Horseradish Peroxidase Research Articles

Construction of Liposome‐Based Extracellular Artificial Organelles on Individual Living Cells

AbstractIntegration of living cells with extrinsic functional entities gives rise to bioaugmented nanobiohybrids, which hold tremendous potential across diverse fields such as cell therapy, biocatalysis, and cell robotics. This study presents a biocompatible method for incorporating multilayered functional liposomes onto the cell surface, creating extracellular artificial organelles or exorganelles. The introduction of various extrinsic functionalities to cells is achieved without comprising their viabilities. The integration of extrinsic enzymatic reactions is exemplified through the cascade reaction involving glucose oxidase and horseradish peroxidase. Furthermore, our protocol offers the design flexibility to customize liposome compositions, thereby providing effective cell modification. The versatility of the liposome‐based exorganelle approach establishes an advanced chemical tool, empowering cells with novel functionalities that surpass or are complementary to their innate capabilities.

Quantitative Intracellular Delivery of Anticancer Nanodrugs Via an Immunoassay Employing Pt-SiO2 Janus-Peroxidase Nanozyme.

The accurate and efficient quantification of nanodrug dosage is crucial for early anticancer therapy. The enzyme-linked immunosorbent assay (ELISA) has emerged as a robust tool for detecting anticancer nanodrug dosage; however, the development of sensing elements to quantify anticancer nanodrugs still poses a challenge. To overcome this problem, we utilize polysuccinimide-loaded curcumin (CUR @PSIOAm) as a model to employ an ELISA based on peroxidase nanozyme Pt-SiO2 Janus nanoparticles (Pt-SiO2 JNPs) for the indirect quantitative analysis of intracellular anticancer nanodrug dosage. This novel approach employs an immunoassay to indirectly quantify the dosage of anticancer nanodrugs while preserving its structural integrity. The silica components of Pt-SiO2 JNPs adsorb intermediates, while the Pt NP components exhibit high catalytic activity. Pt-SiO2 JNPs are functionalized with anti-PSIOAm antibody (Pt-SiO2 JNPs-Ab) to serve as an immunosensor capable of specific recognition of CUR @PSIOAm. Additionally, we employed cytotoxicity assays and confocal imaging techniques to demonstrate the excellent biocompatibility of CUR @PSIOAm, as well as its specific uptake by cancer cells. According to the experimental results, the limit of detection (LOD) for the immunoassay of Pt-SiO2 JNPs as a marker for detecting CUR @PSIOAm is approximately 4.5-fold lower than that of horseradish peroxidase. Therefore, by optimizing the conditions, we established a direct competitive ELISA using Pt-SiO2 JNPs as colorimetric indicators for the quantitative detection of intracellular CUR @PSIOAm. The LOD for this ELISA was determined to be 0.01 ng/mL, while the loaded CUR amount calculated from the drug loading capacity was found to be 0.22 pg/mL. Furthermore, the recoveries obtained from this established ELISA ranged between 94.0 and 108%, demonstrating excellent accuracy. Consequently, the peroxidase mimic Pt-SiO2 JNPs-based ELISA exhibits significant potential for precise quantification of intracellular anticancer nanodrug dosages.

Fusarium graminearum Ste2 and Ste3 Receptors Undergo Peroxidase-Induced Heterodimerization when Expressed Heterologously in Saccharomyces cerevisiae.

