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Rapid and precise detection of hydrogen peroxide (H2O2) holds great significance since it is linked to numerous physiological and inorganic catalytic processes. We herein developed a label-free and washing-free strategy to detect H2O2 by employing a hand-held personal glucose meter (PGM) as a signal readout device. By focusing on the fact that the reduced redox mediator ([Fe(CN)6]4-) itself is responsible for the final PGM signal, we developed a new PGM-based strategy to detect H2O2 by utilizing the target H2O2-mediated oxidation of [Fe(CN)6]4- to [Fe(CN)6]3- in the presence of horseradish peroxidase (HRP) and monitoring the reduced PGM signal in response to the target amount. Based on this straightforward and facile design principle, H2O2 was successfully determined down to 3.63 μM with high specificity against various non-target molecules. We further demonstrated that this strategy could be expanded to identify another model target choline by detecting H2O2 produced through its oxidation promoted by choline oxidase. Moreover, we verified its practical applicability by reliably determining extracellular H2O2 released from the breast cancer cell line, MDA-MB-231. This work could evolve into versatile PGM-based platform technology to identify various non-glucose target molecules by employing their corresponding oxidase enzymes, greatly advancing the portable biosensing technologies.

In this work, it was found that organotin-functionalized tungstophosphates PW9-TM-SnRCOO and P2W15-TM-SnRCOO (PW9 = [PW9O34]9−; P2W15 = [P2W15O56]12−; TM = Mn, Co, Zn; R = CH2CH2) exhibited mimicking horseradish peroxidase (HRP) activity, with the catalytic activity of PW9-TM-SnRCOO being higher than that of P2W15-TM-SnRCOO. Among the polyoxometalates (POMs), those containing Zn ions demonstrated the highest catalytic activity. Furthermore, a recyclable POM-based ZIF-nanocomposite, Na8K2[Zn2{Sn(CH2)2COO}2(B-α-PW9O34)2]·13H2O@ZIF-67 (PW9-Zn-SnRCOO@ZIF-67), was designed and synthesized using a convenient one-step method at room temperature. As a mimic enzyme of HRP for the catalytic oxidation of o-phenylenediamine (OPD), PW9-Zn-SnRCOO@ZIF-67 exhibited enhanced catalytic activity and anti-interference ability. Moreover, the introduction of the POM-estertin derivative significantly improved the acid resistance of ZIF-67. PW9-Zn-SnRCOO@ZIF-67 can be further used as a colorimetric sensor material for the detection of acetone with a wide linear range (0.01–4.0 mM), a low detection limit (0.0074 mM), and good reusability. These results demonstrate that organotin-functionalized POMs@ZIFs, as a new class of non-enzymatic sensor materials, have wide application prospects for the detection of acetone in real water samples. [ABSTRACT FROM AUTHOR]

Targeted cancer therapy is a promising alternative to the currently established cancer treatments, aiming to selectively kill cancer cells while sparing healthy tissues. Hereby, molecular targeting agents, such as monoclonal antibodies, are used to bind to cancer cell surface markers specifically. Although these agents have shown great clinical success, limitations still remain such as low tumor penetration and off-target effects. To overcome this limitation, novel fusion proteins comprised of the two proteins ADAPT6 and Horseradish Peroxidase (HRP) were engineered. Cancer cell targeting is hereby enabled by the small scaffold protein ADAPT6, engineered to specifically bind to human epidermal growth factor receptor 2 (HER2), a cell surface marker overexpressed in various cancer types, while the enzyme HRP oxidizes the nontoxic prodrug indole-3-acetic acid (IAA) which leads to the formation of free radicals and thereby to cytotoxic effects on cancer cells. The high affinity to HER2, as well as the enzymatic activity of HRP, were still present for the ADAPT6-HRP fusion proteins. Further, in vitro cytotoxicity assay using HER2-positive SKOV-3 cells revealed a clear advantage of the fusion proteins over free HRP by association of the fusion proteins directly to the cancer cells and therefore sustained cell killing. This novel strategy of combining ADAPT6 and HRP represents a promising approach and a viable alternative to antibody conjugation for targeted cancer therapy. [Display omitted] • A novel fusion protein for targeted cancer therapeutics is presented. • The fusion protein combines high affinity to HER2 with enzymatic activity. • The therapeutic protein is easily produced in the bacterial host E. coli. • Efficient cancer cell killing through tumor cell-specific binding and activity has been demonstrated. [ABSTRACT FROM AUTHOR]

In this article, we present the first demonstration of a distance-based paper analytical device (dPAD) for uric acid quantification in human urine samples with instrument-free readout and user-friendliness for the rapid diagnosis and prognosis of various related diseases. By employing gold nanoparticles (AuNPs) as a peroxidase-like nanozyme, our proposed technique eliminates the utilization of horseradish peroxidase (HRP), making the device cost-effective and stable. In our dPAD, uric acid in the sample is oxidized by the uricase enzyme and subsequently catalysed with AuNPs in the sample zone, generating hydroxyl radicals (˙OH). Then, the produced ˙OH reacts with 3,3′-diaminobenzidine (DAB) to form poly DAB (oxDAB), resulting in a coloured distance signal in the detection zone of the dPAD. The variation of the distance of the observed red-brown colour is directly proportional to the uric acid concentration. Our sensor exhibited a linear range from 0.50 to 6.0 mmol L−1 (R2 = 0.9922) with a detection limit (LOD) of 0.25 mmol L−1, covering the clinical range of uric acid in urine. Hence, there is no need for additional sample preparation or dilution. Additionally, this assay is highly selective, with no interferences. We also found that this approach could accurately and precisely determine uric acid in human control samples with the recovery ranging from 99.37 to 100.35 with the highest RSD of 4.05%. Our method is comparable with the use of a commercially available uric acid sensor at a 95% confidence interval. Consequently, the developed dPAD offers numerous advantages such as cost-effectiveness, simplicity, and ease of operation with unskilled individuals. Furthermore, this concept can be applied for extensive biosensing applications in monitoring other biomarkers as an alternative analytical point-of-care (POC) device. [ABSTRACT FROM AUTHOR]

In the current work, electrostatic interactions were used to immobilize the horseradish peroxidase (HRP) onto five types of ceramic materials (C) with different concentrations of oxidized metals (C1–C5). The highest immobilization efficiency (70 and 77%) was detected at 6 mg C3 and 18 enzyme units. Scanning Electron Microscope (SEM), Energy Dispersive X-ray (EDX) and Fourier Transform Infrared (FTIR) analysis of C3-HRP confirmed the immobilization of the enzyme. After ten reuses, the reusability analysis showed that (66%) of the C3-HRP enzyme activity was retained. For C3-HRP, the optimum pH and temperature of the soluble enzyme were shifted from 7.0 and 30 °C to 6.0 and 50 °C. Up to 40 °C and 50 °C, respectively, the soluble HRP and C3-HRP remained steady. The kinetic analysis revealed that the Km and Vmax of soluble HRP and C3-HRP were, respectively, 5.5 mM, 0.66 units, and 8 mM, 0.52 units for hydrogen peroxide (H2O2) and 35.5 mM, 3.4 units and 40 mM, 1.1 units for guaiacol. Compared to soluble-HRP, the C3-HRP exhibited a greater oxidizing affinity toward several phenolic compounds (Guaiacol, o-dianisidine, o–phenylenediamine, pyrogallol, p-aminoantipyrine). In comparison with soluble-HRP, the C3-HRP showed increased stress tolerance with Triton X-100, urea, metals, isopropanol, and dimethyl sulfoxide. The C3-HRP removed methyl orange more effectively compared to soluble-HRP. [ABSTRACT FROM AUTHOR]

The catalytic hairpin assembly (CHA)-based miRNA detection methods have garnered significant attention due to their simplicity and acceptable amplification efficiency. However, these methods necessitate improved sensitivity. In this work, we present a colorimetric and ultrasensitive approach for the detection of cancer-related miRNAs which is initiated by CHA-mediated nicking endonuclease-assisted signals recycling. The target initiates the CHA process to expose the functional section in the P2 probe. This section can activate cascaded recycling cycles to produce numerous linker sequences by Nt.AlwI endonuclease-assisted cleavage of two hairpin signal probes. The 3,3',5,5'-tetramethylbenzidine sulfate (TMB)/H2O2-based color reaction is induced by the fixation of the cDNA-HRP on the surface of magnetic beads, which is mediated by the linker sequence. The proposed method demonstrates a sensitivity that is either comparable to or superior to that of previous colorimetric miRNA detection methods, as a result of this design. Furthermore, the method demonstrated a promising potential for clinical applications and a high selectivity to target miRNA. Consequently, it provides a colorimetric assay that is both ultrasensitive and dependable for the visual detection of miRNA, which has the potential to revolutionize the early diagnosis of cancer. [ABSTRACT FROM AUTHOR]

