Probing hot spots of protein-protein interactions mediated by the safety-belt region of REV7.

REV7 is a HORMA (Hop1, Rev7, Mad2) family adaptor protein best known as an accessory subunit of the translesion synthesis (TLS) DNA polymerase ζ (Polζ). In this role, REV7 binds REV3, the catalytic subunit of Polζ, by locking REV7-binding motifs (RBMs) in REV3 underneath the REV7 safety-belt loop. The same mechanism is used by REV7 to interact with RBMs from other proteins in DNA damage response (DDR) and mitosis. Because of the importance of REV7 for TLS and other DDR pathways, targeting REV7:RBM protein-protein interactions (PPIs) with small molecules has emerged as a strategy to enhance cancer response to genotoxic chemotherapy. To identify druggable pockets at the REV7:RBM interface, we performed computational analyses of REV7 complexed with several RBM partners. The contributions of different interface regions to REV7:RBM stabilization were corroborated experimentally. These studies provide insights into key intermolecular interactions and establish targetable regions of REV7 for the design of REV7:RBM PPI inhibitors.

High Affinity Inhibitors of the Macrophage Infectivity Potentiator Protein from Trypanosoma cruzi, Burkholderia pseudomallei, and Legionella pneumophila─A Comparison.

Since Chagas disease, melioidosis, and Legionnaires' disease are all potentially life-threatening infections, there is an urgent need for new treatment strategies. All causative agents, Trypanosoma cruzi, Burkholderia pseudomallei, and Legionella pneumophila, express a virulence factor, the macrophage infectivity potentiator (MIP) protein, emerging as a promising new therapeutic target. Inhibition of MIP proteins having a peptidyl-prolyl isomerase activity leads to reduced viability, proliferation, and cell invasion. The affinity of a series of pipecolic acid-type MIP inhibitors was evaluated against all MIPs using a fluorescence polarization assay. The analysis of structure-activity relationships led to highly active inhibitors of MIPs of all pathogens, characterized by a one-digit nanomolar affinity for the MIPs and a very effective inhibition of their peptidyl-prolyl isomerase activity. Docking studies, molecular dynamics simulations, and quantum mechanical calculations suggest an extended σ-hole of the meta-halogenated phenyl sulfonamide to be responsible for the high affinity.

Applying a Fluorescence Polarization Assay for Detection of Brucellosis in Animals Using the Fluorescently Labeled Synthetic Oligosaccharides as Biosensing Tracer.

Brucellosis in animals is an infectious disease caused by bacteria of the genus Brucella. Known methods for diagnosing brucellosis face some challenges, due to the difficulties in isolating and standardizing the natural brucellosis antigen. In this work, we investigated the possibility of using the fluorescence polarization assay (FPA) with synthetic glycoconjugate biosensing tracers to detect antibodies against Brucella as a new methodology for diagnosing brucellosis. Based on the received results, the synthetic fluorescein-labeled trisaccharide tracer is most effective for Brucellosis detection. This tracer is structurally related to the immune determinant fragment of the Brucella LPS buildup of N-formyl-d-perosamine units, connected via α-(1→3)-linkage at the non-reducing end and α-(1→2)-linkage at the reducing end. The sensitivity and specificity in the case of the use of trisaccharide tracer 3b were 71% and 100% (Yuden's method) and 87% and 88% (Euclidean method), respectively, which is comparable with the diagnostic efficiency of traditionally used serological methods, such as the agglutination test (AT), complement fixation test (CFT), and Rose Bengal test (RBT). Given the known advantages of FPA (e.g., speed, compactness of the equipment, and standard reagents) and the increased specificity of the developed test system, it would be appropriate to consider its widespread use for the diagnosis of brucellosis in animals, including rapid testing in the field.

High-Throughput Screening for LC3/GABARAP Binders Utilizing the Fluorescence Polarization Assay.

The characterization of interactions between autophagy modifiers (Atg8-family proteins) and their natural ligands (peptides and proteins) or small molecules is important for a detailed understanding of selective autophagy mechanisms and for the design of potential Atg8 inhibitors that affect the autophagy processes in cells. The fluorescence polarization (FP) assay is a rapid, cost-effective, and robust method that provides affinity and selectivity information for small molecules and peptide ligands targeting human Atg8 proteins.This chapter introduces the basic principles of FP assays. In addition, a case study on peptide interaction with human Atg8 proteins (LC3/GABARAPs) is described. Finally, data analysis and quality control of FP assays are discussed for the proper calculation of Ki values for the measured compounds.