Fusarium graminearum FgSte2 and FgSte3 are G-protein coupled receptors (GPCRs) shown to play roles in hyphal chemotropism and fungal plant pathogenesis in response to activity arising from host-secreted peroxidases. Here, we follow up on the observation that chemotropism is dependent on both FgSte2 and FgSte3 being present; testing the possibility that this might be due to formation of an FgSte2-FgSte3 heterodimer. Bioluminescence resonance energy transfer (BRET) analyses were conducted in Saccharomyces cerevisiae, where the addition of horse radish peroxidase (HRP) was found to increase the transfer of energy from the inducibly-expressed FgSte3-Nano luciferase donor, to the constitutively-expressed FgSte2-yellow fluorescent protein (YFP) acceptor, compared to controls. A partial response was also detected when an HRP-derived ligand-containing extract was enriched from F. graminearum spores and applied instead of HRP. In contrast, substitution with pheromones or an unrelated bovine GPCR, rhodopsin-YFP used as acceptor, eliminated all BRET responses. Interaction results were validated by affinity pulldown and receptor expression was validated by confocal immunofluorescence microscopy. Taken together these findings demonstrate the formation of HRP and HRP-derived ligand stimulated heterodimers between FgSte2 and FgSte3. Outcomes are discussed from the context of the roles of ligands and reactive oxygen species in GPCR dimerization.

Unlocking of Hidden Mesopores for Enzyme Encapsulation by Dynamic Linkers in Stable Metal-Organic Frameworks.

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.

Unlocking of Hidden Mesopores for Enzyme Encapsulation by Dynamic Linkers in Stable Metal‐Organic Frameworks

AbstractMesoporous 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 (&lt;5 nm) such as cytochrome c (Cyt C) and horseradish peroxidase (HRP), but also enables the immobilization of various large‐sized enzymes (&gt;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.

Enhanced Peroxidase-like Activity of Ruthenium-Modified Single-Atom-Thick A Layers in MAX Phases for Biomedical Applications.

Nanozymes have demonstrated significant potential as promising alternatives to natural enzymes in biomedical applications. However, their lower catalytic activity compared to that of natural enzymes has limited their practical utility. Addressing this challenge necessitates the development of innovative enzymatic systems capable of achieving specific activity levels of natural enzymes. In this study, we focus on enhancing the catalytic performance of nanozymes by introducing Ru atoms into the single-atom-thick A layer of the V2SnC MAX phase, resulting in the formation of V2(Sn0.8Ru0.2)C with Ru single-atom sites. The V2(Sn0.8Ru0.2)C MAX phase demonstrated an exceptional peroxidase-like specific activity of up to 1792.6 U mg-1, surpassing the specific activity of a previously reported horseradish peroxidase (HRP). Through X-ray photoelectron spectroscopy (XPS) and density functional theory (DFT) investigations, it has been revealed that both the V2C atom layers and single-atom-thick Sn readily accept a negative charge from Ru, leading to a reduction of the energy barrier for H2O2 adsorption. This discovery has enabled the successful application of V2(Sn0.8Ru0.2)C in the development of a lateral flow immunoassay for heart failure biomarkers, achieving a detection sensitivity of 4 pg mL-1. Additionally, V2(Sn0.8Ru0.2)C demonstrated exceptional broad-spectrum antibacterial efficacy. This study lays the groundwork for the precise design of MAX phase-based nanozymes with high specific activity, offering a viable alternative to natural enzymes for various applications.

Distance-based analytical device integrated with carbon nanomaterials for sarcosine quantification in human samples.

Astraightforward distance-based paper analytical device (dPAD) was developedfor monitoring sarcosine levels in human samples for the rapid diagnosis and prognosis of prostate cancer and related symptoms. This assay eliminates the need for the expensive horseradish peroxidase (HRP) enzyme by utilizing carbon nanodots (CDs) as a peroxidase-like nanozyme. The proposed dPAD sensor consists of a sample zone pre-deposited with sarcosine oxidase (SOx) and CDs, and a detection zone containing 3,3',5,5'-tetramethylbenzidine (TMB). When a solution containing sarcosine is added to the sample zone, hydroxyl radicals (•OH) are produced through SOx oxidation and subsequent peroxidase catalysis by the CDs. The formed •OH radicals immediately flow to the detection zone via capillary force, where they oxidize TMB, resulting in a visible colour change from colourless to blue. Sarcosine quantification is effortlessly accomplished by measuring the distance of the blue colour in the detection zone. The developed dPAD offers a linear working range between 12.5 and 35.0nmol L-1 (R2 = 0.9959) and a detection limit (LOD) of 10.0nmol L-1. This covers the clinical range for urinary sarcosine determination, thereby suggesting no additional sample preparation or dilution is needed. The sensor shows high precision with the highest relative standard deviation (RSD) of 4.58% and demonstrates excellent selectivity with no observed interferences. Furthermore, recovery studies in human control urine samples ranged from 98.67 to 101.50%, with the highest RSD of 2.03%. Correspondingly, our dPAD method showed a great match with the performance of a commercial ELISA method for detecting sarcosine in human control serum. The sensor is more cost-effective, user-friendly, and accessible than previous methods. Overall, the proposed method represents a promising analytical tool for sarcosine quantification. The concept is also applicable for broader analytical applications in detecting other biomolecules.

