Characterization of nanozyme kinetics for highly sensitive detection.

Nanozymes have been widely used as enzyme substitutes. Based on a comprehensive literature survey of 261 publications, we report the significant differences in the Michaelis-Menten constants (Km) between peroxidase-mimicking nanozymes and horseradish peroxidase (HRP). Further, these differences were not considered in more than 60% of the publications for analytical developments. As a result, nanozymes' catalytic activity is limited, resulting in a potentially higher limit of detection (LOD). We used a peroxidase-mimicking Au@Pt nanozyme, which has Km for TMB comparable with HRP and three orders of magnitude higher Km for H2O2. Using the Au@Pt nanozyme as a label for immunoassays, non-optimized nanozyme substrate concentrations led to 30 times higher LOD compared to optimized conditions. The results confirm the necessity of measuring nanozymes' kinetic parameters and the corresponding adjustment of substrate concentrations for highly sensitive detection.

Simple, Visual, Point-of-Care SARS-CoV-2 Detection Incorporating Recombinase Polymerase Amplification and Target DNA-Protein Crosslinking Enhanced Chemiluminescence.

The ongoing COVID-19 pandemic, driven by persistent SARS-CoV-2 transmission, threatens human health worldwide, underscoring the urgent need for an efficient, low-cost, rapid SARS-CoV-2 detection method. Herein, we developed a point-of-care SARS-CoV-2 detection method incorporating recombinase polymerase amplification (RPA) and DNA-protein crosslinking chemiluminescence (DPCL) (RPADPCL). RPADPCL involves the crosslinking of biotinylated double-stranded RPA DNA products with horseradish peroxidase (HRP)-labeled streptavidin (SA-HRP). Modified products are captured using SA-labeled magnetic beads, and then analyzed using a chemiluminescence detector and smartphone after the addition of a chemiluminescent substrate. Under optimal conditions, the RPADPCL limit of detection (LOD) was observed to be 6 copies (within the linear detection range of 1-300 copies) for a plasmid containing the SARS-CoV-2 N gene and 15 copies (within the linear range of 10-500 copies) for in vitro transcribed (IVT) SARS-CoV-2 RNA. The proposed method is convenient, specific, visually intuitive, easy to use, and does not require external excitation. The effective RPADPCL detection of SARS-CoV-2 in complex matrix systems was verified by testing simulated clinical samples containing 10% human saliva or a virus transfer medium (VTM) spiked with a plasmid containing a SARS-CoV-2 N gene sequence or SARS-CoV-2 IVT RNA. Consequently, this method has great potential for detecting targets in clinical samples.

FeMo2Ox(OH)y-based mineral hydrogels as a novel POD nanozyme for sensitive and selective detection of aromatic amines contaminants via a colorimetric sensor array.

Developing convenient pathways to discriminate and identify multiple aromatic amines (AAs) remains fascinating and critical. Here, a novel three-channel colorimetric sensor array based on FeMo2Ox(OH)y-based mineral (FM) hydrogels is successfully constructed to monitor AAs in tap water. Benefiting from the substantial oxygen vacancies (VO), FM nanozymes exhibit extraordinary peroxidase (POD)-like activities with Km of 0.133 mM and Vmax of 2.518 × 10-2 mM·s-1 toward 3,3',5,5'-tetramethylbenzidine (TMB), which are much better than horseradish peroxidase and most of POD mimics. This reveals that doping Cu and Co into FM (FM-Cu and FM-Co) can change POD activity. Based on various POD activities, TMB and H2O2 are used to generate fingerprint colorimetry signals from the colorimetry sensor array. The analytes can accurately discriminate through linear discriminant analysis, with a detection limit as low as 2.12 × 10-2-0.14 µM. The sensor array can effectively identify and discriminate AA contaminants and their mixtures and has performed well in real sample tests.

Ultrasensitive colorimetric detection of Staphylococcus aureus using wheat germ agglutinin and IgY as a dual-recognition strategy.

