Bioluminescence, a natural phenomenon resulting from enzyme-catalysed oxidation of substrates like luciferin, has emerged as a powerful tool in modern biology, biochemistry, and medicine. Advances in understanding bioluminescent systems, particularly those of bacteria, fireflies, coelenterates, and crustaceans, have facilitated the development of highly sensitive and specific analytical methods. Applications range from intracellular bioimaging to the detection of metabolites and effectors, offering unparalleled insight into cellular and molecular processes. Recently, experimental neuroscience has embraced bioluminescent techniques, leveraging their unique advantages for visualizing and assessing brain cell function and metabolic activity in vivo and in vitro. Furthermore, the potential to control neuronal activity through bioluminescent reactions is opening new frontiers in neurobiology. This review focuses on the principles of bioluminescent assays based on bacterial and firefly luciferases and coelenterazine-derived systems, highlighting their current and prospective applications in experimental brain studies.

Enzymatic inhibitory assay based on the coupled enzyme system NAD(P)·H:FMN oxidoreductase and luciferase (Red + Luc), originally developed for environmental monitoring of soils, water, and air, is proposed as a method for evaluating changes in the properties of active ingredients of pesticide preparations depending on the additional components (formulants), as well as when pesticides are combined in mixtures. Using the commercial pesticide preparations containing glyphosate, it was shown that the degree of inhibition of the coupled enzyme system Red + Luc largely depends on the formulants rather than on the active ingredient in their composition. Moreover, the combined inhibitory effect of the pesticides mixture on the coupled enzyme system Red + Luc was not additive. According to the results of the inhibitory assay, the type of interaction of pesticide preparations in mixtures depends on both the formulants used and the ratio of pesticides in the mixture.

Enzymatic biosensors have been lately gaining popularity in various areas of human life, from medicine to agriculture, owing to their effectiveness and selectivity towards the tested substances. Bioluminescent enzymatic biosensors attract special attention because of the light produced by the enzyme reaction. Researchers designing bioluminescent enzymatic sensors face a number of challenges, mainly associated with the low stability and/or sensitivity of the enzymes used as a biological recognition element of the biosensor. Herein we describe strategies of improving the analytical performance of bioluminescent enzymatic biosensors based on luciferase enzymes. We, first, discuss the increase in biosensors’ sensitivity by optimizing the composition of the biological recognition element, including enzyme and substrate variations and addition of nanoparticles or stabilizing agents. Then, we provide insights into improving the stability of luciferases by immobilization or by the presence of chaperones and via directed mutagenesis. And the last section deals with projections for the future. We give special consideration to results of using these approaches in developing biosensors based on bioluminescent enzyme systems of fireflies and bacteria. Yet, the approaches described in the review are universal and can be effectively used to develop other enzymatic biosensors.

While food additives are widely used in the modern food industry and generally are important in maintaining the ability to provide food for the increasing world population, the progress occurring in this field is much ahead of the evaluation of their possible consequences for human health. The present study suggests a set of single- and multi-enzyme assay systems for revealing toxic effects of the most widely spread food preservatives, such as sorbic acid (E200), potassium sorbate (E202), and sodium benzoate (E211) at the primary molecular level of their interaction with enzymes. The assay is based on the inhibition of enzyme activity by toxic substances proportional to the amount of the toxicants in the sample. The single-enzyme assay system based on NAD(P)H:FMN oxidoreductase (Red) proved to be most sensitive to the impact of food additives, with the IC50 values being 29, 14, and 0.02 mg/L for sodium benzoate, potassium sorbate, and sorbic acid, respectively, which is considerably lower than their acceptable daily intake (ADI). No reliable change in the degree of inhibition of the enzyme assay systems by food preservatives was observed upon elongating the series of coupled redox reactions. However, the inhibition of activity of the multi-enzyme systems by 50% was found at a preservative concentration below the maximum permissible level for food. The inhibition effect of food preservatives on the activity of butyrylcholinesterase (BChE), lactate dehydrogenase (LDH), and alcohol dehydrogenase (ADH) was either absent or found in the presence of food preservatives at concentrations significantly exceeding their ADI. Among the preservatives under study, sodium benzoate is considered to be the safest in terms of the inhibiting effect on the enzyme activity. The results show that the negative effect of the food preservatives at the molecular level of organization of living things is highly pronounced, while at the organismal level it may not be obvious.