Diabetic wound healing presents a persistent clinical challenge, often characterized by prolonged healing times, and can be particularly difficult to achieve in a hyperglycemic environment. In this study, a multi‐functional silver‐enzyme nanogels assembly (Ag‐nGHC) is designed by focusing on the complex diabetic wound environment. Glucose oxidase (GOX), horseradish peroxidase (HRP), and catalase (CAT) are modified within polymeric nanogel layers and assembled into a large enzyme cluster with silver ions. The close attachment of three enzymes ensures fast and continuous consumption of a high level of glucose, generation of oxygen, and hydroxyl radicals (•OH) around the wound site. Meanwhile, the silver ions within the Ag‐nGHC assembly act as an effective agent to kill bacteria. This cascade enzyme system significantly improves the microenvironment of the wound site by reducing bacterium infection and alleviating hypoxia as well as hyperglycemia. Sequentially, the improved environment facilitates the later processes including anti‐inflammatory, re‐epithelialization, and angiogenesis, evidenced by enhancing polarization toward M2 macrophages and increasing CD31 signals in this study. Overall, the Ag‐nGHC materials are proven to achieve multifunctional properties toward complicated diabetic wound healing processes (attributes such as adaptability, hypoxia‐alleviated, anti‐hyperglycemic, antimicrobial, anti‐inflammatory, and angiogenic) and showed great potential for the treatment of chronic diabetic wound. [ABSTRACT FROM AUTHOR]

The adsorption efficiency of Cr(VI) and anionic textile dyes onto MgAl-layered double hydroxides (LDHs) and MgAl-LDH coated on bio-silica (b-SiO2) nanoparticles (MgAl-LDH@SiO2) derived from waste rice husks was studied in this work. The material was characterized using field-emission scanning electron microscopy (FE-SEM/EDS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopic (XPS) techniques. The adsorption capacities of MgAl-LDH@SiO2 were increased by 12.2%, 11.7%, 10.6%, and 10.0% in the processes of Cr(VI), Acid Blue 225 (AB-225), Acid Violet 109 (AV-109), and Acid Green 40 (AG-40) dye removal versus MgAl-LDH. The obtained results indicated the contribution of b-SiO2 to the development of active surface functionalities of MgAl-LDH. A kinetic study indicated lower intraparticle diffusional transport resistance. Physisorption is the dominant mechanism for dye removal, while surface complexation dominates in the processes of Cr(VI) removal. The disposal of effluent water after five adsorption/desorption cycles was attained using enzymatic decolorization, photocatalytic degradation of the dyes, and chromate reduction, satisfying the prescribed national legislation. Under optimal conditions and using immobilized horseradish peroxidase (HRP), efficient decolorization of effluent solutions containing AB-225 and AV-109 dyes was achieved. Exhausted MgAl-LDH@SiO2 was processed by dissolution/precipitation of Mg and Al hydroxides, while residual silica was used as a reinforcing filler in polyester composites. The fire-proofing properties of composites with Mg and Al hydroxides were also improved, which provides a closed loop with zero waste generation. The development of wastewater treatment technologies and the production of potentially marketable composites led to the successful achievement of both low environmental impacts and circular economy implementation. [ABSTRACT FROM AUTHOR]

Engineering multimetallic nanocatalysts with the entropy‐mediated strategy to reduce reaction activation energy is regarded as an innovative and effective approach to facilitate efficient heterogeneous catalysis. Accordingly, conformational entropy‐driven high‐entropy alloys (HEAs) are emerging as a promising candidate to settle the catalytic efficiency limitations of nanozymes, attributed to their versatile active site compositions and synergistic effects. As proof of the high‐entropy nanozymes (HEzymes) concept, elaborate PdMoPtCoNi HEA nanowires (NWs) with abundant active sites and tuned electronic structures, exhibiting peroxidase‐mimicking activity comparable to that of natural horseradish peroxidase are reported. Density functional theory calculations demonstrate that the enhanced electron abundance of HEA NWs near the Fermi level (EF) is facilitated via the self‐complementation effect among the diverse transition metal sites, thereby boosting the electron transfer efficiency at the catalytic interface through the cocktail effect. Subsequently, the HEzymes are integrated with a portable electronic device that utilizes Internet of Things‐driven signal conversion and wireless transmission functions for point‐of‐care diagnosis to validate their applicability in digital biosensing of urinary biomarkers. The proposed HEzymes underscore significant potential in enhancing nanozymes catalysis through tunable electronic structures and synergistic effects, paving the way for reformative advancements in nano‐bio analysis. [ABSTRACT FROM AUTHOR]

[Display omitted] • FOx and HRP were co-immobilized in organic–inorganic hybrid nanoflower. • FOx@HRP showed good catalytic activity owing to spatial localization of enzymes. • FOx@HRP could be applied in the sensitive and colorimetric detection of formate. • FOx@HRP possessed ideal stability and reusability in the detection of formate. Formate is an important environmental pollutant, and meanwhile its concentration change is associated with a variety of diseases. Thus, rapid and sensitive detection of formate is critical for the biochemical analysis of complex samples and clinical diagnosis of multiple diseases. Herein, a colorimetric biosensor was constructed based on the cascade catalysis of formate oxidase (FOx) and horseradish peroxidase (HRP). These two enzymes were co-immobilized in Cu 3 (PO 4) 2 -based hybrid nanoflower with spatial localization, in which FOx and HRP were located in the shell and core of nanoflower, respectively (FOx@HRP). In this system, FOx could catalyze the oxidation of formate to generate H 2 O 2 , which was then utilized by HRP to oxidize 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid to yield blue product. Ideal linear correlation could be obtained between the absorbance at 420 nm and formate concentration. Meanwhile, FOx@HRP exhibited excellent detection performance with low limit of detection (6 μM), wide linear detection range (10–900 μM), and favorable specificity, stability and reusability. Moreover, it could be applied in the detection of formate in environmental, food and biological samples with high accuracy. Collectively, FOx@HRP provides a useful strategy for the simple and sensitive detection of formate and is potentially to be used in biochemical analysis and clinical diagnosis. [ABSTRACT FROM AUTHOR]

Phenolic compounds are expected to be successfully removed from wastewater as pervasive environmental contaminants. For the removal of phenolic chemicals, enzymes such as horseradish peroxidase have shown to have a lot of promise. Horseradish peroxidase (HRP) was immobilized on amino functionalized metal organic frameworks (NH2-MOF-Zr) utilizing covalent bonds. The microstructural and physicochemical properties were investigated using Fourier transform infrared (FT-IR), X-Ray diffraction (XRD) analysis, and scanning electron microscopy (SEM). The free HRP and NH2-MOF-Zr@HRP were characterized by determining the activity profile as a function of kinetic behavior, pH, storage stability, effect of organic solvent, and temperature. The Km and Vmax for NH2-MOF-Zr@HRP were estimated to be 34.7 mM, 0.811 U/mL for guaiacol and 12.58 mM, 0.930 U/mL for H2O2, respectively. The optimum pH values of the enzyme activity were found as 7 and 7–7.5 for the free HRP and NH2-MOF-Zr@HRP, respectively. The optimum temperature profile of the free HRP and NH2-MOF-Zr@HRP revealed as 40 °C and 50 °C, respectively. NH2-MOF-Zr@HRP maintained 74% of its initial activity after 6 weeks of storage and 59% of its initial activity after ten consecutive cycles of the guaiacol oxidation. A substantial degree of the phenolic compounds was removed by HRP immobilized on the NH2-MOF-Zr. About 87%, 53%, and 49% of 4-methoxyphenol (4-MEP), bisphenol A (BPA), and phenol were removed by NH2-MOF-Zr@HRP during a 6-h reaction. The removal efficiency of phenol, BPA, and 4-MEP after five reuse cycles was 48%, 51%, and 62%, respectively. [ABSTRACT FROM AUTHOR]

In this study, a mathematical model was formulated to characterize the functioning of third‐generation enzyme‐based amperometric biosensors. This study utilized a horseradish peroxidase (HRP)‐based, screen‐printed biosensor. The model parameters were determined using the Environment for Modeling Simulation and Optimization (EMSO), validated with experimental data, and exhibited high determination coefficients, indicating the accuracy of the models in capturing biosensor behavior. Simulations based on this model emphasize diffusion as a limiting factor. Further sensitivity analysis was conducted using simulated response surfaces and biosensor performance optimization, which confirmed that higher concentrations of HRP in the thinnest polymeric film resulted in enhanced biosensor sensitivity. [ABSTRACT FROM AUTHOR]

