Free Thiol Groups Research Articles

Effect of pH and temperature on fibrous structure formation of plant proteins during high-moisture extrusion processing

This study investigated the effect of pH on fibrous structure formation (protein alignment) during high-moisture extrusion processing of gluten, rice protein, as well as pea protein concentrate, and isolate. The pH of the raw material was shifted to 5 and 7 in water suspension with an acid or base and freeze-dried, after which, conductivity, solubility, water-holding capacity, particle size, and pH were measured. The pH-shifted raw materials were extruded at various temperatures (95–160 °C) and the extrudates were analysed for protein alignment (macro and microstructure), tensile strength, free thiol groups, and cooking properties. In general, all raw materials generated fibrous structure at lower temperatures (115–140 °C) at pH 7 than at pH 5 (135–160 °C). Higher pH and temperature values led to an increased tensile strength and pronounced protein alignment. No such unambiguous link could be observed between the raw material properties and enhanced structure formation. This study showed that the structure formation of the extrudate can be positively influenced by increasing the pH of the raw material, which facilitates the plant protein structuring into appealing meat analogue products.

Plasma glycation adducts and various RAGE isoforms are intricately associated with oxidative stress and inflammatory markers in type 2 diabetes patients with vascular complications

BackgroundThe secondary vascular complications in diabetes mellitus (DM) are contributed by acute as well as inflammatory responses which get activated due to interaction between glycation adducts and respective receptors. AimThe present work was performed to understand the relationship between Advanced glycation end products (AGEs)-receptor for advanced glycation end products (RAGE) interaction with oxidative stress and inflammation in vascular complications. MethodsFor the present work we recruited 103 controls, 200 patients with type 2 DM, and 200 patients with Diabetic complications. Different Plasma glycation adducts (fructosamine, carbonyls, AGEs, β-amyloid content, free amino groups, and free thiol groups); RAGE isoforms, level of antioxidant such as glutathione, catalase activity, nitric oxide level, total antioxidant capacity, and superoxide dismutase activity, as well as oxidative markers, and expression of Nε-carboxymethyl-lysine (CML), different isoforms of RAGE, NF-κB, and inflammatory markers were analyzed. ResultsGlycation adducts were higher in DM patients and more elevated in nephropathy patients where free amino groups and thiol groups lowered as compared to controls. sRAGE levels and expression were increased mainly in nephropathy. CML expression was higher in nephropathy patients. The antioxidant profile indicates a reduced level of different antioxidants while increased lipid peroxidation and intracellular ROS generation in DM and much higher in nephropathy patients. Expression of membrane RAGE, NF-κB, and inflammatory markers showed a remarkably increased level in DM patients with nephropathy. ConclusionThis work provides the first evidence of four different RAGE isoforms in diabetes and in complications. The glycation via the activation of RAGE, oxidative stress, and resultant inflammation plays a crucial role in the development of diabetic complications.

Building Polyvalent DNA-Functionalized Anisotropic AuNPs using Poly-Guanine-Mediated In-Situ Synthesis for LSPR-Based Assays: Case Study on OncomiR-155.

DNA-functionalized gold nanoparticles (DNA-AuNPs) hold great promise for numerous biomedical applications, especially the building of well-defined nanosystems. Previously reported methods for the preparation of DNA-AuNPs all rely on the use of DNA-bearing free thiol or disulfide groups at their 3'/5' ends. But here we report a novel polyvalent DNA-AuNPs conjugation approach by in-situ fast synthesis of AuNPs at the polyguanine (G12 ) strands. As confirmed by both TEM images and gel electrophoresis analysis, many poly G strand can form an individual anisotropic AuNP and so each AuNP functionalized with a dense layer of DNA, resulting in the formation of polyvalent (p)DNA-AuNPs. The general applicability of this novel approach was further verified in hybridization test and UV-Vis spectroscopy results show that pDNA-AuNPs conjugation is more attractive in biomedical diagnosis and specific sequence detection like microRNA-155 by using an extra-strand poly G with "sticky end" that are complementary to the target sequence.

Lymph-Directed Self-Immolative Nitric Oxide Prodrug for Inhibition of Intractable Metastatic Cancer.