Novel Quinazoline Derivatives as Highly Effective A2A Adenosine Receptor Antagonists.

The adenosine A2A receptor (A2AR) has been identified as a therapeutic target for treating neurodegenerative diseases and cancer. In recent years, we have highlighted the 2-aminoquinazoline heterocycle as an promising scaffold for designing new A2AR antagonists, exemplified by 6-bromo-4-(furan-2-yl)quinazolin-2-amine 1 (Ki (hA2AR) = 20 nM). Here, we report the synthesis of new 2-aminoquinazoline derivatives with substitutions at the C6- and C7-positions, and the introduction of aminoalkyl chains containing tertiary amines at the C2-position to enhance antagonist activity and solubility properties. Compound 5m showed a high affinity for hA2AR with a Ki value of 5 nM and demonstrated antagonist activity with an IC50 of 6 µM in a cyclic AMP assay. Introducing aminopentylpiperidine and 4-[(piperidin-1-yl)methyl]aniline substituents maintained the binding affinities (9x, Ki = 21 nM; 10d, Ki = 15 nM) and functional antagonist activities (9x, IC50 = 9 µM; 10d, IC50 = 5 µM) of the synthesized compounds while improving solubility. This study provides insights into the future development of A2AR antagonists for therapeutic applications.

Optimized methods for measuring competitive binding of chemical substances to thyroid hormone distributor proteins transthyretin and thyroxine binding globulin.

Transthyretin (TTR) and thyroxine-binding globulin (TBG) are two major thyroid hormone (TH) distributor proteins in human plasma, playing important roles in stabilizing the TH levels in plasma, delivery of TH to target tissues, and trans-barrier transport. Binding of xenobiotics to these distributor proteins can potentially affect all these three important roles of distributor proteins. Therefore, fast and cost-effective experimental methods are required for both TTR and TBG to screen both existing and new chemicals for their potential binding. In the present study, the TTR-binding assay was therefore simplified, optimized and pre-validated, while a new TBG-binding assay was developed based on fluorescence polarization as a readout. Seven model compounds (including positive and negative controls) were tested in the pre-validation study of the optimized TTR-binding assay and in the newly developed TBG-binding assay. The dissociation constants of the natural ligand (thyroxine, T4) and potential competitors were determined and compared between two distributor proteins, showing striking differences for perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA).

Click chemistry-based fluorescence polarization sensor for sensitive detection of ampicillin.

Ampicillin (AMP) is a ß-lactam antibiotic that can inhibit bacterial wall synthesis. The overuse and misuse of AMP makes it micropollutant that commonly found in food and various environmental media. In this work, a fluorescence polarization sensor was designed to sensitive detection of trace ampicillin based on click chemistry, using graphene oxide (GO) as a fluorescence polarization (FP) signal enhancement element. First, when ampicillin binds to its aptamer (apt), the adjacent alkyne and azide groups are separated, hindering the click-linking reaction. When Carboxyfluorescein (FAM) fluorophore-labeled probe (C-FAM) is added, its protruding 3-terminal FAM is recognized and cleaved by exonuclease I (EXO I), releasing fluorophores free that could not be adsorbed on GO, resulting in a lo0wer polarization signal. If there is no AMP in the system, aptamer probe is connected to its complementary chain ends by a click reaction. After C-FAM hybridizes with apt, the apt/P duplex is opened and the prominent single-stranded ends adsorb on the GO, leading a significantly enhanced FP signal. According to the relationship between the difference in FP values and the concentrations of AMP, the limit of detection of proposed method is as low as 80 pg/mL. This assay has a wide linear range plus excellent selectivity, and has been applied to detect AMP in milk and river water samples with satisfactory results, which demonstrates that the FP sensor has great potential for practical applications in food safety and environmental protection fields.

Deep learning-based cell segmentation for rapid optical cytopathology of thyroid cancer.