The 3-phenoxybenzoic acid (3-PBA) residues in environment are posing a significant challenge to our daily lives. To establish a more sensitive and rapid detection method, anti-3-PBA nanobodies (Nbs) were immobilized onto magnetosomes (bacterial magnetic nanoparticles, termed as BMPs), forming a robust BMP-Nb complex. The 3-PBA derivative was labeled with horseradish peroxidase (HRP) and further associated with gold nanoparticles (AuNPs) to create a highly sensitive probe (3-PBA-HRP-AuNP). An innovative immunoassay that combined BMP-Nb complex with 3-PBA-HRP-AuNP was developed for determinaton of 3-PBA. This method enabled the determination of 3-PBA with a half-maximum signal inhibition concentration (IC50) of 1.03 ng/mL, which was more sensitive than that of using 3-PBA-HRP as tracer with an IC50 of 2.18 ng/mL. The reliability of the assay was evidenced by the quantitative recovery of 3-PBA from water and soil samples ranging from 76.85 to 95.64%. The 3-PBA residues determined by this assay in actual water samples were between < LOD and 2.54 ng/mL and were between < LOD and 11.25 ng/g (dw) in real soils, respectively, which agreed well with those of liquid chromatography mass spectrometry (LC–MS). Collectively, the BMP-Nb and 3-PBA-HRP-AuNP-based immunoassay provides a powerful tool for the precise detection of 3-PBA residues in environment matrices, reinforcing our capacity to monitor and mitigate potential ecological and health impacts associated with this prevalent pollutant. [ABSTRACT FROM AUTHOR]

The enhanced and direct immobilization of the enzyme horseradish peroxidase on poly(methyl methacrylate) (PMMA) microchannel surfaces to create a miniaturized enzymatic reactor for the biocatalytic oxidation of phenols is demonstrated. Enzyme immobilization occurs by physical adsorption after oxygen plasma treatment, which micro-nanotextures the PMMA surfaces. A five-fold enhancement in immobilized enzyme activity was observed, attributed to the increased surface area and, therefore, to a higher quantity of immobilized enzymes compared to an untreated PMMA surface. The enzymatic reaction yield reached 75% using a flow rate of 2.0 μL/min for the reaction mixture. Additionally, the developed microreactor was reused more than 16 times without affecting the enzymatic conversion yield. These results demonstrate the potential of microchannels with plasma micro/nanotextured surfaces for the rapid and facile fabrication of microfluidic enzymatic microreactors with enhanced catalytic activity and stability. [ABSTRACT FROM AUTHOR]

Laser-induced graphene (LIG) possesses characteristics of easy handling, miniaturization, and unique electrical properties. We modified the surface of LIG by electropolymerizing β-cyclodextrin (β-CD), which was used to immobilize antibodies on the electrode surface for highly sensitive detection of targets. N6-methyladenosine (m6A) is the most prevalent reversible modification in mammalian messenger RNA and noncoding RNA, influencing the development of various cancers. Here, β-CD was electropolymerized to immobilize the anti-m6A antibody, which subsequently recognized the target m6A. This was integrated into the catalytic hydrogen peroxide–hydroquinone (H2O2-HQ) redox system using phos-tag-biotin to generate electrochemical signals from streptavidin-modified horseradish peroxidase (SA-HRP). Under optimal conditions, the biosensor exhibited a linear range from 0.1 to 100 nM with a minimum detection limit of 96 pM. The method was successfully applied to the recovery analysis of m6A from HeLa cells through spiking experiments and aims to inspire strategies for point-of-care testing (POCT). [ABSTRACT FROM AUTHOR]

Early diagnosis and treatment of gastric cancer (GC) play a vital role in improving efficacy, reducing mortality and prolonging patients' lives. Given the importance of early detection of gastric cancer, an electrochemical biosensor was developed for the ultrasensitive detection of miR-19b-3p by integrating MoS2-based nanozymes, hybridization chain reaction (HCR) with enzyme catalyzed reaction. The as-prepared MoS2-based nanocomposites were used as substrate materials to construct nanoprobes, which can simultaneously load probe DNA and HCR initiator for signal amplification. Moreover, the MoS2-based nanocomposites are also employed as nanozymes to amplify electrochemical response. The presence of miR-19b-3p induced the assembly of MoS2-based nanoprobes on the electrode surface, which can activate in-situ HCR reaction to load a large number of horseradish peroxidase (HRP) for signal amplification. Coupling with the co-catalytic ability of HRP and MoS2-based nanozymes, the designed electrochemical biosensor can detect as low as 0.7 aM miR-19b-3p. More importantly, this biosensor can efficiently analyze miR-19b-3p in clinical samples from healthy people and gastric cancer patients due to its excellent sensitivity and selectivity, suggesting that this biosensor has a potential application in early diagnosis of disease. [ABSTRACT FROM AUTHOR]

The immobilized horseradish peroxidase (P1-HRP) was prepared based on the affinity interaction between the phenylborate group in mVBA-b-p (AAm co AN) polymer (P1) and the adjacent dihydroxy group in horseradish peroxidase (HRP). The immobilization conditions of P1-HRP were optimized. Under the conditions of P1 concentration of 20 mg/mL, pH 8, temperature of 50 ℃, and immobilization time of 2 h, the HRP was immobilized to obtain the optimal immobilization amount (84.7 mg/g). The successful preparation of P1-HRP was demonstrated through characterizations such as laser scanning confocal microscopy (LCSM), scanning electron microscope (SEM), dynamic light scattering (DLS) and thermogravimetric analyzer (TGA). P1-HRP exhibited excellent pH stability, thermal stability and storage stability compared to the free horseradish peroxidase. P1-HRP had shown good application value in the degradation of phenol pollutants. After 10 repetitions of phenol degradation, the relative enzyme activity of P1-HRP could still be retained by 55.62%, indicating its good reusability. The optimal conditions for the catalytic degradation of phenol by P1-HRP were optimized. After catalytic degradation of phenol for 60 min under the same conditions, the degradation rate of P1-HRP (92.33%) was higher than that of free horseradish peroxidase (85.32%). [ABSTRACT FROM AUTHOR]

Hydrogels have garnered significant interest in the biomedical field owing to their tissue-like properties and capability to incorporate various fillers. Among these, injectable hydrogels have been highlighted for their unique advantages, especially their minimally invasive administration mode for implantable use. These injectable hydrogels can be utilized in their pristine forms or as composites by integrating them with therapeutic filler materials. Given their primary application in implantable platforms, enzymatically crosslinked injectable hydrogels have been actively explored due to their excellent biocompatibility and easily controllable mechanical properties for the desired use. This review introduces the crosslinking mechanisms of such hydrogels, focusing on those mediated by horseradish peroxidase (HRP), transglutaminase (TG), and tyrosinase. Furthermore, several parameters and their relationships with the intrinsic properties of hydrogels are investigated. Subsequently, the representative biomedical applications of enzymatically crosslinked-injectable hydrogels are presented, including those for wound healing, preventing post-operative adhesion (POA), and hemostasis. Furthermore, hydrogel composites containing filler materials, such as therapeutic cells, proteins, and drugs, are analyzed. In conclusion, we examine the scientific challenges and directions for future developments in the field of enzymatically crosslinked-injectable hydrogels, focusing on material selection, intrinsic properties, and filler integration. [ABSTRACT FROM AUTHOR]

Cell wall peptidoglycan binding domains (CBDs) of cell lytic enzymes, including bacteriocins, autolysins and bacteriophage endolysins, enable highly selective bacterial binding, and thus, have potential as biorecognition molecules for nondestructive bacterial detection. Here, a novel design for a self-complementing split fluorescent protein (FP) complex is proposed, where a multimeric FP chain fused with specific CBDs ((FP-CBD) n) is assembled inside the cell, to improve sensitivity by enhancing the signal generated upon Staphylococcus aureus or Bacillus anthracis binding. Flow cytometry shows enhanced fluorescence on the cell surface with increasing FP stoichiometry and surface plasmon resonance reveals nanomolar binding affinity to isolated peptidoglycan. The breadth of function of these complexes is demonstrated through the use of CBD modularity and the ability to attach enzymatic detection modalities. Horseradish peroxidase-coupled (FP-CBD) n complexes generate a catalytic amplification, with the degree of amplification increasing as a function of FP length, reaching a limit of detection (LOD) of 103 cells/droplet (approximately 0.1 ng S. aureus or B. anthracis) within 15 min on a polystyrene surface. These fusion proteins can be multiplexed for simultaneous detection. Multimeric split FP-CBD fusions enable use as a biorecognition molecule with enhanced signal for use in bacterial biosensing platforms. [Display omitted] • Multimeric fluorescent protein (FP) chain with cell wall binding domains (CBDs). • Self-complementing split FP complexation with specific CBDs inside the cell. • Enhanced signal generation upon target bacteria binding induced by multimeric CBDs. • Amplified colorimetric signals of HRP-coupled FP-CBD fusions for bacteria sensing. • Multiple FP-CBD fusions for simultaneous detection of different bacteria. [ABSTRACT FROM AUTHOR]

