A "2-in-1" Bioanalytical System Based on Nanocomposite Conductive Polymers for Early Detection of Surface Water Pollution.

This work proposes an approach to the formation of receptor elements for the rapid diagnosis of the state of surface waters according to two indicators: the biochemical oxygen demand (BOD) index and toxicity. Associations among microorganisms based on the bacteria P. yeei and yeast S. cerevisiae, as well as associations of the yeasts O. polymorpha and B. adeninivorans, were formed to evaluate these indicators, respectively. The use of nanocomposite electrically conductive materials based on carbon nanotubes, biocompatible natural polymers-chitosan and bovine serum albumin cross-linked with ferrocenecarboxaldehyde, neutral red, safranin, and phenosafranin-has made it possible to expand the analytical capabilities of receptor systems. Redox polymers were studied by IR spectroscopy and Raman spectroscopy, the contents of electroactive components were determined by atomic absorption spectroscopy, and electrochemical properties were studied by electrochemical impedance and cyclic voltammetry methods. Based on the proposed kinetic approach to modeling individual stages of bioelectrochemical processes, the chitosan-neutral red/CNT composite was chosen to immobilize the yeast association between O. polymorpha (ks = 370 ± 20 L/g × s) and B. adeninivorans (320 ± 30 L/g × s), and a bovine serum albumin (BSA)-neutral composite was chosen to immobilize the association between the yeast S. cerevisiae (ks = 130 ± 10 L/g × s) and the bacteria P. yeei red/CNT (170 ± 30 L/g × s). After optimizing the composition of the receptor systems, it was shown that the use of nanocomposite materials together with associations among microorganisms makes it possible to determine BOD with high sensitivity (with a lower limit of 0.6 mg/dm3) and detect the presence of a wide range of toxicants of both organic and inorganic origin. Both receptor elements were tested on water samples, showing a high correlation between the results of biosensor analysis of BOD and toxicity and the results of standard analytical methods. The results obtained show broad prospects for creating sensitive and portable bioelectrochemical sensors for the early warning of environmentally hazardous situations based on associations among microorganisms and nanocomposite materials.

Novel Conductive Polymer Composite PEDOT:PSS/Bovine Serum Albumin for Microbial Bioelectrochemical Devices.

A novel conductive composite based on PEDOT:PSS, BSA, and Nafion for effective immobilization of acetic acid bacteria on graphite electrodes as part of biosensors and microbial fuel cells has been proposed. It is shown that individual components in the composite do not have a significant negative effect on the catalytic activity of microorganisms during prolonged contact. The values of heterogeneous electron transport constants in the presence of two types of water-soluble mediators were calculated. The use of the composite as part of a microbial biosensor resulted in an electrode operating for more than 140 days. Additional modification of carbon electrodes with nanomaterial allowed to increase the sensitivity to glucose from 1.48 to 2.81 µA × mM-1 × cm-2 without affecting the affinity of bacterial enzyme complexes to the substrate. Cells in the presented composite, as part of a microbial fuel cell based on electrodes from thermally expanded graphite, retained the ability to generate electricity for more than 120 days using glucose solution as well as vegetable extract solutions as carbon sources. The obtained data expand the understanding of the composition of possible matrices for the immobilization of Gluconobacter bacteria and may be useful in the development of biosensors and biofuel cells.

Electrochemical Biosensors for Express Analysis of the Integral Toxicity of Polymer Materials.

Biosensors based on an oxygen electrode, a mediator electrode, and a mediator microbial biofuel cell (MFC) using the bacteria Gluconobacter oxydans B-1280 were formed and tested to determine the integral toxicity. G. oxydans bacteria exhibited high sensitivity to the toxic effects of phenol, 2,4-dinitrophenol, salicylic and trichloroacetic acid, and a number of heavy metal ions. The system "G. oxydans bacteria-ferrocene-graphite-paste electrode" was superior in sensitivity to biosensors formed using an oxygen electrode and MFC, in particular regarding heavy metal ions (EC50 of Cr3+, Mn2+, and Cd2+ was 0.8 mg/dm3, 0.3 mg/dm3 and 1.6 mg/dm3, respectively). It was determined that the period of stable functioning of electrochemical systems during measurements was reduced by half (from 30 to 15 days) due to changes in the enzyme system of microbial cells when exposed to toxicants. Samples of the products made from polymeric materials were analyzed using developed biosensor systems and standard biotesting methods based on inhibiting the growth of duckweed Lemna minor, reducing the motility of bull sperm, and quenching the luminescence of the commercial test system "Ecolum". The developed bioelectrocatalytic systems were comparable in sensitivity to commercial biosensors, which made it possible to correlate the results and identify, by all methods, a highly toxic sample containing diphenylmethane-4,4'-diisocyanate according to GC-MS data.

