Genetic engineering approach has been used to create a collection of recombinant E. coli strains-producers of bacterial enzymes beta-lactamases related both to the existing natural analogues and to the mutant forms of natural enzymes. Currently, the collection includes beta-lactamases of the TEM (molecular class A), CTX-M (molecular class A), VIM (molecular class B), and NDM (molecular class B) types. A set of recombinant beta-lactamases can be used for screening new antibacterial drugs and inhibitors in vitro. The collection of beta-lactamase producers is included in the National Depository Bank of Live Systems “Noah’s Ark” of the Lomonosov Moscow State University.

The widespread prevalence of antibiotic-resistant pathogenic microorganisms that cause infectious diseases in humans and animals severely limits the possibilities of chemotherapy. Large families of enzymes play a key role in the various mechanisms of bacterial antibiotic resistance; in particular, resistance to β-lactam antibiotics in Gram-negative pathogens is due to the production of β-lactamases involving serine hydrolases and metalloenzymes. Due to the high cost of developing new antibacterial drugs, the main approach to overcoming bacterial antibiotic resistance is to combine β-lactams with β-lactamase inhibitors. This review analyzes information about two generations of inhibitors of this superfamily of enzymes: first-generation inhibitors are structural analogs of β-lactams and inhibit only class A serine β-lactamases. Second-generation inhibitors include compounds of the diazabicyclooctane class and boronic acid derivatives. They form complexes with β-lactamases that mimic the structure of acyl-enzyme complexes with antibiotics or the structure of the transition state of these complexes. Second-generation inhibitors are characterized by broader inhibitory specificity compared to first-generation inhibitors. Derivatives of cyclic boronic acids (oxoborines) have the broadest specificity for serine and metallo-β-lactamases. The development of new combinations of antibiotics with inhibitors that target various β-lactamases will restore the therapeutic efficacy of β-lactam antibacterial agents.

Drug resistance caused by single-point amino acid substitutions in target proteins represents a serious problem in modern therapy of oncological, infectious and viral diseases. This review systematizes modern computational modeling methods that allow studying the molecular mechanisms of such resistance and predicting its emergence. Contemporary databases and machine learning tools used for resistance prediction from genomic data are analyzed. The potential of the emerging direction of physics-informed artificial intelligence models for enhancing the interpretability of predictions is noted.

To develop highly sensitive biosensors based on field-effect transistors with a silicon nanowire channel (FET), single interactions of antibodies with prostate-specific antigen (PSA) on the surface of pure silicon modified with 5 nm gold nanoparticles were studied. A digital immunocomplex registration method using scanning electron microscopy was employed, where 25 nm gold nanoparticles served as antibody visualizing labels. A specialized algorithm was developed to calculate the nanoparticle density on the silicon surface. Various methods of chemical silicon modification using silanes (3-glycidoxypropyltrimethoxysilane (GOPS), 3-mercaptopropyltrimethoxysilane (GOPS-SH), and 3-aminopropyltriethoxysilane (APTES)), bifunctional reagents, and polyethylene glycol were applied to investigate covalent antibody immobilization. It has been shown that chemical modification methods using GOPS are characterized by a lower detection limit for prostate-specific antigen (PSA)—a biomarker of prostate tumors. Biosensor structures based on field-effect transistors with nanowire channels, whose surfaces were modified by two different methods using GOPS, were fabricated, and their pH sensitivity was studied. It has been demonstrated that the modification method using GOPS-SH is characterized by a maximum pH sensitivity of 70 mV/pH and is the most promising for the development of highly sensitive biosensors for biomarker detection.

The wide spread of antibiotic-resistant bacterial pathogens has attracted great interest in the development of new drugs to suppress resistance emergence. They include inhibitors of β-lactamases (β-Ls), which confer bacterial resistance to β-lactam antibiotics. This class of antibiotics targets penicillin-binding proteins (PBPs), the inhibition of which leads to the disruption of the bacterial cell wall synthesis. β-Lactamases catalyze the hydrolysis of the amide bond of the β-lactam ring, causing inactivation of the antibiotic. Due to the structural diversity of β-Ls, it is relevant to search for their inhibitors characterized by broad specificity. Boronic acids are a promising class of compounds capable of inhibiting not only different molecular classes of β-Ls but also PBPs. The review considers the data on the mechanisms of interaction of β-lactams with PBPs and β-Ls, as well as on the effect of the structure of boronic acid derivatives on their inhibitory activity against various bacterial enzymes. Combinations of antibacterial drugs with β-L inhibitors that simultaneously inhibit PBPs are promising for overcoming antibiotic resistance of different pathogens.