In this work, a horseradish peroxidase (HRP)‐encapsulated metal organic framework (MOF)@MOF nanocomposite is developed for detecting H2O2 converted by dismutation of superoxide anions released from live HeLa mitochondria. Initially, an HRP‐polyacrylic acid cluster is incorporated on a mesoporous, peroxidase‐like Cu/Co‐1,4‐benzenedicarboxylate (BDC) MOF platform to avoid structural change and deactivation of HRP through its interactions with MOF metal ions. Additionally, a Cu/Co‐BDC(HRP)@1,3,5‐benzenetricarboxyate (BTC) core‐shell MOF/MOF structure, also with peroxide‐like properties, serves as a protective matrix for HRP. Then, ultrathin porous carbon shells (UPCS) are adopted to improve the electrical conductivity of the MOF@MOF. The Cu/Co‐BDC(HRP)@BTC|UPCS sensing platform exhibits two linear ranges of 0.05–1 µM and 1–1000 µM with a sensitivity of 172 mA mM−1 cm−2 and 1.63 mA mM−1 cm−2, respectively. A limit of detection of 0.057 µM, good selectivity and stability over 35 days for H2O2 detection are also achieved. After treating the mitochondrial complex with specific inhibitors, amperometric results at the sensing platform confirmed complex I and III within mitochondria as the main electron leakage sites in the electron transfer chains. Therefore, this sensing platform provides a tool that may aid in predicting and even developing treatments for some oxidative stress diseases caused by mitochondrial abnormalities. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus (S. aureus) infection is a primary cause of otitis media (OM), the most common disease for which children are prescribed antibiotics. However, the abuse of antibiotics has led to a global increase in antimicrobial resistance (AMR). Nanozymes, as promising alternatives to traditional antibiotics, are being extensively utilized to combat AMR. Here, we synthesize a series of single-atom nanozymes (metal-C3N4 SANzymes) by loading four metals (Ag, Fe, Cu, Ru) with antibacterial properties onto a crystalline g-C3N4. These metal-C3N4 display a rob-like morphology and well-dispersed metal atoms. Among them, Ru-C3N4 demonstrates the optimal peroxidase-like activity (285.3 U mg-1), comparable to that of horseradish peroxidase (267.7 U mg-1). In vitro antibacterial assays reveal that Ru-C3N4 significantly inhibits S. aureus growth compared with other metal-C3N4 even at a low concentration (0.06 mg mL-1). Notably, Ru-C3N4 acts as a narrow-spectrum nanoantibiotic with relative specificity against Gram-positive bacteria. Biofilms formed by S. aureus are easily degraded by Ru-C3N4 due to its high peroxidase-like activity. In vivo, Ru-C3N4 effectively eliminates S. aureus and relieves ear inflammation in OM mouse models. However, untreated OM mice eventually develop hearing impairment. Due to its low metal load, Ru-C3N4 does not exhibit significant toxicity to blood, liver, or kidney. In conclusion, this study presents a novel SANzyme-based antibiotic that can effectively eliminate S. aureus and treat S. aureus-induced OM. [ABSTRACT FROM AUTHOR]

Japanese encephalitis virus (JEV) is a mosquito-borne virus that can infect pigs, horses, and other mammals, including humans. Sero-epidemiological investigations of JEV have been performed using hemagglutination inhibition (HI), virus neutralization (VN) tests and enzyme-linked immunosorbent assay (ELISA). A need exists for a new ELISA that can detect JEV antibodies in the sera of several animal species. We aimed to develop a blocking ELISA (B-ELISA) for detecting JEV antibodies in pig and horse serum samples. JEV antibodies in 218 pig and 315 horse serum samples were measured using HI and VN tests. The purified KV1899-306 strain was used as an antigen for B-ELISA. The purified antibody (7A13) was conjugated with horseradish peroxidase and used as a detector antibody. The sera of pigs and horses to measure antibody against JEV were subjected to B-ELISA and analyzed. The B-ELISA had a diagnostic sensitivity of 94.6% to 100%, a specificity of 91.2 to 100%, and an accuracy of 94.9 to 98.6% compared with those of the HI and VN tests in pig and horse sera. The B-ELISA had a higher correlation with pig sera (r = 0.89 and 0.90 for VN and HI) than with horse sera (r = 0.75 and to 0.79). The new B-ELISA could be useful in the sero-surveillance of JEV in pig and horse sera and replace indirect ELISA. [ABSTRACT FROM AUTHOR]

An innovative supramolecular architecture is reported for bienzymatic glucose biosensing based on the use of a nanohybrid made of multi-walled carbon nanotubes (MWCNTs) non-covalently functionalized with a Schiff base modified with two phenylboronic acid residues (SB-dBA) as platform for the site-specific immobilization of the glycoproteins glucose oxidase (GOx) and horseradish peroxidase (HRP). The analytical signal was obtained from amperometric experiments at − 0.050 V in the presence of 5.0 × 10−4 M hydroquinone as redox mediator. The concentration of GOx and HRP and the interaction time between the enzymes and the nanohybrid MWCNT–SB-dBA deposited at glassy carbon electrodes (GCEs) were optimized through a central composite design (CCD)/response surface methodology (RSM). The optimal concentrations of GOx and HRP were 3.0 mg mL−1 and 1.50 mg mL−1, respectively, while the optimum interaction time was 3.0 min. The bienzymatic biosensor presented a sensitivity of (24 ± 2) × 102 µA dL mg−1 ((44 ± 4) × 102 µA M−1), a linear range between 0.06 mg dL−1 and 21.6 mg dL−1 (3.1 µM–1.2 mM) (R2 = 0.9991), and detection and quantification limits of 0.02 mg dL−1 (1.0 µM) and 0.06 mg dL−1 (3.1 µM), respectively. The reproducibility for five sensors prepared with the same MWCNT–SB-dBA nanohybrid was 6.3%, while the reproducibility for sensors prepared with five different nanohybrids and five electrodes each was 7.9%. The GCE/MWCNT–SB-dBA/GOx-HRP was successfully used for the quantification of glucose in artificial human urine and commercial human serum samples. [ABSTRACT FROM AUTHOR]

Enzymatic modification, compared with traditional starch (St) chemical modification methods, avoids using toxic solvents and toxic catalysts. The horseradish peroxidase (HRP), being the most commonly employed natural green initiator, has found extensive application in starch grafting modification as a substitute for chemical initiators. HRP‐catalyzed grafting of starch with vinyl zwitterionic monomers has not been reported. Here, modified starch (St‐PSBMA) is first synthesized by grafting zwitterionic sulfobetaine [2‐(Methacryloyloxy)ethyl]dimethyl‐(3‐sulfopropyl) (SBMA) onto starch by HRP/Acetylacetone(ACAC) binary initiation system catalyzed. The modified starch‐based hydrogel is then prepared by the repeated freeze‐thaw method. Compared with native starch‐based hydrogels, which bind water molecules only through hydrogen bonding, the ability of modified starch‐based hydrogels to bind water molecules is enhanced by ionic solvation. The modified starch hydrogels also have excellent antiprotein adsorption and antibacterial properties due to the presence of a strong hydration layer. The modified starch‐based hydrogel also has excellent sensitivity and stability when used as a sensor for detecting joint motion. The binary HRP initiating system, employed in this study instead of the traditional three‐component HRP initiating system, offers a novel and promising approach for grafting modification of starch without the need for strong oxidant hydrogen peroxide. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) bioprinting has emerged as an important technique for fabricating tissue constructs with precise structural and compositional control. However, developing suitable bioinks with biocompatible crosslinking mechanisms remains a significant challenge. This study investigates extrusion-based bioprinting (EBB) using uniaxial or coaxial nozzles with enzymatic crosslinking (EC) to produce 3D tissue constructs in vitro. Initially, low-molecular-weight dextran-tyramine and hyaluronic acid-tyramine (LMW Dex-TA/HA-TA) bioink prepolymers were evaluated. Enzymatically pre-crosslinking these prepolymers, achieved by the addition of horseradish peroxidase and hydrogen peroxide, produced viscous polymer solutions. However, this approach resulted in inconsistent bioprinting outcomes (uniaxial) due to inhomogeneous crosslinking, leading to irreproducible properties and suboptimal shear recovery behavior of the hydrogel inks. To address these challenges, we explored a one-step coaxial bioprinting system consisting of enzymatically crosslinkable high-molecular-weight hyaluronic acid-tyramine conjugates (HMW HA-TA) mixed with horseradish peroxidase (HRP) in the inner core and a mixture of Pluronic F127 and hydrogen peroxide in the outer shell. This configuration resulted in nearly instantaneous gelation by diffusion of the hydrogen peroxide into the core. Stable hydrogel fibers with desirable properties, including appropriate swelling ratios and controlled degradation rates, were obtained. The optimized bioink and printing parameters included 1.3% w/v HMW HA-TA and 5.5 U/mL HRP (bioink, inner core), and 27.5% w/v Pluronic F127 and 0.1% H2O2 (sacrificial ink, outer shell). Additionally, optimal pressures for the inner core and outer shell were 45 and 80 kPa, combined with a printing speed of 300 mm/min and a bed temperature of 30 °C. The extruded HMW HA-TA core filaments, containing bovine primary chondrocytes (BPCs) or 3T3 fibroblasts (3T3 Fs), exhibited good cell viabilities and were successfully cultured for up to seven days. This study serves as a proof-of-concept for the one-step generation of core filaments using a rapidly gelling bioink with an enzymatic crosslinking mechanism, and a coaxial bioprinter nozzle system. The results demonstrate significant potential for developing designed, printed, and organized 3D tissue fiber constructs. [ABSTRACT FROM AUTHOR]

