Chromogenic Substrate Research Articles

Portable Hydrogel Kits Made with Bimetallic Nanozymes for Point-of-Care Testing of Perfluorooctanesulfonate.

Perfluorooctanesulfonate (PFOS), an emerging organic contaminant, necessitates robust on-site detection strategies to safeguard human health and ecological balance. This study introduces a novel point-of-care testing (POCT) platform, combining a hydrogel kit with nanozymes and smartphone technology, for the highly sensitive detection of PFOS. The strategy utilizes copper-substituted cobalt-based Prussian blue analogue nanoboxes (CuCo-PBA NBs), which exhibit intricate hollow structures and remarkable peroxidase-like catalytic activity, efficiently catalyzing the oxidation of chromogenic substrates with hydrogen peroxide (H2O2). Density functional theory calculations elucidate the adsorption dynamics of H2O2 on CuCo-PBA NBs, identifying the factors that improve the catalytic efficiency. The colorimetric POCT platform, integrating the hydrogel kit with a smartphone interface, demonstrates practical utility and achieves a detection limit of 1.43 × 10-8 mol L-1 for PFOS. This research not only presents a new nanozyme design for PFOS detection in diverse matrices, such as lake water, whole blood, urine, and milk, but also paves the way for developing a portable and efficient POCT platform for a variety of emerging contaminants.

Thrombin-targeted screening of anticoagulant active components from Polygonum amplexicaule D. Don var. sinense Forb by affinity ultrafiltration coupled with UPLC-Q-TOF-MS.

Polygonum amplexicaule D. Don var. sinense Forb (PAF), a medicinal plant, has the effect of promoting blood circulation and removing blood stasis. However, the active compounds and targets of its anticoagulant effect are still unclear. This study aims to establish an effective reversely thrombin-targeted screening method for anticoagulant active components in PAF by affinity ultrafiltration (AUF) coupled with ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectroscopy (UPLC-Q-TOF-MS). Different polar parts of PAF were screened for potential thrombin ligands by AUF-HPLC and identified by UPLC-Q-TOF-MS. After studying the affinity between ligands and thrombin by molecular docking, the antithrombotic activity of ligands was detected in vivo by zebrafish thrombus model, and in vitro by chromogenic substrate method. The mechanism of such ligands on thrombin was further studied by coagulation factor assay. Eleven potential thrombin ligands from PAF were screened by the AUF-UPLC-Q-TOF-MS method, and two compounds (butyl gallate and β-sitosterol) with significant anticoagulant activity were discovered via in vitro and in vivo activity testing. A method system based on AUF-UPLC-Q-TOF-MS, molecular docking and in vivo and in vitro experiments also provided a powerful tool for further exploration of anticoagulant active components in PAF.

Borophene Quantum Dots as Novel Peroxidase-Mimicking Nanozyme: A Dual-Mode Assay for the Detection of Oxytetracycline and Tetracycline Antibiotics.

The greater advantages and wide applications of zero-dimensional nanodots inspire researchers to develop new materials. Therefore, novel borophene quantum dots (QDs) were prepared by a hydrothermal liquid exfoliation technique using water medium. The borophene QDs proved to be highly stable in water medium for more than 120 days. The synthesized borophene QDs revealed intrinsic peroxidase mimetic activity using two chromogenic substrates, 3,3',5,5'-tetramethylbenzidine (TMB) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS). The excellent intrinsic peroxidase activity toward TMB and ABTS substrates was executed using optimal reaction conditions (pH, borophene QDs' concentration, incubation time, and temperature). The formation of hydroxyl radicals in the presence of H2O2 upon TMB and ABTS oxidation played a significant role in the peroxidase reaction. The borophene QDs further proved to be successful for the colorimetric detection of antibiotics (oxytetracycline and tetracycline) using both TMB and ABTS peroxidase substrates. The limit of detection (LOD) for oxytetracycline and tetracycline was found to be 1.10 and 1.02 μM using TMB and 1.03 and 1.02 μM using ABTS chromogenic substrates, respectively. In addition, the fluorescence sensing of oxytetracycline and tetracycline over borophene QDs was also examined. The high fluorescence of borophene QDs (turn ON) was quenched (turn OFF) by oxytetracycline and tetracycline through the inner filter effect mechanism. The LOD of the fluorescence sensing of oxytetracycline and tetracycline was 1.14 and 1.08 μM, respectively. Interestingly, the borophene QDs could be used for the sensitive and selective colorimetric and fluorometric sensing of oxytetracycline and tetracycline after 120 days of storage. The synthesized borophene QDs with long-term stability and real sample analysis provide new insight as nanozymes with higher peroxidase mimetic and fluorescence performance and can be further exploited for medical diagnosis and environmental toxicants' detection.

