Horseradish Peroxidase Research Articles

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 <12%, with a TE% < 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.

Integrated triple signal amplification strategy for ultrasensitive electrochemical detection of gastric cancer-related microRNA utilizing MoS2-based nanozyme, hybridization chain reaction, and horseradish peroxidase

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.

A dye-decolorizing peroxidase from Vibrio cholerae can demetallate heme

Iron is an essential element for bacterial survival. Bacterial pathogens have therefore developed methods to obtain iron. Vibrio cholerae, the intestinal pathogen that causes cholera, utilizes heme as an iron source. DyP from V. cholerae (VcDyP) is a dye-decolorizing peroxidase. When VcDyP was expressed in Escherichia coli and purified, it was found to contain protoporphyrin IX (PPIX) but not heme, indicating that the protein possesses deferrochelatase activity. Here, we examined the demetallation reaction of VcDyP using fluorescence spectroscopy. Treatment of heme-reconstituted VcDyP with sodium dithionite under anaerobic conditions led to an increase in the fluorescence intensity at 624 nm, suggesting the formation of PPIX. Although the same reaction was conducted using myoglobin, horseradish peroxidase and hemin, no increase in the fluorescence was observed. Therefore, demetallation of heme is specific to VcDyP. This reaction was faster at lower pH, but the amplitudes of the fluorescence increase were larger at pH 6.5–7.5, in clear contrast to the dye-decolorizing activity with the optimal pH of 4.5. In contrast to HutZ from V. cholerae, which is a heme-degrading enzyme that cleaves the heme macrocycle to release iron, VcDyP can remove iron from heme without degradation. To our knowledge, VcDyP is the first enzyme whose demetallation activity has been confirmed at neutral pH. Our results show that VcDyP is a bifunctional protein that degrades anthraquinone dyes and demetallates heme.

Dual responsive polymersomes as versatile, intelligent labeling system in biosensing

The ability to manipulate artificial nanosystems is still one of the current challenges in devel-oping multifunctional nanomaterials. Those based on the photochromic effect are particularly interesting because of structural and functional control by the transient activation/inactivation with high spatiotemporal resolution. To mimic the photocontrol of natural nanosystems, we developed the first photochromic polymersomes from the novel poly(ethylene-alt-maleic an-hydride)-random-aminoazobenzene copolymer (PEMA-r-AAB) and its derivatives. Ultraviolet (UV)-triggered azobenzene (AZO) moieties’ medium-dependent isomerization from non-polar trans- to polar cis-AZO controls polymersome’s bilayer selective permeability. As a result, small hydrophilic molecules loaded inside polymersomes can be released according to the release profiles, while those outside the polymersome bilayer can eventually penetrate it. Remarkably, as a new look, cargo photorelease from the nanopolymersomes was studied in real-time by cyclic voltammetry (CV). Finally, as a proof-of-concept, horseradish peroxidase (HRP)-loaded polymersomes were assembled as an intelligent labeling system of an optical biosensor of interleukin-6 (IL-6). This synthesis route can be exploited with other photo-switching AZO derivatives, paving the way toward new AZO compound-based nanocarrier systems.

Simple and efficient enrichment and separation of glycoprotein by teamed boronate affinity magnetic carbon nanospheres

Protein glycosylation is a key regulatory mechanism that controls many physiological and pathological processes. Analyses of glycoproteins in biological samples require challenging enrichment steps in light of sample complexity and low glycoprotein abundance. Here, in order to develop an efficient method to enrich glycoproteins, a magnetic carbon nanospheres (MCs) rich in amino group and polyboric acid sites was designed and prepared. The teamed boronate affinity was formed by the self-assembled of amino group and boric acid, which can specifically capture glycoprotein containing cis-diol under neutral conditions. Firstly, a simple green hydrothermal method led to the production of a carbon nanospheres matrix with abundant surface hydroxyl and carboxyl moieties. Then, MCs were synthesized by solvothermal method. Subsequently, the MCs were modified with polyethyleneimine and boric acid in turn to obtain the proposed composite. The hydrophilicity of amino group and its hydrogen bond with glycoprotein, as well as the cyclization reaction of boric acid with cis-1,2-diol, are utilized to specifically bind to glycoprotein. The prepared magnetic nanospheres exhibited binding to glycoproteins with rapid kinetics and strong affinity. Adsorption equilibrium was reached within 40 min, and the adsorption capacities for the glycoproteins ovalbumin, transferrin and horseradish peroxidase were found to be 493, 346 and 326 mg/g, respectively, while the adsorption capacities for the non-glycoproteins bovine serum protein and lysozyme were 60 and 30 mg/g, respectively. This novel nanoparticle provides a simple and efficient method for the enrichment and separation of glycoproteins from biological samples.

