The interactions of dsDNA with alpha-lipoic acid (ALA, α-lipoic acid) and ALA embedded into phospholipid nanoparticles (ALA-NPhs) were studied electrochemically by square-wave voltammetry technique. Screen-printed electrodes (SPE) modified with a freshly prepared water dispersion of single-walled carbon nanotubes (SPE/CNT), were used for the quantitative electrochemical analysis of ALA and ALANPhs. SPE/CNTs effectively registered direct electrochemical oxidation of guanine, adenine, and thymine heterocyclic bases of dsDNA, and their changes in the presence of ALA or ALA-NPhs. The electrochemical oxidation signal of adenine was chosen as the most pronounced detecting signal registered in the presence of ALA or ALA-NPhs. From the obtained experimental data on the decrease in the amplitude of the adenine oxidation currents, the electrochemical coefficient of the toxic effect was calculated as the ratio of the unbound dsDNA signal to the dsDNA signal in the presence of ALA or ALA-NPhs. Binding constants (Kb) of the DNA–drug complexes were calculated in accordance with the decline of adenine oxidation signals. Based on our experiments, we have proven that incorporating ALA–Loaded into Phospholipid Nanoparticles (ALA-NPhs) changes the moderately toxic effect of lipoic acid on DNA to a non-toxic effect. The calculated Kb = 2.30 M− 1 value for DNA–ALA-NPhs complex also confirmed this suggestion. The Kb of the DNA–ALA complex is about 1.45 × 103 M− 1 and reflects non-covalent interaction with DNA through hydrogen bonding within the minor groove.

L-asparaginase (L-Aspase) enzyme has found applications in medicine for treatment of various cancers. Herein, we report single-molecule study of thermal denaturation of L-Aspase within 25°C to 60°C temperature range by atomic force microscopy (AFM) and by single-molecule sensing with a (solid state nanopore)-based electrical detector (SSNPED). AFM has allowed us to reveal a thermally induced changes in aggregation state of L-Aspase and in its adsorbability on mica. At the same time, the configuration of the enzyme’s globule spatial conformation has been found to alter according to data obtained with the SSNPED. Our results reported open up opportunities for further development of anti-cancer drugs.

The expression of beta-lactamases, hydrolyzing beta-lactams, is the main mechanism of Gram-negative bacteria resistance to this class of antibiotics. To select the tactics for chemotherapy, it is necessary to determine rapidly whether pathogenic bacteria produce expressed extended-spectrum beta-lactamases (ESBLs). These enzymes are hazardous because they can hydrolyze the most widely used antibiotics, 3rd-generation cephalosporins. Electrochemical sensors, characterized by fast response and minimal sample handling, are promising for these purposes. For rapid screening of ESBL-producing bacteria, electrochemical sensors nanostructured by single-wall carbon nanotubes (CNT) have been developed that detect the changes of cefotaxime (CTX) concentration due to enzymatic degradation. Modification of the electrodes with CNT significantly improves electrochemical characteristics. In the presence of a sample obtained from E. coli expressing CTX-M-type ESBL, the antibiotic is hydrolyzed, which is recorded as a decrease in the peak of CTX oxidation current. The addition of sulbactam, an inhibitor of ESBLs, restores the oxidation current. A sample obtained from E. coli expressing beta-lactamase TEM-1, which does not hydrolyze CTX, was used as a negative control. The applicability of the developed sensors was further demonstrated using several clinical strains of K. pneumonia and E. coli resistant to antibiotics, and a 40-50% decrease in the peak of CTX oxidation current was detected. The developed electrochemical mediatorfree sensors can be used for rapid screening of bacterial strains for ESBL expression, as well as for screening of novel beta-lactamase inhibitors.