Aptamer-Based Electrochemical Biosensing Platform for Analysis of Cardiac Biomarkers.

Monitoring biomarkers secreted by cardiomyocytes is critical to evaluate anticancer drug-induced myocardial injury (MI). Cardiac troponin I (cTnI) is considered the gold standard biomarker for MI. Herein, an electrochemical aptasensor is engineered for cTnI detection based on lanthanide europium metal-organic frameworks (Eu-MOFs) and a hybridization chain reaction-directed DNAzyme strategy. Three types of Eu-MOF morphologies were easily synthesized by changing the solvent, and the Eu-MOF modulated by mixing the solvent of dimethylformamide and H2O (D-Eu-MOF) exhibited the best performance compared to other morphologies of the Eu-MOFs. Multifunctional nanoprobes were constructed from D-Eu-MOF@Pt loaded with natural horseradish peroxidase and combined with an aptamer-initiated nuclear acid hybridization chain reaction to form G-quadruplex/hemin DNAzymes for signal amplification. A novel capture probe is constructed on the basis of DNA nanotetrahedrons modified on screen-printed gold electrodes to enhance the capture of the target and multifunctional nanoprobes for signal amplification. It exhibits a detection limit of 0.17 pg mL-1 and a linear range from 0.5 pg mL-1 to 15 ng mL-1. The practicality of the platform is evaluated by measuring cTnI in real samples and secreted by cardiomyocytes after drug treatment, which provides great potential in drug-induced MI evaluation for clinical application.

A New Activity Assay Method for Diamine Oxidase Based on Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Copper-containing diamine oxidases are ubiquitous enzymes that participate in many important biological processes. These processes include the regulation of cell growth and division, programmed cell death, and responses to environmental stressors. Natural substrates include, for example, putrescine, spermidine, and histamine. Enzymatic activity is typically assayed using spectrophotometric, electrochemical, or fluorometric methods. The aim of this study was to develop a method for measuring activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry based on the intensity ratio of product to product-plus-substrate signals in the reaction mixtures. For this purpose, an enzyme purified to homogeneity from pea (Pisum sativum) seedlings was used. The method employed α-cyano-4-hydroxycinnamic acid as a matrix with the addition of cetrimonium bromide. Product signal intensities with pure compounds were evaluated in the presence of equal substrate amounts to determine intensity correction factors for data processing calculations. The kinetic parameters kcat and Km for the oxidative deamination of selected substrates were determined. These results were compared to parallel measurements using an established spectrophotometric method, which involved a coupled reaction of horseradish peroxidase and guaiacol, and were discussed in the context of data from the literature and the BRENDA database. It was found that the method provides accurate results that are well comparable with parallel spectrophotometry. This method offers advantages such as low sample consumption, rapid serial measurements, and potential applicability in assays where colored substances interfere with spectrophotometry.

Thread-Based Bienzymatic Biosensor for Linoleic Acid Detection.