Fluorescence polarization (Fpol) imaging of methylene blue (MB) is a promising quantitative approach to thyroid cancer detection. Clinical translation of MB Fpol technology requires reduction of the data analysis time that can be achieved via deep learning-based automated cell segmentation with a 2D U-Net convolutional neural network. The model was trained and tested using images of pathologically diverse human thyroid cells and evaluated by comparing the number of cells selected, segmented areas, and Fpol values obtained using automated (AU) and manual (MA) data processing methods. Overall, the model segmented 15.8% more cells than the human operator. Differences in AU and MA segmented cell areas varied between - 55.2 and + 31.0%, whereas differences in Fpol values varied from - 20.7 and + 10.7%. No statistically significant differences between AU and MA derived Fpol data were observed. The largest differences in Fpol values correlated with greatest discrepancies in AU versus MA segmented cell areas. Time required for auto-processing was reduced to 10 s versus one hour required for MA data processing. Implementation of the automated cell analysis makes quantitative fluorescence polarization-based diagnosis clinically feasible.

A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated CD4 downregulation.

Therapeutic inhibition of the viral protein Nef is an intriguing direction of antiretroviral drug discovery-it may revitalize immune mechanisms to target, and potentially clear, HIV-1-infected cells. Of the many cellular functions of Nef, the most conserved is the downregulation of surface CD4, which takes place through Nef hijacking the clathrin adaptor protein complex 2 (AP2)-dependent endocytosis. Our recent crystal structure has unraveled the molecular details of the CD4-Nef-AP2 interaction. Guided by the new structural knowledge, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef's activity on CD4. In our assay, AP2 is included along with Nef to facilitate the proper formation of the CD4-binding pocket and a fluorescently labeled CD4 cytoplasmic tail binds competently to the Nef-AP2 complex generating the desired polarization signal. The optimized assay has a good signal-to-noise ratio, excellent tolerance of dimethylsulfoxide and detergent, and the ability to detect competitive binding at the targeted Nef pocket, making it suitable for high-throughput screening.

A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated MHC-I downregulation.

The multifunctional, HIV-1 accessory protein Nef enables infected cells to evade host immunity and thus plays a key role in viral pathogenesis. One prominent function of Nef is the downregulation of major histocompatibility complex class I (MHC-I), which disrupts antigen presentation and thereby allows the infected cells to evade immune surveillance by the cytotoxic T cells. Therapeutic inhibition of this Nef function is a promising direction of antiretroviral drug discovery as it may revitalize cytotoxic T cells to identify, and potentially clear, hidden HIV-1 infections. Guided by the crystal structure of the protein complex formed between Nef, MHC-I, and the hijacked clathrin adaptor protein complex 1, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef's activity on MHC-I. The optimized assay has a good signal-to-noise ratio, substantial tolerance of dimethylsulfoxide, and excellent ability to detect competitive inhibition, indicating that it is suitable for high-throughput screening.

Fluorescence polarization assay for screening FANCM-RMI inhibitors to target the alternative lengthening of telomeres.

Alternative Lengthening of Telomeres (ALT) is a mechanism used by 10-15% of all cancers to achieve replicative immortality, bypassing the DNA damage checkpoint associated with short telomeres that leads to cellular senescence or apoptosis. ALT does not occur in non-cancerous cells, presenting a potential therapeutic window for cancers where this mechanism is active. Disrupting the FANCM-RMI interaction has emerged as a promising therapeutic strategy that induces synthetic ALT lethality in genetic studies on cancer cell lines. There are currently no chemical inhibitors reported in the literature, in part due to the lack of reliable biophysical or biochemical assays to screen for FANCM-RMI disruption. Here we describe the development of a robust competitive fluorescence polarization (FP) assay that quantifies target binding at the FANCM-RMI interface. The assay employs a labeled peptide tracer TMR-RaMM2 derived from the native MM2 binding motif, which binds to recombinant RMI1-RMI2 and can be displaced by competitive inhibitors. We report the methods for recombinant production of RMI1-RMI2, design and evaluation of the tracer TMR-RaMM2, along with unlabeled peptide inhibitor controls to enable ALT-targeted drug discovery.

Determining the Affinity and Kinetics of Small Molecule Inhibitors of Galectin-1 Using Surface Plasmon Resonance.