A novel colorimetric platform was designed for the determination of S. aureus by utilizing a dual-recognition strategy, where wheat germ agglutinin (WGA)-functionalized magnetic beads were served as separation elements to capture and enrich S. aureus efficiently from the matrix. Horseradish peroxidase (HRP) labeled chicken anti-protein A IgY (HRP-IgY) was used to label the captured S. aureus. A chicken IgY was introduced as a signal tracer to bind with staphylococcal protein A (SPA) on the surface of S. aureus, which can circumvent the interference from protein G-producing Streptococcus. Subsequently, the colorimetric signal was achieved by an HRP-catalyzed reaction, which was amplified by HRP-IgY bound by approximately 80,000 SPA molecules on one S. aureus. The entire detection process could be accomplished within 90 min. Under optimal conditions, the linear response of different S. aureus concentrations ranged from 7.8 × 102 to 2.0 × 105 CFU/mL and the limit of detection reached down to 3.9 × 102 CFU/mL. Some common non-target bacteria yielded negative results, indicating the excellent specificity of the method. The developed strategy was successfully applied to the determination of S. aureus in various types of samples with satisfactory recoveries. Therefore, the novel dual-recognition strategy possessed the advantages of high sensitivity, specificity, and low cost and exhibited considerable potential as a promising tool to defend public health.

Electrochemical bioplatform to manage alpha-gal syndrome by tracking the carbohydrate allergen in meat.

This work presents the first bioplatform described to date for the determination of galactose-α-1,3-galactose (α-Gal), a non-primate mammalian oligosaccharide responsible for almost all cases of red meat allergy. The bioplatform is based on the implementation of an indirect competitive immunoassay and enzymatic labeling with the enzyme horseradish peroxidase (HRP) built on the surface of magnetic microparticles (MBs) and amperometric transduction on screen-printed carbon electrodes (SPCEs) using the H2O2/hydroquinone (HQ) system. The target α-Gal competed with biotinylated α-Gal immobilized on the surface of neutravidin-modified MBs for the limited immunorecognition sites of a detection antibody enzymatically labeled with an HRP-conjugated secondary antibody. The resulting magnetic immunoconjugates were trapped on the surface of the SPCE working electrode and amperometric transduction was performed, providing a cathodic current variation inversely proportional to the concentration of α-Gal in the analyzed sample. The developed biotool was optimized, characterized and applied with satisfactory results to the determination of the target allergen in different samples of raw and processed meats.

Kinetic detection of hydrogen peroxide in single horseradish peroxidase-concentrated silica particle using confocal fluorescence microspectroscopic measurement.

In the present study, we propose a scheme for detecting H2O2 by using horseradish peroxidase (HRP) adsorbed onto single silica particles and fluorescence microspectroscopy. When the silica particles were immersed in an HRP solution, the HRP concentration in the silica particles increased by a factor of 690 compared to that in the bulk aqueous solution because HRP was adsorbed on the silica surface. When a single particle containing HRP was added to a mixed solution of H2O2 and Amplex Red, fluorescence from resorufin, which was produced by the reaction of HRP, H2O2, and Amplex Red, was observed. The fluorescence from the resorufin in the particles increased after a single particle was added to the solution, and the release of resorufin was observed. As the concentration of H2O2 (CH2O2) decreased, the time it takes for fluorescence intensity to reach its maximum was shorter. The detection limit for H2O2 in the present system was 980 nM. The reaction behavior of a single silica particle was evaluated using a spherical diffusion model, which explains the approximate concentration change of resorufin in the silica particle. The proposed method has the advantages of simple sample preparation and detection, low sample consumption, and a short detection time.

Enzyme-Mediated Bioorthogonal Cascade Catalytic Reaction for Metabolism Intervention and Enhanced Ferroptosis on Neuroblastoma.

It remains a tremendous challenge to explore effective therapeutic modalities against neuroblastoma, a lethal cancer of the sympathetic nervous system with poor prognosis and disappointing treatment outcomes. Considering the limitations of conventional treatment modalities and the intrinsic vulnerability of neuroblastoma, we herein develop a pioneering sequential catalytic therapeutic system that utilizes lactate oxidase (LOx)/horseradish peroxidase (HRP)-loaded amorphous zinc metal-organic framework, named LOx/HRP-aZIF, in combination with a 3-indole-acetic acid (IAA) prodrug. On the basis of abnormal lactate accumulation that occurs in the tumor microenvironment, the cascade reaction of LOx and HRP consumes endogenous glutathione and a reduced form of nicotinamide adenine dinucleotide to achieve the first stage of killing cancer cells via antioxidative incapacitation and electron transport chain interference. Furthermore, the generation of reactive oxygen species induced by HRP and IAA through bioorthogonal catalysis promotes ferritin degradation and lipid peroxidation, ultimately provoking self-enhanced ferroptosis with positive feedback by initiating an endogenous Fenton reaction. This work highlights the superiority of the natural enzyme-dependent cascade and bioorthogonal catalytic reaction, offering a paradigm for synergistically enzyme-based metabolism-ferroptosis anticancer therapy.