A system for monitoring the operation of a laser interferometer in a reactive ion etching setup has been developed. For the precise calibration of the etching rate of the top silicon layer in silicon-on-insulator materials, a series of chips with identical structures were fabricated. The thickness of the structure on each chip varied depending on the etching time. The heights of the resulting steps were measured using the tapping mode of an atomic force microscope. For the etching mode in a plasma of CF _4 and O _2 gases (flow ratio 20:5 , pressure 4 Pa, power 40 W), the silicon etching rate was determined to be 0.31± 0.1 nm/s. The adduce parameters allow stopping silicon etching at a depth of ∼5 to 120 nm with an accuracy of no worse than 2 nm. The obtained results make it possible to address a number of tasks in the fabrication of various silicon nanoelectronic devices. In particular, the process of forming silicon channel nanowires for field-effect transistors requires high-precision control of the silicon layer thickness during reactive ion etching.

Immunosensors based on field-effect transistors with nanowire channels (NWFETs) provide fast and real-time detection of a variety of biomarkers without the need for additional labels. The key feature of the developed immunosensor is the coating of silicon NWs with multilayers of polyelectrolytes (polyethylenimine (PEI) and polystyrene sulfonate (PSS)). By causing a macromolecular crowding effect, it ensures the “soft fixation” of the antibodies into the 3-D matrix of the oppositely charged layers. We investigated the interaction of prostate-specific antigen (PSA), a biomarker of prostate cancer, and antibodies adsorbed in the PEI and PSS matrix. In order to visualize the formation of immune complexes between polyelectrolyte layers using SEM and AFM techniques, we employed a second clone of antibodies labeled with gold nanoparticles. PSA was able to penetrate the matrix and concentrate close to the surface layer, which is crucial for its detection on the nanowires. Additionally, this provides the optimal orientation of the antibodies’ active centers for interacting with the antigen and improves their mobility. NWFETs were fabricated from SOI material using high-resolution e-beam lithography, thin film vacuum deposition, and reactive-ion etching processes. The immunosensor was characterized by a high sensitivity to pH (71 mV/pH) and an ultra-low limit of detection (LOD) of 0.04 fg/mL for PSA. The response of the immunosensor takes less than a minute, and the measurement is carried out in real time. This approach seems promising for further investigation of its applicability for early screening of prostate cancer and POC systems.

Antibiotic-resistant bacteria represent a global issue that calls for novel approaches to diagnosis and treatment. Given the variety of genetic factors that determine resistance, multiplex methods hold promise in this area. We developed a novel method to covalently attach oligonucleotide probes to the wells of polystyrene plates using photoactivation with 4-azidotetrafluorobenzaldehyde. Then, it was used to develop the technique of microarrays in the wells. It consists of the following steps: activating polystyrene, hybridizing the probes with biotinylated target DNA, and developing the result using a streptavidin–peroxidase conjugate with colorimetric detection. The first microarray was designed to identify 11 different gene types and 16 single-nucleotide polymorphisms (SNPs) of clinically relevant ESBLs and carbapenemases, which confer Gram-negative bacteria resistance to β-lactam antibiotics. The detection of bla genes in 65 clinical isolates of Enterobacteriaceae demonstrated the high sensitivity and reproducibility of the technique. The highly reproducible spot staining of colorimetric microarrays allowed us to design a second microarray that was intended to quantify four different types of bla mRNAs in order to ascertain their expressions. The combination of reliable performance, high throughput in standard 96-well plates, and inexpensive colorimetric detection makes the microarrays suitable for routine clinical application and for the study of multi-drug resistant bacteria.