Phenotypic and genetic analysis of a Chinese pedigree affected with Hereditary antithrombin deficiency due to a novel variant of SERPINC1 gene

To analyze the clinical phenotype and genetic characteristics of a Chinese pedigree affected with Hereditary antithrombin deficiency. A pedigree diagnosed at the the Second Affiliated Hospital of Wenzhou Medical University, Yuying Children's Hospital in June, 2020 was selected as the study subject. Plasma prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), and thrombin time (TT) of the probands and their pedigree members were determined using a STA-R automatic coagulation analyzer. Antithrombin activity (AT: A) and antithrombin antigen (AT: Ag) in plasma were determined with chromogenic substrate and immunonephelometry assays. All exons and flanking sequences of the anticoagulant protein gene SERPINC1 were amplified by PCR and subjected to Sanger sequencing. Candidate variants were verified with bioinformatic tools (PolyPhen-2, SIFT, Mutation Taster and PYMOL) to explore their effect on the function and structural conformation of the protein. The probands (II-2, II-10), their brother (II-5) and sons (III-1, III-8) had shown normal PT, APTT, FIB, and TT, but significantly decreased AT: A and AT: Ag, with their levels being 34%, 57%, 56%, 48%, 53% and 13.51 mg/dL, 13.44 mg/dL, 18.39 mg/dL, 17.36 mg/dL, 17.71 mg/dL, respectively. The remaining pedigree members had normal values. Sanger sequencing revealed that the probands and all affected pedigree members had harbored a heterozygous c.851T>C (p.Met284Thr) missense variant in exon 5 of the SERPINC1 gene. Bioinformatic analysis and simulation suggested that the variant has resulted in alteration of hydrogen bonds at the c.851 position, which may affect the structure of the protein. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as pathogenic (PS1+PM1+PM5+PP1+PP4). The probands and other affected members were all diagnosed with type I hereditary AT deficiency, for which the c.851T>C (p.Met284Thr) variant of the SERPINC1 gene may be accountable.

The peroxidase-like activity of Au NPs deposited inverse opal CeO2 nanozyme for rapid and sensitive H2O2 sensing

In this study, the hybrid nanozyme gold nanoparticles deposited CeO2 inverse opal photonic crystals (IOPCs) on the glass slide was fabricated by bestowing the hybrid with the beneficial porous morphology with its unique advantages: its surface valence states, high surface area, and copious active sites. The oxidase and peroxidase-mimicking activity was studied by observing the catalytic oxidation of chromogenic substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) in the presence of H2O2, which can be visualized by the eyes. Au NPs and CeO2 IOPCs consisting of Ce3+ and Ce4+ can oxidize ABTS in the presence of H2O2 by mimicking peroxidase enzymes. The peroxidase-like activity of nanozyme was substantially improved by introducing light irradiation due to the photosensitive nature of the hybrid. The peroxidase-like activity of nanozyme enabled H2O2 linear detection over the range 9 to 500 μM, with a LOD of 2.7 µM. The acceptable nanozyme activity is probably owing to the easy access of H2O2 to the active sites of nanozyme. The porous architecture led to the easy channeling of reactants and products. The periodic architecture of the nanozyme also led to decent reproducibility. Without functionalization, the nanozyme can be a promising alternative to peroxidase enzymes for hydrogen peroxide measurements.