The concentration of nonesterified fatty acids (NEFAs) in biological media is associated with metabolic and cardiovascular disorders (e.g., diabetes, cancer, and cystic fibrosis) and in food products is indicative of their quality. Therefore, the early identification of NEFAs is crucial for both medical diagnosis and food quality assessment. However, the development of a portable and scalable sensor capable of detecting these compounds at a low cost presents challenges due to their considerable chemical and physical stability. This research endeavors to illustrate the viability of detecting linoleic acid using a chemiresistive bienzymatic sensor constructed with cotton thread. The sensor's design incorporates the conductive polymer poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) within the thread, alongside the enzymes horseradish peroxidase (HRP) and lipoxygenase (LOX). By implementing this technology, a sensitive detection range spanning from 161 nM to 16.1 μM is achieved when the PEDOT:PSS/HRP/LOX system is integrated into a single thread. The sensor exhibits exceptional selectivity toward linoleic acid, owing to the specific enzymatic reaction between LOX and linoleic acid. This selectivity is upheld even in the presence of other unsaturated fatty acids. This system can be used for future designs with the capability to detect polyunsaturated fatty acids and other intricate biomolecules.

Enantiospecific profiling of D-amino acid for gastric cancer diagnosis by using a biocatalytic MHOF nanoreactor

The dynamic variation of D-amino acids (D-AAs) in human saliva has great potential as the indicator for individual physiological states and disease progression; however, enantiospecific profiling of D-AAs is a great challenge. In this work, a nanoreactor based on mesoporous hydrogen-bonded organic framework (MHOF) has been fabricated for the enantiospecific profiling of D-AAs. The nanoreactor is prepared by using MHOFs as the scaffold to organize the biocatalytic cascade of D-AA oxidase (DAAO) and horseradish peroxidase (HRP). In the meanwhile, the large, stable, and robust transport channels of MHOFs allow the unimpeded transport of the involved biocatalytic substrates. Once D-AA substrates efficiently transport into MHOFs, the chromogenic reactions of 3,3′,5,5′-tetramethylbenzidine (TMB) and H2O2 can be activated. So, using this MHOF nanoreactor, enantiospecific and visual detection of D-AAs can be achieved. Moreover, the stable nanoreactor can be operated in complex solution and utilized for the profiling of D-AA levels in saliva samples collected from gastric cancer patients, suggesting that D-AAs may be a powerful fingerprint for the diagnosis of gastric cancer. Therefore, the fabricated MHOF-based nanoreactor may provide an emerging tool for the enantiospecific profiling of D-AAs and supports the D-AA fingerprint as the indicator of gastric cancer diagnosis.

A novel double antibody sandwich quantitative ELISA for detecting porcine epidemic diarrhea virus infection.

Porcine epidemic diarrhea (PED), a contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), has caused significant economic losses to the global pig farming industry due to its rapid course and spread and its high mortality among piglets. In this study, we prepared rabbit polyclonal antibody and monoclonal antibody 6C12 against the PEDV nucleocapsid (N) protein using the conserved and antigenic PEDV N protein as an immunogen. A double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) was established to detect PEDV using rabbit polyclonal antibodies as capture antibodies and horseradish peroxidase (HRP)-labeled 6C12 as the detection antibody. Using DAS-qELISA, recombinant PEDV N protein, and virus titer detection limits were approximately 0.05 ng/mL and 103.02 50% tissue culture infective dose per mL (TCID50/mL), respectively. There was no cross-reactivity with porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (PoRV), porcine pseudorabies virus (PRV), porcine deltacoronavirus (PDCoV), or porcine circovirus (PCV). The reproducibility of DAS-qELISA was verified, and the coefficient of variation (CV) for intra- and inter-batch replicates was less than 10%, indicating good reproducibility. When testing anal swab samples from PEDV-infected piglets using DAS-qELISA, the coincidence rate was 92.55% with a kappa value of 0.85 when using reverse transcription-polymerase chain reaction (RT-PCR) and 94.29% with a kappa value of 0.88 when using PEDV antigen detection test strips, demonstrating the reliability of the method. These findings provide fundamental material support for both fundamental and practical studies on PEDV and offer a crucial diagnostic tool for clinical applications. KEY POINTS: • A new anti-PEDV N protein monoclonal antibody strain was prepared • Establishment of a more sensitive double antibody sandwich quantitative ELISA • DAS-qELISA was found to be useful for controlling the PEDV spread.