"3-in-1" Hybrid Biocatalysts: Association of Yeast Cells Immobilized in a Sol-Gel Matrix for Determining Sewage Pollution.

This study presents a novel ″3-in-1″ hybrid biocatalyst design that combines the individual efficiency of microorganisms while avoiding negative interactions between them. Yeast cells of Ogataea polymorpha VKM Y-2559, Blastobotrys adeninivorans VKM Y-2677, and Debaryomyces hansenii VKM Y-2482 were immobilized in an organosilicon material by using the sol-gel method, resulting in a hybrid biocatalyst. The catalytic activity of the immobilized microorganism mixture was evaluated by employing it as the bioreceptor element of a biosensor. Optical and scanning electron microscopies were used to examine the morphology of the biohybrid material. Elemental distribution analysis confirmed the encapsulation of yeast cells in a matrix composed of methyltriethoxysilane (MTES) and tetraethoxysilane (TEOS) (85 and 15 vol %, respectively). The resulting heterogeneous biocatalyst exhibited excellent performance in determining the biochemical oxygen demand (BOD) index in real surface water samples, with a sensitivity coefficient of 50 ± 3 × 10-3·min-1, a concentration range of 0.3-31 mg/L, long-term stability for 25 days, and a relative standard deviation of 3.8%. These findings demonstrate the potential of the developed hybrid biocatalyst for effective pollution monitoring and wastewater treatment applications.

Conductive Polymers and Their Nanocomposites: Application Features in Biosensors and Biofuel Cells.

Conductive polymers and their composites are excellent materials for coupling biological materials and electrodes in bioelectrochemical systems. It is assumed that their relevance and introduction to the field of bioelectrochemical devices will only grow due to their tunable conductivity, easy modification, and biocompatibility. This review analyzes the main trends and trends in the development of the methodology for the application of conductive polymers and their use in biosensors and biofuel elements, as well as describes their future prospects. Approaches to the synthesis of such materials and the peculiarities of obtaining their nanocomposites are presented. Special emphasis is placed on the features of the interfaces of such materials with biological objects.

A Two-Mediator System Based on a Nanocomposite of Redox-Active Polymer Poly(thionine) and SWCNT as an Effective Electron Carrier for Eukaryotic Microorganisms in Biosensor Analyzers.

Electropolymerized thionine was used as a redox-active polymer to create a two-mediated microbial biosensor for determining biochemical oxygen demand (BOD). The electrochemical characteristics of the conducting system were studied by cyclic voltammetry and electrochemical impedance spectroscopy. It has been shown that the most promising in terms of the rate of interaction with the yeast B. adeninivorans is the system based on poly(thionine), single-walled carbon nanotubes (SWCNT), and neutral red (kint = 0.071 dm3/(g·s)). The biosensor based on this system is characterized by high sensitivity (the lower limit of determined BOD concentrations is 0.4 mgO2/dm3). Sample analysis by means of the developed analytical system showed that the results of the standard dilution method and those using the biosensor differed insignificantly. Thus, for the first time, the fundamental possibility of effectively using nanocomposite materials based on SWCNT and the redox-active polymer poly(thionine) as one of the components of two-mediator systems for electron transfer from yeast microorganisms to the electrode has been shown. It opens up prospects for creating stable and highly sensitive electrochemical systems based on eukaryotes.

Multicationic Quaternary Ammonium Compounds: A Framework for Combating Bacterial Resistance.