PdMoPtCoNi High Entropy Nanoalloy with d Electron Self-Complementation-Induced Multisite Synergistic Effect for Efficient Nanozyme Catalysis.

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.

Redox-mediated dsDNA-dye photooxidase mimic enable catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine by dissolved O2 at neutral pH for improved biosensing

Catalytic oxidation of 3,3′,5,5′-Tetramethylbenzidine (TMB, an excellent chromogenic substrate) at neutral pH is critically important for amplified bioanalysis. Although some nanozymes exhibited the peroxidase activity at neutral pH, it is difficult to modulate their activity for homogeneous detection of biomolecules. In this work, we developed a redox-mediated dsDNA-dye photooxidase mimic that enables catalytic oxidation of TMB by dissolved O2 at neutral pH for improved biosensing. During illumination, the double-stranded DNA-SYBR Green I (dsDNA-SG) photogenerated singlet oxygen (1O2) can oxidized Mn2+ to Mn3+ that can efficiently oxidize TMB to produce a distinct blue within 4 min under neutral conditions. The catalytic oxidation of TMB can be readily modulated by the formation or dissociation of dsDNA during the sensing. After investigating a series of redox mediators, we found that only the Mn3+/Mn2+ redox mediator can lead to the oxidation of TMB at neutral pH. The maximum reaction rate of Mn2+-mediated dsDNA-SG photooxidase mimic under neutral conditions (pH 7.0) was 1.7×10-4 mM/s, even higher than that of horseradish peroxidase (HRP, 8.0×10-5 mM/s). The redox-mediated dsDNA-SG photooxidase mimic was used for detection of APE1 at pH 7.0 with over 130-fold higher sensitivity than that at 4.0, owing to the high enzymatic activity of APE1 at neutral pH. Meanwhile, we further extended this photooxidase mimic for the sensitive detection of DNA (LOD, 8 pM) and heavy metal ions at neutral pH. The redox-mediated dsDNA-dye photooxidase mimic with the ease of modulating its enzymatic activity and working at neutral pH is quite appealing for biosensing.

Development of a blocking ELISA for detection of Japanese encephalitis virus antibodies in pig and horse sera

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.

Fabrication of a Heptapeptide-Modified Poly(glycidyl Methac-Rylate) Nanosphere for Oriented Antibody Immobilization and Immunoassay

Oriented antibody immobilization has been widely employed in immunoassays and immunodiagnoses due to its efficacy in identifying target antigens. Herein, a heptapeptide ligand, HWRGWVC (HC7), was coupled to poly(glycidyl methacrylate) (PGMA) nanospheres (PGMA-HC7). The antibody immobilization behavior and antigen recognition performance were investigated and compared with those on PGMA nanospheres by nonspecific adsorption and covalent coupling via carbodiimide chemistry. The antibodies tested included bovine, rabbit, and human immunoglobulin G (IgG), while the antigens included horseradish peroxidase (HRP) and β-2-Microglobulin (β2-MG). The nanospheres were characterized using zeta potential and particle size analyzers, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and reversed-phase chromatography, proving each synthesis step was succeeded. Isothermal titration calorimetry assay demonstrated the strong affinity interaction between IgG and PGMA-HC7. Notably, PGMA-HC7 achieved rapid and extremely high IgG adsorption capacity (~3 mg/mg) within 5 min via a specific recognition via HC7 without nonspecific interactions. Moreover, the activities of immobilized anti-HRP and anti-β2-MG antibodies obtained via affinity binding were 1.5-fold and 2-fold higher than those of their covalent coupling counterparts. Further, the oriented-immobilized anti-β2-MG antibody on PGMA-HC7 exhibited excellent performance in antigen recognition with a linear detection range of 0–5.3 μg/mL, proving its great potential in immunoassay applications.