Support Materials of Organic and Inorganic Origin as Platforms for Horseradish Peroxidase Immobilization: Comparison Study for High Stability and Activity Recovery.

In the presented study, a variety of hybrid and single nanomaterials of various origins were tested as novel platforms for horseradish peroxidase immobilization. A thorough characterization was performed to establish the suitability of the support materials for immobilization, as well as the activity and stability retention of the biocatalysts, which were analyzed and discussed. The physicochemical characterization of the obtained systems proved successful enzyme deposition on all the presented materials. The immobilization of horseradish peroxidase on all the tested supports occurred with an efficiency above 70%. However, for multi-walled carbon nanotubes and hybrids made of chitosan, magnetic nanoparticles, and selenium ions, it reached up to 90%. For these materials, the immobilization yield exceeded 80%, resulting in high amounts of immobilized enzymes. The produced system showed the same optimal pH and temperature conditions as free enzymes; however, over a wider range of conditions, the immobilized enzymes showed activity of over 50%. Finally, a reusability study and storage stability tests showed that horseradish peroxidase immobilized on a hybrid made of chitosan, magnetic nanoparticles, and selenium ions retained around 80% of its initial activity after 10 repeated catalytic cycles and after 20 days of storage. Of all the tested materials, the most favorable for immobilization was the above-mentioned chitosan-based hybrid material. The selenium additive present in the discussed material gives it supplementary properties that increase the immobilization yield of the enzyme and improve enzyme stability. The obtained results confirm the applicability of these nanomaterials as useful platforms for enzyme immobilization in the contemplation of the structural stability of an enzyme and the high catalytic activity of fabricated biocatalysts.

Clinically Approved Ferric Maltol: A Potent Nanozyme with Added Effect for High-Efficient Catalytic Disinfection.

Nanozyme has been proven to be an attractive and promising candidate to alleviate the current pressing medical problems. However, the unknown clinical safety and limited function beyond the catalysis of the most reported nanozymes cannot promise an ideal therapeutic outcome in further clinical application. Herein, we find that ferric maltol (FM), a clinically approved iron supplement synthesized through a facile scalable method, exhibits excellent peroxidase-like activity than natural horseradish peroxidase-like (HRP) and commonly reported Fe-based nanozymes, and also shows high antibacterial performance for methicillin-resistant Staphylococcus aureus (MRSA) elimination (100%) and wound disinfection. In addition, with added effects inherited from contained maltol, FM can accelerate skin barrier recovery. Therefore, the exploration of FM as a safe and desired nanozyme provides a timely alternative to current antibiotic therapy against drug-resistant bacteria.

A self-assembled 3D nanoflowers based nano-ELISA platform for the sensitive detection of pyridaben.

Biomimetic methods are invariably employed to synthesize hybrid organic-inorganic multilevel structure nanoflowers with self-assembly processes in aqueous solutions, which is an ideal way to meet the challenges of immobilizing antibodies or enzymes in nanomaterial based enzyme-linked immunosorbent assay (nano-ELISA). In this study, we developed protein-inorganic hybrid 3D nanoflowers composed of bovine serum albumin (BSA), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (IgG-HRP) and copper(Ⅱ) phosphate (BSA-(IgG-HRP)-Cu3(PO4)2) using a self-assembly biomimetic method. The preparation process avoided the use of any organic solvent and protein immobilization did not require covalent modifications. Additionally, the unique hierarchical structure enhances the thermal and storage stability of HRP. The BSA-(IgG-HRP)-Cu3(PO4)2 hybrid 3D nanoflower was then applied to a nano-ELISA platform for pyridaben detection, achieving a 50% inhibition concentration of 3.90 ng mL-1. The nano-ELISA achieved excellent accuracy for pyridaben detection. Such a novel BSA-(IgG-HRP)-Cu3(PO4)2 hybrid 3D nanoflower provide an excellent reagent for small molecule immunoassay.

Biomimetic Mineralization of Large Enzymes Utilizing a Stable Zirconium-Based Metal-Organic Frameworks.