Portable hydrogel kit driven by bimetallic carbon dots nanozyme for H2O2-self-supplying dual-modal monitoring of atmospheric CH3SH

Due to the typical volatility of gaseous pollutant methyl mercaptan (CH3SH), the development of a facile, reliable, and accurate onsite environmental surveillance of highly toxic CH3SH faces many challenges, but it is critical to environmental atmosphere assessment and safeguarding public health. Here, we prepared a novel bimetallic carbon dots (Fe&Cu@CDs) nanozyme with high peroxidase-mimicking activity to design a portable hydrogel kit for onsite visual H2O2-self-supplying enzymatic cascade catalytic colorimetric and photothermal signal synergistic amplification dual-modal monitoring of CH3SH in atmospheric environment. Assisted by alcohol oxidase (AOX), CH3SH could be specifically converted into H2O2 for oxidizing chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB) catalyzed by Fe&Cu@CDs to produce dark blue ox-TMB with absorption at 652 nm and photothermal characters. Consequently, a CH3SH concentration-dependent change both in naked-eye color and photothermal effect-triggered temperature were observed. By hybridizing AOX-assisted Fe&Cu@CDs + TMB with agarose, a H2O2-self-supplying colorimetric and photothermal signal synergistic amplification sensory hydrogel kit integrated with Color Picker APP-installed smartphone and 660 nm laser-equipped handheld thermal imager for CH3SH was proposed with acceptable results in atmospheric environment around wastepile (e.g., solid waste and food waste piles), which exhibited great potentials to further develop commercial onsite monitoring platforms in warning-early abnormal atmospheric CH3SH for safeguarding environmental health.

New Triazole-Based Potent Inhibitors of Human Factor XIIa as Anticoagulants.

Factor XIIa (FXIIa) functions as a plasma serine protease within the contact activation pathway. Various animal models have indicated a substantial role for FXIIa in thromboembolic diseases. Interestingly, individuals and animals with FXII deficiency seem to maintain normal hemostasis. Consequently, inhibiting FXIIa could potentially offer a viable therapeutic approach for achieving effective and safer anticoagulation without the bleeding risks associated with the existing anticoagulants. Despite the potential, only a limited number of small molecule inhibitors targeting human FXIIa have been documented. Thus, we combined a small library of 32 triazole and triazole-like molecules to be evaluated for FXIIa inhibition by using a chromogenic substrate hydrolysis assay under physiological conditions. Initial screening at 200 μM involved 18 small molecules, revealing that 4 molecules inhibited FXIIa more than 20%. In addition to being the most potent inhibitor identified in the first round, inhibitor 8 also exhibited a substantial margin of selectivity against related serine proteases, including factors XIa, Xa, and IXa. However, the molecule also inhibited thrombin with a similar potency. It also prolonged the clotting time of human plasma, as was determined in the activated partial thromboplastin time and prothrombin time assays. Subsequent structure-activity relationship studies led to the identification of several inhibitors with submicromolar activity, among which inhibitor 22 appears to demonstrate significant selectivity not only over factors IXa, Xa, and XIa, but also over thrombin. In summary, this study introduces novel triazole-based small molecules, specifically compounds 8 and 22, identified as potent and selective inhibitors of human FXIIa. The aim is to advance these inhibitors for further development as anticoagulants to provide a more effective and safer approach to preventing and/or treating thromboembolic diseases.

The optimization of gold nanoparticles–horseradish peroxidase as peroxidase mimic using central composite design for the detection of hydrogen peroxide

The oxidizing agent, hydrogen peroxide (H2O2), which is a part of reactive oxygen species (ROS) is well-known to contribute to oxidative stress-induced damage to biological molecules. An excess of free radicals can harm health and is associated with human diseases. Gold nanotechnology, a highly relevant nanomaterial, has been utilized as a new material in advanced sensor detection. In this study, colorimetric methods based on peroxidase enzymes were developed for measuring H2O2. The synthesized gold nanoparticles (AuNPs) showed a concentration of approximately 1.73 nM at a wavelength of 520 nm. The average diameter displayed a uniform size distribution, estimated at 18 nm, and an increase in the shell thickness of AuNPs-horseradish peroxidase (HRP) was observed in the TEM images. The AuNPs-HRP system demonstrated remarkable catalytic activity in the reaction of the chromogenic substrate tetramethylbenzidine (TMB) with H2O2, resulting in the production of an oxide product. The optimal conditions for the AuNPs-HRP system, as determined by central composite design (CCD), were a temperature of 25 °C and a pH of 7 within an 8 h period. A strong linear relationship was observed between different absorbance values and the H2O2 concentration, with a coefficient of determination of 0.9956. A portable platform was successfully used to determine H2O2 levels in beverages with recoveries ranging from 95.51% to 118.85%. These findings suggest that the AuNPs-HRP system could be applied to detect H2O2 in beverages.