The present study describes the method of preparing reagents containing firefly luciferase (FLuc) and its substrate, D-luciferin, immobilized into gelatin gel separately or together. The addition of stabilizers dithiothreitol (DTT) and bovine serum albumin (BSA) to the reagent is a factor in achieving higher activity of reagents and their stability during storage. The use of immobilized reagents substantially simplifies the procedure of assay for microbial contamination. The mechanism of action of the reagents is based on the relationship between the intensity of the bioluminescent signal and the level of ATP contained in the solution of the lysed bacterial cells. The highest sensitivity to ATP is achieved by using immobilized FLuc or reagents containing separately immobilized FLuc and D-luciferase. The limit of detection of ATP by the developed reagents is 0.3 pM, which corresponds to 20,000 cells·mL−1. The linear response range is between 0.3 pM and 3 nM ATP. The multicomponent reagent, containing co-immobilized FLuc and D-luciferin, shows insignificantly lower sensitivity to ATP—0.6 pM. Moreover, the proposed method of producing an immobilized firefly luciferin-luciferase system holds considerable promise for the development of bioluminescent biosensors intended for the analysis of microbial contamination.

In this work, we considered the influence of viscogenic agents (glycerol, sucrose) as well as the temperature on the fluorescent characteristics of fluorescein at pH 6.5 in order to describe the acid-base status of local environment in terms of a spectrally detectable dianion-anion equilibrium. The protolytic equilibrium of fluorescein was found to depend on the solvent viscosity in a complex way. Whereas in the presence of sucrose the ratiometric signal of fluorescein (I488/I435) remains rather unchanged, the addition of glycerol (up to 40% w/w) results in the increase of the signal (up to 19%), that can be attributed to the different mechanisms of cosolvents effects on dye molecules in the ground state. Molecular dynamics of the dye in the presence of glycerol and sucrose revealed that the cosolvents preferentially interact with fluorescein monoanion and dianion, displacing water molecules from the local environment which in turn reduces the average number of the hydrogen bonds between xanthene ring of the dye and water molecules. The ratiometric signal demonstrates linear growth with the temperature in the range of 10–80 °C regardless of the presence of viscogenic agents. A linear correlation between the temperature sensitivity of the ratiometric signal and the change in the molar enthalpy of the proton dissociation reaction in buffer and viscous media was determined.

The present work is a review of the research on using hydrogels based on natural biodegradable polymers, starch, and gelatin for enzyme immobilization. This review addresses the main properties of starch and gelatin that make them promising materials in biotechnology for producing enzyme preparations stable during use and storage and insensitive to chemical and physical impacts. The authors summarize their achievements in developing the preparations of enzymes immobilized in starch and gelatin gels and assess their activity, stability, and sensitivity for use as biorecognition elements of enzyme inhibition-based biosensors.