The review analyzes recent advances, challenges, and practical applications in the field of enzymes within the framework of chemical enzymology and enzyme engineering. The achievements in the fundamental understanding of molecular mechanisms of the catalytic cycle of enzymatic reactions made using quantum mechanics/molecular mechanics methods with supercomputer technologies and bioinformatic approaches are considered. The design of protein biocatalysts with new properties is a fundamentally significant methodology of the bioengineering approach to solving practical problems, which is demonstrated by a number of examples. The increasing role of biocatalysis in medicine and biomedical research is illustrated by addressing the problems of antibiotic synthesis and overcoming antibiotic resistance of bacteria, mechanisms of neurodegenerative diseases and development of drugs to treat Alzheimer's disease, biocatalytic processes of DNA repair and the role of mechanisms of functioning of heme peroxidases in the human body. The use of enzymes to degrade endogenous and exogenous toxicants has been greatly developed in recent decades. The advances and problems of using enzymes in therapy and drug delivery are analyzed. The fundamental role of enzymes in modern analysis and diagnosis is noted. The review considers a new trend in the development of bioanalytical methods using aptamers, multi-analysis systems on biochips, surface-enhanced Raman scattering systems, and bioelectroanalysis. The bibliography includes 460 references.

Inhibition of β-lactamases involved in the development of bacterial resistance to β-lactam antibiotics is promising for restoring their effectiveness as antibacterial drugs. In this work, we screened the inhibitory activity against some serine β-lactamases of class A among humic substances of various origins, which represent complex organic matrices, as well as their narrow fractions obtained by separating preparations by acidity and polarity. The structural and group composition of broad fractions was characterized by 13C NMR spectroscopy; the molecular composition of narrow fractions was characterized by Fourier transform-ion cyclotron resonance mass spectrometry. The widest specificity of inhibition against various ESBL (TEM-12, TEM-17, TEM-18) and the inhibitor-resistant β-lactamase TEM-34 was possessed by the fraction of humic acids of brown coal CHA-I, isolated at pH 9: the values of residual activity of TEM -12, TEM-17, TEM-18, and TEM-34 were (

A new approach is proposed based on the use of electrodes modified with carbon nanomaterials to determine enzymatic activity and screening for inhibitors of serine β-lactamases such as extended spectrum β-lactamases (ESBLs). These enzymes are responsible for the development of antibiotic resistance of pathogenic bacteria to β-lactam antibiotics. Electrochemical oxidation of cephalosporin antibiotic cefotaxime was effectively registered at a potential E from +596 to +625 mV (relative to Ag/AgCl). This property makes it possible to determine the change in cefotaxime concentration in solution upon interaction with serine β-lactamases. By analyzing the electrochemical characteristics of the cefotaxime oxidation reaction, the kinetic parameters of its hydrolysis catalyzed by the serine β-lactamase CTX-M-116 were determined. The Michaelis constant was KM = 50 µM and the maximum rate of the catalytic reaction was 1.67∙10–6 M/min. A comparative analysis of the electrochemical parameters of the enzyme/substrate cefotaxime and enzyme/substrate cefotaxime/inhibitor sulbactam (SBT) systems was carried out. Inhibition of β-lactamase by sulbactam was characterized by an IC50 value of 2.5 μM. The proposed approach can be used for screening new substrates and inhibitors of β-lactamases.

