ABSTRACT Nitration is an essential process for the synthesis of energetic materials. However, conventional nitration processes are prone to exothermic decomposition, which can lead to runaway reactions and catastrophic explosions. In addition, the use of strong acid causes corrosion of equipment and poses environmental contamination. This study examined enzymatic nitration as a safer and environmentally friendly method for synthesizing these materials. An enzymatic process using horseradish peroxidase (HRP) was adopted to nitrate a triazole, 3-amino-1,2,4-triazole. The reaction proceeded under mild aqueous conditions without the use of strong acids or extreme temperatures, highlighting the safety advantages of this enzymatic method. Ultraviolet-visible (UV-Vis) spectrophotometry, and quantitative nuclear magnetic resonance (qNMR) analyses confirmed the generation of 3-nitro-1,2,4-triazole from 3-amino-1,2,4-triazole. The temperature-dependent behavior was observed: the yield of 3-nitro-1,2,4-triazole was calculated to be 91.4%. These findings demonstrate that HRP-catalyzed nitration can serve as a practical and environmentally benign alternative to traditional acid-based nitration systems.

Surface ligands play a critical role in the preparation of stable, covalently bound nanoparticle-protein conjugates. However, large surface ligands often introduce steric and antifouling effects that reduce protein conjugation yields, whereas small ligands tend to induce nanoparticle aggregation during activation. Here, we report Alkyne Nitro Tag (ANT), a compact heterobifunctional ligand that represents a design principle for nanoparticle surface chemistry. ANT presents a preinstalled reactive nitrophenyl ester that undergoes nucleophilic acyl substitution with protein residues under mild aqueous conditions, while its nitro substituent imparts colloidal stability to AuNPs through electrostatic repulsion. In ANT, the terminal alkyne is essential for strong attachment to the AuNP surface, as thiol groups are incompatible with the reactive ester. Robust conjugation of ANT-capped AuNPs (Au@ANT) with streptavidin (SA), horseradish peroxidase (HRP), and immunoglobulin G (IgG) was demonstrated by lateral flow assays, Western blotting, enzymatic sensing, and immunoprecipitation. Compared to the conventional physical adsorption and EDC/NHS chemistry, Au@ANT conjugates consistently exhibited higher yields, improved stability under physiological and denaturing conditions, and greater retention of protein activity. These results establish ANT as a generalizable and high-performance strategy for generating stable and active AuNP-protein conjugates, offering significant advantages for nanomedicine and advanced materials science.

FFF as a microfluidic platform for the streamlined optimization of ready-to-use nanozyme-labelled probes to enable robust and ultra-sensitive chemiluminescent bioassays.

Divanillin synthesis from vanillin by horseradish peroxidase in consideration of mathematical model and morphology of product

A universal protein immobilization method to construct responsive photonic hydrogels with enhanced sensing performance.

On-exosome-membrane DNA polymerization-powered magneto-electrochemical aptasensing for osteosarcoma.

Rational design of Ag@Fe3O4 core-shell nanozymes with peroxidase-mimic activity: shell thickness-dependent kinetics for the colorimetric detection of dopamine.

We report the formation of an artificial cytoskeleton within droplet-based protocells, which dramatically increases the efficiency of a cascade reaction. The cytoskeleton is formed via self-assembly of a short peptide phenylalanine-phenylalanine-methionine (FFM) in the confinement of cell-sized water-in-oil droplets. FFM undergoes coacervation, followed by fiber formation when increasing the pH value from 5.3 to 8, resulting in the generation of a fibrous network within the droplets, resembling a cytoskeleton. This cytoskeleton can bind proteins and enzymes on it, such as bovine albumin serum, glucose oxidase and horseradish peroxidase, resulting in the co-localization of the enzymes on the fiber network, which leads to the enhancement of cascade reaction efficiency. The efficiency of the cascade is even further increased when reducing the size of the microreactors from 86 µm to 49 µm and 31 µm. This artificial cytoskeleton mimics an important feature of the natural cytoskeleton, providing anchorage and colocalization for enzymes involved in cascade reactions.