There has been a significant clinical demand for lymph‐directed anti‐metastatic therapy as tumor‐draining lymph nodes play pivotal roles in cancer metastasis which accounts for more than 90% of tumor‐related deaths. Despite the high potential of nitric oxide (NO) in anti‐cancer therapy owing to its biocompatibility and tumor cell‐specific cytotoxicity, the poor stability and lack of target specificity of present NO donors and delivery systems have limited its clinical applications. Herein, a redox‐triggered self‐immolative NO prodrug that can be readily conjugated to various materials containing free thiol groups such as albumin, is reported. The prodrug and its conjugates demonstrate smart release of NO donor via intramolecular cyclization under reductive conditions, followed by spontaneously generating NO in physiological conditions. The albumin‐prodrug conjugate inhibits tumor metastasis by inducing cytotoxicity preferentially on tumor cells after efficiently draining into lymph nodes. This novel prodrug can contribute to the development of on‐demand NO delivery systems for anti‐metastatic therapy and other treatments.

Pickering emulsions by regulating the molecular interactions between gelatin and catechin for improving the interfacial and antioxidant properties

Molecular interaction forces regulate the interfacial properties and play a leading role in the distribution of particles on the oil-water interface and the stability of emulsions. In this work, gelatin/catechin nanoparticles (GCNPs) were prepared at different pH and mass ratios. The GCNPs showed excellent interfacial properties when the mass ratio of gelatin to catechin was 8:1 at pH 7. The contents of free thiol and amino groups were 0.36 μmol/g and 0.38 ± 0.03 mmol/mL, respectively, and the oil-water contact angle (θow) was 61.59° at 0 min. GCNPs (1% w/v) were applied as Pickering emulsifiers to stabilize the pecan oil emulsions, the turbiscan stability index (TSI), rheological properties, and lipid peroxidation inhibition capacity were measured. The emulsion at the oil fraction (φ) value of 0.7 (v/v) had a good thixotropic recovery of 74%, lower ROOH value, and the higher inhibition rate of thiobarbituric acid reactive substance (TBARS). It was demonstrated that the φ addition enlarged the viscosity and modulus of Pickering emulsions, led to a gel-like structure and improved the emulsions stability. Our findings on regulating the non-covalent and covalent interactions between gelatin and catechin, provided novel insights about the formation of protein-polyphenol nanoparticles. These nanoparticles might serve as excellent food-grade Pickering emulsifiers for enhancing the interfacial and antioxidant properties of Pickering emulsions with a high φ.

A New Practice to Monitor the Fabrication Process of Fab-Targeting Ligands from Bevacizumab by LC-MS: Preparation and Analytical Characterization

The objective of this study was to qualitatively evaluate a Fab-targeting ligand preparation containing free thiol groups in the hinge region by using bevacizumab as a model. The evaluation focused on the purification of fragments through a nonaffinity-based process using a centrifugal ultrafiltration technique and mild reduction conditions for the intact production of F(ab’) fragments with specific inter-heavy-chain disulfide bonds cleavage. Under these conditions, F(ab’) fragments with a defined chemical composition were successfully obtained via proteolytic digestion followed by a controlled reduction reaction process maintaining the integrity of the binding sites. The ultrafiltration purification technique appears to be suitable for the removal of the digestive enzyme but inefficient for the removal of Fc fragments, thus requiring additional processing. A suitable analytical strategy was developed, allowing us to demonstrate the reformation of disulfide bridges between the two reduced cysteines within F(ab’) fragments.

Attenuation of Liver Injury via Bicyclol–Bovine Serum Albumin Nanopreparation using a Green Synthetic Approach

Bicyclol (BIC) is a traditional antihepatitis drug that is used to treat chronic hepatitis B infections by improving liver function and reducing transaminase levels. Because the extensive use of BIC for treating liver injury is limited by its poor bioavailability, the course of treatment with oral BIC lasted for >6 months. This study aimed to develop a nano‐BIC injection to improve its bioavailability and anti‐hepatitis efficacy. We used a green synthetic approach to prepare BIC‐bovine serum albumin (BSA) nanoparticles (BIC‐NPs). Moderate protein denaturation is required to release free thiol groups in the intramolecular reactions of BSA, which may form an intermolecular disulfide network to stabilize the BIC–BSA nanoassembly. Our results showed that the administration of BIC‐NPs in rats resulted in a 2.42‐fold increase in drug concentration in plasma. Further, nano‐BIC injection showed high bioavailability and rapidly achieved therapeutic concentrations. BIC can restore mitochondrial function by scavenging reactive oxygen species. Moreover, we observed that 70 nm NPs can accumulate in the liver, and the nano‐BIC injection protected mice from methotrexate‐induced liver injury. This study provides an improved strategy for developing a liver‐protecting agent to meet various clinical needs of patients.