Enzymes are natural catalysts for a wide range of metabolic chemical transformations, including selective hydrolysis, oxidation, and phosphorylation. Herein, we demonstrate a strategy for the encapsulation of enzymes within a highly stable zirconium-based metal-organic framework. UiO-66-F4 was synthesized under mild conditions using an enzyme-compatible amino acid modulator, serine, at a modest temperature in an aqueous solution. Enzyme@UiO-66-F4 biocomposites were then formed by an in situ encapsulation route in which UiO-66-F4 grows around the enzymes and, consequently, provides protection for the enzymes. A range of enzymes, namely, lysozyme, horseradish peroxidase, and amano lipase, were successfully encapsulated within UiO-66-F4. We further demonstrate that the resulting biocomposites are stable under conditions that could denature many enzymes. Horseradish peroxidase encapsulated within UiO-66-F4 maintained its biological activity even after being treated with the proteolytic enzyme pepsin and heated at 60 °C. This strategy expands the toolbox of potential metal-organic frameworks with different topologies or functionalities that can be used as enzyme encapsulation hosts. We also demonstrate that this versatile process of in situ encapsulation of enzymes under mild conditions (i.e., submerged in water and at a modest temperature) can be generalized to encapsulate enzymes of various sizes within UiO-66-F4 while protecting them from harsh conditions (i.e., high temperatures, contact with denaturants or organic solvents).

Highly Sensitive Measurement of Horseradish Peroxidase Using Surface-Enhanced Raman Scattering of 2,3-Diaminophenazine.

The development of various enzyme-linked immunosorbent assays (ELISAs) coupled with surface-enhanced Raman scattering (SERS) detection is a growing area in analytical chemistry due to their potentially high sensitivity. A SERS-based ELISA with horseradish peroxidase (HRP) as an enzymatic label, an o-phenylenediamine (oPD) substrate, and a 2,3-diaminophenazine (DAP) enzymatic product was one of the first examples of such a system. However, the full capabilities of this long-known approach have yet to be revealed. The current study addresses a previously unrecognized problem of SERS detection stage performance. Using silver nanoparticles and model mixtures of oPD and DAP, the effects of the pH, the concentration of the aggregating agent, and the particle surface chloride stabilizer were extensively evaluated. At the optimal mildly acidic pH of 3, a 0.93 to 1 M citrate buffer, and AgNPs stabilized with 20 mM chloride, a two orders of magnitude advantage in the limits of detection (LODs) for SERS compared to colorimetry was demonstrated for both DAP and HRP. The resulting LOD for HRP of 0.067 pmol/L (1.3 amol per assay) underscores that the developed approach is a highly sensitive technique. We suppose that this improved detection system could become a useful tool for the development of SERS-based ELISA protocols.

Visual detection of H2O2 and glucose by HBcAb-HRP fluorescence-enhanced CdTe QDs/CDs ratiometric fluorescence sensing platform.

This study presents the development of a sensitive and simple enhanced ratiometric fluorescence sensing platform in the consist of CdTe quantum dots (QDs), carbon dots (CDs), and hepatitis B core antibody labeled with horseradish peroxidase (HBcAb-HRP) for the visual analysis of H2O2 and glucose. The sulfur atoms in HBcAb-HRP have a strong affinity for Cd(II), which effectively enhances the fluorescence intensity of the CdTe QDs due to the generation of more radiative centers at the CdTe/Cd-SR complex. In the presence of H2O2, the Cd-S bonds are oxidized to form disulfide products and results in linear fluorescence quenching, while CDs maintain stable. Becasue glucose can be converted into H2O2 with the aid of glucose oxidase, this sensing platform can also be used for analyzing glucose. The detection limits for H2O2 and glucose are 2.9 µmol L-1 with RSD of 2.6% and 1.6 µmol L-1 with RSD of 2.4% respectively. In addition, under UV lamp irradiation, the orange-yellow CdTe QDs gradually quench with increasing H2O2 and glucose, while the blue CDs remain unchanged. A color change from orange-yellow to blue enables a visual semi-quantitative determination of H2O2 in commercial contact lens solution and glucose in human serum without any pretreatment. Thus, this CdTe QDs/CDs ratiometric sensing platform has significant potential for the rapid analysis of H2O2 and glucose in actual application.

Highly Light-Harvesting MOF-on-MOF Heterostructure: Cascading Functionality to Flexible Photogating of Organic Photoelectrochemical Transistor and Bienzyme Cascade Detection.

