Immobilization Of Horseradish Peroxidase Research Articles

Design of a Peroxidase‐Based Gas Diffusion Electrode for Bioelectroenzymatic Applications

We report here the design and application of an all‐in‐one gas diffusion electrode (GDE) combined with surface immobilized horseradish peroxidase (HRP) capable of in situ generation of hydrogen peroxide (H2O2) from air and simultaneous oxidation of a model substrate (ABTS) within a single‐cell electrochemical reactor. Carboxyl‐functionalized multiwalled carbon nanotubes (MWCNT‐COOH) were employed as an electrocatalyst for oxygen reduction reaction producing 719 ± 97 µM h−1 H2O2 at −0.2 V (vs Ag/AgCl) as an electron acceptor for HRP. We investigated two immobilization strategies to obtain HRP‐modified biocathodes using covalent amide conjugation between primary amine groups of HRP and carboxyl groups of MWCNT‐COOH (GDE/MWCNT‐COOH/HRP) and entrapment into a cross‐linked pyrene‐modified linear poly(ethylenimine) matrix (GDE/MWCNT‐COOH/Py‐LPEI/HRP). Fourier transform infrared spectroscopy (FT‐IR) and scanning electron microscopy (SEM) were used to characterize such surface modifications. The apparent catalytic activity achieved by HRP‐modified biocathodes via either covalent conjugation or entrapment into a polymer film was 311 ± 31 U mg−1 and 174 ± 17 U mg−1, respectively, as compared to the activity of freely diffusing 188 ± 23 U mg−1. The interfaces were reused showing 55% and 82% residual activity after 5 consecutive cycles for GDE/MWCNT‐COOH/HRP and GDE/MWCNT‐COOH/Py‐LPEI/HRP, respectively. Our findings illustrate prospects for integrating GDE and surface‐bound peroxidases for H2O2‐dependent electroenzymatic reactions, offering a promising platform for diverse applications in bioelectrosynthesis.

Immobilization of horseradish peroxidase on UiO-66-NH2 for colorimetric and fluorometric sensing of nitrite.

Immobilized enzymes play a crucial role in analytical sensing due to their exceptional stability and considerable commercial importance. In this study, a stable Zr-based metal-organic framework (UiO-66-NH2) was prepared as an immobilization platform for horseradish peroxidase (HRP) through covalent binding. HRP@UiO-66-NH2 retained 75% of its activity after 10 cycles. Subsequently, a colorimetric/fluorometric dual-mode sensing strategy using HRP@UiO-66-NH2 was developed for nitrite detection. HRP@UiO-66-NH2 facilitated the transformation of the non-colored compound 3,3',5,5'-tetramethylbenzidine (TMB) into its blue oxidized form. When nitrite is added, oxidized TMB (ox-TMB) specifically engaged with nitrite (NO2-) to generate diazotized TMB, leading to a color shift from blue to yellow. Concurrently, NO2- reacted with the amino groups of HRP@UiO-66-NH2, forming diazonium compounds and suppressing the fluorescence of HRP@UiO-66-NH2. The limits of detection were 0.21 μM and 0.19 μM for the colorimetric and fluorometric strategies, respectively. Furthermore, a portable kit for detecting nitrite was created by integrating gelatin with HRP@UiO-66-NH2. The kit could visually identify nitrite from 10-400 μM in colorimetric mode and 0-400 μM in fluorometric mode. This method provides an innovative approach for nitrite sensing, paving the way for new research into multifunctional immobilized enzymes and their potential uses in biochemical sensing applications.

