Enzyme Horseradish Peroxidase Research Articles

Single-site iron-anchored amyloid hydrogels as catalytic platforms for alcohol detoxification.

Constructing effective antidotes to reduce global health impacts induced by alcohol prevalence is a challenging topic. Despite the positive effects observed with intravenous applications of natural enzyme complexes, their insufficient activities and complicated usage often result in the accumulation of toxic acetaldehyde, which raises important clinical concerns, highlighting the pressing need for stable oral strategies. Here we present an effective solution for alcohol detoxification by employing a biomimetic-nanozyme amyloid hydrogel as an orally administered catalytic platform. We exploit amyloid fibrils derived from β-lactoglobulin, a readily accessible milk protein that is rich in coordinable nitrogen atoms, as a nanocarrier to stabilize atomically dispersed iron (ferrous-dominated). By emulating the coordination structure of the horseradish peroxidase enzyme, the single-site iron nanozyme demonstrates the capability to selectively catalyse alcohol oxidation into acetic acid, as opposed to the more toxic acetaldehyde. Administering the gelatinous nanozyme to mice suffering from alcohol intoxication significantly reduced their blood-alcohol levels (decreased by 55.8% 300 min post-alcohol intake) without causing additional acetaldehyde build-up. Our hydrogel further demonstrates a protective effect on the liver, while simultaneously mitigating intestinal damage and dysbiosis associated with chronic alcohol consumption, introducing a promising strategy in effective alcohol detoxification.

Human and camel cystic echinococcosis - a polyclonal antibody-based sandwich ELISA for its serodiagnosis with molecular identification.

Cystic echinococcosis (CE) is an emergent neglected disease affecting human and animals in Egypt with a wide distribution and incidence. This study aimed to evaluate the use of a polyclonal antibody-based sandwich ELISA in the detection of Echinococcus granulosus antigen in human and camel sera. Hydatid cyst protoscoleces antigen (PsAg) was isolated from hydatid cysts collected from naturally infected camel livers and lungs. PsAg was used for immunization of rabbits to raise IgG polyclonal antibodies (IgG PsAb). IgG PsAb were then precipitated, purified using Protein-A Sepharose gel and labeled with horseradish peroxidase enzyme. We assayed the purity of the IgG PsAb, and the two prepared E. granulosus antigens CPsAg from camel cysts and HPsAg from human cysts by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The resulted protein bands of the prepared CPsAg appeared at different molecular weights: 180, 90, 68, 54, 42 and 22kDa while, HPsAg shared with it in 4 common bands at 68, 54, 42, and 22kDa. The purified IgG PsAb had been resolved at two bands at 52kDa and at 32kDa. Sandwich ELISA were performed for the detection of circulating E. granulosus antigens in sera of human (n = 183) and camels (n = 190). The purified IgG PsAb showed strong reactivity against E. granulosus infected human and camel samples and no cross reactivity neither with free-healthy negative sera nor with others parasitic diseases (Schistosomiasis, Fascioliasis, Toxoplasmosis, Ancylostomiasis for human samples and Fascioliasis, ticks' infestation, Eimeriosis, Cryptosporidiosis, Nasal myiasis, Toxoplasmosis for camel samples). The sensitivity of the assay was 98.25% (56/57) and 96.9% (31/32) against human and camel samples, respectively. Specificity was 100% in both human and camel samples. Sandwich ELISA detected CE in 33.3% (24/72) and 55.6% (50/90) random human and camel samples, respectively. Indirect ELISA, using CPsAg, was used for detection of antibodies in positive human and camels' sera and detected 96.5% (55/57) and 93.8% (30/32) of human and camel samples, respectively. In our study, Genomic DNA was extracted from protoscoleces fluid of human liver hydatid cysts to identify the Echinococcus sp. isolate based on NADH dehydrogenase subunit 1 (NAD1) gene by Polymerase Chain Reaction (PCR) and the isolate (GenBank: OP785689.1) were identified as E. granulosus sensu lato genotype. In conclusion, Sandwich ELISA technique was found to be a potent and sensitive assay for detection of hydatid antigen in both human and camel samples.

Innovative Detection of Biomarkers Based on Chemiluminescent Nanoparticles and a Lensless Optical Sensor.