Recently, organic photoelectrochemical transistor (OPECT) bioanalysis has become a prominent technique for the high-performance detection of biomolecules. However, as a sensitive index of the OPECT, the dynamic regulation transconductance (gm) is still severely deficient. Herein, this work reports a new photosensitive metal-organic framework (MOF-on-MOF) heterostructure for the effective modulation of maximum gm and natural bienzyme interfacing toward choline detection. Specifically, the bidentate ligand MOF (b-MOF) was assembled onto the UiO-66 MOF (u-MOF) by a modular assembly method, which could facilitate the charge separation and generate enhanced photocurrents and offer a biophilic environment for the immobilization of choline oxidase (ChOx) and horseradish peroxidase (HRP) through hydrogen-bonded bridges. The transconductance of the OPECT could be flexibly altered by increased light intensity to maximal value at zero gate bias, and sensitive choline detection was achieved with a detection limit of 0.2 µM. This work reveals the potential of MOF-on-MOF heterostructures for futuristic optobioelectronics.

Electron Lock Manipulates the Catalytic Selectivity of Nanozyme.

Nanomaterials with enzyme-mimicking functions, termed nanozymes, offer attractive opportunities for biocatalysis and biomedicine. However, manipulating nanozyme selectivity poses an insurmountable hurdle. Here, we propose the concept of an energy-governed electron lock that controls electron transfer between nanozyme and substrates to achieve selectivity manipulation of enzyme-like catalysis. An electron lock can be constructed and opened, via modulating the nanozyme's electron energy to match the energy barrier of enzymatic reactions. An iron-doped carbon dot (FeCD) nanozyme with easy-to-regulate electron energy is selected as a proof of concept. Through regulating the conduction band which dominates electron energy, activatable oxidase and selective peroxidase (POD) with substrate affinity 123-fold higher than that of natural horseradish peroxidase (HRP) is achieved. Furthermore, while maintaining selectivity, FeCDs exhibit catalytic kinetics comparable to that of HRP upon transforming photons into electrons. Superior selectivity, efficient catalysis, and undetectable biotoxicity energize FeCDs as potent targeted drugs on antibiotic-resistant bacterial abscesses. An electron lock provides a robust strategy to manipulate selectivity toward advanced nanozymes.

Rational Biomimetic Construction of Lignin-based Carbon Nanozyme for Identification of Uric Acid in Human Urine.

Nanozymes have made remarkable progress in the field of sensing assays by replacing native enzyme functions. However, it is still a challenge to rationally design active centers from molecular structure to enhance the catalytic performance and develop low-cost nanozymes. In this work, guided by the catalytic site of horseradish peroxidase (HRP), iron source and histidine were coupled to the main chain of aminated sodium lignosulfonate (SL) through the self-assembly biomimetic strategy to construct His-SL-Fe with peroxidase activity. The inherent functional groups and basic framework of aminated SL provide a robust environment and promote the formation of active sites. His-SL-Fe shows excellent robustness over multiple test cycles and has a strong affinity for the substrate compared to HRP. His-SL-Fe had been effectively integrated in the sensing system for catalytic detection of uric acid (UA) to achieve accurate recognition of UA in the range of 0.5-100 µM with the limit of detection as low as 0.18 µM. The recovery of human urine samples is in the range of 96.8%-106.1 % and the error is within 4 %. This work not only provides a new approach for the directed design of high-performance nanozymes, but also demonstrates promising ideas for the refined application of biomass resources.

Tyramine-Invertase Bioconjugate-Amplified Personal Glucose Meter Signaling for Ultrasensitive Immunoassay.

Highly sensitive and facile detection of low levels of protein markers is of great significance for the early diagnosis and efficacy monitoring of diseases. Herein, aided by an efficient tyramine-signal amplification (TSA) mechanism, we wish to report a simple but ultrasensitive immunoassay with signal readout on a portable personal glucose meter (PGM). In this study, the bioconjugates of tyramine and invertase (Tyr-inv), which act as the critical bridge to convert and amplify the protein concentration information into glucose, are prepared following a click chemistry reaction. Then, in the presence of a target protein, the sandwich immunoreaction between the immobilized capture antibody, the target protein, and the horseradish peroxidase (HRP)-conjugated detection antibody is specifically performed in a 96-well microplate. Subsequently, the specifically loaded HRP-conjugated detection antibodies will catalyze the amplified deposition of a large number of Tyr-inv molecules onto adjacent proteins through highly efficient TSA. Then, the deposited invertase, whose dosage can faithfully reflect the original concentration of the target protein, can efficiently convert sucrose to glucose. The amount of finally produced glucose is simply quantified by the PGM, realizing the highly sensitive detection of trace protein markers such as the carcinoembryonic antigen and alpha fetoprotein antigen at the fg/mL level. This method is simple, cost-effective, and ultrasensitive without the requirement of sophisticated instruments or specialized laboratory equipment, which may provide a universal and promising technology for highly sensitive immunoassay for in vitro diagnosis of diseases.