Biomedical Application of Enzymatically Crosslinked Injectable Hydrogels.

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.

Enhanced Immobilization of Enzymes on Plasma Micro-Nanotextured Surfaces and Microfluidics: Application to HRP

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.

β-Cyclodextrin-Modified Laser-Induced Graphene Electrode for Detection of N6-Methyladenosine in RNA.

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).

B-293 Enzyme linked immunosorbent assay development and full validation for the quantification of the antibody conjugated fraction of an antiFAP ADC in human serum

Abstract Background Cancer remains a global public health issue. Despite recent progress in early diagnosis and treatment strategies, development of anti-tumor therapies with reduced systemic toxicity remains a challenge. Antibody-drug conjugates (ADCs), a new, highly-potent drug class are synthesized by linking a small molecule cytotoxic drug to a tumor specific antibody, are an emerging treatment option for many cancers. Despite advances in ADCs, study of the pharmacokinetics (PK) is challenging. A complete PK profile requires at least three analytical methods to determine: conjugated antibody, total antibody, and free drug by Ligand Binding Assays (LBA) and liquid chromatography/tandem mass spectrometry (LC-MS/MS) respectively. Objective To develop and validate an ELISA method to quantify the conjugated antibody fraction of an anti-fibroblast activation protein (FAP) ADC against solid tumors in human serum. Methods Conjugated antibody assay: This indirect sandwich ELISA utilizes human FAP as capture antigen (Abcam) with a proprietary rabbit polyclonal anti-drug IgG as detection antibody. The signal is amplified by the use of a second antibody conjugated to Horse Radish Peroxidase (Abcam). Absorbance is measured at 450nm. Immunoassay method validation was done in compliance with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) M10 which establishes the limit for precision in CV% and accuracy in Relative Error (RE%) at ± 20% each (± 25% for LLOQ and ULOQ). Additionally, the total error (TE%) for accuracy and precision batches, should be ≤ 30% (≤ 40% at LLOQ and ULOQ). Results The weighted calibration curves were linear over a concentration range 0.5 to 100 µg/mL. A four-parameter regression model with a 1/Y2 weighting factor was adopted to best fit the OD450 values and concentrations. The intra- and inter-assay accuracy and precision for RE% and CV% was &amp;lt;12%, with a TE% &amp;lt; 23% for all concentrations tested. The quantification limit of 0.5 µg/mL complies with ICH criteria with a RE%, CV% and TE% of -10%, 8,5% and 15% respectively. Selectivity and specificity test fall within the criteria indicating that no individual matrix or co-medication affects the samples. The results from the dilution linearity obtained were an RE% of 11, -16 and -6 for 1:50, 1:100 and 1:200 respectively. Furthermore, not hook effect was observed as the dilution QC results gave above the quantification limit. Finally, the analyte was found to be stable at -20° and -80°for 148 days, at room temperature for 24hr and after 5 freeze/thaw cycles with a maximum RE and CV of 12 and 14% respectively in all the stability samples. Conclusions We have developed and optimized an ELISA method for measuring the antibody conjugated fraction of the ADC in serum. This method complies with the criteria of the ICH50 for selectivity, specificity, accuracy and precision, dilution linearity and hook effect in a concentration range between 0.5 - 100 µg/mL. Further, serum specimens are stable for 24h at room temperature, 5 freeze/thaw cycles and 148 days at -20 and -80 °C.

Directionally co-immobilizing glucose oxidase and horseradish peroxidase on three-pronged DNA scaffold and the regulation of cascade activity.