During previous stages of research, high biocidal activity toward microorganism archival strains has been used as the main indicator in the development of new antiseptic formulations. Although this factor remains one of the most important characteristics of biocide efficiency, the scale of antimicrobial resistance spread causes serious concern. Therefore, focus shifts toward the development of formulations with a stable effect even in the case of prolonged contact with pathogens. Here, we introduce an original isocyanuric acid alkylation method with the use of available alkyl dichlorides, which opened access to a wide panel of multi-QACs with alkyl chains of various lengths between the nitrogen atoms of triazine and pyridine cycles. We used a complex approach for the resulting series of 17 compounds, including their antibiofilm properties, bacterial tolerance development, and antimicrobial activity toward multiresistant pathogenic strains. As a result of these efforts, available compounds have shown higher levels of antibacterial activity against ESKAPE pathogens than widely used commercial QACs. Hit compounds possessed high activity toward clinical bacterial strains and have also demonstrated a long-term biocidal effect without significant development of microorganism tolerance. The overall results indicated a high level of antibacterial activity and the broad application prospects of multi-QACs based on isocyanuric acid against multiresistant bacterial strains.

From Antibacterial to Antibiofilm Targeting: An Emerging Paradigm Shift in the Development of Quaternary Ammonium Compounds (QACs).

In a previous development stage, mostly individual antibacterial activity was a target in the optimization of biologically active compounds and antiseptic agents. Although this targeting is still valuable, a new trend has appeared since the discovery of superhigh resistance of bacterial cells upon their aggregation into groups. Indeed, it is now well established that the great majority of pathogenic germs are found in the environment as surface-associated microbial communities called biofilms. The protective properties of biofilms and microbial resistance, even to high concentrations of biocides, cause many chronic infections in medical settings and lead to serious economic losses in various areas. A paradigm shift from individual bacterial targeting to also affecting more complex cellular frameworks is taking place and involves multiple strategies for combating biofilms with compounds that are effective at different stages of microbiome formation. Quaternary ammonium compounds (QACs) play a key role in many of these treatments and prophylactic techniques on the basis of both the use of individual antibacterial agents and combination technologies. In this review, we summarize the literature data on the effectiveness of using commercially available and newly synthesized QACs, as well as synergistic treatment techniques based on them. As an important focus, techniques for developing and applying antimicrobial coatings that prevent the formation of biofilms on various surfaces over time are discussed. The information analyzed in this review will be useful to researchers and engineers working in many fields, including the development of a new generation of applied materials; understanding biofilm surface growth; and conducting research in medical, pharmaceutical, and materials sciences. Although regular studies of antibacterial activity are still widely conducted, a promising new trend is also to evaluate antibiofilm activity in a comprehensive study in order to meet the current requirements for the development of highly needed practical applications.

Oligohexamethylene Guanidine Derivative as a Means to Prevent Biological Fouling of a Polymer-Based Composite Optical Oxygen Sensor.

The use of biocidal agents is a common practice for protection against biofouling in biomass-rich environments. In this paper, oligohexamethyleneguanidine (OHMG) polymer, known for its biocidal properties, was further modified with para-aminosalicylic acid (PAS) to enhance its properties against microorganisms coated with a lipid membrane. The structure of the product was confirmed by 1H NMR, 13C NMR, and FTIR spectroscopy. The values of the minimum inhibitory concentration (MIC) against Mycobacterium smegmatis ATCC 607 and Pseudomonas chlororaphis 449 were found to be 1.40 and 1.05 µg/mL, respectively. The synthesized substance was used as an additive to the polymer matrix of the composite optical oxygen sensor material. A series of samples with different contents of OHMG-PAS was prepared using a co-dissolution method implying the fabrication of a coating from a solution containing both polymers. It turned out that the mutual influence of the components significantly affects the distribution of the indicator in the matrix, surface morphology, and contact angle. The optimal polymer content turned out to be wt.3%, at which point the water contact angle reaches almost 122°, and the fouling rate decreases by almost five times, which is confirmed by both the respiratory MTT assay and confocal microscopy with staining. This opens up prospects for creating stable and biofouling-resistant sensor elements for use in air tanks or seawater.

Bioanalytical System for Determining the Phenol Index Based on Pseudomonas putida BS394(pBS216) Bacteria Immobilized in a Redox-Active Biocompatible Composite Polymer "Bovine Serum Albumin-Ferrocene-Carbon Nanotubes".