The identification and quantification of biomarkers with innovative technologies is an urgent need for the precise diagnosis and follow up of human diseases. Body fluids offer a variety of informative biomarkers, which are traditionally measured with time-consuming and expensive methods. In this context, lateral flow tests (LFTs) represent a rapid and low-cost technology with a sensitivity that is potentially improvable by chemiluminescence biosensing. Here, an LFT based on gold nanoparticles functionalized with antibodies labeled with the enzyme horseradish peroxidase is combined with a lensless biosensor. This biosensor comprises four Silicon Photomultipliers (SiPM) coupled in close proximity to the LFT strip. Microfluidics for liquid handling complete the system. The development and the setup of the biosensor is carefully described and characterized. C-reactive protein was selected as a proof-of-concept biomarker to define the limit of detection, which resulted in about 0.8 pM when gold nanoparticles were used. The rapid readout (less than 5 min) and the absence of sample preparation make this biosensor promising for the direct and fast detection of human biomarkers.

Enhanced peroxidase-like activity of Cu–Cu2O composite film through PtPd immobilization for colorimetric glucose detection

In this study, Cu–Cu2O/PtPd nanocomposites were synthesized and characterized for their peroxidase-like enzyme activity. X-ray diffraction and energy dispersive X-ray spectroscopy analyses confirmed the successful synthesis of the nanocomposites, which exhibited a flower-like morphology and a more uniform dispersion than Cu–Cu2O. The catalytic activity of Cu–Cu2O/PtPd was evaluated using the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB), finding that Cu–Cu2O/PtPd outperformed Cu–Cu2O. The optimal temperature and pH for the catalytic activity of Cu–Cu2O/PtPd were determined to be 40 °C and pH 4.0, respectively. A kinetic analysis revealed that Cu–Cu2O/PtPd followed Michaelis–Menten kinetics and exhibited a higher affinity toward TMB than the horseradish peroxidase enzyme. The catalytic mechanism of Cu–Cu2O/PtPd involved the generation of hydroxyl radicals, which facilitated the oxidation of TMB. Furthermore, the Cu–Cu2O/PtPd nanocomposite was successfully applied for the colorimetric detection of glucose, demonstrating a linear range of 8–90 μM, a detection limit of 2.389 μM, and high selectivity for glucose over other sugars.

Biodegradability of Carbon Nano‐Onions by Human Myeloperoxidase and Photo‐Fenton Process

The biodegradability of carbon nanomaterials (CNMs) in the environment and organisms needs to be understood, and it is highly essential to estimate their in vivo fate, especially for clinical applications. It is also important to investigate their metabolites formation to fully understand their biocompatibility. Herein, we explored the biodegradability of multilayer fullerenes, better known as carbon nano‐onions (CNOs), by treating them with human myeloperoxidase (hMPO) isolated from the neutrophils, plant enzyme horseradish peroxidase (HRP) and the photo‐Fenton (PF) reaction. High‐resolution electron microscopy (HRTEM), Raman spectroscopy, X‐ray photoelectron spectroscopy (XPS), and mass spectrometry (MS) results confirmed that CNOs are not bio‐persistent. Moreover, these results indicate that the oxidized (functionalized) CNOs can undergo faster biodegradation than the pristine CNOs. The possible by‐products of biodegradation of CNOs were characterized using mass spectrometry. An understanding of the biodegradation properties of CNOs and their metabolites is fundamental for future biomedical applications.

Bringing to Light the Importance of the miRNA Methylome in Colorectal Cancer Prognosis Through Electrochemical Bioplatforms.

This work reports the first electrochemical bioplatforms developed for the determination of the total contents of either target miRNA or methylated target miRNA. The bioplatforms are based on the hybridization of the target miRNA with a synthetic biotinylated DNA probe, the capture of the formed DNA/miRNA heterohybrids on the surface of magnetic microcarriers, and their recognition with an antibody selective to these heterohybrids or to the N6-methyladenosine (m6A) epimark. The determination of the total or methylated target miRNA was accomplished by labeling such secondary antibodies with the horseradish peroxidase (HRP) enzyme. In both cases, amperometric transduction was performed on the surface of disposable electrodes after capturing the resulting HRP-tagged magnetic bioconjugates. Because of their increasing relevance in colorectal cancer (CRC) diagnosis and prognosis, miRNA let-7a and m6A methylation were selected. The proposed electrochemical bioplatforms showed attractive analytical and operational characteristics for the determination of the total and m6A-methylated target miRNA in less than 75 min. These bioplatforms, innovative in design and application, were applied to the analysis of total RNA samples extracted from cultured cancer cells with different metastatic profiles and from paired healthy and tumor tissues of patients diagnosed with CRC at different stages. The obtained results demonstrated, for the first time using electrochemical platforms, the potential of interrogating the target miRNA methylation level to discriminate the metastatic capacities of cancer cells and to identify tumor tissues and, in a pioneering way, the potential of the m6A methylation in miRNA let-7a to serve as a prognostic biomarker for CRC.