Two-dimensional (2D) borophene, a single-atom-thick allotrope of boron, exhibits exceptional conductivity, anisotropy, and chemical reactivity, yet very little is known about its electrochemical properties. Here, we delineate its enzymatic and electrochemical behavior under biologically relevant redox conditions. Spectroscopic and microscopic studies reveal concentration-dependent degradation of borophene by hydrogen peroxide (H2O2), yielding boronic acids, confirmed by a curcumin-rosocyanine assay. Enzymatic cascades employing glucose oxidase and horseradish peroxidase establish borophene as both an electrocatalyst and a chemically responsive transducer, generating dual colorimetric and electrochemical outputs. Electroanalytical measurements (cyclic voltammetry, chronoamperometry, and differential pulse voltammetry) show that borophene efficiently wires peroxidase reactions by sensitizing H2O2 detection through borophene-HRP interfaces. In contrast, glucose sensing displays diminished currents due to reactive oxygen species-mediated passivation. These results position borophene as a unique platform for catalytic wiring of enzyme cascades in which ROS flux dynamically regulates signal output, enabling transient, self-reporting biosensors and motivating stabilization strategies for long-term bioelectronic integration.

Precise control over macromolecular chirality is critical for molecular recognition and targeted biological interventions. Herein, we report a pair of chiral gold nanozymes (l-/d-AuNEs) that mimic horseradish peroxidase (HRP)-like activity to catalyze the oxidative coupling of chiral phenolic monomers in a cellular environment. By introducing peptide ligands with both amino-acid-based configurational chirality and secondary structural chirality, the nanozymes enable high-yield enantioselective polymerization of the chiral Ritodrine (RITO), leading to the formation of enantiomeric dimers and high-molecular-weight polymers. The resulting polymers, Poly-l-RITO and Poly-d-RITO, exhibit significantly enhanced cytotoxicity relative to the monomer, with Poly-d-RITO showing a notably higher pro-apoptotic effect. Mechanistic studies reveal that Poly-d-RITO preferentially accumulates at the cell membrane and induces more severe membrane damage. These findings reveal a chirality-dependent biointerface effect and highlight the potential of nanozyme-driven asymmetric polymerization as an alternative strategy for chiral drug development and targeted cancer therapy.

Biofilms formed by bacterial symbiosis significantly strengthen bacterial resistance to external interference and cause chronic infections. Herein, a chemodynamic therapy (CDT) and photodynamic therapy (PDT) coarmed bacteriophage cocktail was developed to eradicate Staphylococcus aureus biofilms by conjugating aggregation-induced emission photosensitizer (AIE PSs), glucose oxidase (GOx), and horseradish peroxidase (HRP) on the bacteriophage surface. Leveraging the particular specificity of the bacteriophage toward host bacteria, the three conjugates can penetrate the biofilm and colocalize on the inner bacterial surface. When thus enriched, AIE PSs exhibited intensified fluorescence, enabling labeling and killing pathogens via photoirradiation-generated singlet oxygen. After combining AIE PSs with GOx/HRP, which can convert glucose nutrients into H2O2 and ultimately to hydroxyl radicals via cascade catalysis, the bactericidal efficiency was dramatically improved compared to individual phage-CDT (>468%) or phage-PDT (>290%) at the same PFU concentration of phage. The colocalized PSs and enzymes on the confined space of the bacterial surface are mutually promoted in the microenvironment of the biofilm, realizing synergistic enhancement. This strengthened bacteriophage cocktail offers an effective strategy for treating biofilm-related clinical superbug infections.

Development of HRP-based 3D intercellular exchange assay for high throughput screening.

A novel lignin peroxidase-mimicking by CoO-Co2VO4/C nanocomposite and its application in sensing fungal metabolite veratryl alcohol.

Precisely engineered honeycomb-like C-ZIF67 aptasensor array for integrated detection of multiple cardiac biomarkers in AMI diagnosis.

Oral (poly)peptide delivery: On the emulsifying properties of inverted lipid phases under gastrointestinal conditions.

Comparative evaluation of SARS-CoV-2 antigens as capture and detection elements in an in-house antigen-based ELISA for COVID-19 total antibody detection.

Analysis of critical biomarkers like L-lactate is extremely important in clinical practice. Herein, a non-invasive and sensitive colorimetric biosensor for accurate L-lactate determination has been developed. The proposed method demonstrates the ability of Fe3+ ions of iron(III) chloride to substitute the traditional horseradish peroxidase enzyme in the colorimetric determination of L-Lactate. The biosensor is based on the release of H2O2 by lactate oxidase enzyme (LOx) after 30min incubation in a 37°C water bath. Subsequently, H2O2 reacts with 3,3',5,5'-tetramethylbenzidine substrate (TMB) catalyzed by Fe3+ ion utilizing its peroxidase-mimetic activity. Fe3+ ion has peroxidase-like activity which could rapidly catalyze the oxidation reaction of TMB by H2O2, producing a characteristic blue colored product at 30°C water bath for 15min. Based on the catalytic mechanism of fast electron transfer between TMB and H2O2 with the assistance of the intrinsic peroxidase-like activity of Fe3+ ion, a colorimetric biosensor for determination of L-lactate was developed. The obtained colored product of oxidized TMB could be measured spectrophotometrically at λmax 652nm. The biosensor yielded a reproducible response over a linear range of 5 µM-20 µM of L-lactate with a limit of detection of 1.278 µM. Furthermore, satisfactory results were obtained upon application of the method to artificial saliva samples.