Background/Objectives: Our study brings a new method to properly evaluating drug efficacy at the non-invasive in vitro level. Methods: In this work, the electrochemical mediator-free and reagent-free analysis of cell lines based on the registration of electrochemical profiles of membrane proteins was developed. We studied the specificity of cell lines Wi-38 and HepG2 and the toxic effects of drugs on cell-on-electrode systems. Results: A linear dependence of the peak current on the concentration of cells applied to the electrode in the range from 1 × 105 to 6 × 105 cells/electrode was registered (R2 0.932 for Wi-38 and R2 0.912 for HepG2). The water-soluble form of phosphatidylcholine (wPC) nanoparticles recommended for atherosclerosis treatment and prevention of cardiovascular diseases did not show a toxic effect on the human fibroblast cells, Wi-38, or the human hepatocellular carcinoma cells, HepG2, at sufficiently high concentrations (such as 0.1–1 mg/mL). The antitumor drug doxorubicin, at concentrations of 3 and 10 μg/mL, showed a pronounced toxic effect on the tested cell lines, where the percentage of living cells was 50–55%. Conclusions: A comparative analysis of the cytotoxicity of wPC (0.1–1 mg/mL) and doxorubicin (3–10 μg/mL) on the cell lines Wi-38 and HepG2 using the MTT test and electrochemical approach for the registration of cells showed their clear adequacy.

The proposed approach for determining catalytic activity of the SARS-CoV-2 main protease (Mpro) is based on registration of the peak area of electrochemical oxidation of tyrosine residue in the model peptide substrate CGGGAVLQSGY immobilized on the surface of a graphite screen-printed electrode (SPE) modified with gold nanoparticles (AuNP). The AuNP were obtained by electrosynthesis. Steady state kinetic parameters of Mpro in the reaction with the model peptide were determined: catalytic constant (kcat) was (3.1 ± 0.1) × 10−3 s−1; Michaelis constant (KM) was (358 ± 32) × 10−9 M; catalytic efficiency (kcat/KM) was 8659 s−1/M. The limit of detection (LOD) determined for Mpro using the proposed electrochemical system was 44 nM. The proposed approach is a promising tool to search for new Mpro inhibitors as drugs for treatment of coronavirus infections.

Aim . Determination of the catalytic activity of a single molecule of horseradish peroxidase (HRP) in the oxidation reaction of the substrate 2,2ʹ-azino-bis-[3-ethylbenzthiazoline-6- sulfonate] (ABTS) with hydrogen peroxide. Methodology . To determine (monitor) the catalytic activity of HRP, pore technology has been used; it has allowed us to analyze the activity of a single HRP molecule without introducing additional components into the system to enhance the signal. For this purpose, a solid-state pore of about 5 nm in size, formed by electron-beam drilling in a silicon nitride plate of ~40 nm thickness, has been used. A HRP molecule has been embedded in this pore, after which the catalytic activity of the molecule embedded in the pore in the presence of ABTS and H 2 O 2 has been analyzed by measuring the ion current through the pore with the HRP molecule embedded in it. Results. A pore detector has been proposed to study the catalytic activity of HRP in the reaction of ABTS oxidation. It has been found that this detector made it possible to monitor the activity of this enzyme by registering of ion current through the pore. Research implications . It has been shown that the manufactured pore can be used to monitor HRP activity. The results obtained are important for the development of work in the field of enzyme research at the level of single molecules.

An electrochemical analysis of the interaction of double-stranded DNA (dsDNA) with bovine serum albumin (BSA) as model protein was carried out. A decrease in the electrochemical oxidation peak current of DNA nucleobases was evaluated in the DNA-BSA complex at a BSA concentrations above 0.1 mu M, which indicated compaction of the DNA structure with a formation of partial clew and shielding of guanine, adenine, and thymine available for electrode reactions. At a BSA concentration below 0.1 mu M in DNA-BSA complex, an increase in the intensity of heterocyclic bases electro oxidation was observed, indicating partial unwinding of the DNA double strand. Using a calorimetric assay, the enthalpy of Delta H = + 2620.4 J/g was registered for DNA-BSA complex with low BSA concentration (below 0.1 mu M), and Delta H = + 82.7 J/g for high BSA concertation (above 0.1 mu M). The binding constants of the DNA-BSA complex based on adenine and thymine electrochemical signals were Kb = 4.52 x 106 M- 1 and Kb = 3.99 x 106 M- 1, respectively. The spectral binding constant of the DNA-BSA complex was determined as an integral parameter and corresponded to Kb = 3.98 x 106 M- 1, which in good agreement with electrochemical Kb values.