Accurate HER2 determination in breast cancer: a prominent COF-immobilized enzyme-enhanced electrochemical aptasensor employing 4-acetamidophenol as an efficient mediator

An effective strategy for enzyme-enhanced electrochemical detection of human epidermal growth factor receptor 2 (HER2) is proposed for breast cancer diagnosis. This strategy utilizes a three-dimensional mesoporous covalent organic framework (COF), immobilized horseradish peroxidase (HRP), and a novel redox mediator, 4-acetamidophenol (APAP). The mesoporous structure, with encapsulation effect, and good biocompatibility of COF, makes the functionalized COF an efficient carrier for HRP immobilization (HRP-Ab-AuNPs@COF). It demonstrates superior catalytic activity, stability, and electrochemical performance compared to free HRP, thus making it an ideal probe for simultaneous target recognition and signal amplification. APAP is screened from four candidate phenolic compounds based on its high formal potential (0.32 V vs. Ag/AgCl), rapid electron transfer activity (kapp = 2.80 × 105 M− 1 s− 1), excellent solubility and stability. These properties prove significantly better than the conventional mediator hydroquinone (HQ), achieving a higher signal-to-background ratio. By integrating decorated multi-walled carbon nanotubes as substrate materials, the electrochemical aptasensor achieves a low HER2 detection limit (0.418 pg mL− 1) with high specificity. This method’s selectivity surpasses that of the HQ-mediated method by 59–73%. Moreover, the aptasensor can effectively distinguish breast cancer patients and healthy individuals, as well as patients at different stages of the disease with high accuracy (AUC = 0.928). This performance exceeds traditional biomarkers CEA and CA15-3. This work paves novel avenues for innovative applications of COF-immobilized enzymes and the novel mediator APAP in electrochemical biosensing, thus holding significant promise for individualized breast cancer diagnosis and treatment.

Biochemical properties of immobilized horseradish peroxidase on ceramic and its application in removal of azo dye

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

Immobilized Horseradish Peroxidase on Enriched Diazo-Activated Silica Gel Harnessed High Biocatalytic Performance at a Steady State in Organic Solvent.

Dimethyldichlorosilane (DMDCS), an efficient silane coupling reagent appearing between the -OH groups of silica gel (SG) and picric acid, instantaneously produces a derivative enriched with nitro groups. The nitro group acting as an end-cap terminates the reaction and subsequently was converted into diazo to couple tyrosine's phenol ring via its O-carbon, the inert center to immobilize horseradish peroxidase (HRP) in a multipoint mode. It maintains the status quo of the native enzyme's protein folding and the entire protein groups' chemistry. The molecular formula of the synthesized material was verified and appeared as {Si(OSi)4 (H2O)x}n{-O-Si(CH3)2-O-C6H2(N+≡N)3(HRP)}4·yH2O; the parameters were evaluated as x = 0.5, n = 1158, and y = 752. The immobilized biocatalyst's activity in organic solvents was 1.5 times better than that in an aqueous medium; it worked smoothly, wherein the activity in both solvents stabilized at six months and continued up to nine months at 63 ± 3% compared to the initial.

MgAl-Layered Double Hydroxide-Coated Bio-Silica as an Adsorbent for Anionic Pollutants Removal: A Case Study of the Implementation of Sustainable Technologies.

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

Construction of hierarchical porous MIL-100(Fe) for horseradish peroxidase immobilization and its degradation performance of bisphenol A

Bisphenol A (BPA) is an emerging endocrine disruptor commonly found in industrial wastewater. Horseradish peroxidase (HRP) is a plant enzyme that is effective in removing BPA in the presence of hydrogen peroxide, and immobilization techniques can improve its stability in use as well as its catalytic efficiency. This study constructs the MIL-100(Fe) materials with hierarchical pores and peroxidase-like activity as carriers for immobilizing HRP. The relevant characterization proved that the crystal structure and chemical composition of HP-MIL-100(Fe) (Hierarchically Porous MIL-100(Fe)) were the same as that of standard MIL-100(Fe), and the specific surface area reached 940.88 m2 with an average mesopore size of 5.12 nm. HRP was immobilized by diffusion and covalent binding method and the immobilized amount was obtained as 78.49 mg/g. Compared to HRP, HRP@HP-MIL-100(Fe) has better chemical stability as well as substrate affinity. The results of degradation experiments showed that HRP@HP-MIL-100 (Fe) degraded BPA 71% more than free HRP at the fifth minute, reaching 90.83%. This research demonstrates the potential of immobilized enzyme technology for future application in the sustainable degradation of phenolic effluents, contributing to cleaner production in industry as well as environmental protection.