Hydrogels are widely used in tissue engineering due to their ability to form three-dimensional (3D) structures that support cellular functions and mimic the extracellular matrix (ECM). Despite their advantages, dextran-based hydrogels lack intrinsic biological activity, limiting their use in this field. Here, we present a strategy for developing bioactive hydrogels through sequential thiol–maleimide bio-functionalization and enzyme-catalyzed crosslinking. The hydrogel network is formed through the reaction of tyramine moieties in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2), allowing for tunable gelation time and stiffness by adjusting H2O2 concentrations. Maleimide groups on the hydrogel backbone enable the coupling of thiol-containing bioactive molecules, such as arginylglycylaspartic acid (RGD) peptides, to enhance biological activity. We examined the effects of hydrogel stiffness and RGD concentration on human mesenchymal stem cells (hMSCs) during differentiation and found that hMSCs encapsulated within these hydrogels exhibited over 88% cell viability on day 1 across all conditions, with a slight reduction to 60–81% by day 14. Furthermore, the hydrogels facilitated adipogenic differentiation, as evidenced by positive Oil Red O staining. These findings demonstrate that DexTA–Mal hydrogels create a biocompatible environment that is conducive to cell viability and differentiation, offering a versatile platform for future tissue engineering applications. [ABSTRACT FROM AUTHOR]

Estrogenic chemicals are widely distributed and structurally diverse. They primarily disrupt estrogen-related metabolism in animals or humans by mimicking the agonistic receptor effects of natural estrogens, thereby influencing the transcription of estrogen receptors to regulate their quantity and sensitivity. This disruption of estrogen-related metabolism can lead to estrogen-related effects, posing risks to biological health, emphasizing the urgent need for simple and effective methods to screen compounds with estrogenic effects. Herein, a new electrochemical biological effect biosensor based on human estrogen receptor α (hERα) is developed, which uses hERα as the biorecognition element and employs the electroactive horseradish peroxidase (HRP) labeled 17β-estradiol (E2) multifunctional conjugate HRP-E2 as the signal-boosting element and ligand competition agent. Based on the specific ligand–receptor interaction principle between the target and nuclear receptor, by allowing the test compound to compete with HRP-E2 conjugate for binding to hERα and testing the electrocatalytic signal of the conjugate that fails to bind to the hERα estrogen receptor, rapid screening and quantitative detection of chemical substances with estrogenic effect have been achieved. The biosensor shows a wide linear range of 40 pM to 40 nM with a detection limit of 17 pM (S/N = 3) for E2, and the detection limit is 2 orders of magnitude better than that of the previously reported sensors. The biosensor based on ligand–receptor binding can not only quantitatively analyze the typical estrogen E2, but also evaluate the relative estrogen effect strength of other estrogen compounds, which has good stability and selectivity. This electrochemical sensing platform displays its promising potential for rapid screening and quantitative detection of chemicals with estrogenic effects. [ABSTRACT FROM AUTHOR]

Abstract In the current work, electrostatic interactions were used to immobilize the horseradish peroxidase (HRP) onto five types of ceramic materials (C) with different concentrations of oxidized metals (C1–C5). The highest immobilization efficiency (70 and 77%) was detected at 6 mg C3 and 18 enzyme units. Scanning Electron Microscope (SEM), Energy Dispersive X-ray (EDX) and Fourier Transform Infrared (FTIR) analysis of C3-HRP confirmed the immobilization of the enzyme. After ten reuses, the reusability analysis showed that (66%) of the C3-HRP enzyme activity was retained. For C3-HRP, the optimum pH and temperature of the soluble enzyme were shifted from 7.0 and 30 °C to 6.0 and 50 °C. Up to 40 °C and 50 °C, respectively, the soluble HRP and C3-HRP remained steady. The kinetic analysis revealed that the Km and Vmax of soluble HRP and C3-HRP were, respectively, 5.5 mM, 0.66 units, and 8 mM, 0.52 units for hydrogen peroxide (H2O2) and 35.5 mM, 3.4 units and 40 mM, 1.1 units for guaiacol. Compared to soluble-HRP, the C3-HRP exhibited a greater oxidizing affinity toward several phenolic compounds (Guaiacol, o-dianisidine, o–phenylenediamine, pyrogallol, p-aminoantipyrine). In comparison with soluble-HRP, the C3-HRP showed increased stress tolerance with Triton X-100, urea, metals, isopropanol, and dimethyl sulfoxide. The C3-HRP removed methyl orange more effectively compared to soluble-HRP.

Abstract The catalytic hairpin assembly (CHA)-based miRNA detection methods have garnered significant attention due to their simplicity and acceptable amplification efficiency. However, these methods necessitate improved sensitivity. In this work, we present a colorimetric and ultrasensitive approach for the detection of cancer-related miRNAs which is initiated by CHA-mediated nicking endonuclease-assisted signals recycling. The target initiates the CHA process to expose the functional section in the P2 probe. This section can activate cascaded recycling cycles to produce numerous linker sequences by Nt.AlwI endonuclease-assisted cleavage of two hairpin signal probes. The 3,3’,5,5’-tetramethylbenzidine sulfate (TMB)/H2O2-based color reaction is induced by the fixation of the cDNA-HRP on the surface of magnetic beads, which is mediated by the linker sequence. The proposed method demonstrates a sensitivity that is either comparable to or superior to that of previous colorimetric miRNA detection methods, as a result of this design. Furthermore, the method demonstrated a promising potential for clinical applications and a high selectivity to target miRNA. Consequently, it provides a colorimetric assay that is both ultrasensitive and dependable for the visual detection of miRNA, which has the potential to revolutionize the early diagnosis of cancer.

Peroxidases are essential elements in many biotechnological applications. An especially interesting concept involves split enzymes, where the enzyme is separated into two smaller and inactive proteins that can dimerize into a fully active enzyme. Such split forms were developed for the horseradish peroxidase (HRP) and ascorbate peroxidase (APX) already. Both peroxidases have a high potential for biotechnology applications. In the present study, we performed biophysical comparisons of these two peroxidases and their split analogues. The active site availability is similar for all four structures. The split enzymes are comparable in stability with their native analogues, meaning that they can be used for further biotechnology applications. Also, the tertiary structures of the two peroxidases are similar. However, differences that might help in choosing one system over another for biotechnology applications were noticed. The main difference between the two systems is glycosylation which is not present in the case of APX/sAPEX2, while it has a high impact on the HRP/sHRP stability. Further differences are calcium ions and cysteine bridges that are present only in the case of HRP/sHRP. Finally, computational results identified sAPEX2 as the systems with the smallest structural variations during molecular dynamics simulations showing its dominant stability comparing to other simulated proteins. Taken all together, the sAPEX2 system has a high potential for biotechnological applications due to the lack of glycans and cysteines, as well as due to high stability.