The activity of cytochrome P450 enzymes decreases in older adults, which can lead to toxic effects from polypharmacy. Cytochromes P450 are the most significant enzymes involved in the metabolism of foreign compounds, including pharmaceutical substances. Vitamin B2, or riboflavin (RF), is a potent antioxidant that is vital for the body and participates in numerous enzyme-catalyzed redox reactions. RF is phosphorylated intracellularly to form flavin mononucleotide (FMN), which is further metabolized into flavin adenine dinucleotide (FAD). The active site of the NADPH-dependent cytochrome P450 reductase (CPR), a redox partner of CYP enzymes, is necessary for the catalytic functions of cytochromes P450. The active site of reductase is a complex formed by two types of vitamin B2, such as flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). In our study, we investigated the impact of the phosphorylated form of vitamin B2, FAD, and FMN on the catalytic activity of cytochrome P450 2C9 (CYP2C9) towards non-steroidal anti-inflammatory medications diclofenac and naproxen. It was shown that FAD significantly enhanced the catalytic efficiency of CYP2C9. The 4-hydroxylation of diclofenac was enhanced by 148 ± 10%. The O-demethylation of naproxen showed an increase of 120 ± 14%. Based on these data, we can assume that intake of vitamin B2 (riboflavin) improves catalytic efficiency of CYP2C9. This finding is essential for the modulation of catalytic activity of CYP2C9. The proposed electroanalytic approach is a sensitive and robust method for drug metabolism assay.

Nano-carbon quantum dots (nano-CQDs) were prepared by a hydrothermal method using chitosan and melamine and characterized by FTIR, TEM, and SEM. The maximum excitation and emission wavelengths of the carbon dots were determined to be 338 nm and 412 nm, respectively, indicating their strong fluorescence. The fluorescence properties of complexes of nano-CQDs with dsDNA and bovine serum albumin (BSA) were investigated. It was shown that DNA and albumin acquire fluorescent properties upon complexation with CQDs. DNA and albumin labelled with CQDs exhibit electrochemical properties comparable to unlabeled biomolecules. DNA and DNA/CQD complexes showed well-separated irreversible waves with peak potentials corresponding to guanine, adenine, and thymine bases. BSA and BSA/CQD complexes exhibited one broad peak corresponding to electrochemical oxidation of tyrosine amino acids of the polypeptide backbone. This indicates the biocompatibility of CQDs and the preservation of the spatial structures of DNA and bovine serum albumin during the formation of fluorogenic complexes. The studied electrochemical behavior of nucleic acid or protein complexes with CQDs allows the use of these structures for the visualization of biological objects and their simultaneous electrochemical analysis.

This study aimed to investigate whether the water-soluble pharmaceutical form of phosphatidylcholine nanoparticles (wPC) stimulated the catalytic activity of CYP enzymes 2C9 and 2D6. We have shown that electroenzymatic CYP2C9 catalysis to nonsteroidal anti-inflammatory drug naproxen as a substrate was enhanced from 100% to 155% in the presence of wPC in media. Electroenzymatic CYP2D6 activity in the presence of the adrenoceptor-blocking agent bisoprolol as a substrate was elevated significantly from 100% to 144% when wPC was added to potassium phosphate buffer solution. These results indicate the ability of wPC in the form of the phospholipid ultra-small nanoparticles to work as a membrane additive and crowding agent to accelerate the electroenzymatic reactions of cytochrome P450.

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.