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.

Enhanced conversion of methane to liquid-phase oxygenates via hollow ferrite nanotube@horseradish peroxidase based photoenzymatic catalysis

A novel photoenzymatic catalytic system employing hollow ferrite nanotubes and horseradish peroxidase (HRP) has been developed to significantly enhance the conversion of methane to liquid-phase oxygenates. The 1.5 % HRP-Fe3O4 HNTs demonstrated an exceptional liquid product yield of 177.87 μmol g−1 h−1 and exhibited reusability for methane photo-oxidation under ambient conditions. The physicochemical characterization revealed that the immobilization of HRP enhanced the visible light absorption and charge carrier separation efficiency. The Fe3O4 HNTs facilitated methane (CH4) molecule adsorption, while photogenerated electrons and H2O2 participated in the Fe2+-porphyrin and Fe3+-porphyrin cycle of HRP, enabling continuous generation of hydroxyl radicals, which activated CH4 to methyl radicals. In situ EPR and FTIR results indicated that CH3OH and CH3CH2OH were produced via the formation and consumption of •CH3O and •CH2 intermediates. This work presents an efficient strategy and provides new insights into the direct photo-oxidation of methane reaction through a photoenzyme catalytic system.

Fabrication of hierarchical 3D layered yttrium hydroxide nanocomposites: Their use for enzyme immobilization and catalysis of H2O2

3D layer metal hydroxides (LMHs) have attracted significant interest due to their distinctive structural features. Unfortunately, the limited electrical conductivity hinders the potential utilization of LMHs in electrochemical analysis. To address this issue, herein, gold nanoparticles (AuNPs) were introduced to layered yttrium hydroxides by a facile in-situ electrodeposition method. Subsequently, the hierarchical AuNPs/LYH nanocomposite was employed as solid supports for immobilization of horseradish peroxidase, generating an enzymatic biosensor for hydrogen peroxide detection. As a result, a linear range of 1–500 μM with a remarkably low detection limit of 0.33 μM (S/N=3) was achieved. Moreover, the biosensor also demonstrated excellent selectivity, reproducibility and stability. The superior electrochemical responses of the HRP/AuNPs/LYH nanocomposite towards H2O2 can be ascribed to the synergistic effect arising from the flower-like structures of LYHs and the high conductivity of AuNPs. Our study provides a significant approach for the development of enzyme immobilized supports through the construction of layer metal hydroxide nanocomposites.

Cyanobacterial Phycocyanin-Based Electrochemical Biosensor for the Detection of the Free Radical Hydrogen Peroxide.

Cyanobacteria are photosynthetic prokaryotes that inhabit extreme environments by modifying their photosensitive chemoreceptors called cyanobacteriochromes (CBCRs) which are linear tetrapyrrole-linked phycobilin molecules. These light-sensitive phycobilin from Spirulina platensis is recognized as a potential photoreceptor tool in optogenetics for monitoring cellular morphogenesis. We prepared and extracted highly fluorescent cyanobacterial phycocyanin (C-PC) by irradiating the culture with ambient red light. The crude phycocyanin was subjected to ion exchange chromatography, and its purity was monitored using UV-visible, fluorescence, and FT-IR spectroscopy methods. In the conventional method, red light-induced C-PC exhibited strong antioxidant activity against H2O2, with 88.7% in vitro scavenging activity without requiring any other preservatives. Interestingly, this red light-acclimated phycocyanin was applied as a biosensing material for the detection of the free radical hydrogen peroxide (H2O2) using the enzyme horseradish peroxidase (HRP) as a mediator. The modified C-PC-HRP glassy carbon electrode (GCE) can detect H2O2 from 0.1 to 1600µM. The lowest possible detection limit of the electrode for H2O2 was 19nM. This electrode was used to detect free radical H2O2 in blood serum samples. The microstructure of the lyophilized PC under SEM showed a flat crystal pattern, which enabled the immobilization of HRP on the electrode surface and electron transfer.