Objective: This study compares the brain protective effects of L-borneolum and its main components (a combined application of L-borneol and L-camphor) on the rat model of middle cerebral artery occlusion/reperfusion (MCAO/R). It also makes clear the intrinsic regulatory mechanisms that link the neuroprotective effects of these compounds on IS to the blood-brain barrier (BBB), based on network pharmacology predictions. Furthermore, the study investigates the relationship between these compounds and the Major Facilitator Superfamily Domain-containing Protein 2A (MFSD2A)/Caveolin-1 (Cav-1) signaling axis. Methods: The MCAO/R model in rats was established to evaluate the therapeutic effect of L-borneolum (200 mg/kg) and its main components combination of L-borneol and L-camphor (6:4 ratio, 200 mg/kg). Neurological scores, 2,3,5-triphenyl tetrazolium chloride (TTC) staining, hematoxylin-eosin (HE) staining, and Nissl staining were performed to evaluate the neurological damage in the rats. Cerebral blood flow Doppler was applied to monitor the cerebral blood flow changes. Immunofluorescence analysis of albumin leakage and transmission electron microscopy (TEM) were conducted to evaluate blood-brain barrier (BBB) integrity. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the optimal drug concentration. Trans-epithelial electrical resistance (TEER) and horseradish peroxidase (HRP) assays were employed to confirm the successful establishment of an in vitro BBB co-culture model. Network pharmacology was utilized to predict the biological processes, molecular functions, and cellular components involved in the treatment of ischemic stroke (IS) by the main components of L-borneolum (L-borneol and L-camphor). Finally, immunofluorescence, real-time fluorescent quantitative PCR (RT-qPCR) and western blot analyses were performed to detect the expression of Major Facilitator Superfamily Domain Containing 2A (MFSD2A), caveolin-1 (CAV-1), sterol regulatory element-binding protein 1 (SREBP1) in brain tissue and hCMEC/D3 cells. Results: Network pharmacology prediction indicated that L-borneolum and its main components (L-borneol and L-camphor) in the treatment of IS are likely associated with vesicle transport and neuroprotection. Treatment of IS with L-borneolum and its main components significantly decreased neurological function scores and cerebral infarction area, while alleviating pathological morphological changes and increasing the number of Nissl bodies in the hippocampus. Additionally, it improved cerebral blood flow, reduced albumin leakage, and decreased vesicle counts in the brain. The trans-epithelial electrical resistance (TEER) of the co-culture model stabilized on the fifth day after co-culture, and the permeability to horseradish peroxidase (HRP) in the co-culture model was significantly lower than that of the blank chamber at this time. RT-qPCR and Western blot results demonstrated that, compared to the model group, the expression of SREBP1 and MFSD2A significantly increased, while the expression of Cav-1 decreased. Conclusions: L-borneolum and its main components combination (L-borneol/L-camphor, 6:4 ratio) may exert a protective effect in rats with IS by improving BBB transport function through modulation of the MFSD2A/Cav-1 signaling pathway.

Small enzymes (10-50kDa) encounter challenges of unstable enzyme-carrier interactions and low activity retention during immobilization, with existing strategies lacking specificity. Unlike previous industrial enzyme-focused studies, a rigid-flexible layered immobilization strategy integrating networked metal-organic frameworks (MOFs) into flexible hydrogels is proposed. This innovative strategy stabilizes enzyme conformation, strengthens enzyme-matrix interactions, and prevents enzyme leakage via a dual-pore system. This synergistic system, composed of MOF micropores (50-150nm, confinement) and hydrogel macropores (800-900nm, mass transfer), resolves the stability-accessibility trade-off. Molecular docking shows MOFs reduce substrate-enzyme distance by 34.2% and enhance rigidity. In industrial biocatalysis, immobilized horseradish peroxidase maintains robust conversion efficiency for the reaction of 1M o-phenylenediamine over 40 cycles, effectively promoting the production efficiency of related chemical products. In the biomedical field, immobilizing thrombin reduces murine wound bleeding by 55%, providing a new solution to wound hemostasis. This scalable platform integrates nanoscale precision with macroscale responsiveness, holding great promise for advancing sustainable biocatalysis and biomedicine.

Multi-functionalization of hemin with gelatin and histamine to enhance the horseradish peroxidase catalytic activities for in situ hydrogelation