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

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

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

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

Self-assembled copper nanoclusters used to mimic peroxidase for glucose detection.

A sensing system for glucose was established based on a self-assembled copper nanoclusters (Cu NCs)-based nano-enzyme and glucose oxidase (GOD). The assembled copper nanosheets (Cu NSs) were prepared in a one-step method using 2,3,4,5,6-pentafluorothiophenol (PFTP) as a reducing agent and protecting ligand. Cu NSs could be used to mimic the enzyme horseradish peroxidase. Cu NSs were endowed with excellent enzymatic catalytic activity in the oxidation of o-phenyldiamine (OPD) in the presence of H2O2. The latter could be generated in the aerobic oxidation of glucose catalyzed by GOD. Therefore, a detection method for glucose was constructed based on a Cu NSs-OPD-GOD catalytic system. This proposed sensing platform showed a standard linear range from 10 μM to 5 mM towards glucose, and the limit of detection was 5.5 μM. Finally, practical application of a sensor based on the Cu NSs nano-enzyme was verified in three sugared beverages as real samples. Our data reveal that the prepared Cu NSs could mimic peroxidase and be applied to a mixed catalytic system with GOD for glucose detection.

Development of enzyme-linked immunosorbent assay (ELISA) based on covalent immobilization of antibody on plate for measurement of digoxin

Objectives: The aim of this study was to design an enzyme immunoassay based on a modified ELISA with high sensitivity for detecting digoxin.
 Methods: The first step in development involved the conjugation of digoxin to the HRP (Horse Radish Peroxidase) enzyme via sodium metaperiodate oxidation. Surface modification, and thus assay modification, was achieved by the covalent immobilization of an anti-digoxin monoclonal antibody on a functional plate using 3-ATPES (3-aminopropyltriethoxysilane).
 Results: The developed ELISA demonstrated superior sensitivity (0.026μg/ml) and lower variability in measurements repeated throughout a day, compared to a conventional ELISA (0.051μg/ml). This assay detected the exact amount of digoxin. The sensitivity and specificity of the modified ELISA surpassed other methods, and measurements were performed within a few hours.
 Conclusion: An efficient ELISA kit was produced, characterized by its affordability, ease of learning, and absence of a hand-washing step.

Production of flower‐shaped nanobiocatalysts from green tea and investigation of their peroxidase mimicking activity on the polymerization of phenol derivatives

AbstractEnzyme catalyzed reactions are known to be environmental friendly and easy method for many applications. However, utilization of enzymes in a variety of reactions is strictly limited due to their high cost, instability in aqueous solutions, denaturation in organic solvents and high temperatures. For this reason, it is important to discover new generation catalyst systems indicating enzyme‐like catalytic activity. Here, we report hybrid organic–inorganic flower‐shaped green tea‐Cu2+ nanobiocatalyst synthesized from green tea extract as an organic component and copper (II) ions (Cu2+) as inorganic component. The effect of the peroxidase‐mimicking activity of green tea‐Cu2+ nanobiocatalyst was investigated on the polymerization of phenol and derivatives (guaiacol and salicylic acid) through Fenton‐like reaction mechanism. Obtained successful outcomes showed that the synthesized nanobiocatalyst showed very high catalytic activity upon polymerization of phenol and guaiacol. The slight solubility of salicylic acid in water limited to achieve its polymerization under‐performed reaction conditions. The yields and molecular weights of the obtained polymers were found to be quite high. While free peroxidase enzymes like horseradish peroxidase (HRP) enzyme loses its catalytic activity at 60°C and above temperatures, green tea‐Cu2+ nanobiocatalyst exhibited very high catalytic activity upon polymerization reactions even at 60°C reaction temperature. This outcome provides significant advantages in some reactions requiring high temperatures. In order to understand the origin of the catalytic activity of the green tea‐Cu2+ nanoflowers, similar biocatalysts were also synthesized from caffeine and catechin alkaloids which are the active components of green tea. Caffeine‐Cu2+ and catechine‐Cu2+ nanobiocatalysts also exhibited quite high catalytic activity toward polymerization of phenol and derivatives. We suggest that green tea‐Cu2+ and similar types of nanobiocatalysts may expand their utilization in polymer chemistry as promising catalytic agents for radicalic polymerizations.