Synthesis of Spherical Mn2O3 Nanozymes from Different Green Precursors for their Innovative Applications in Catalytic Properties and Bioactivity.

Here, spherical Mn2O3 nanozymes were synthesized via a one-step green method using different green precursors, and their physicochemical properties and biological activities were monitored with various green precursors. Powder X-ray diffraction (PXRD) was performed to determine the crystalline properties and phases involved in the formation of cubic Mn2O3 nanozymes. The synthesized nanozymes were spherical and examined by SEM and FESEM studies. All of the samples synthesized using different green precursors exhibited different sizes but similar spherical shapes. Moreover, all green-synthesized nanozymes catalyzed the oxidation reaction of the chromogenic substrate 3,3'5,5' tetramethylbenzidine (TMB) in the absence of H2O2, and A2 (lemon-mediated Mn2O3 nanozymes), which the followed Michaelis-Menten kinetics, showed the best activity. Therefore, A2 (lemon-mediated nanozyme) showed oxidase-mimicking activity with distinct Km and Vmax values calculated by the Lineweaver-Burk plot. Furthermore, the current nanozymes demonstrated a significant ability to kill both Gram-negative and Gram-positive bacteria as well as effectively destroy biofilms under physiological conditions. Moreover, the green-mediated nanozymes also displayed ROS-scavenging activity. Our nanozymes exhibited scavenging activity toward OH and O2-• radicals and metal chelation activity, which were investigated colorimetrically. Therefore, these nanozymes might be used as effective antibacterial agents and also for the consumption of reactive oxygen species.

Iron-Doped Polymer Dots with Enhanced Fluorescence and Dual Enzyme Activity for Versatile Bioassays.

Nanozymes with multiple functionalities endow biochemical sensing with more sensitive and efficient analytical performance by widening the sensing modes. Meanwhile, the target-oriented design of multifunctional nanozymes for certain biosensing remains challenging. Herein, a constructive strategy of doping iron into polymer dots (PDs) to achieve nanozymes with excellent oxidase-mimicking and peroxidase-mimicking activity is proposed. Compared with the Fe-free PDs prepared under the same mild condition, the Fe-doped PDs (Fe-PDs) exhibit greatly boosted fluorescence at 500 nm. While applying 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic substrate, the fluorescence of the Fe-PDs can be further quenched by oxTMB due to the inner filter effect (IFE). Inspired by this, a simple but efficient colorimetric and fluorometric dual-mode sensing platform is developed for monitoring the reducing substances ascorbic acid (AA), α-glucosidase (α-Glu), and its inhibitors (AGIs). We believe that such multifunctional enzyme-mimic materials will provoke the exploration of multimode sensing strategy with strong practicality to serve as a versatile tool in biochemical sensing.

Plasmon-Stimulated Colorimetry Biosensor Array for the Identification of Multiple Metabolites.

Rationally designing highly catalytic and stable nanozymes for metabolite monitoring is of great importance because of their huge potential in early disease diagnosis. Herein, a novel nanozyme based on hierarchically structured CuS/ZnS with a highly efficient peroxidase (POD)-mimic capability was developed and synthesized for multiple metabolite determination and recognition via the plasmon-stimulated biosensor array strategy. The designed nanozyme can simultaneously harvest plasmon triggered hot electron-hole pairs and generate photothermal properties, leading to a sharply boosted POD-mimic capability under 808 nm laser irradiation. Interestingly, because of the interaction diversity of the metabolite with POD-like nanomaterials, the unique inhibitory effect of metabolites on the POD-mimic activity could be the signal response as the differentiation. Thus, utilizing TMB as a typical chromogenic substrate in the addition of H2O2, the designed colorimetric biosensor array can produce diverse fingerprints for the three vital metabolisms (cysteine (Cys), ascorbic acid (AA), and glutathione (GSH)), which can be precisely identified by principal component analysis (PCA). Notably, a distinct fingerprint of a single metabolite with different levels and metabolite mixtures is also achieved with a detection limit of 1 μM. Most importantly, cell lysis could be effectively discriminated by the biosensor assay, implying its great potential in clinical diagnosis.