Dynamic hyaluronic acid hydrogel with covalent linked gelatin as an anti-oxidative bioink for cartilage tissue engineering

In the past decade, cartilage tissue engineering has arisen as a promising therapeutic option for degenerative joint diseases, such as osteoarthritis, in the hope of restoring the structure and physiological functions. Hydrogels are promising biomaterials for developing engineered scaffolds for cartilage regeneration. However, hydrogel-delivered mesenchymal stem cells or chondrocytes could be exposed to elevated levels of reactive oxygen species (ROS) in the inflammatory microenvironment after being implanted into injured joints, which may affect their phenotype and normal functions and thereby hinder the regeneration efficacy. To attenuate ROS induced side effects, a multifunctional hydrogel with an innate anti-oxidative ability was produced in this study. The hydrogel was rapidly formed through a dynamic covalent bond between phenylboronic acid grafted hyaluronic acid (HA-PBA) and poly(vinyl alcohol) and was further stabilized through a secondary crosslinking between the acrylate moiety on HA-PBA and the free thiol group from thiolated gelatin. The hydrogel is cyto-compatible and injectable and can be used as a bioink for 3D bioprinting. The viscoelastic properties of the hydrogels could be modulated through the hydrogel precursor concentration. The presence of dynamic covalent linkages contributed to its shear-thinning property and thus good printability of the hydrogel, resulting in the fabrication of a porous grid construct and a meniscus like scaffold at high structural fidelity. The bioprinted hydrogel promoted cell adhesion and chondrogenic differentiation of encapsulated rabbit adipose derived mesenchymal stem cells. Meanwhile, the hydrogel supported robust deposition of extracellular matrix components, including glycosaminoglycans and type II collagen, by embedded mouse chondrocytes in vitro. Most importantly, the hydrogel could protect encapsulated chondrocytes from ROS induced downregulation of cartilage-specific anabolic genes (ACAN and COL2) and upregulation of a catabolic gene (MMP13) after incubation with H2O2. Furthermore, intra-articular injection of the hydrogel in mice revealed adequate stability and good biocompatibility in vivo. These results demonstrate that this hydrogel can be used as a novel bioink for the generation of 3D bioprinted constructs with anti-ROS ability to potentially enhance cartilage tissue regeneration in a chronic inflammatory and elevated ROS microenvironment.

Effect of wooden breast degree on lipid and protein oxidation and citrate synthase activity of chicken pectoralis major muscle

In this study, 154 pectoralis major breast muscles from three chicken age groups were separated into different severity levels of wooden breast condition by manual palpation to investigate the changes of lipid and protein oxidation and citrate synthase activity. TBARS values, carbonyl contents and loss of free thiol groups were greater (P < 0.05) in the superficial (ventral) part of severe wooden breast samples, whereas no clear change was found in the deep (dorsal) part. Mitochondrial citrate synthase activity declined with increasing severity of wooden breast myopathy. Both bird age and wooden breast degree showed significant main effects on these indicators, while no interaction effect was observed. Moreover, there were significant correlations between lipid and protein oxidation and citrate synthase activity. Here, we propose a hypothesis that lipid and protein damages and mitochondrial dysfunction is linked to the aggravation of the wooden breast myopathy.

The interaction of allicin with bovine serum albumin and its influence on the structure of protein

The interaction of allicin with bovine serum albumin (BSA) and the effect of allicin binding on the structure of protein were investigated. The BSA fluorescence displayed decreased fluorescence strength after binding with allicin, which quenched BSA fluorescence in a concentration-dependent manner. The number of binding sites and binding constants were measured by fluorescent quenching method. The result of reverse phase HPLC showed that the peak of BSA shifted to higher retention time, which indicated that allicin had bound to BSA. 88.4 % of total free thiol groups of BSA have bound to allicin at the allicin/BSA molar ratio of 0.1. Allicin could destabilize the secondary and tertiary structure of BSA. At the allicin/BSA ratio of 0.1, the BSA's circular dichroism spectrum showed that allicin caused a decrease of α-helix content and a significant increase of the β-sheet content (p < 0.05). Changes in surface hydrophobicity indicated that more hydrophobic groups were exposed due to the action of allicin. These results provide important insight into the study of prevailing role of protein in drug pharmacokinetics and pharmacodynamics.