We describe a bielectrode system for evaluation of the electrocatalytic activity of cytochrome P450 2E1 (CYP2E1) towards chlorzoxazone. One electrode of the system was employed to immobilize Bactosomes with human CYP2E1, cytochrome P450 reductase (CPR), and cytochrome b5 (cyt b5). The second electrode was used to quantify CYP2E1-produced 6-hydroxychlorzoxazone by its direct electrochemical oxidation, registered using square-wave voltammetry. Using this system, we determined the steady-state kinetic parameters of chlorzoxazone hydroxylation by CYP2E1 of Bactosomes immobilized on the electrode: the maximal reaction rate (Vmax) was 1.64 ± 0.08 min−1, and the Michaelis constant (KM) was 78 ± 9 μM. We studied the electrochemical characteristics of immobilized Bactosomes and have revealed that electron transfer from the electrode occurs both to the flavin prosthetic groups of CPR and the heme iron ions of CYP2E1 and cyt b5. Additionally, it has been demonstrated that CPR has the capacity to activate CYP2E1 electrocatalytic activity towards chlorzoxazone, likely through intermolecular electron transfer from the electrochemically reduced form of CPR to the CYP2E1 heme iron ion.

The review discusses electrochemical methods for analysis of drug interactions with DNA. The electroanalysis method is based on the registration of interaction-induced changes in the electrochemical oxidation potential of heterocyclic nitrogenous bases in the DNA molecule and in the maximum oxidation current amplitude. The mechanisms of DNA–drug interactions can be identified based on the shift in the electrooxidation potential of heterocyclic nitrogenous bases toward more negative (cathodic) or positive (anodic) values. Drug intercalation into DNA shifts the electrochemical oxidation potential to positive values, indicating thermodynamically unfavorable process that hinders oxidation of nitrogenous bases in DNA. The potential shift toward the negative values indicates electrostatic interactions, e.g., drug binding in the DNA minor groove, since this process does not interfere with the electrochemical oxidation of bases. The concentration-dependent decrease in the intensity of electrochemical oxidation of DNA bases allows to quantify the type of interaction and calculate the binding constants.

The electrocatalytic properties of cytochrome P450 2C9 and the cytochrome P450 2C9/FAD and cytochrome P450 2C9/FMN complexes have been studied using a two-electrode system. The system consisted of an enzymatic catalyst electrode modified by the membrane-like compound didodecyldimethylammonium bromide (SPE/DDAB) and a measuring electrode, modified with carbon nanotubes (SPE/CNT). To study the effectiveness of electroenzymatic reactions catalyzed by cytochrome P450 2C9, the nonsteroidal anti-inflammatory drug diclofenac was used as a substrate. Cytochrome P450 2C9 catalyzes the stereospecific hydroxylation reaction to form 4′-hydroxydiclofenac. The metabolite 4′-hydroxydiclofenac was recorded at a potential E=+0.12 (relative to Ag/AgCl).The use of FAD and FMN as low-molecular mediators made it possible to increase the efficiency of electrocatalysis of the SPE/DDAB/CYP2C9/FAD system to 148±10% and SPE/DDAB/CYP2C9/FMN to 113±6% compared to SPE/DDAB/CYP2C9 (100±5%), and also increase the rate of the enzymatic reaction by 1.5 and 1.13 times, respectively.

The review is devoted to new highly effective methods for analyzing the catalytic activity of enzymes of medical significance, such as cytochromes P450, trypsin, asparaginase, beta-lactamase, and nucleases. The methods are based on registration the specific activity of enzymes using electroanalytical methods. The review analyzes the experimental data obtained by the authors. Two platforms have been developed that allow quantitative measurement of catalytic activity based on the electrochemical properties of the enzyme (cytochrome P450, bactosomes, asparaginase) or substrate (trypsin, nucleases, restriction enzymes, beta-lactamase).