Catalytic Activity of Horseradish Peroxidase Immobilized on Pristine and Two‐Photon Oxidized Graphene

AbstractBiosensors based on graphene and bio‐graphene interfaces have gained momentum in recent years due to graphene's outstanding electronic and mechanical properties. By introducing the patterning of a single‐layer graphene surface by two‐photon oxidation (2PO), the surface hydrophobicity/hydrophilicity and doping can be varied at the nanoscale while preserving the carbon network, thus opening possibilities to design new devices. In this study, the effect of 2PO on the catalytic activity of the noncovalently immobilized enzyme horseradish peroxidase (HRP) on single‐layer graphene‐coated Si/SiO2 chips is presented. To monitor the activity continuously, a simple well‐plate setup is introduced. Upon controllable 1–2‐layer immobilization, the catalytic activity decreases to a maximum value of 7.5% of the free enzyme. Interestingly, the activity decreases with increasing 2PO area on the samples. Hence, the HRP catalytic activity on the graphene surface is locally controlled. This approach can enable the development of graphene‐bio interfaces with locally varying enzyme activity.

Enzymatic Dimerization-Induced Self-Assembly of Alanine-Tyramine Conjugates into Versatile, Uniform, Enzyme-Loaded Organic Nanoparticles.

Herein, we report a novel enzymatic dimerization-induced self-assembly (e-DISA) procedure that converts alanine-tyramine conjugates into highly uniform enzyme-loaded nanoparticles (NPs) or nanocontainers by the action of horseradish peroxidase (HRP) in an aqueous medium under ambient conditions. The NP formation was possible with both enantiomers of alanine, and the average diameter could be varied from 150 nm to 250 nm (with a 5-12 % standard deviation of as-prepared samples) depending on the precursor concentration. About 60 % of the added HRP enzyme was entrapped within the NPs and was subsequently utilized for post-synthetic modification of the NPs with phenolic compounds such as tyramine or tannic acid. One-pot multi-enzyme entrapment of glucose oxidase (GOx) and peroxidase (HRP) within the NPs was also achieved. These GOx-HRP loaded NPs allowed multimodal detection of glucose, including that present in human saliva, with a limit of detection (LoD) of 740 nM through fluorimetry. The NPs exhibited good cytocompatibility and were stable to changes in pH (acidic to basic), temperature, ultrasonication, and even the presence of organic solvent (EtOH) to a certain extent, since they are stabilized by intermolecular hydrogen bonding, π-π, and CH-π interactions. The proposed e-DISA procedure can be widely expanded through the design of diverse enzyme-responsive precursors.

Enzymatic Dimerization‐Induced Self‐Assembly of Alanine‐Tyramine Conjugates into Versatile, Uniform, Enzyme‐Loaded Organic Nanoparticles

AbstractHerein, we report a novel enzymatic dimerization‐induced self‐assembly (e‐DISA) procedure that converts alanine‐tyramine conjugates into highly uniform enzyme‐loaded nanoparticles (NPs) or nanocontainers by the action of horseradish peroxidase (HRP) in an aqueous medium under ambient conditions. The NP formation was possible with both enantiomers of alanine, and the average diameter could be varied from 150 nm to 250 nm (with a 5–12 % standard deviation of as‐prepared samples) depending on the precursor concentration. About 60 % of the added HRP enzyme was entrapped within the NPs and was subsequently utilized for post‐synthetic modification of the NPs with phenolic compounds such as tyramine or tannic acid. One‐pot multi‐enzyme entrapment of glucose oxidase (GOx) and peroxidase (HRP) within the NPs was also achieved. These GOx‐HRP loaded NPs allowed multimodal detection of glucose, including that present in human saliva, with a limit of detection (LoD) of 740 nM through fluorimetry. The NPs exhibited good cytocompatibility and were stable to changes in pH (acidic to basic), temperature, ultrasonication, and even the presence of organic solvent (EtOH) to a certain extent, since they are stabilized by intermolecular hydrogen bonding, π‐π, and CH‐π interactions. The proposed e‐DISA procedure can be widely expanded through the design of diverse enzyme‐responsive precursors.

Soft Sub-Structured Multi-Material Biosensor Hydrogels with Enzymes Retained by Plant Viral Scaffolds.