Hemp globulin forms colloidal nanocomplexes with sodium caseinate during pH-cycling

Seed from industrial hemp (Cannabis sativa L.) contains around 25% protein (mainly globulins) which is easily digested, but the low solubility of hemp globulins (HG) limits their application in many food systems. In this study, the solubility of HG was improved by blending HG with sodium caseinate (SC) and treating with a pH-cycling process. The pH-cycling involved adjusting the pH to 12 and reacting for 1 hr, followed by neutralisation to pH 7. Nanoparticles composed of HG and SC (Z-average diameter ≈ 130 nm) were formed after the pH-cycling, and the solubility of HG increased to > 80% when there was more than 1% of SC for 1% of HG. These HG|SC nanoparticles were monodisperse (PDI < 0.17) and ζ-potential was ≈ −17 mV. Hydrogen bonding is the main forces that assembles HG|SC nanoparticles because the nanoparticles dissociated by heat treatment (up to 60 °C) or urea, which is an effective hydrogen bond breaker. HG|SC nanoparticles will aggregate irreversibly above 60 °C, possibly due to thiol-disulphide exchange. The nanoparticles were heat-stable as the Z-average diameter was only 229 nm after heating (90 °C, 30 min). N-ethylmaleimide blocked free thiol groups on HG and resulted in less disulphide-linked HG aggregation after pH- cycling, which in turn lead to smaller HG|SC nanoparticles and a bimodal particle size distribution, indicating the importance of disulphide bond for the formation of monodisperse HG|SC nanoparticles. The soluble and heat-stable HG|SC nanoparticles could be used to increase the hemp protein content in beverages and emulsions.

Antiglycoxidative Properties of Extracts and Fractions from Reynoutria Rhizomes.

Hyperglycemia, when sustained over a long time in diabetes mellitus (DM), leads to biochemical and cellular abnormalities, primarily through the formation of advanced glycation end-products (AGEs). In the treatment of diabetes, beside blood-sugar-lowering medications, a consumption of herbal products that can inhibit the AGEs’ formation is recommended. This study investigated the in vitro antiglycoxidative potential of extracts and fractions from the rhizomes of Japanese, Giant, and Bohemian knotweeds (Reynoutria japonica (Houtt.), R. sachalinensis (F. Schmidt) Nakai, and R.× bohemica Chrtek et Chrtkova). Their effects on glycooxidation of bovine and human serum albumin were evaluated by incubation of the proteins with a mixture of glucose and fructose (0.5 M) and 150 µg/mL of extract for 28 days at 37 °C, followed by measuring early and late glycation products, albumin oxidation (carbonyl and free thiol groups), and amyloid-β aggregation (thioflavin T and Congo red assays). The highest antiglycoxidative activity, comparable or stronger than the reference drug (aminoguanidine), was observed for ethyl acetate and diethyl ether fractions, enriched in polyphenols (stilbenes, phenylpropanoid disaccharide esters, and free and oligomeric flavan-3-ols). In conclusion, the antiglycoxidative compounds from these three species should be further studied for potential use in the prevention and complementary treatment of DM.

Structural insights into the enhanced thermostability of cysteine substitution mutants of L-histidine decarboxylase from Photobacterium phosphoreum.

Enzymatic amino acid assays are important in physiological research and clinical diagnostics because abnormal amino acid concentrations in biofluids are associated with various diseases. L-histidine decarboxylase from Photobacterium phosphoreum (PpHDC) is a pyridoxal 5'-phosphate-dependent enzyme and a candidate for use in an L-histidine quantitation assay. Previous cysteine substitution experiments demonstrated that the PpHDC C57S mutant displayed improved long-term storage stability and thermostability when compared with those of the wild-type enzyme. In this study, combinational mutation experiments of single cysteine substitution mutants of PpHDC were performed, revealing that the PpHDC C57S/C101V/C282V mutant possessed the highest thermostability. The stabilizing mechanism of these mutations was elucidated by solving the structures of PpHDC C57S and C57S/C101V/C282V mutants by X-ray crystallography. In the crystal structures, two symmetry-related PpHDC molecules form a domain-swapped homodimer. The side chain of S57 is solvent exposed in the structure, indicating that the C57S mutation eliminates chemical oxidation or disulfide bond formation with a free thiol group, thereby providing greater stability. Residues 101 and 282 form hydrophobic interactions with neighboring hydrophobic residues. Mutations C101V and C282V enhanced thermostability of PpHDC by filling a cavity present in the hydrophobic core (C101V) and increasing hydrophobic interactions.