Synthetization and Characterization of Zinc Oxide Nanoparticles by X- Ray Diffractometry (XRD), Fourier Transforms, Infra-Red Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM) and Antibacterial Activity Test

Purpose: Nanomaterials with their derivable potentials offer wide obtain ability and have recently aroused much attention for biomedical applications. Nowadays, nanomaterials-based colorimetric sensing is a quickly emerging field of sensing applications. Nanomaterials are considered as the main component of colorimetric determination of hydrogen peroxide to replace the natural enzyme-based sensors because of some associated intrinsic drawbacks. Considering the advantageous properties of ionic liquid (IL) for various applications, significant attention has been made to the use of ionic liquid stabilized metal NPs which may serve as a regulator to enhance the catalytic performance of the metal nanoparticles in the different IL reaction medium. 
 Methodology: The peroxidase-like activity of IL coated metal NPs (IL-MNPs) have been considered for the catalytic oxidation reaction of chromogenic substrate 3,3,5,5- tetramethylbenzidine (TMB) in the presence of H2O2 at an estimated wavelength of 652 nm.
 Results: The synthesized metal nanoparticles (Ag) were produced using a chemical reduction method. Various characterization techniques like FTIR, UV-Visible spread Reflectance Spectroscopy [UV-VIS DRS], were employed, which verified the structure, nano-size and successful combination of metal dopant ion into the samples. The molecular structure of ionic liquid with varying cations was produced and confirmed by 1H-NMR spectroscopy. The ionic liquid was coated on metal nanoparticles to enhance their conductivity. 
 Unique Contribution to Theory, Practice and Policy: Optimized reaction conditions like pH, temperature and catalyst dosage affect catalytic activity and color sensing properties. The coating of [Min] Ac on Ag achieved low detection limits and colorimetric detection of [Pyr] based Ag.

CRISPR-Cas12a-based colorimetric aptasensor for aflatoxin M1 detection based on oxidase-mimicking activity of flower-like MnO2 nanozymes

Given the highly mutagenic and carcinogenic nature of Aflatoxin M1 (AFM1), the quantity assessment of AFM1 residues in milk and dairy products is necessary to maintain consumer health and food safety. Herein, CRISPR-Cas12a-based colorimetric aptasensor was developed using the catalytic activity of flower-like nanozymes of MnO2 and trans-cleavage property of CRISPR-Cas12a system to quantitatively detect AFM1. The basis of the developed colorimetric aptasensor relies on whether or not the CRISPR-Cas12a system is activated, as well as the contrast in oxidase-mimicking capability exhibited by flower-like MnO2 nanozymes when AFM1 is absent or present. When AFM1 is not present in the sample, single-stranded DNA (ssDNA) is degraded by the activated CRISPR-Cas12a, and the solution turns into yellow due to the catalytic activity of the nanozymes. While, in the attendance of AFM1, ssDNA degradation does not occur due to the inactivation of the CRISPR-Cas12a. Therefore, with the adsorption of the ssDNA on the MnO2 nanozymes, their catalytic activity decreases, and the solution color becomes pale yellow due to less oxidation of the chromogenic substrate. In this aptasensor, the relative absorbance changes increased linearly from 6 to 160 ng L−1, and the detection limit was 2.1 ng L−1. The developed aptasensor displays a selective detection performance and a practical application for quantitative analysis of AFM1 in milk samples. The results of the introduced aptasensor open up the way to design other selective and sensitive aptasensors for the detection of other mycotoxins by substitution of the used sequences.