Exploitation of Luminescent Lanthanide Nanoparticles for a Sensitivity-Enhanced ELISA Detection Method.

A new detection method based on the photoluminescence properties of dye-sensitized lanthanide nanoparticles (Ln NPs) was developed for enzyme-linked immunosorbent assays (ELISAs). In this method, the horseradish peroxidase (HRP) enzyme catalyzes the oxidation of phenol derivatives in the presence of hydrogen peroxide, providing dimers that are able to interact with the Ln NP surface and to efficiently photosensitize the Ln ions. Due to the very long emission lifetime of Ln, the time-gated detection of Ln NP luminescence allows the elimination of background noise due to the biological environment. After a comparison of the enzyme-catalyzed oxidation of various phenol derivatives, methyl 4-hydroxyphenyl acetate (MHPA) was selected as the most promising substrate, as the highest Ln emission intensity was observed following its HRP-catalyzed oxidation. After a meticulous optimization of the conditions of both the enzymatic reaction and the Ln sensitization (buffer, pH, concentration of the reactants, NP type, etc.), this new detection method was successfully implemented in a commercial insulin ELISA kit as a proof-of-concept, with an increased sensitivity compared to the commercial detection method.

Development of chia gum/alginate-polymer support for horseradish peroxidase immobilization and its application in phenolic removal.

Chia gum's molecular structure with distinctive properties as well as the alginate-based hydrogel's three-dimensionally cross-linked structure can provide a potent matrix for immobilization of enzyme. Herein, chia gum (CG)/alginate (A)-polymeric complex was synthesized and employed as a support material for the immobilization of horseradish peroxidase (HRP). HRP was successfully immobilized on the developed ACG-polymeric support, and the highest immobilization recovery (75%) was observed at 1.0% CG and 2% A, pH 7.0, and 50 units of the enzyme. The structure, morphology, and thermal properties of the prepared ACG-HRP were demonstrated using Fourier Transform Infrared (FTIR), Scanning Electron Microscope, and Thermogravimetric (TGA) analyses. ACG-HRP showed a good reusability (60%) over ten reuses. The immobilized ACG-HRP displayed an acidic pH optimum (6.0), a higher temperature optimum (50 °C), and improved thermal stability (30-50 °C) compared to the soluble HRP at pH 7.0, 40 °C and (30-40 °C), respectively. ACG-HRP has a lower affinity for hydrogen peroxide (H2O2) and guaiacol and a higher oxidizing affinity for a number of phenolic substrates. The ACG-HRP demonstrated greater resistance to heavy metals, isopropanol, urea, Triton X-100, and urea, as well as improved efficiency for eliminating phenol and p-chlorophenol. The developed ACG-polymeric support provided improved enzyme properties, allowed the reuse of the immobilized HRP in 10 cycles, and made it promising for several biotechnological applications.

Carbon dots derived from expired drugs based ratiometric fluorescent sensor for horseradish peroxidase in fruits and vegetables and screening inhibitors.

Household storage of pharmaceuticals to extract raw materials synthesized from carbon points facilitates the utilization of solid waste resources. A novel ratiometric fluorescence sensing technique was developed to ascertain the presence of horseradish peroxidase (HRP) in fruits and vegetables. The method employed a fluorescent probe, synthesized from expired amoxicillin (referred to as carbon dots, or A-CDs), serving as a reference fluorophore. Additionally, 2,3-diaminophenazine (DAP) was utilized as a specific response signal. DAP resulted from a catalytic reaction system involving phenylenediamine and hydrogen peroxide under the catalysis of HRP. The fluorescence intensity corresponding to DAP at 562 nm exhibited a substantial increase, simultaneous with the fluorescence quenching of A-CDs at 450 nm. The ratiometric fluorescence nanosensors displayed a broad linear range and high sensitivity for the detection of HRP. Across the concentration range 0.01 to 6 U L-1, the fluorescence intensity ratio between DAP and A-CDs demonstrated a proportional increase with rising HRP concentration, achieving an impressive detection limit of 0.002 U L-1. The recovery of HRP in fruit and vegetable samples ranged from 96.1 to 103%, with an RSD value of less than 3.8%. The proposed method facilitated the screening of inhibitors of HRP enzyme activity, contributing to the preservation of freshness in fruits and vegetables.