The beta-galactoside-binding mammalian lectin galectin-1 can bind, via its carbohydrate recognition domain (CRD), to various cell surface glycoproteins and has been implicated in a range of cancers. As a consequence of binding to sugar residues on cell surface receptors, it has been shown to have a pleiotropic effect across many cell types and mechanisms, resulting in immune system modulation and cancer progression. As a result, it has started to become a therapeutic target for both small and large molecules. In previous studies, we used fluorescence polarization (FP) assays to determine KD values to screen and triage small molecule glycomimetics that bind to the galectin-1 CRD. In this study, surface plasmon resonance (SPR) was used to compare human and mouse galectin-1 affinity measures with FP, as SPR has not been applied for compound screening against this galectin. Binding affinities for a selection of mono- and di-saccharides covering a 1000-fold range correlated well between FP and SPR assay formats for both human and mouse galectin-1. It was shown that slower dissociation drove the increased affinity at human galectin-1, whilst faster association was responsible for the effects in mouse galectin-1. This study demonstrates that SPR is a sound alternative to FP for early drug discovery screening and determining affinity estimates. Consequently, it also allows association and dissociation constants to be measured in a high-throughput manner for small molecule galectin-1 inhibitors.

Polycations as Aptamer-Binding Modulators for Sensitive Fluorescence Anisotropy Assay of Aflatoxin B1.

Fluorescence induced by the excitation of a fluorophore with plane-polarized light has a different polarization depending on the size of the fluorophore-containing reagent and the rate of its rotation. Based on this effect, many analytical systems have been implemented in which an analyte contained in a sample and labeled with a fluorophore (usually fluorescein) competes to bind to antibodies. Replacing antibodies in such assays with aptamers, low-cost and stable oligonucleotide receptors, is complicated because binding a fluorophore to them causes a less significant change in the polarization of emissions. This work proposes and characterizes the compounds of the reaction medium that improve analyte binding and reduce the mobility of the aptamer-fluorophore complex, providing a higher analytical signal and a lower detection limit. This study was conducted on aflatoxin B1 (AFB1), a ubiquitous toxicant contaminating foods of plant origins. Eight aptamers specific to AFB1 with the same binding site and different regions stabilizing their structures were compared for affinity, based on which the aptamer with 38 nucleotides in length was selected. The polymers that interact reversibly with oligonucleotides, such as poly-L-lysine and polyethylene glycol, were tested. It was found that they provide the desired reduction in the depolarization of emitted light as well as high concentrations of magnesium cations. In the selected optimal medium, AFB1 detection reached a limit of 1 ng/mL, which was 12 times lower than in the tris buffer commonly used for anti-AFB1 aptamers. The assay time was 30 min. This method is suitable for controlling almond samples according to the maximum permissible levels of their contamination by AFB1. The proposed approach could be applied to improve other aptamer-based analytical systems.

Establishment of an at-line nanofractionation-based screening platform by coupling HPLC-MS/MS with high-throughput fluorescence polarization bioassay for natural SARS-CoV-2 fusion inhibitors.

In this study, a novel at-line nanofractionation platform was established for screening SARS-CoV-2 fusion inhibitors from natural products for the first time by combining HPLC-MS/MS with high-throughput fluorescence polarization (FP) bioassay. A time-course FP bioassay in 384 well-plates was conducted in parallel with MS/MS to simultaneously obtain chemical and biological information of potential fusion inhibitors in Lonicerae Japonicae Flos (LJF) and Lianhua Qingwen capsules (LHQW). Semi-preparative liquid chromatography and orthogonal HPLC separation were employed to enrich and better identify the co-eluted components. After comprehensive evaluation and validation, 28 potential SARS-CoV-2 fusion inhibitors were screened out and identified. Several compounds at low micromolar activity were validated by in vitro inhibitory assay, molecular docking, cytotoxicity test, and pseudovirus assay. Moreover, four potential dual-target inhibitors against influenza and COVID-19 were discovered from LJF using this method, offering novel insights for the development of future pharmaceuticals targeting epidemic respiratory diseases.

The role of the size of affinity ligands in the detection and characterization of extracellular vesicles.