The possibility of using the microorganisms Pseudomonas sp. 7p-81, Pseudomonas putida BS394(pBS216), Rhodococcus erythropolis s67, Rhodococcus pyridinivorans 5Ap, Rhodococcus erythropolis X5, Rhodococcus pyridinivorans F5 and Pseudomonas veronii DSM 11331T as the basis of a biosensor for the phenol index to assess water environments was studied. The adaptation of microorganisms to phenol during growth was carried out to increase the selectivity of the analytical system. The most promising microorganisms for biosensor formation were the bacteria P. putida BS394(pBS216). Cells were immobilized in redox-active polymers based on bovine serum albumin modified by ferrocenecarboxaldehyde and based on a composite with a carbon nanotube to increase sensitivity. The rate constants of the interaction of the redox-active polymer and the composite based on it with the biomaterial were 193.8 and 502.8 dm3/(g·s) respectively. For the biosensor created using hydrogel bovine serum albumin-ferrocene-carbon nanotubes, the lower limit of the determined phenol concentrations was 1 × 10-3 mg/dm3, the sensitivity coefficient was (5.8 ± 0.2)∙10-3 µA·dm3/mg, Michaelis constant KM = 230 mg/dm3, the maximum rate of the enzymatic reaction Rmax = 217 µA and the long-term stability of the bioanalyzer was 11 days. As a result of approbation, it was found that the urban water phenol content differed insignificantly, measured by creating a biosensor and using the standard photometric method.

Optical Oxygen Sensing and Clark Electrode: Face-to-Face in a Biosensor Case Study.

In the last decade, there has been continuous competition between two methods for detecting the concentration of dissolved oxygen: amerometric (Clark electrode) and optical (quenching of the phosphorescence of the porphyrin metal complex). Each of them has obvious advantages and disadvantages. This competition is especially acute in the development of biosensors, however, an unbiased comparison is extremely difficult to achieve, since only a single detection method is used in each particular study. In this work, a microfluidic system with synchronous detection of the oxygen concentration by two methods was created for the purpose of direct comparison. The receptor element is represented by Saccharomyces cerevisiae yeast cells adsorbed on a composite material, previously developed by our scientific group. To our knowledge, this is the first work of this kind in which the comparison of the oxygen detection methods is carried out directly.

The Use of Diethoxydimethylsilane as the Basis of a Hybrid Organosilicon Material for the Production of Biosensitive Membranes for Sensory Devices.

Biomembranes based on an organosilica sol-gel matrix were used to immobilize bacteria Paracoccus yeei VKM B-3302 as part of a biochemical oxygen demand (BOD) biosensor. Diethoxydimethylsilane (DEDMS) and tetraethoxysilane (TEOS) were used as precursors to create the matrix in a 1:1 volume ratio. The use of scanning electron microscopy (SEM) and the low-temperature nitrogen adsorption method (BET) showed that the sol-gel matrix forms a capsule around microorganisms that does not prevent the exchange of substrates and waste products of bacteria to the cells. The use of DEDMS as part of the matrix made it possible to increase the sensitivity coefficient of the biosensor for determining BOD by two orders of magnitude compared to a biosensor based on methyltriethoxysilane (MTES). Additionally, the long-term stability of the bioreceptor increased to 68 days. The use of such a matrix neutralized the effect of heavy metal ions on the microorganisms' catalytic activity in the biosensor. The developed biosensor was used to analyze water samples from water sources in the Tula region (Russia).

Microbial Biosensors for Rapid Determination of Biochemical Oxygen Demand: Approaches, Tendencies and Development Prospects.

One of the main indices of the quality of water is the biochemical oxygen demand (BOD). A little over 40 years have passed since the practical application of the first microbial sensor for the determination of BOD, presented by the Japanese professor Isao Karube. This time span has brought new knowledge to and practical developments in the use of a wide range of microbial cells based on BOD biosensors. At present, this field of biotechnology is becoming an independent discipline. The traditional BOD analysis (BOD5) has not changed over many years; it takes no less than 5 days to carry out. Microbial biosensors can be used as an alternative technique for assessing the BOD attract attention because they can reduce hundredfold the time required to measure it. The review examines the experience of the creation and practical application of BOD biosensors accumulated by the international community. Special attention is paid to the use of multiple cell immobilization methods, signal registration techniques, mediators and cell consortia contained in the bioreceptor. We consider the use of nanomaterials in the modification of analytical devices developed for BOD evaluation and discuss the prospects of developing new practically important biosensor models.