An all-soft multi-material combination consisting of a hydrogel based on poly(ethylene glycol) (PEG) coated with spatially defined spots of gelatin methacryloyl (GM) containing selectively addressable viral nanorods is presented, and its basic application as a qualitative biosensor with reporter enzymes displayed on the tobacco mosaic virus (TMV) bioscaffolds within the GM is demonstrated. Biologically inert PEG supports are equipped with GM spots serving as biological matrix for enzymes clustered on TMV particles preventing diffusion out of the gel. For this multi-material combination, i) the PEG-based hydrogel surface is modified to achieve a clear boundary between coated and non-coated regions by introducing either isothiouronium or thiol groups. ii) Cross-linking of the GM spots is studied to achieve anchoring to the hydrogel surface. iii) The enzymes horseradish peroxidase or penicillinase (Pen) are conjugated to TMV and integrated into the GM matrix. In contrast to free enzymes, enzyme-decorated TMVs persist in GM spots and show sustained enzyme activity as evidenced by specific color reaction after 7 days of washing, and for Pen after 22 months after dry storage. Therefore, the integration of enzyme-coupled TMV into hydrogel matrices is a promising and versatile approach to obtaining reusable and analyte-specific sensor components.

Alkaline surface treatment and time-resolved reading of mn-doped nanocrystal signal transducer for enhanced bioassay sensitivity

Recently, Mn-doped semiconductor nanocrystals (NCs) with high brightness, long lifetimes, and low-energy excitation are emerging for time-resolved luminescence biosensing/imaging. Following our previous work on Mn-doped NCs, in this work we developed poly(styrene-co-maleic anhydride) (PSMA)-encapsulated Mn-doped AgZnInS/ZnS NCs as signal transducers for immunoassay of capsular polysaccharide (CPS), a surface antigen and also a biomarker of Burkholderia pseudomallei which causes a fatal disease called melioidosis. To enhance the assay sensitivity, a surface treatment for PSMA-encapsulated NCs (NC-probes) was performed to promote the presence of carboxyl groups that help conjugate more anti-CPS antibodies to the surface of NC-probes and thus enhance bioassay signals. Meanwhile, time-resolved reading on the luminescence of NC-probes was adopted to minimize the assay background autofluorescence. Both strategies essentially enhance the assay signal-to-background ratio (or equivalently the assay sensitivity) by increasing the signal and decreasing the background, respectively. Through performing and comparing immunoassays with different NC-probes (with and without surface treatment) and different signal reading methods (time-resolved reading and non-time-resolved reading), it was proven that the immunoassay adopting surface-treated NC-probes and time-resolved reading achieved a lower limit-of-detection (LOD) than the ones adopting non-surface-treated NC-probes or non-time-resolved reading. Moreover, the achieved LOD is comparable to the LOD of immunoassay using enzyme horseradish peroxidase as a signal transducer.

A sensitive biosensor for cyanide detection using modified glassy carbon electrode with reduced graphene oxide and immobilization of horseradish peroxidase

AbstractCyanide is a highly poisonous and hazardous substance which may release into the environment from natural sources or industrial effluent; therefore, cyanide detection is a fundamental step to prevent environmental pollution and secure health and safety. In this study, we prepared a sensitive amperometric inhibition biosensor for cyanide detection by immobilization of horseradish peroxidase (HRP) enzyme and reduced graphene oxide (rGO) on the surface of glassy carbon electrode (GCE). To do so, we performed the amperometric measurement by modified GCE to test its efficiency in detecting cyanide. The optimum conditions of pH equal to 7.5, −100 mV applied potential, 0.7 μM mediator concentration, and 0.5 mM substrate concentration were found. Then, experiments were performed at different boundary conditions in a range of 0.1 to 10 μM cyanide concentration at optimal conditions and a low detection limit of 0.01 μM was obtained. Also, the possible mechanism of inhibition was analyzed based on the Michalis–Menten equation and non‐competitive inhibition was observed. Due to high sensitivity, low detection limit, and low cost, this biosensor is proposed as a useful method for cyanide determination in real samples.

Development of a new affinity chromatography method for purification of horseradish peroxidase enzyme.