Pesticides can affect the health of individual organisms and the function of the entire ecosystem. Therefore, thorough assessment of the risks associated with the use of pesticides is a high-priority task. An enzyme inhibition-based assay is used in this study as a convenient and quick tool to study the effects of pesticides at the molecular level. The contribution of formulants to toxicological properties of the pesticide formulations has been studied by analyzing effects of 7 active ingredients of pesticides (AIas) and 10 commercial formulations based on them (AIfs) on the function of a wide range of enzyme assay systems differing in complexity (single-, coupled, and three-enzyme assay systems). Results have been compared with the effects of AIas and AIfs on bioluminescence of the luminous bacterium Photobacterium phosphoreum. Mostly, AIfs produce a considerably stronger inhibitory effect on the activity of enzyme assay systems and bioluminescence of the luminous bacterium than AIas, which confirms the contribution of formulants to toxicological properties of the pesticide formulation. Results of the current study demonstrate that "inert" ingredients are not ecotoxicologically safe and can considerably augment the inhibitory effect of pesticide formulations; therefore, their use should be controlled more strictly. Circular dichroism and fluorescence spectra of the enzymes used for assays do not show any changes in the protein structure in the presence of commercial pesticide formulations during the assay procedure. This finding suggests that pesticides produce the inhibitory effect on enzymes through other mechanisms.

Chemoenzymatic assay systems are widely used to detect toxicants in various samples, including food and environment specimens. These methods are based on the ability of various types of toxicant to specifically inhibit/activate the functions of individual enzymes or enzyme systems. The present study examines the possibility of using the proteolytic enzyme trypsin as a specific marker to detect protease inhibitors in different samples. The study shows that trypsin activity is not affected by various heavy metals, pesticides, or quinones at levels considerably greater than their maximum allowable concentrations (MACs) in water bodies. At the same time, the IC50 value for the food preservative potassium sorbate (E202) is 15 mg/L, which is substantially lower than its acceptable daily intake (ADI). The quenching of trypsin fluorescence in the presence of potassium sorbate suggests that inhibition could occur due to the binding of the preservative to the enzyme in the region adjacent to the active center. The trypsin was immobilized in starch gel to ensure its stability in the enzyme inhibition based assay. Single-use reagents were prepared as dry starch disks that could be stored over long periods. Their sensitivity to copper (II) chloride, potassium sorbate, and chromium (III) chloride was similar to the sensitivity of the free trypsin.

A complex heterogeneous intracellular environment seems to affect enzymatic catalysis by changing the mobility of biomolecules, their stability, and their conformational states, as well as by facilitating or hindering continuously occurring interactions. The evaluation and description of the influence of the cytoplasmic matrix components on enzymatic activity are problems that remain unsolved. In this work, we aimed to determine the mechanisms of action of two-component media with cosolvents of various molecular sizes on the complex multi-stage bioluminescent reaction catalyzed by bacterial luciferase. Kinetic and structural effects of ethylene glycol, glycerol, sorbitol, glucose, sucrose, dextran, and polyethylene glycol on bacterial luciferase were studied using stopped-flow and fluorescence spectroscopy techniques and molecular dynamics simulations. We have found that diffusion limitations in the presence of cosolvents promote the stabilization of flavin substrate and peroxyflavin intermediate of the reaction, but do not provide any advantages in bioluminescence quantum yield, because substrate binding is slowed down as well. The catalytic constant of bacterial luciferase has been found to be viscosity-independent and correlated with parameters of water-cosolvent interactions (Norrish constant, van der Waals interaction energies). Crowding agents, in contrast to low-molecular-weight cosolvents, had little effect on peroxyflavin intermediate decay and enzyme catalytic constant. We attributed specific kinetic effects to the preferential interaction of the cosolvents with enzyme surface and their penetration into the active site.

Pesticides are commonly used in agriculture and are an important factor of food security for humankind. However, the overuse of pesticides can harm non-target organisms, and, thus, it is vital to comprehensively study their effects on the different metabolic pathways of living organisms. In the present study, enzyme-inhibition-based assays have been used to investigate the effects of commercial pesticide formulations on the key enzymes of the organisms, which catalyze a wide variety of metabolic reactions (protein catabolism, lactic acid fermentation, alcohol metabolism, the conduction of nerve impulses, etc.). Assay conditions have been optimized, and the limitations of the methods used in the study, which are related to the choice of the solvent for commercial pesticide formulations and optical effects occurring when commercial pesticide formulations are mixed with solutions of enzymes and substrates of assay systems, have been revealed. The effects of commercial pesticide formulations on simple chemoenzymatic assay systems (single-enzyme reactions) have been compared to their effects on complex multicomponent molecular systems (multi-enzyme reactions) and organisms (luminescent bacterium). The in vitro assay systems have shown higher sensitivity to pesticide exposure than the in vivo assay system. The sensitivity of the in vitro assay systems increases with the elongation of the chain of conjugated chemoenzymatic reactions. The effects exerted by commercial pesticide formulations with the same active ingredient but produced by different manufacturers on assay system functions have been found to differ from each other.