Serine β-lactamase TEM-1 is the first β-lactamase discovered and is still common in Gram-negative pathogens resistant to β-lactam antibiotics. It hydrolyzes penicillins and cephalosporins of early generations. Some of the emerging TEM-1 variants with one or several amino acid substitutions have even broader substrate specificity and resistance to known covalent inhibitors. Key amino acid substitutions affect catalytic properties of the enzyme, and secondary mutations accompany them. The occurrence of the secondary mutation M182T, called a “global suppressor”, has almost doubled over the last decade. Therefore, we performed saturating mutagenesis at position 182 of TEM-1 to determine the influence of this single amino acid substitution on the catalytic properties, thermal stability, and ability for thermoreactivation. Steady-state parameters for penicillin, cephalothin, and ceftazidime are similar for all TEM-1 M182X variants, whereas melting temperature and ability to reactivate after incubation at a higher temperature vary significantly. The effects are multidirectional and depend on the particular amino acid at position 182. The M182E variant of β-lactamase TEM-1 demonstrates the highest residual enzymatic activity, which is 1.5 times higher than for the wild-type enzyme. The 3D structure of the side chain of residue 182 is of particular importance as observed from the comparison of the M182I and M182L variants of TEM-1. Both of these amino acid residues have hydrophobic side chains of similar size, but their residual activity differs by three-fold. Molecular dynamic simulations add a mechanistic explanation for this phenomenon. The important structural element is the V159-R65-E177 triad that exists due to both electrostatic and hydrophobic interactions. Amino acid substitutions that disturb this triad lead to a decrease in the ability of the β-lactamase to be reactivated.

In this work we present an automatic thermoregulation system for biosensors based on field-effect transistors with a nanowire channel, which provides full control on the required temperature regime in bioanalytical analises. The system elements, including field-effect transistors with a nanowire channel, temperature sensors and heaters, were fabricated on a single silicon cristal using electron beam lithography, reactive ion etching and high-vacuum deposition techniques. Unicue electronics have been developed to control and maintain temperature. The dependence of thermometer readout on heating power was measured, which is in good agreement with the results of numerical simulation. A demonstration of a thermoregulation system with PID-feedback was carried out, ensuring the establishment of a desiered temperature in the range of 30-70◦ C in 18 s in liquid. A demonstration of a thermoregulation system for detecting nucleic acids was carried out using synthetic single-stranded DNA, which is a gene fragment from the bacterium Escherichia coli. The minimal detectable response was observed for a sample with a concentration of 3 fM.

Multiplex analysis as an immunochip-in-a well format for simultaneous detection of post-vaccinal antibodies to three poultry infections (Newcastle disease, infectious bronchitis and bursal disease) in one chicken sera was developed. The immunochip had a microarray format printed on the bottom of a standard microtiter plate well and consisted of 36 microspots (d = 400 μm each) with three lines of viral antigens absorbed in a gradient of five decreasing concentrations. Optimization of assay conditions revealed the necessity of careful choice of the reaction buffer due to the high tendency of chicken IgY to exhibit unspecific binding. The best results were obtained for PBS buffer (pH 6.0) supplied with 0.1

We present a CMOS compatible technique for fabrication a sensor system based on field-effect transistors with a nanowire channel with an integrated thermoregulation elements. The proposed system provides the necessary temperature regimes for many bioanalytical studies. Field-effect transistors with a nanowire channel were fabricated using of reactive-ion etching of the upper layer of a silicon on insulator through a mask formed by electron beam lithography. Titanium thermoresistive strips for temperature control were located on the surface of the chip nearby to the nanowire transistors. Their fabrication is carried out simultaneously with the formation of contact pads to the transistor electrodes, which made it possible to avoid additional technological steps. A demonstration of a system with a built-in temperature controller for the determination of nucleic acids was carried out on model oligonucleotides. Increasing the operating temperature of the device to the ranges at which DNA hybridization occurs most efficiently allows increasing specificity and avoiding false positive results, as well as reducing analysis time. The possibility of heating up to 85–90∘C allows you to reuse such devices.

An electrochemical immunosensor based on screen-printed graphite electrodes is developed for the determination of the antibiotic chloramphenicol in water and milk samples. It is shown that the immobilization of chloramphenicol-specific antibodies in the liquid-crystal layer of the membrane-like didodecyldimethylammonium bromide preserves the mobility and accessibility of active centers of antibodies, and the addition of gold nanoparticles improves the electron transfer from the electrode surface to the redox centers of horseradish peroxidase, which is used as a label. The limit of detection of chloramphenicol is 0.02 μg/L in water and 0.04 μg/L in milk. This method can be used to determine the residual amounts of chloramphenicol in animal products.