Preparation of Hybrid Sol-Gel Materials Based on Living Cells of Microorganisms and Their Application in Nanotechnology.

Microorganism-cell-based biohybrid materials have attracted considerable attention over the last several decades. They are applied in a broad spectrum of areas, such as nanotechnologies, environmental biotechnology, biomedicine, synthetic chemistry, and bioelectronics. Sol-gel technology allows us to obtain a wide range of high-purity materials from nanopowders to thin-film coatings with high efficiency and low cost, which makes it one of the preferred techniques for creating organic-inorganic matrices for biocomponent immobilization. This review focuses on the synthesis and application of hybrid sol-gel materials obtained by encapsulation of microorganism cells in an inorganic matrix based on silicon, aluminum, and transition metals. The type of immobilized cells, precursors used, types of nanomaterials obtained, and their practical applications were analyzed in detail. In addition, techniques for increasing the microorganism effective time of functioning and the possibility of using sol-gel hybrid materials in catalysis are discussed.

Development of Nanocomposite Materials Based on Conductive Polymers for Using in Glucose Biosensor.

Electropolymerized neutral red, thionine, and aniline were used as part of hybrid nanocomposite conductive polymers, to create an amperometric reagent-less biosensor for glucose determination. The structure of the obtained polymers was studied using infrared (IR) spectroscopy and scanning electron microscopy. Electrochemical characteristics were studied by cyclic voltammetry and impedance spectroscopy. It was shown that, from the point of view of both the rate of electron transfer to the electrode, and the rate of interaction with the active center of glucose oxidase (GOx), the most promising is a new nanocomposite based on poly(neutral red) (pNR) and thermally expanded graphite (TEG). The sensor based on the created nanocomposite material is characterized by a sensitivity of 1000 ± 200 nA × dm3/mmol; the lower limit of the determined glucose concentrations is 0.006 mmol/L. The glucose biosensor based on this nanocomposite was characterized by a high correlation (R2 = 0.9828) with the results of determining the glucose content in human blood using the standard method. Statistical analysis did not reveal any deviations of the results obtained using this biosensor and the reference method. Therefore, the developed biosensor can be used as an alternative to the standard analysis method and as a prototype for creating sensitive and accurate glucometers, as well as biosensors to assess other metabolites.

A kinetic approach to the formation of two-mediator systems for developing microbial biosensors as exemplified by a rapid biochemical oxygen demand assay.

This work proposes a method of forming a microorganism-mediator(s) receptor system, in which the rates of separate stages of mediator bioelectrocatalysis are used as the basis for the development of biosensors for the biochemical oxygen demand (BOD) rapid assay. In the presence of a ferrocene mediator, the yeast Blastobotrys adeninivorans was shown to enable oxidation of a larger range of substrates as compared with other investigated microorganisms-bacteria Escherichia coli and yeast Ogataea polymorpha. The rate constants of the interaction of the yeast B. adeninivorans with nine compounds, electron transfer mediators, were determined; the best mediator for these microorganisms was found to be neutral red (k int = 0.681 ± 0.009 dm3/g s). Neutral red possesses a high rate of interaction with the ferrocene mediator (14,200 ± 100 dm3/mol s) shown earlier to be the most promising acceptor of electrons at a carbon paste electrode (0.4 ± 0.1 cm/s). These features enabled the formation of a two-mediator ferrocene-neutral red system to be used in a biosensor. A two-mediator-based biosensor had a higher sensitivity (the lower limit of detected BOD concentrations, 0.16 mg/dm3) than that of a one-mediator system based on neutral red and ferrocene. Analysis of ten samples from surface water reservoirs showed the combination of ferrocene, neutral red and the yeast B. adeninivorans to enable the data that highly correlated (R = 0.9693) with those of the standard method.