Novel enzyme-linked immunoassay based on nanoceria as colorimetric substrate for foodborne pathogen detection

Rapid and reliable detection of foodborne pathogens is a key requirement for food safety. Traditional rapid ELISA methods assessed by the ability of horseradish peroxidase to oxidize a chromogenic substrate such as 3, 3′, 5, 5′-tetramethylbenzidine in the presence of H2O2 may result in the high rate of negative responses because of the instability of H2O2 and product. Here, we report the use of redox active nanoceria as colorimetric indicator in immunoassay taking Staphylococcus aureus as a model. The nanoceria is fully water-soluble and can be stored as least 90 days in the refrigerator while maintaining the same performance. Moreover, it does not require external H2O2, as glucose oxidase used in the immunoassay can catalyze the oxidation of glucose to produce H2O2in situ, which is more robust in signal readout. Taken together, these results demonstrate that nanoceria are potentially suitable as colorimetric substrate in the development of novel enzyme immunoassays.

Enhanced biomimetic catalysis via self-cascade photocatalytic hydrogen peroxide production over modified carbon nitride nanozymes for total antioxidant capacity evaluation

The peroxidase mimics usually requires the addition of exogenous hydrogen peroxide (H2O2), which greatly hinder their practical applications. Herein, through rational co-modification of multiple elements (potassium (K), chlorine (Cl) and iodine (I)), the modified carbon nitride nanomaterials (KCl/KI-CN) could serve as efficient bifunctional catalysts. The multiple elements doping and the incorporation of cyano groups (CN) are deemed to enhance their photocatalytic and peroxidase-like activity, respectively. Based on the photocatalytic function, H2O2 can be produced continuously and steadily via two-electron oxygen reduction over modified carbon nitride under visible light irradiation. Subsequently, the KCl/KI-CN could catalyze the chromogenic substrate by the in-situ produced H2O2. Taking advantage of the bifunctional properties of modified carbon nitride, we for the first time demonstrate a self-cascade catalytic process and apply successfully for the ascorbic acid (AA) detection and versatile total antioxidant capacity (TAC) evaluation. This paper not only prepares an efficiently bifunctional catalyst but also provides a new self-cascade photocatalytic H2O2 production strategy for the peroxidase-like application.

A separation-free paper-based hydrogel device for one-step reactive oxygen species determination by a smartphone.

Paper-based analytical devices (PADs) are very convenient for determining biomarkers in point-of-care (POC) diagnosis while requiring sample pre-treatment or impurity separation. This study reports a novel hydrogel-coupled, paper-based analytical device (PAD) for separation-free H2O2 colorimetric detection in both aqueous solution and cell lysis with sample-to-answer analysis by directly loading into the sample test zone. By encapsulating an inorganic mimic enzyme and chromogenic substrate into the sodium alginate (SA) hydrogel, amplification of the color signal after catalyzing the substrate could be achieved. Taking advantage of the nanoscale porous structure of the hydrogel and the lateral flow channel of the PAD, large interference fragments or bio-macromolecules are prevented from diffusing into the chromogenic reaction, whereas the small target molecules enter the sensing region to trigger the catalytic reaction. This method demonstrated a rapid and accurate analysis with a limit of detection as low as 0.06 mM and detection selectivity. Our proposed device requires no enzyme and is separation-free, portable, easy-to-fabricate, and low-cost, and may offer a platform for quantitative or qualitative analysis of other analytes in body fluids for POC applications.

A Chromogenic Quantification of Protein Expression in Zebrafish Larvae.

Western blot is a versatile and widely used technique in many areas of molecular biology and biotechnology for studying different protein characteristics. In general, the Western blot technique involves the extraction of proteins from the samples such as cells or tissues, which, after denaturation, are separated by molecular size using electrophoresis. The protein is then transferred to a membrane, typically PVDF or nitrocellulose, which, after blocking, is probed with specific antibodies labeled with a detection agent. Overall, this allows the recognition and binding to the target protein allowing the visualization of bands, a step called immunodetection. Over the years, new approaches to the Western blotting technique have been proposed to overcome performance limitations.This chapter describes a routine procedure for protein evaluation in zebrafish (Danio rerio) larvae, a widely used animal model for predicting the toxicity of drugs, by using a chromogenic substrate and allowing the proper execution of the technique without the costly equipment needed for detection.

