monitoring and control of biotechnological processes].

Results of the research performed at the Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, on designing immunobiosensors for detection of toxic compounds and microbial cells enzyme-based biosensors for detection of hydrocarbons and alcohols, and microbial biosensors for aromatic compounds, surfactants, and biological oxygen consumption are briefed. Parameters of the mediator electrodes involving microbial cells and data on the properties of microbial biofuel cells--devices based on biosensor principle and representing alternative sources of electric energy--are given.

[Degradation of p-toluenesulfonate by immobilized Comamonas testosteroni BS1310 (pBS1010) cells].

Parameters of degradation of p-toluenesulfonate (TS) by free and agar-embedded Comamonas testosteroni BS1310 (pBS1010) cells were determined. The maximum rate of TS degradation was 25% lower in by immobilized than free cells, equaling 11 nmol x min(-1) x mg(-1) cells. Degradation of TS by both free and immobilized cells was associated with molecular oxygen consumption (molar ratio, 1 : 2). In a plug-flow reactor, the degradation rate was 10.4 nmol x min(-1) x mg(-1) cells. The results can be applied to designing reactors for TS degradation in sewage and developing biosensors.

[Evaluation of substrate specificity of biosensor models based on strains degrading polycyclic aromatic compounds].

Models of microbial biosensors based on 11 strains of degrading surface-active substances (SASs) and polycyclic aromatic hydrocarbons (PAHs) were studied. Substrate specificity, sensitivity, and stability of biosensor models were comparatively evaluated.