The aim of this work was to develop an electrochemical approach for the analysis of DNA degradation and fragmentation in apoptotic cells. DNA damage is considered one of the major causes of human diseases. We analyzed the cleavage processes of the circular plasmid pTagGFP2-N and calf thymus DNA, which were exposed to restriction endonucleases (the restriction endonucleases BstMC I and AluB I and the nonspecific endonuclease I). Genomic DNA from the leukemia K562 cell line was used as a marker of the early and late (mature) stages of apoptosis. Registration of direct electrochemical oxidation of nucleobases of DNA molecules subjected to restriction endonuclease or apoptosis processes was proposed for the detection of these biochemical events. Label-free differential pulse voltammetry (DPV) has been used to measure endonuclease activities and DNA damage using carbon nanotube-modified electrodes. The present DPV technique provides a promising platform for high-throughput screening of DNA hydrolases and for registering the efficiency of apoptotic processes. DPV comparative analysis of the circular plasmid pTagGFP2-N in its native supercoiled state and plasmids restricted to 4 and 23 parts revealed significant differences in their electrochemical behavior. Electrochemical analysis was fully confirmed by means of traditional methods of DNA analysis and registration of apoptotic process, such as gel electrophoresis and flow cytometry.

This article describes the approaches developed by the authors with the aim to increase the efficiency of electro enzymatic reactions catalyzed by cytochrome P450 3A4. A comparative analysis of cytochrome P450 3A4 systems was carried out during the formation of the functional complexes hemoprotein-flavin nucleotides as low-molecular models of NAD(P)H-dependent cytochrome P450 reductase. The formation of a productive enzyme-substrate complex before the stage ofaccepting electrons from the modified electrode was studied from the electocatalytic viewpoint. Incorporation of the enzyme into nanopores of different nature on the electrode (2D-3D transition) was also studied. The results on the electrochemical reduction of bactosomes as the functionally active models of the microsomal monooxygenase system are also considered. The electrochemical and electrocatalytic parameters of cytochrome P450 3A4 were compared for different models of the electrocatalytic generation of metabolites.

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.

We discuss the diverse functions of proteases in the context of their biotechnological and medical significance, as well as analytical approaches used to determine the functional activity of these enzymes. An insight into modern approaches to studying the kinetics and specificity of proteases, based on spectral (absorption, fluorescence), mass spectrometric, immunological, calorimetric, and electrochemical methods of analysis is given. We also examine in detail electrochemical systems for determining the activity and specificity of proteases. Particular attention is given to exploring innovative electrochemical systems based on the detection of the electrochemical oxidation signal of amino acid residues, thereby eliminating the need for extra redox labels in the process of peptide synthesis. In the review, we highlight the main prospects for the further development of electrochemical systems for the study of biotechnologically and medically significant proteases, which will enable the miniaturization of the analytical process for determining the enzymatic activity of these enzymes.

Objectives: The main aim of our experiments was to demonstrate the suitability of cell-based biosensors for searching for new anticancer medicinal preparations. Methods: The effect of the substance doxorubicin, doxorubicin embedded in phospholipid nanoparticles, and doxorubicin with phospholipid nanoparticles modified by targeting vectors (cRGD and folic acid) on dsDNA and breast cancer cell lines (MCF-7, MDA-MB-231) was studied. Results: In the obtained doxorubicin nanoforms, the particle size was less than 60 nm. Our study of the percentage of doxorubicin inclusion showed the almost complete embeddability of the substance into nanoparticles for all samples, with an average of 95.4 ± 4.6%. The calculation of the toxicity index of the studied doxorubicin samples showed that all substances were moderately toxic drugs in terms of adenine and guanine. The biosensor analysis using electrodes modified with carbon nanotubes showed an intercalation interaction between doxorubicin and its derivatives and dsDNA, except for the composition of doxorubicin with folic acid with a linker length of 2000 (NPh-Dox-Fol(2.0)). The results of the electroanalysis were normalized to the total cell protein (mg) and cell concentration. The highest intensity of the electrochemical signals was observed in intact control cells of the MCF-7 and MDA-MB-231 cell lines. Conclusions: The proposed electrochemical approach is useful for the analysis of cell line responses to the medicinal preparations.