In situ Hg2+ improved the peroxidase-like activity and triggered "ON" the oxidase-like activity of yolk-shell Co3S4 microspheres for the detection of Hg2.

Exploring highly active nanozymes is an important task to realize the real-time detection of some heavy metal ions in water. In this work, yolk-shell Co3S4 microspheres have been verified to possess excellent peroxidase-like activity, which can be further improved by adding Hg2+. Very interestingly, Hg2+ can trigger "ON" the oxidase-like activity of Co3S4 microspheres. The dual peroxidase-/oxidase-like activity of the yolk-shell Co3S4 microspheres is evaluated by using the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB). Furthermore, comprehensive studies verify that the enhanced peroxidase-like activity, together with the "ON" oxidase-like activity of the yolk-shell Co3S4 microspheres, is attributed to the in situ generation of HgS on the surface of Co3S4 microspheres and then the release of more active sites. Importantly, the in situ generated HgS on the surface of Co3S4 microspheres can form a heterojunction, which also accelerates the catalytic process. During the catalytic reaction, some active species (O2- and h+) can be detected by ESR. Thus, a colorimetric sensing platform based on Hg2+-triggered signal amplification has been successfully constructed, which can be validated by the detection of Hg2+ residue in environmental water.

CRISPR-Cas12a-based ultrasensitive assay for visual detection of SARS-CoV-2 RNA.

The development of ultrasensitive and visual methods is of great significance for molecular diagnosis at the point-of-care. In this study, we have integrated recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system to design an ultrasensitive strategy for visual nucleic acid testing. RPA is utilized to amplify the target nucleic acid, producing amplicons that activate the single-stranded DNase property of CRISPR-Cas12a. The activated CRISPR-Cas12a then degrades the single-stranded DNA on magnetic nanoparticles (MNPs), releasing immobilized GOx from the MNPs which catalyses the chromogenic substrate. The developed method exhibits remarkable sensitivity, successfully detecting as low as 10 aM (∼6 copies per μL) of the target nucleic acid by visual colour changes in solution. The instrumental limit of detection is calculated to be 2.86 aM (∼2 copies per μL), comparable to the sensitivity of polymerase chain reaction (PCR). Importantly, this approach only requires isothermal incubation operation and does not involve costly instruments. The method has been validated by visually detecting the SARS-CoV-2 RNA gene fragment within 50 minutes. With its ultrasensitivity, simplicity of operation, and potential for integration into a point-of-care detection kit, this strategy holds great promise for nucleic acid testing in various settings.

Biochemical Properties and Antithrombotic Effect of a Serine Protease Isolated from the Medicinal Mushroom Pycnoporus coccineus (Agaricomycetes).

The purification of a fibrinolytic enzyme from the fruiting bodies of wild-growing medicinal mushroom, Pycnoporus coccineus was achieved through a two-step procedure, resulting in its homogeneity. This purification process yielded a significant 4.13-fold increase in specific activity and an 8.0% recovery rate. The molecular weight of P. coccineus fibrinolytic enzyme (PCFE) was estimated to be 23 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. PCFE demonstrated its optimal activity at a temperature of 40 °C and pH 8. Notably, the enzymatic activity was inhibited by the presence of zinc or copper metal ions, as well as serine protease inhibitors, such as phenylmethylsulfonyl fluoride and 4-amidinophenylmethanesulfonyl fluoride. PCFE exhibited remarkable specificity towards a synthetic chromogenic substrate for thrombin. The enzyme demonstrated the Michaelis-Menten constant (Km), maximal velocity (V ), and catalytic rate constant (Kcat) values of 3.01 mM, 0.33 mM min-1 μg-1, and 764.1 s-1, respectively. In vitro assays showed PCFE's ability to effectively degrade fibrin and blood clots. The enzyme induced alterations in the density and structural characteristics of fibrin clots. PCFE exhibited significant effects on various clotting parameters, including recalcification time, activated partial thromboplastin time, prothrombin time, serotonin secretion from thrombin-activated platelets, and thrombin-induced acute thromboembolism. These findings suggest that P. coccineus holds potential as an antithrombotic biomaterials and resources for cardiovascular research.