2D chitosan modified Mo x W 1−x Se 2 transformed conformational nanoarchitectonics loaded with chemotherapeutic drug mitoxantrone was utilized to explore the synergistic therapeutic effects of triple PTT/CDT/CT, thereby achieving the personalised therapy of breast carcinoma. [Display omitted] Two-dimensional transition metal dichalcogenides (TMDCs) exhibit promising photothermal therapy (PTT) and chemodynamic therapy (CDT) for anti-tumour treatment. Herein, we proposed an engineering strategy to regulate the lattice structure of tungsten-doped molybdenum selenide (Mo x W 1−x Se 2) transformed conformational nanoarchitectonics using a microwave-assisted solvothermal method for enhancing peroxidase (POD)-like catalytic performance by adjusting the ratio of molybdenum (Mo) and tungsten (W). Furthermore, the optimised Mo 0.8 W 0.2 Se 2 nanoflakes surface was modified with chitosan (CHI) for improved biocompatibility and nanocatalytic efficacy, then the obtained CHI-Mo 0.8 W 0.2 Se 2 subsequently loaded the chemotherapeutic drug mitoxantrone (MTO) for enhanced 4 T1 cells killing ability, shortly denoted as CHI-Mo 0.8 W 0.2 Se 2 -MTO for PTT-augmented CDT and chemotherapy (CT). A series of performance validations successfully showed that electrons tend to transfer from W to Mo in CHI-Mo 0.8 W 0.2 Se 2 , which resulted in superior POD-like activity (K m = 0.038 mM) of CHI-Mo 0.8 W 0.2 Se 2 compared with that of horseradish peroxidase. Furthermore, CHI-Mo 0.8 W 0.2 Se 2 -MTO with excellent photothermal conversion efficiency (PCE=63.2 %) in the near-infrared (NIR) region could further promote endogenous •OH generation and MTO controlled release within solid tumours. In vivo studies confirmed the successful achievement of synergistic therapeutic effects (tumour inhibition rate of over 90 %) with minimised side effects. Versatile therapeutic nanoagents hold great potential for personalised therapy of breast cancer and will find their way to the pharmaceutical field. [ABSTRACT FROM AUTHOR]

The fibroblast growth factor-2 (FGF-2) is a critical protein for biological processes such as angiogenesis and tissue regeneration. Recently, hydrogels based on semi-synthetic sulfated polysaccharides have been developed for the controlled delivery of FGF-2. These affinity-based FGF-2 carriers utilizing hydrogels based on sulfated polysaccharides enable sustained delivery of FGF-2, yet choice of materials is limited. Here, we demonstrate a novel synthetic sulfated polysaccharide-based hydrogel based on bacterial polyglucuronic acid (PGU). We synthesized phenol-grafted sulfated PGU (PGUS-Ph), an enzymatically cross-linkable PGU derivative that exhibited an enhanced affinity for FGF-2. The aqueous solution of PGUS-Ph, when combined with FGF-2, could be injected into affected sites and form a hydrogel in a minimally invasive manner. The FGF-2 released from the PGUS-Ph hydrogel induced blood vessel formation, as proven by a chick embryo-based angiogenesis assay. Our results indicate that the PGUS-Ph has the potential as an enzymatically cross-linkable and minimally invasively injectable affinity-based FGF-2 delivery system. [ABSTRACT FROM AUTHOR]

This study focuses on the immobilization of horseradish peroxidase (HRP) on modified acrylic fabrics incorporating gold and silver nanoparticles. The process involves treating the acrylic fabrics with hydroxylamine hydrochloride and then coating them with silver and gold nanoparticles. Both obtained materials, treated acrylic fabrics-coated with silver nanoparticles (AgNPs@TA-HAC) and treated acrylic fabrics-coated with gold nanoparticles (AuNPs@TA-HAC), were utilized as supporters for HRP. The physicochemical properties of modified acrylic fabrics were investigated using FTIR, SEM, and zeta potential. HRP immobilized on AgNPs@TA-HAC displayed an activity of 69 units/g support with a specific activity of 4.55 units/mg protein, whereas HRP immobilized on AuNPs@TA-HAC demonstrated an activity of 76 units/g support with a specific activity of 4.75 units/mg protein. After the 15 repetitive cycles, the immobilized HRP on AuNPs@TA-HAC and AgNPs@TA-HAC retained 75 and 59% of their enzymatic activity, respectively. The immobilized HRP on both material supporters retained its activity better compared to the free HRP when exposed to all tested organic solvents. The Michaelis constant (Km) for free HRP and HRP immobilized on AuNPs@TA-HAC and AgNPs@TA-HAC were determined to be 6.23, 8.65, and 9.11 mM, respectively. The maximum reaction rates (Vmax) for the immobilized HRP on both supports were slightly reduced at 0.71 and 0.69 U/mL, compared to 0.74 U/mL for free HRP. This approach of utilizing acrylic fabrics and metal nanoparticles provides a promising method for enzyme immobilization, with potential applications in various industries. [ABSTRACT FROM AUTHOR]

Abstract The present study aimed to develop a system using a combination of enzymatic and microbial degradation techniques for removing phenol from contaminated water. In our prior research, the HRP enzyme extracted from horseradish roots was utilized within a core-shell microcapsule to reduce phenolic shock, serving as a monolayer column. To complete the phenol removal process, a second column containing degrading microorganisms was added to the last column in this research. Phenol-degrading bacteria were isolated from different microbial sources on a phenolic base medium. Additionally, encapsulated calcium peroxide nanoparticles were used to provide dissolved oxygen for the microbial population. Results showed that the both isolated strains, WC1 and CC1, were able to completely remove phenol from the contaminated influent water the range within 5 to 7 days, respectively. Molecular identification showed 99.8% similarity for WC1 isolate to Stenotrophomonas rizophila strain e-p10 and 99.9% similarity for CC1 isolate to Bacillus cereus strain IAM 12,605. The results also indicated that columns using activated sludge as a microbial source had the highest removal rate, with the microbial biofilm completely removing 100% of the 100 mg/L phenol concentration in contaminated influent water after 40 days. Finally, the concurrent use of core-shell microcapsules containing enzymes and capsules containing Stenotrophomonas sp. WC1 strain in two continuous column reactors was able to completely remove phenol from polluted water with a concentration of 500 mg/L for a period of 20 days. The results suggest that a combination of enzymatic and microbial degrading systems can be used as a new system to remove phenol from polluted streams with higher concentrations of phenol by eliminating the shock of phenol on the microbial population.

Nanomaterials excel in mimicking the structure and function of natural enzymes while being far more interesting in terms of structural stability, functional versatility, recyclability, and large‐scale preparation. Herein, the story assembles hemin, histidine analogs, and G‐quadruplex DNA in a catalytically competent supramolecular assembly referred to as assembly‐activated hemin enzyme (AA‐heminzyme). The catalytic properties of AA‐heminzyme are investigated both in silico (by molecular docking and quantum chemical calculations) and in vitro (notably through a systematic comparison with its natural counterpart horseradish peroxidase, HRP). It is found that this artificial system is not only as efficient as HRP to oxidize various substrates (with a turnover number kcat of 115 s−1) but also more practically convenient (displaying better thermal stability, recoverability, and editability) and more economically viable, with a catalytic cost amounting to <10% of that of HRP. The strategic interest of AA‐heminzyme is further demonstrated for both industrial wastewater remediation and biomarker detection (notably glutathione, for which the cost is decreased by 98% as compared to commercial kits). [ABSTRACT FROM AUTHOR]

Mesoporous metal‐organic frameworks (MOFs) are promising supports for the immobilization of enzymes, yet their applications are often limited by small pore apertures that constrain the size of encapsulated enzymes to below 5 nm. In this study, we introduced labile linkers (4,4′,4′′‐(2,4,6‐boroxintriyl)‐tribenzoate, TBTB) with dynamic boroxine bonds into mesoporous PCN‐333, resulting in PCN‐333‐TBTB with enhanced enzyme loading and protection capabilities. The selective breaking of B−O bonds creates defects in PCN‐333, which effectively expands both window and cavity sizes, thereby unlocking hidden mesopores for enzyme encapsulation. Consequently, this strategy not only increases the adsorption kinetics of small enzymes (<5 nm) such as cytochrome c (Cyt C) and horseradish peroxidase (HRP), but also enables the immobilization of various large‐sized enzymes (>5 nm), such as glycoenzymes. The glycoenzymes@PCN‐333‐TBTB platform was successfully applied to synthesize thirteen complex oligosaccharides and polysaccharides, demonstrating high activity and enhanced enzyme stability. The dynamic linker‐mediated enzyme encapsulation strategy enables the immobilization of enzymes exceeding the inherent pore size of MOFs, thus broadening the scope of enzymatic catalytic reactions achievable with MOF materials. [ABSTRACT FROM AUTHOR]

The treatment of intervertebral disc degeneration (IVDD) using bone marrow mesenchymal stem cell‐derived exosomes has shown success in alleviating inflammation and restoring the extracellular matrix (ECM), however, challenges persist due to the deficiency in mechanical support and controlled release. Herein, a carbon‐carbon double bond modified keratin (KeMA) is synthesized by 2‐isocyanatoethyl modification for exosomes wrapping. This injectable KeMA hydrogel, initiated by a biocompatible glucose/ glucose oxidase/ horse radish peroxidase enzymatic cascade reaction with acetylacetone and N‐vinylpyrrolidone, displayed rapid gelation, resembling nucleus pulposus (NP) elasticity, and excellent cytocompatibility. In vitro studies showcased that the exosomes‐loaded KeMA hydrogel (Exo@KeMA) enhanced exosome release kinetics, suppressed inflammation, fostered extracellular matrix (ECM) regeneration, and reinstated NP biomechanics. RNA‐seq analysis indicated Exo@KeMA's effects involved PI3K‐Akt signaling for matrix regeneration and NF‐κB signaling inhibition for anti‐inflammation. In vivo IVDD rat models demonstrated Exo@KeMA attenuated inflammation, maintained NP water content, preserved disc height, and promoted structural regeneration. This research introduces an injectable KeMA hydrogel as a promising therapy for IVDD, by facilitating biomechanics restoration, anti‐inflammatory response, and ECM regeneration. [ABSTRACT FROM AUTHOR]