In this study, benzohydroxamic acid molecules were synthesized from methyl 4-amino-2-methoxy, methyl 4-amino-3-nitro, methyl 4-amino-3-methyl, and methyl 4-amino-3-chloro benzoate molecules, and the horseradish peroxidase (HRP) enzyme was purified in one step using the affinity chromatography technique for the first time. The IC50 and Ki values for the 4-amino 3-methyl benzohydroxamic acid molecule were 0.136 and 0.132 ± 0.054μM, respectively, while the IC50 and Ki values for the 4-amino-3-nitro benzohydroxamic acid molecule were 56.00 and 51.90 ± 9.90μM, respectively. It was found that the IC50 and Ki values for the 4-amino-3-chloro benzohydroxamic acid molecule were 218.33 and 175.67 ± 43.78μM, respectively, whereas the IC50 and Ki values for the 4-amino-2-methoxy benzohydroxamic acid molecule were 306.00 and 218.00 ± 68.80μM, respectively. The HRP enzyme was synthesized from 4-amino-2-methoxy hydroxamic acid column with a 35.97% yield 601.13 times, 4-amino-3-nitro hydroxamic acid column, with a 14.00% yield 404.11 times, 4-amino-3-methyl hydroxamic acid column with an 8.70% yield 394.88 times, and 4-amino-3-chloro hydroxamic acid column with a 4.48% yield 284.85 times. Thus, the HRP enzyme was purified in a single step with hydroxamic acids, and its molecular weight was found to be 44kDa. The optimum pH was 8.0, the optimum temperature was 15°C, and the optimum ionic strength was 0.4M for the purified HRP enzyme.

Registration of Functioning of a Single Horseradish Peroxidase Macromolecule with a Solid-State Nanopore

Currently, nanopore-based technology for the determination of the functional activity of single enzyme molecules continues its development. The use of natural nanopores for studying single enzyme molecules is known. At that, the approach utilizing artificial solid-state nanopores is also promising but still understudied. Herein, we demonstrate the use of a nanotechnology-based approach for the investigation of the enzymatic activity of a single molecule of horseradish peroxidase with a solid-state nanopore. The artificial 5 nm solid-state nanopore has been formed in a 40 nm thick silicon nitride structure. A single molecule of HRP has been entrapped into the nanopore. The activity of the horseradish peroxidase (HRP) enzyme molecule inserted in the nanopore has been monitored by recording the time dependence of the ion current through the nanopore in the course of the reaction of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) oxidation reaction. We have found that in the process of ABTS oxidation in the presence of 2.5 mM hydrogen peroxide, individual HRP enzyme molecules are able to retain activity for approximately 700 s before a decrease in the ion current through the nanopore, which can be explained by structural changes of the enzyme.

Bimetallic Cu-Zn Zeolitic Imidazolate Frameworks as Peroxidase Mimics for the Detection of Hydrogen Peroxide: Electrochemical and Spectrophotometric Evaluation.

A copper incorporated zeolitic imidazolate framework-8 (ZIF-8) has been synthesized and demonstrated to be a potential material for a peroxidase mimic. The resultant bimetallic Cu-Zn incorporated MOF is used for the dual mode sensing of hydrogen peroxide by following electrochemical as well as spectrophotometric methods. Using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic substrate, spectrophotometric studies are carried out, and the steady state kinetic parameters are determined for two different concentrations of Cu incorporated ZIF-8 (viz Cu@ZIF-8-1 and Cu@ZIF-8-2). It is found that both Cu@ZIF-8-1 and Cu@ZIF-8-2 exhibit more affinity toward the TMB substrate than the horseradish peroxidase (HRP) enzyme as indicated by the low Km values obtained for the substrate. Also, as the concentration of incorporated Cu increases, Vmax values are also found to be enhanced. Electrochemically, the Cu@ZIF-8 modified glassy carbon electrode (GCE) showed a good response for peroxide detection in the concentration range from 0.5 mM to 5 mM at a working potential of -0.25 V in PBS (pH 7.0) with a limit of detection (LOD) value of 0.46 mM and a sensitivity of 20.25 μA/mM. Further, the chromogenic substrate TMB is successfully immobilized on the electrode surface and subsequently used for the peroxide detection along with Cu@ZIF-8. Here, TMB acts as a mediator and shifted the working potential to 0.1 V in acetate buffer (pH 5.0) in the concentration range from 0.5 mM to 5 mM with an LOD value of 0.499 mM and a sensitivity of 0.097 μA/mM. Interestingly, the same electrode in PBS of pH 7.0 showed a response to peroxide at a working potential of -0.1 V in the concentration range from 0.5 mM to 5 mM with an LOD value of 0.143 mM and a sensitivity of 0.33 μA/mM. Moreover, the applicability of this material for peroxide sensing is evaluated using milk samples, and the proposed material is able to recover the peroxide present in milk. Thus, the bimetallic Cu-Zn MOF can be utilized for the dual mode sensing of peroxide and can be extended for various real time applications.