Topical Delivery of Rapamycin by Means of Microenvironment-Sensitive Core-Multi-Shell Nanocarriers: Assessment of Anti-Inflammatory Activity in an ex vivo Skin/T Cell Co-Culture Model.

IntroductionRapamycin (Rapa) is an immunosuppressive macrolide that inhibits the mechanistic target of rapamycin (mTOR) activity. Thanks to its anti-proliferative effects towards different cell types, including keratinocytes and T cells, Rapa shows promise in the treatment of skin diseases characterized by cell hyperproliferation. However, Rapa skin penetration is limited due to its lipophilic nature (log P = 4.3) and high molecular weight (MW = 914 g/mol). In previous studies, new microenvironment-sensitive core multishell (CMS) nanocarriers capable of sensing the redox state of inflamed skin were developed as more efficient and selective vehicles for macrolide delivery to inflamed skin.MethodsIn this study, we tested such redox-sensitive CMS nanocarriers using an inflammatory skin model based on human skin explants co-cultured with Jurkat T cells. Serine protease (SP) was applied on skin surface to induce skin barrier impairment and oxidative stress, whereas phytohaemagglutinin (PHA), IL-17A, and IL-22 were used to activate Jurkat cells. Activation markers, such as CD45 and CD69, phosphorylated ribosomal protein S6 (pRP-S6), and IL-2 release were monitored in activated T cells, whereas pro-inflammatory cytokines were measured in skin extracts and culture medium.ResultsWe found that alteration of skin barrier proteins corneodesmosin (CDSN), occludin (Occl), and zonula occludens-1 (ZO-1) as well as oxidation-induced decrease of free thiol groups occurred upon SP-treatment. All Rapa formulations exerted inhibitory effects on T cells after penetration across ex vivo skin. No effects on skin inflammatory markers were detected. The superiority of the oxidative-sensitive CMS nanocarriers over the other formulations was observed with regard to drug delivery as well as downregulation of IL-2 release.ConclusionOverall, our results demonstrate that nanocarriers addressing features of diseased skin are promising approaches to improve the topical delivery of macrolide drugs.

Hierarchically Porous Microspheres by Thiol-ene Photopolymerization of High Internal Phase Emulsions-in-Water Colloidal Systems.

A facile method for the preparation of hierarchically porous spherical particles using high internal phase water-in-oil-in-water (w/o/w) double emulsions via the photopolymerization of the water-in-oil high internal phase emulsion (w/o HIPE) was developed. Visible-light photopolymerization was used for the synthesis of microspherical particles. The HIP emulsion had an internal phase volume of 80% and an oil phase containing either thiol pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) or trimethylolpropane tris(3-mercaptopropionate) (TMPTMP) and acrylate trimethylolpropane triacrylate (TMPTA). This enabled the preparation of microspheres with an open porous morphology, on both the surface and within the microsphere, with high yields in a batch manner. The effect of the thiol-to-acrylate ratio on the microsphere diameter, pore and window diameter, and degradation was investigated. It is shown that thiol has a minor effect on the microsphere and pore diameter, while the acrylate ratio affects the degradation speed, which decreases with increasing acrylate content. The possibility of free thiol group functionalization was demonstrated by a reaction with allylamine, while the microsphere adsorption capabilities were tested by the adsorption of methylene blue.

Serum Albumin in Health and Disease: Esterase, Antioxidant, Transporting and Signaling Properties.

Being one of the main proteins in the human body and many animal species, albumin plays a decisive role in the transport of various ions—electrically neutral and charged molecules—and in maintaining the colloidal osmotic pressure of the blood. Albumin is able to bind to almost all known drugs, as well as many nutraceuticals and toxic substances, largely determining their pharmaco- and toxicokinetics. Albumin of humans and respective representatives in cattle and rodents have their own structural features that determine species differences in functional properties. However, albumin is not only passive, but also an active participant of pharmacokinetic and toxicokinetic processes, possessing a number of enzymatic activities. Numerous experiments have shown esterase or pseudoesterase activity of albumin towards a number of endogeneous and exogeneous esters. Due to the free thiol group of Cys34, albumin can serve as a trap for reactive oxygen and nitrogen species, thus participating in redox processes. Glycated albumin makes a significant contribution to the pathogenesis of diabetes and other diseases. The interaction of albumin with blood cells, blood vessels and tissue cells outside the vascular bed is of great importance. Interactions with endothelial glycocalyx and vascular endothelial cells largely determine the integrative role of albumin. This review considers the esterase, antioxidant, transporting and signaling properties of albumin, as well as its structural and functional modifications and their significance in the pathogenesis of certain diseases.