Surface proteins on the membrane of nano-sized extracellular vesicles (EVs) not only play crucial roles in cell-to-cell communication, but also are specific binding targets for EV detection, isolation and tracking. The low abundance of protein biomarkers on EV surface, the formation of clusters and the complex EV surface network impose significant challenges to the study of EVs. Employing bulky sized affinity ligands, such as antibodies, in the detection and characterization of these vesicles often result in reduced sensitivity of detection or poor quantification of proteins on the EV surface. By virtue of their small size and high specificity, Affibody molecules emerge as a potential alternative to their monoclonal antibody counterparts as robust affinity ligands in EV research. In this study, we present a theoretical framework on the superiority of anti-HER2 Affibodies over anti-HER2 antibodies in labeling and detecting HER2-positive EVs, followed by the demonstration of the advantages of HER2 Affibodies in accessing EV surface and the detection of EVs through multiple types of approaches including fluorescence intensity, colorimetry, and fluorescence polarization. HER2 Affibodies outperformed by 10-fold over three HER2 antibody clones in accessing HER2-positive EVs derived from different human cancer cell lines. Furthermore, HRP-Affibody molecules could detect EVs from cancer cells spiked into human serum with at least a 2-fold higher sensitivity compared with that of their antibody counterparts. In addition, in fluorescence polarization assays in which no separation of free from bound ligand is required, FITC-labeled HER2 Affibodies could sensitively detect HER2-positive EVs with a clinically relevant limit of detection, whilst HER2 antibodies failed to detect EVs in the same conditions. With the demonstrated superiority in accessing and detecting surface targets over bulky-sized antibodies in EVs, Affibodies may become the next-generation of affinity ligands in the precise characterization and quantification of molecular architecture on the surface of EVs.

Discovery and validation of a novel inhibitor of HYPE-mediated AMPylation.

Adenosyl monophosphate (AMP)ylation (the covalent transfer of an AMP from Adenosine Triphosphate (ATP) onto a target protein) is catalyzed by the human enzyme Huntingtin Yeast Interacting Partner E (HYPE)/FicD to regulate its substrate, the heat shock chaperone binding immunoglobulin protein (BiP). HYPE-mediated AMPylation of BiP is critical for maintaining proteostasis in the endoplasmic reticulum and mounting a unfolded protein response in times of proteostatic imbalance. Thus, manipulating HYPE's enzymatic activity is a key therapeutic strategy toward the treatment of various protein misfolding diseases, including neuropathy and early-onset diabetes associated with two recently identified clinical mutations of HYPE. Herein, we present an optimized, fluorescence polarization-based, high-throughput screening (HTS) assay to discover activators and inhibitors of HYPE-mediated AMPylation. After challenging our HTS assay with over 30,000 compounds, we discovered a novel AMPylase inhibitor, I2.10. We also determined a low micromolar IC50 for I2.10 and employed biorthogonal counter-screens to validate its efficacy against HYPE's AMPylation of BiP. Further, we report low cytotoxicity of I2.10 on human cell lines. We thus established an optimized, high-quality HTS assay amenable to tracking HYPE's enzymatic activity at scale, and provided the first novel small-molecule inhibitor capable of perturbing HYPE-directed AMPylation of BiP in vitro. Our HTS assay and I2.10 compound serve as a platform for further development of HYPE-specific small-molecule therapeutics.

Dsg2 ectodomain organization increases throughout desmosome assembly.

Desmosomes are intercellular junctions that regulate mechanical integrity in epithelia and cardiac muscle. Dynamic desmosome remodeling is essential for wound healing and development, yet the mechanisms governing junction assembly remain elusive. While we and others have shown that cadherin ectodomains are highly organized, how this ordered architecture emerges during assembly is unknown. Using fluorescence polarization microscopy, we show that desmoglein 2 (Dsg2) ectodomain order gradually increases during 8 h of assembly, coinciding with increasing adhesive strength. In a scratch wound assay, we observed a similar increase in order in desmosomes assembling at the leading edge of migratory cells. Together, our findings indicate that cadherin organization is a hallmark of desmosome maturity and may play a role in conferring adhesive strength.

Discovery of a potent inhibitor, D-132, targeting AsfvPolX, via protein-DNA complex-guided pharmacophore screening and in vitro molecular characterizations.