In traditional multienzyme random co-immobilization, it is difficult to precisely locate and regulate the relative positions between two enzyme molecules, resulting in low cascade efficiency between the two enzymes and limiting the application of multienzyme cascade catalysis technology. This study prepared PVAC@Y-dsDNA@GOD/HRP magnetic co-immobilized multienzyme by constructing a three-pronged DNA scaffold for co-coupling glucose oxidase (GOD) and horseradish peroxidase (HRP), which achieved directional co-immobilization of dual enzymes and precise regulation of inter-enzyme distance. Compared with traditional random co-immobilization of multienzyme, PVAC@Y-dsDNA@GOD/HRP could shorten the distance between GOD and HRP to the nanoscale and form substrate channeling, which greatly improved the cascade activity between the two enzymes. The inter-enzyme spacing between GOD and HRP could be precisely regulated by changing the length of DNA strands. When the inter-enzyme spacing was 10.08 nm, PVAC@Y-dsDNA@GOD/HRP exhibited high cascade activity of 707 U/mg. The inter-enzyme spacing that was too large or too small would reduce the cascade activity, indicating a distance-dependence of multienzyme cascade activity. PVAC@Y-dsDNA@GOD/HRP showed good reusability, indicating that the three-pronged DNA scaffold constructed by DNA double strands hybridization could firmly immobilize enzyme on carrier, with less enzyme leakage.

Revisiting the catalytic activity of single horseradish peroxidase clusters through electrochemical collision technique: Effect of electrolyte and substrate

Horseradish peroxidase (HRP) is a versatile biosensing label and signal reporter owing to its broad-spectrum catalytic ability. In present work, we characterized HRP's catalytic performance with various substrates using electrochemical collision technique and analyzed the associated electron transfer processes. Different electrolyte solutions greatly affected enzyme dispersibility and zeta potential, thereby impacting HRP collision dynamics in single H2O2 substrate system. The maximum turnover number (kcat) for single HRP molecules was calculated to be 3.611 ± 0.149 × 103 s−1 in 0.85 % NaCl and 2.967 ± 0.286 × 103 s−1 in 0.1 M PBS solution, reflecting differences in cluster size induced by the electrolyte conditions. More severe agglomeration of HRP molecules was observed in double-substrate systems, where the hydrophilic mediator (K4Fe(CN)6) and lipophilic mediator (ABTS) served as electron donors and signal reporters. The calculated kcat value of single HRP molecules in ABTS-H2O2 was 7.6 times higher than that in K4Fe(CN)6–H2O2. This difference is attributed to mediators' solubility, lipophilicity, and HRP's affinity for different substrates, with HRP demonstrated stronger affinity for ABTS-H2O2 substrates, which realized more efficient electron transfer and compensated for the low diffusion coefficient of ABTS. This work provides a comprehensive analysis of the effects of electrolytes and substrates on HRP collision and catalytic behavior, offering valuable insights for the advanced design of HRP-based biosensors and diagnostic platforms.

Star-Like Polypeptides as Simplified Analogues of Horseradish Peroxidase (HRP) Metalloenzymes.

Peroxidases, like horseradish peroxidase (HRP), are heme metalloenzymes that are powerful biocatalysts for various oxidation reactions. By using simple grafting-from approach, ring-opening polymerization (ROP), and manganese porphyrins, star-shaped polypeptides analogues of HRP capable of catalyzing oxidation reactions with H2O2 is successfully prepared. Like their protein model, these simplified analogues show interesting Michaelis-Menten constant (KM) in the mM range for the oxidant. Interestingly, the polymer structures are more resistant to denaturation (heat, proteolysis and oxidant concentration) than HRP, opening up interesting prospects for their use in catalysis or in biosensing devices.

Facile synthesis of nanoflowers of immobilized enzyme using layered rare earth hydroxides as carriers and their application for detection of H2O2 and phenol

This study reports a simple method for synthesizing the nanoflower-immobilized enzyme (LYH-HRP) using horseradish peroxidase (HRP) as the bioenzyme and layered yttrium hydroxide (LYH) as the inorganic carrier. Utilizing the structural advantage of LYH and the catalytic properties of HRP, a nanoflower-based colorimetric platform was newly designed and applied for sensitively detecting H2O2 and phenol with a detection time of as fast as 5 min. The limits of detection (LODs) for H2O2 and phenol are as low as 0.046 μM and 0.778 μM, respectively. The activity and stability tests showed that the activity of LYH-HRP was 1.52 times that of free HRP, and it maintained 75% of the initial activity after 60 days.