Formation of whey protein aggregates by partial hydrolysis and reduced thermal treatment

Thermal treatment to form functional, soluble whey protein aggregates often generates undesired off-flavours. The aim of the present study was to generate soluble aggregates of whey protein isolates (WPI) at lower temperature than traditionally used by introducing partial hydrolysis of WPI prior to thermal treatment. WPI was partially hydrolyzed either by Bacillus licheniformis protease (BLP) or by a trypsin type protease (TP) at pH 7.0 and 65 °C for 1–10 min prior to thermal treatment at 80 °C for 5–10 min to generate soluble protein aggregates. Partial hydrolysis by BLP and TP induced structural changes and exposure of free thiol groups, but no change of surface hydrophobicity of WPI. After incubation with BLP or TP and thermal treatment, the yield of protein aggregates was 36–48%, which was similar to the reference sample obtained by thermal treatment at 90 °C for 10 min. In addition to non-covalent interactions, disulfide bonds also contributed to the association of protein aggregates. The soluble protein aggregates obtained by BLP partial hydrolysis and thermal treatment, had particle size range of 10–100 nm in radius which was in the same range as observed for the reference sample; and samples obtained by TP treatment had a particle size range of 7–50 nm in radius. However, aggregates obtained by BLP or TP and thermal treatment were not UHT (140 °C/5 s) stable.

Transient Receptor Potential channels (TRP) in GtoPdb v.2021.3

Transient Receptor Potential channels (TRP) in GtoPdb v.2021.3

In Vitro Investigation of Thiolated Chitosan Derivatives as Mucoadhesive Coating Materials for Solid Lipid Nanoparticles.

In the present study, chitosan (CS) was thiolated by introducing l-cysteine via amide bond formation. Free thiol groups were protected with highly reactive 6-mercaptonicotinic acid (6-MNA) and less-reactive l-cysteine, respectively, via thiol/disulfide-exchange reactions. Unmodified CS, l-cysteine-modified thiolated CS (CS-Cys), 6-MNA-S-protected thiolated CS (CS-Cys-MNA), and l-cysteine-S-protected thiolated CS (CS-Cys-Cys) were applied as coating materials to solid lipid nanoparticles (SLN). The strength of mucus interaction followed the rank order plain < CS < CS-Cys-Cys < CS-Cys < CS-Cys-MNA, whereas mucus diffusion followed the rank order CS-Cys < CS-Cys-Cys < CS < CS-Cys-MNA < plain. In accordance with lower reactivity, CS-Cys-Cys-coated SLN were immobilized to a lower extent than CS-Cys-coated SLN, while CS-Cys-MNA-coated SLN dissociated from their coating material resulting in a similar diffusion behavior as plain SLN. Consequently, CS-Cys-Cys-coated SLN and CS-Cys-MNA-coated SLN showed the highest retention on porcine intestinal mucosa by enabling a synergism of efficient mucus diffusion and strong mucoadhesion.

Thiol-sensitive probe enables dynamic electrochemical assembly of serum protein for detecting SARS-Cov-2 marker protease in clinical samples

The poor situational awareness about the spreading of the virus especially in the underdeveloped regions calls for novel virus assays of low cost and simple operation. Currently, such assays are exclusively restricted to nucleic acid detection. In this investigation, a virus protein serum assay has been proposed in a one-step and reagent-less route. Specifically, in this assay, the main protease of the virus is targeted by a short probe mimicking its substrate. While the probe-protein interaction brings them together, a fluorescent thiol targeting molecule reacts with the free thiol groups on the target protein near the probe, generating a fluorescence signal proportional to the concentration of the target. This induces an electroactive 2D peptide nano-network on the sensing surface only in the presence of the target protein. The sensitivity of the method is enhanced through potential electrochemical scanning during incubation with serum samples. The successful detection of the virus marker protein in the serum of the infected patients encourages further development of incorporation of this method into clinical practice.