The rapid detection of pollutants in water can be performed with enzymatic probes, the catalytic light-emitting activity of which decreases in the presence of many types of pollutants. Herein, we present a microfluidic system for continuous chemoenzymatic biosensing that generates emulsion droplets containing two enzymes of the bacterial bioluminescent system (luciferase and NAD(P)H:FMN–oxidoreductase) with substrates required for the reaction. The developed chip generates “water-in-oil” emulsion droplets with a volume of 0.1 μL and a frequency of up to 12 drops per minute as well as provides the efficient mixing of reagents in droplets and their distancing. The bioluminescent signal from each individual droplet was measured by a photomultiplier tube with a signal-to-noise ratio of up to 3000/1. The intensity of the luminescence depended on the concentration of the copper sulfate with the limit of its detection of 5 μM. It was shown that bioluminescent enzymatic reactions could be carried out in droplet reactors in dispersed streams. The parameters and limitations required for the bioluminescent reaction to proceed were also studied. Hereby, chemoenzymatic sensing capabilities powered by a droplet microfluidics manipulation technique may serve as the basis for early-warning online water pollution systems.

A new method of producing butyrylcholinesterase (BChE) preparations, stable in storage and use, has been proposed. The BChE preparation is the enzyme co-immobilized with 0.2 M 5-5′-dithiobis (2-nitrobenzoic acid) in starch or gelatin gel. All experimental preparations retain enzyme activity for at least 300 d. The preparations based on gelatin gel show higher activity but lower sensitivity to the toxicants tested in this study compared to the starch gel-based preparations. A method has been proposed for integrated detection of anti-cholinesterase substances in aqueous solutions using the experimental preparation with immobilized BChE. After the additional incubation of the preparation with the immobilized enzyme in the solution of the analyte, the detection limits of malathion and pirimiphos-methyl determined using the IC20 values were below their maximum allowable concentrations—0.005 µM and 0.03 µM, respectively.

The presence of potentially toxic xenobiotics in complex matrices has become rather the rule than the exception. Therefore, there is a need for highly sensitive inexpensive techniques for analyzing environmental and food matrices for toxicants. Enzymes are selectively sensitive to various toxic compounds, and, thus, they can be used as the basis for detection of contaminants in complex matrices. There are, however, a number of difficulties associated with the analysis of complex matrices using enzyme assays, including the necessity to take into account properties and effects of the natural components of the test media for accurate interpretation of results. The present study describes the six-stage procedure for designing new enzyme sensors intended for assessing the quality of complex matrices. This procedure should be followed both to achieve the highest possible sensitivity of the biosensor to potentially toxic substances and to minimize the effect of the uncontaminated components of complex mixtures on the activity of the biosensor. The proposed strategy has been tested in designing a bioluminescent biosensor for integrated rapid assessment of the safety of fruits and vegetables. The biosensor is based on the coupled enzyme system NAD(P)H:FMN-oxidoreductase and luciferase as the biorecognition element. The study describes methods and techniques for attaining the desired result in each stage. The proposed six-stage procedure for designing bioluminescent enzyme biosensors can be used to design the enzymatic biosensors based on other enzymes.