Phenol Removal through Horseradish Peroxidase Immobilization on Ultrafiltration Membranes: Comparative Analysis of Immobilization Methods and Fouling Patterns

This research investigates the removal of phenol using pure peroxidase from horseradish grade I in conjunction with a dead-end ultrafiltration membrane. Various horseradish peroxidase (HRP) immobilization techniques— physical adsorption, covalent bonding, and cross-linking with glutaraldehyde—were applied to a regenerated cellulose (RC) membrane with a surface area of 44 m2 and a molecular weight cut-off of 30 kDa. The investigation examined factors influencing phenol removal, including phenol concentration, membrane fouling, and the reusability of immobilized enzymes. Results indicated that covalent bonding was the most suitable enzyme immobilization technique, achieving a remarkable 90.1% immobilization yield. Phenol removal efficiency reached 100% at 30 min under specific conditions: phenol concentration of 1 mg/L, pH 6.0, hydrogen peroxide concentration of 0.5 mM, and operating pressure set at 3 psig, with temperature maintained at 28 ± 3 °C. Membrane fouling resulted in a decrease in flux. The performance of fouling models was found to be influenced by phenol concentration, with the Cake Formation Model (CFM) proving most effective at low concentrations, while the Complete Pore Blocking Model (CBM) emerged as more suitable at higher concentrations. The immobilized enzyme exhibited reusability for five cycles, maintaining a phenol removal efficiency exceeding 50%. These findings contribute to understanding the enzymatic phenol removal process and the use of appropriate enzyme immobilization techniques for the effective and sustainable treatment of phenol-contaminated water.

Preparation of Thermosensitive Polymer Immobilized Horseradish Peroxidase and its Application in Catalytic Degradation of Phenol

Preparation of Thermosensitive Polymer Immobilized Horseradish Peroxidase and its Application in Catalytic Degradation of Phenol

Increasing Multienzyme Cascade Efficiency and Stability of MOF via Partitioning Immobilization.

Enhancing the stability of multienzyme cascade reactions in metal-organic frameworks (MOFs) is a challenging task in the fields of biotechnology and chemistry. However, addressing this challenge could yield far-reaching benefits across the application range in the biomedical, food, and environmental sectors. In this study, multienzyme partitioning immobilization that sequentially immobilizes cascade enzymes with hierarchical MOFs is proposed to reduce substrate diffusion resistance. Conversion results of ginsenosides indicate that this strategy improves the cascade efficiency up to 1.26 times. The substrate diffusion model is used to investigate the dual-interenzyme mass transfer behavior of substrates in the restricted domain space and evaluate the substrate channeling effect under partitioning immobilization. Molecular docking and kinetic simulations reveal that the MOFs effectively limit the conformational changes of cascade enzymes at high temperatures and in organic solvents while maintaining a large pocket of active centers. This phenomenon increased efficient substrate docking to the enzyme molecules, further optimizing cascade efficiency. The results of the immobilization of GOX and horseradish peroxidase as model enzymes indicate that the partitioned MOF immobilization strategy could be used for universal adaptation of cascade enzymes.

Smartphone-assisted portable paper-based biosensors for rapid and sensitive detection of biomarkers in urine

In the increasing demand for real-time testing, the imperative for a straightforward, rapid, portable, and effective biosensor is evident. In this study, an intelligent paper-based biosensor was developed, employing cost-effective and readily available filter paper as a matrix to prepare boronate affinity paper-based materials. The immobilization of horseradish peroxidase (HRP) from crude horseradish extract onto the material surface was realized. Ultimately, a cold assembly technique was employed to construct this intelligent paper-based biosensor, which combined with a smartphone APP, facilitated the easy, accurate, and simultaneous detection of the content of glucose (GLU), uric acid (UA), and sarcosine (SAR) in urine, significantly enhancing the convenience and practicality of real-time monitoring. Additionally, UV spectroscopy served as an auxiliary method to affirm the reliability of the detection outcomes. For the detection of GLU, UA, and SAR, the biosensor exhibited linear ranges of 2–200, 2–500, and 2–500 µmol L–1, respectively, with detection limits (LODs) of 1 µmol L–1 for all analytes. In practical urine sample analysis, the recovery rates of GLU, UA, and SAR detected by the intelligent paper-based biosensors were within a reasonable range, with relative standard deviations below 3.5 %. This fully demonstrates the high accuracy and reliability of the intelligent paper-based biosensor. The development of this low-cost, portable biosensor provides a new method for the detection of biomarkers related to H2O2.