The heightened transmissibility and capacity of African swine fever virus (ASFV) induce fatal diseases in domestic pigs and wild boars, posing significant economic repercussions and global threats. Despite extensive research efforts, the development of potent vaccines or treatments for ASFV remains a persistent challenge. Recently, inhibiting the AsfvPolX, a key DNA repair enzyme, emerges as a feasible strategy to disrupt viral replication and control ASFV infections. In this study, a comprehensive approach involving pharmacophore-based inhibitor screening, coupled with biochemical and biophysical analyses, were implemented to identify, characterize, and validate potential inhibitors targeting AsfvPolX. The constructed pharmacophore model, Phar-PolX-S, demonstrated efficacy in identifying a potent inhibitor, D-132 (IC50 = 2.8 ± 0.2 µM), disrupting the formation of the AsfvPolX-DNA complex. Notably, D-132 exhibited strong binding to AsfvPolX (KD = 6.9 ± 2.2 µM) through a slow-on-fast-off binding mechanism. Employing molecular modeling, it was elucidated that D-132 predominantly binds in-between the palm and finger domains of AsfvPolX, with crucial residues (R42, N48, Q98, E100, F102, and F116) identified as hotspots for structure-based inhibitor optimization. Distinctively characterized by a 1,2,5,6-tetrathiocane with modifications at the 3 and 8 positions involving ethanesulfonates, D-132 holds considerable promise as a lead compound for the development of innovative agents to combat ASFV infections.

Establishment, optimization and validation of a fluorescence polarization-based high-throughput screening assay targeting cathepsin L inhibitors.

Cathepsin L (CTSL), a lysosomal cysteine proteinase, is primarily dedicated to the metabolic turnover of intracellular proteins. It is involved in various physiological processes and contributes to pathological conditions such as viral infection, tumor invasion and metastasis, inflammatory status, atherosclerosis, renal disease, diabetes, bone diseases, and other ailments. The coronavirus disease 2019 (COVID-19), with its rapid global spread and significant mortality, has been a worldwide epidemic since the late 2019s. Notably, CTSL plays a role in the processing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, providing a potential avenue to block coronavirus host cell entry and thereby inhibit SARS-CoV-2 infection in humans. In this study, we have developed a novel method using fluorescence polarization (FP) for screening CTSL inhibitors in a high-throughput format. The optimized assay demonstrated its appropriateness for high-throughput screening (HTS) with a Z-factor of 0.9 in a 96-well format. Additionally, the IC50 of the known inhibitor, Z-Phe-Tyr-CHO, was determined to be 188.50 ± 46.88 nM. Upon screening over 2000 small molecules, we identified, for the first time, the anti-CTSL properties of a benzothiazoles derivative named IMB 8015. This work presents a novel high-throughput approach and its application in discovering and evaluating CTSL inhibitors.

Fluorescence Polarization Assays for Organic Compounds in Food Safety.

Elevated concentrations of toxic organic compounds observed in food products pose serious dangers to human health. Both natural and artificial pollutants can cause food contamination. The stages of food production, packaging, transportation, and storage can also largely cause the appearance of undesirable substances in food products. The health consequences of ingesting food containing toxic contaminants range from mild gastroenteritis to deaths resulting from dysfunctional internal organs and neurological syndromes. The World Health Organization (WHO) sets recommendations for the content of such chemicals in food, including a minimum allowable concentration considered safe for human consumption. However, the control of food products from chemical pollutants is necessary. Moreover, fast, sensitive, and inexpensive methods are needed to detect them at the point of need. Currently, immune analysis methods are most widely used to determine pollutants in food. The development of fluorescence polarization immunoassay (FPIA) methods in a competitive format is a powerful and modern tool for detecting organic molecules in various matrices, thereby making FPIA methods useful for food safety applications. Due to the availability of portable devices for measuring the fluorescence polarization signal, FPIA methods can be used at the point of need. The variety of fluorescent labels and recognizing elements (receptors, monoclonal and polyclonal antibodies, and nanobodies) permits fluorescence polarization (FP) assays to detect significantly lower limits of organic substances. The FP assay is a homogeneous, fast, and quantitative method. The development of various formats of FP assays makes them promising in determining food pollutants. This review summarizes publications on FP analyses for detecting organic contaminants (pesticides, hormones, toxins, antibiotics, and other pharmaceuticals) in food products during 2018-2023. Further, it demonstrates the prospects for using this method to determine pollutants at the point of need and for detecting high molecular weight substances, fungi, and bacterial infections during food safety inspections.