The present study demonstrates a simple approach to enhancing thermal stability of butyrylcholinesterase (BChE) by using natural polymers. Analysis of thermal inactivation of the tetrameric BChE in starch and gelatin gels at 50–64 °C showed that thermal inactivation followed second-order kinetics and involved two alternating processes of BChE inactivation, which occurred at different rates (fast and slow processes). The activation enthalpy ΔH# and the activation entropy ΔS# for BChE in starch and gelatin gels were evaluated. The values of ΔH# for the fast and the slow thermal inactivation of BChE in starch gel were 61 ± 3, and 22 ± 2 kcal/mol, respectively, and the values of ΔS# were 136 ± 12 and −2.03 ± 0.05 cal∙K−1∙mol−1, respectively. Likewise, the values of ΔH# for BChE in gelatin gel were 58 ± 6 and 109 ± 11 kcal/mol, and the values of ΔS# were 149 ± 16 and 262 ± 21 cal∙K−1∙mol−1, respectively. The values of the activation parameters obtained in this study suggest that starch gel produced a stronger stabilizing effect on BChE exposed to elevated temperatures over long periods compared with gelatin gel.

Pesticides are commonly used in agriculture to enhance crop production and control pests. Therefore, pesticide residues can persist in the environment and agricultural crops. Although modern formulations are relatively safe to non-target species, numerous theoretical and experimental data demonstrate that pesticide residues can produce long-term negative effects on the health of humans and animals and stability of ecosystems. Of particular interest are molecular mechanisms that mediate the start of a cascade of adverse effects. This is a review of the latest literature data on the effects and consequences of contamination of agricultural crops by pesticide residues. In addition, we address the issue of implicit risks associated with pesticide formulations. The effects of pesticides are considered in the context of the Adverse Outcome Pathway concept.

Early detection of pollutants in wastewater, water coming out of treatment facilities, drinking water, and water for agricultural needs is a challenging problem. Effective water quality monitoring requires development of new methods for express detection of pollutants. Enzymes from bioluminescent bacteria can be used for the development of new express enzyme-based bioassay systems. This work demonstrates, for the first time, a microfluidic chip to generate emulsion droplets containing two enzymes of the bacterial bioluminescent system (luciferase and NAD(P)H:FMN-oxidoreductase) with reaction substrates. The developed chip generated “water-in-oil” emulsion droplets with a volume of 0.1 μL and a frequency of up to 12 droplets per second. A portable photomultiplier tube (PMT) was used to measure the bioluminescent signal in each individual droplet; the signal-to-noise ratio was 3000/1. The intensity of luminescence in droplets depended on the concentration of copper ions. The limit of detection (LOD) for copper sulfate was 1 mg/L. We showed that bioluminescent enzymatic reactions can be carried out in droplet reactors that can be applied for online monitoring of water quality. Thus, the suggested method of biological measurements has a good perspective for biosensing in general.

The regularities of the functioning of a number of enzymes in a viscous environment created by natural polymers, starch and gelatin are examined. Based on the analysis of kinetic curves of thermal inactivation, mechanisms of thermal inactivation of enzymes in a viscous microenvironment are proposed. Using the example of butyrylcholinesterase, NAD(P)H:FMN oxidoreductase, and coupled system of the luminous bacteria (NAD(P)H:FMN oxidoreductase + luciferase), the conditions, under which starch and gelatin have a stabilizing effect on enzyme activity during storage and exposure to various physical and chemical environmental factors, were found. A significant increase in the stabilizing effect is achieved by eliminating water during drying the enzyme preparations immobilized in starch and gelatin polymer gels.

The functioning of NAD(P)H:FMN‑oxidoreductase (Red) from Vibrio fischeri under conditions of macromolecular crowding (MMC) simulated in vitro by adding biopolymers (starch and gelatin) was studied. The dissociation rate constants and the activation energies of dissociation of Red to the subunits were calculated, and the process of denaturation of Red was analyzed. It is shown that the functioning of Red both under conditions of MMC and in diluted solutions is the same. This result refutes the common belief that the native conformation of enzymes in vivo is stabilized due to MMC as compared to the in vitro conditions.