A search for novel sources of biologically active compounds is at the top of the agenda for biomedical technologies. Natural humic substances (HSs) contain a large variety of different chemotypes, such as condensed tannins, hydrolyzable tannins, terpenoids, lignins, etc. The goal of this work was to develop an efficient separation technique based on solid-phase extraction (SPE) for the isolation of narrow fractions of HS with higher biological activity compared to the initial material. We used lignite humic acid as the parent humic material, which showed moderate inhibition activity toward beta-lactamase TEM 1 and antioxidant activity. We applied two different SPE techniques: the first one was based on a gradient elution with water/methanol mixtures of the humic material sorbed at pH 2, and the second one implied separation by a difference in the pKa value by the use of sequential sorption of HS at pH from 8 to 3. SPE cartridges Bond Elute PPL (Agilent) were used in the fractionation experiments. The first and second techniques yielded 9 and 7 fractions, respectively. All fractions were characterized using high-resolution mass spectrometry and biological assays, including the determination of beta-lactamase (TEM 1) inhibition activity and antioxidant activity. The acidity-based separation technique demonstrated substantial advantages: it enabled the isolation of components, outcompeting the initial material at the first step of separation (sorption at pH 8). It showed moderate orthogonality in separation with regard to the polarity-based technique. Good perspectives are shown for developing a 2D separation scheme using a combination of polarity and acidity-based approaches to reduce structural heterogeneity of the narrow fractions of HS.

A technique for lateral ow hybridization analysis of speci c bacterial beta-lactamase mRNAs has been developed, including the preparation of a biotinylated target DNA and its analysis on a test strip with immobilized oligonucleotide probes. When the DNA target and probe structures are complementary, DNA duplexes are formed in the test zone of the strip, which interact with the streptavidin conjugate with gold nanoparticles. The method was used to determine the transcripts of the TEM-type betalactamase genes isolated from the culture of E. coli - producers of the corresponding recombinant betalactamase. It has been shown that singlestranded DNA target of 330 bases was revealed most effectively in lateral ow analysis.

Gold nanoparticles (AuNPs) are popular labels for colorimetric detection of various analytes, involving proteins, nucleic acids, viruses, and whole cells because of their outstanding optical properties, inertness, and modification variability. In this work, we present an improved approach for enhancement of color intensity for DNA membrane microarrays based on seed-mediated growth of AuNP labels. Biotin-labeled DNA is hybridized with capture oligonucleotide probes immobilized on the microarrays. Then biotin is revealed by a streptavidin–AuNP conjugate followed by the detection of AuNPs. Optimization of seed-mediated enlargement of AuNPs by the reduction of tetrachloroauric acid with hydroxylamine made it possible to change the coloring of specific spots on the microarrays from pink to a more contrasting black with minor background staining. Mean size of the resulting AuNPs was four times larger than before the enhancement. Adjusting the pH of HAuCl4 solution to 3.5 and use of a large excess of hydroxylamine increased the signal/background ratio by several times. The method’s applicability was demonstrated for quantification of a short oligonucleotide of 19 bases and full-length TEM-type β-lactamase genes of 860 bp responsible for the development of bacterial resistance against β-lactam antibiotics. Improved protocol for AuNP enlargement may be further transferred to any other membrane-based assays of nucleic acids with both instrumental and visual colorimetric detection.

Methods of molecular genetic analysis based on the colorimetric biochip technology have shown their effectiveness in identifying antibiotic resistance genes in bacteria. For the quantitative determination of nucleic acids, a comparative study of methods for converting digital color images of biochips into monochrome black-and-white versions using RGB and CMYK color models has been carried out. A 19-mer single-stranded oligonucleotide and two model mRNAs corresponding to the genes of two types of clinically relevant beta-lactamases (CTX-M and NDM) were studied as objects. The widest range of staining intensity and the best analytical characteristics for the determination of all types of studied nucleic acids were obtained using the red channel of the RGB color model. The detection limits were 0.10 ± 0.02 pmol/μL for the 19-mer oligonucleotide, and 3.0 ± 0.2 and 8.0 ± 0.6 amol/μL for mRNA of beta-lactamases CTX-M-116 and NDM-1, respectively. The developed method can be used for the quantification of genes responsible for the multiple resistance of bacteria to antimicrobial drugs.