Despite the critical role of hydrogels in material science and biotechnology, current methods for analyzing their formation lack real‐time monitoring and require complex sample preparation and instrumentation. In this work, an innovative methodology is introduced for the real‐time analysis of enzymatically catalyzed hydrogelation. Electrochemical impedance spectroscopy (EIS) coupled with interdigitated electrodes (IDEs) to sense and transduce the gelation reaction of model precursor carboxymethyl cellulose‐tyramine (CMC‐TA) conjugates that undergoes enzymatic cross‐linking by horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). Real‐time monitoring involves single‐frequency analyses at 3 × 105 Hz, where the measured impedance consists solely of a resistive component, and the admittance equates to solution conductance. The gelation trajectories for all tested enzymatically cross‐linked hydrogel component combinations are determined by substituting the conductance data in the modified Michaelis–Menten kinetic model. Specifically, for CMC‐TA cross‐linked by HRP, the authors calculate apparent KM and kcat values of 82.1 µM and 95.5 s−1, respectively. These findings are further validated through rheological characterization, including oscillatory shear measurements and microrheology. Overall, this research paves the way for a streamlined, accurate, and cost‐effective approach to controllable enzymatically initiated hydrogel synthesis, enhancing their successful application in various fields ranging from material science to biotechnology. [ABSTRACT FROM AUTHOR]

Single‐walled carbon nanotubes (SWCNTs) are desirable nanoparticles for sensing biological analytes due to their photostability and intrinsic near‐infrared fluorescence. Previous strategies for generating SWCNT nanosensors have leveraged nonspecific adsorption of sensing modalities to the hydrophobic SWCNT surface that often require engineering new molecular recognition elements. An attractive alternate strategy is to leverage pre‐existing molecular recognition of proteins for analyte specificity, yet attaching proteins to SWCNT for nanosensor generation remains challenging. Toward this end, a generalizable platform is introduced to generate protein‐SWCNT‐based optical sensors and use this strategy to synthesize a hydrogen peroxide (H2O2) nanosensor by covalently attaching horseradish peroxidase (HRP) to the SWCNT surface. A concentration‐dependent response is demonstrated to H2O2, confirming the nanosensor can image H2O2 in real‐time, and assess the nanosensor's selectivity for H2O2 against a panel of biologically relevant analytes. Taken together, these results demonstrate successful covalent attachment of enzymes to SWCNTs while preserving both intrinsic SWCNT fluorescence and enzyme function. It is anticipated this platform can be adapted to covalently attach other proteins of interest including other enzymes for sensing or antibodies for targeted imaging and cargo delivery. [ABSTRACT FROM AUTHOR]

Electrochemical biosensor based on horseradish peroxidase (HRP) modified gold nanoparticle/sulfur-doped graphene (AuNP/SG) has been developed for the quantification of trans-resveratrol (Res). Gold nanoparticle (AuNP) was selected as the carrier for HRP due to its excellent electrochemical catalytic activity, biocompatibility, and conductivity, while sulfur-doped graphene (SG) was selected as the support for AuNP due to the defects introduced in the carbon network by sulfur doping can facilitate the high dispersion state of AuNP and HRP. The results show that the excellent sensing performance of the HRP/AuNP/SG is attributed to the high charge polarization of material after sulfur dropping and the co-catalysis of HRP and AuNP. For the detection of Res, linear response can be observed with high sensitivity and low detection limit (4 × 10−8 M). Further, this electrochemical biosensor presents strong reproducibility, stability, and anti-interference capability in detecting Res. These unique properties make it an excellent choice for Res reliable detection. [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA), as a typical endocrine disruptor, can cause serious harm to human health. In order to solve the limitation of biological enzyme method in the degradation of BPA in actual water, we successfully prepared a photo-enzyme-coupled catalyst CN-HRP@ZIP by in situ generation of a metal–organic skeleton coated with HRP on the carbon nitride surface. The photo-enzymatic synergy of the material and the low mass transfer resistance caused by the two-dimensional ZIP make CN-HRP@ZIP can achieve 100% degradation of BPA at 40 min. The degradation rate was 3.92 times that of CN and 1.65 times that of CN-HRP@ZIF, respectively. The immobilization of the enzyme broadened the degradation pathway and enhanced the resistance to anionic interference by enzyme specificity. The direct conversion of hydrogen peroxide by the enzyme and the indirect enhancement of photocatalytic superoxide radical production increased the concentration of superoxide radicals in the system, and CN-HRP@ZIP maintained a high degradation rate of more than 90% in five cycles. The stability of the enzymatic activity of the material was much higher than that of the free enzyme under extreme pH, high temperature, and organic solvent immersion. These studies provide a new reference for the application of photo-enzymatic synergistic degradation in industrial wastewater treatment. [ABSTRACT FROM AUTHOR]

Interferon gamma (IFN‐γ) released by T lymphocytes, plays an important role in immune regulation and is also an important biomarker for the early diagnosis of bovine tuberculosis. In this work, an electrochemical immunosensor based on gold nanoparticles (AuNPs)/chitosan composite was developed for highly sensitive determination of bovine IFN‐γ. AuNPs/chitosan composite shows high surface area and good hydrophilicity. The bovine IFN‐γ immunosensor was prepared by immobilizing IFN‐γ antibody on AuNPs/chitosan composite‐modified glassy carbon electrode. The immunosensors were characterized by scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry (CV). Using horseradish peroxidase (HRP)‐labeled IFN‐γ antibody as amplification probe, the concentration of bovine IFN‐γ can be monitored via the increasing current signal, which can be attributed to the specific immune binding between bovine IFN‐γ antigen and HRP‐labeled IFN‐γ antibody, as well as the efficient catalysis reaction between thionine and HRP with the presence of H2O2. The developed electrochemical immunosensor exhibited a wide linear range of 0.01 to 30 ng/mL, and low detection limit of 0.003 ng/mL with good specificity and stability. This study provides a simple and promising method for diagnosis of bovine tuberculosis, which can be expanded to determine other biomarkers. [ABSTRACT FROM AUTHOR]

The influence of organic solvents such as methanol and acetonitrile on the results of butachlor immunoassay in samples of rice and rice products was studied. The schemes of enzyme immunoassay using: (a) native antiserum containing specific antibodies to butachlor and antispecies antibodies labeled with horseradish peroxidase, and (b) biotinylated specific antibodies and streptavidin labeled with peroxidase are considered. The close values of IC10 (0.77 and 0.68 ng/mL, respectively) and working range (2.6–165 and 2.4–192 ng/mL, respectively) were established for the two schemes, when analyzing in a mixture of phosphate buffer and methanol in a percentage ratio of 85 : 15, respectively. For the second scheme, the detection of butachlor in butachlor in rice-containing products is shown at 80-132% of the added analyte. A comparison of sample preparation methods allows us to recommend: (a) extraction of butachlor with hexane and re-dissolution of the dry residue in a buffer with 15% methanol or (b) preparation of a methanol extract followed by 6-fold dilution with a buffer solution. [ABSTRACT FROM AUTHOR]

The current study demonstrates an easy utilization of glycine soja sensu auct green extract as a power source for the desired synthesis of copper oxide (CuO) nanoparticles via green synthesis process to acquire significant detection of hydrogen peroxide (H2O2) and monosaccharide glucose through an uncomplicated colorimetric method. Due to sustainability issues, synthesis of the metal or metal oxide nanoparticles has shifted towards eco-safe reducing agents. This research focuses on the utilization of the reducing agent via green technology, namely glycine soja sensu auct extract, for the synthesis of CuO nanoparticles by co-precipitation synthesis. Through XRD studies, it can be depicted that the material is monoclinic. FESEM and HRTEM images reveals spherical shape of nanoparticles. HRTEM images also indicate that the size of the particles falls between 10–30 nm. The particle distribution curve reveals the z-average size of 135.1 nm. The Fourier transform infrared spectroscopy peak at 493.14, 522.01, 557.89 and 649.39 cm-1 refer to the vibrations between Cu and O. Clearly, the peaks indicate the formation of CuO nanostructure. These green CuO NPs successfully catalyzed the oxidation reaction of the exposed peroxidase substrate 3, 3′, 5, 5′- tetramethylbenzidine (TMB) dye by hydrogen peroxide (H2O2) to develop a bluish colour reaction with the absorption maxima centred at 652 nm. The CuO NPs exhibited better kinetics over horseradish peroxidase (HRP), the natural enzyme, with a lower Km value. Linear response range for glucose, was 10 – 100 µM and a limit of detection (5.06 µM) was obtained. [ABSTRACT FROM AUTHOR]

Horseradish peroxidase (HRP) was immobilized in hybrid calcium alginate-gelatin hydrogel beads by entrapment. Then, organic solubility, metal ion compatibility, pH/temperature studies of the synthesized materials have been carried out. Hybrid beads (Ca-Alg@gel-HRP) were synthesized under optimum synthesis conditions with high yield and specific activity. The synthesized bead size was determined to be approximately 0.29 mm in diameter, hydrogel structure and transparent appearance. Accordingly, the optimum pH of free HRP and Ca-Alg@gel HRP was found to be pH 6 and 5 and the optimum temperature was found to be 40 °C and 60 °C respectively. For all identified metal ions except Fe2+, the Ca-Alg@gel HRP had a higher activity than the free HRP. In the presence of 1 mM Na+, Ca2+, Mg2+, Ba2+, Cu2+, Ni2+, Mn2+, Hg2+ ions, the activity increased by more than 20%. The relative activity was also approximately 153%, 150% and 145% in 2.5 mM chloroform, glycerol and DMF. Free enzyme activity was reduced by more than 30%. The Ca-Alg@gel HRP at room temperature and 4 °C retained most of its activity after 14 days. In cycle 5, it had half the activity. As a conclusion, it may have significant potential in a number of industrial processes, particularly in organic synthesis, because it is tolerant to metal ions and organic solvents and is a cheap, practical and simple immobilization method. [ABSTRACT FROM AUTHOR]

Bacterial infections are a serious threat to wound healing and skin regeneration. In recent years, photothermal therapy (PTT) has become one of the most promising tools in the treatment of infectious diseases. However, wound dressings with photo-responsive properties are currently still limited by the difficulties of biosafety and thermal stability brought by the introduction of photosensitizers or photothermal agents. Therefore, how to improve the therapeutic efficiency and biosafety from material design is still a major challenge at present. In this study, the carboxymethyl chitosan (CMCS) and protocatechuic aldehyde (PA) hydrogels based on horseradish peroxidase (HRP) and hydrogen peroxide (H 2 O 2) enzymatic catalysis was developed. Therein, HRP and H 2 O 2 catalyzed cross-linking while polymerizing PA, which not only endowed the hydrogels with photothermal responsiveness but also with good biosafety through this enzyme-catalyzed green approach. Meanwhile, the hydrogels possessed highly efficient bacteriostatic ability with the assistance of near infrared (NIR). Moreover, the ultra-rapid gelation, strong tissue adhesion, high swelling ability, good antioxidant property and hemostatic property of the CMCS-PA hydrogels based on HRP/H 2 O 2 enzymatic catalysis were suitable for the treatment of skin wounds. Meanwhile, NIR-assistant CMCS-PA hydrogels based on HRP/H 2 O 2 enzymatic catalysis reduced inflammation, decreased bacterial infection, and promoted collagen deposition and angiogenesis, which showed remarkable therapeutic effects in a skin wound infection model. All results indicate that this green approach to introduce photothermal property by HRP-catalyzed PA polymerization endows the hydrogels with efficient photothermal conversion efficiency, suggesting that they are promising to provide new options for replacing photothermal agents and photosensitizers. In recent years, wound dressings with photo-responsive properties are currently still limited by the difficulties of biosafety and thermal stability brought by the introduction of agent photosensitizers or photothermal agents. In this study, the carboxymethyl chitosan and protocatechuic aldehyde hydrogels based on horseradish peroxidase and hydrogen peroxide enzymatic catalysis was developed. The photothermal properties of hydrogels were transformed from absent to present just by horseradish peroxidase-catalyzed protocatechuic aldehyde polymerization in a green approach. Meanwhile, the hydrogels possessed highly efficient bacteriostatic ability with the assistance of near infrared. The green approach of introducing photothermal properties from material design solves the biosafety challenge. Therefore, this study is expected to provide new options for alternative photothermal agents and photosensitizers. [Display omitted] [ABSTRACT FROM AUTHOR]

We revealed that the encapsulation of enzyme-immobilized silica particles in hollow-type spherical bacterial cellulose (HSBC) gels enables the use of the inside of HSBC gels as a reaction field. The encapsulation of horseradish peroxidase (HRP)-immobilized silica particles (Si-HRPs, particle size: 40–50 μm) within HSBC gels was performed by using a BC gelatinous membrane produced at the interface between Komagataeibacter xylinus suspension attached onto an alginate gel containing Si-HRPs and silicone oil. After the biosynthesis of the BC gelatinous membrane, formed from cellulose nanofiber networks, the alginate gel was removed via immersion in a phosphate-buffered solution. Si-HRP encapsulated HSBC gels were reproducibly produced using our method with a yield of over 90%. The pore size of the network structure of the BC gelatinous membrane was less than 1 μm, which is significantly smaller than the encapsulated Si-HRPs. Consequently, the encapsulated Si-HRPs could neither pass through the BC gelatinous membrane nor leak from the interior cavity of the HSBC gel. The activity of the encapsulated HRPs was detected using the 3,3′,5,5′-tetramethylbenzidine (TMB)-H2O2 system, demonstrating that this method can encapsulate the enzyme without inactivation. Since HSBC gels are composed of a network structure of biocompatible cellulose nanofibers, immune cells cannot enter the hollow interior, thus, the enzyme-immobilized particles encapsulated inside the HSBC gel are protected from immune-cell attacks. The encapsulation technique demonstrated in this study is expected to facilitate the delivery of enzymes and catalysts that are not originally present in the in vivo environment. [ABSTRACT FROM AUTHOR]

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N-Glycosidase (PNGase) is a type of glycoside hydrolase whose main function is to release N-glycan chains from glycopeptides or glycoproteins, holding significant application value in fields such as food and biomedicine. Currently, commercial PNGases exhibit certain limitations in their application. Therefore, developing new PNGases with superior performance holds research value. This study successfully obtained a novel PNGase, named PNGase Dc, by expressing the gene encoding PNGase from Dyella caseinilytica in Escherichia coli. The molecular weight of PNGasse Dc was 63.01 kDa. Its enzymatic characteristics, functionality, and potential applications were then analyzed. The enzymatic characterization results indicated that the optimal pH and temperature for PNGase Dc are 2.0 and 37 °C, respectively, and its catalytic reaction did not depend on metal ions.The enzyme also demonstrated excellent stability, retaining its activity even after storage at 4 °C for 45 days. Molecular docking analysis indicated that PNGase Dc interacts with its substrate N'N-diacetylchitobiose through hydrogen bonds and van der Waals forces. Site-directed mutagenesis identified Asp102 and Glu236 as key amino acids affecting the enzyme's activity.PNGase Dc exhibited effective deglycosylation on standard glycoproteins, including horseradish peroxidase and lactoferrin. Lastly, PNGase Dc's application potential was evaluated.Arabidopsis thaliana and mouse plasma was used as substrates, confirming its promising applicability in the study of glycoproteins from both animal and plant sources.In summary, this research introduced a new enzymatic tool, PNGase Dc, for the study of glycoproteins derived from animal and plant sources, thereby laying a theoretical foundation for the enzyme's development and utilization. [ABSTRACT FROM AUTHOR]

The present study aimed to develop a system using a combination of enzymatic and microbial degradation techniques for removing phenol from contaminated water. In our prior research, the HRP enzyme extracted from horseradish roots was utilized within a core-shell microcapsule to reduce phenolic shock, serving as a monolayer column. To complete the phenol removal process, a second column containing degrading microorganisms was added to the last column in this research. Phenol-degrading bacteria were isolated from different microbial sources on a phenolic base medium. Additionally, encapsulated calcium peroxide nanoparticles were used to provide dissolved oxygen for the microbial population. Results showed that the both isolated strains, WC1 and CC1, were able to completely remove phenol from the contaminated influent water the range within 5 to 7 days, respectively. Molecular identification showed 99.8% similarity for WC1 isolate to Stenotrophomonas rizophila strain e-p10 and 99.9% similarity for CC1 isolate to Bacillus cereus strain IAM 12,605. The results also indicated that columns using activated sludge as a microbial source had the highest removal rate, with the microbial biofilm completely removing 100% of the 100 mg/L phenol concentration in contaminated influent water after 40 days. Finally, the concurrent use of core-shell microcapsules containing enzymes and capsules containing Stenotrophomonas sp. WC1 strain in two continuous column reactors was able to completely remove phenol from polluted water with a concentration of 500 mg/L for a period of 20 days. The results suggest that a combination of enzymatic and microbial degrading systems can be used as a new system to remove phenol from polluted streams with higher concentrations of phenol by eliminating the shock of phenol on the microbial population. [ABSTRACT FROM AUTHOR]