Immobilization of Horseradish Peroxidase onto Montmorillonite/Glucosamine-Chitosan Composite for Electrochemical Biosensing of Polyphenols.

Glucosamine-chitosan synthesized by the Maillard reaction was combined with montmorillonite to obtain a nanohybrid composite to immobilize horseradish peroxidase. The material combines the advantageous properties of clay with those of the chitosan derivative; has improved water solubility and reduced molecular weight and viscosity; involves an eco-friendly synthesis; and exhibits ion exchange capacity, good adhesiveness, and a large specific surface area for enzyme adsorption. The physicochemical characteristics of the composite were analyzed by infrared spectroscopy and X-ray diffraction to determine clay-polycation interactions. The electrochemical response of the different polyphenols to glassy carbon electrodes modified with the composite was evaluated by cyclic voltammetry. The sensitivity and detection limit values obtained with the biosensor toward hydroquinone, chlorogenic acid, catechol, and resorcinol are (1.6 ± 0.2) × 102 µA mM-1 and (74 ± 8) nM; (1.2 ± 0.1) × 102 µA mM-1 and (26 ± 3) nM; (16 ± 2) µA mM-1 and (0.74 ± 0.09) μM; and (3.7± 0.3) µA mM-1 and (3.3 ± 0.2) μM, respectively. The biosensor was applied to quantify polyphenols in pennyroyal and lemon verbena extracts.

Acrylic modification as an environmentally acceptable supporter for improving peroxidase enzyme: stability and reusability

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

Sweat lactate biosensor based on lactate oxidase immobilized with flower-like NiCo2O4 and carbon nanotubes

We design a portable and non-invasive lactate biosensor that can conveniently monitor lactate levels in real human sweat. The proposed biosensor is constructed through the immobilization of lactate oxidase (LOx) and horseradish peroxidase (HRP) on flower-like NiCo2O4 microspheres coupled with single-walled carbon nanotubes (SWCNTs). The synergistic effect between NiCo2O4 microspheres and SWCNTs can increase the conductivity and the active surface area of the electrode. Using ferrocene–methanol (FcMeOH) as the electron transfer mediator in buffer, the developed biosensor can detect lactate in a wide linear range of 0.1 mM to 30 mM with a detection limit of 39.9 µM under optimal conditions. Additionally, the biosensor exhibits excellent selectivity and stability. As proof of concept, the biosensor was applied to monitor lactate level in real human sweat samples, and the results were in good agreement with those obtained by high-performance liquid chromatography (HPLC) analysis. The lactate biosensor is cost effective and can be as a non-invasive alternative to blood analysis, which is suitable for rapid analysis of lactate levels in the fields of fitness, health monitoring and clinical diagnosis.

Immobilization of Horseradish Peroxidase and Myoglobin Using Sodium Alginate for Treating Organic Pollutants

Removing organic pollutants from wastewater is crucial to prevent environmental contamination and protect human health. Immobilized enzymes are increasingly being explored for wastewater treatment due to their specific catalytic activities, reusability, and stability under various environmental conditions. Peroxidases, such as horseradish peroxidase (HRP) and myoglobin (Mb), are promising candidates for immobilized enzymes utilized in wastewater treatment due to their ability to facilitate the oxidation process of a wide range of organic molecules. However, the properties of the carrier and support materials greatly influence the stability and activity of immobilized HRP and Mb. In this research, we developed immobilized HRP and Mb using support material composed of sodium alginate and CaCl2 as carriers and glutaraldehyde as a crosslinking agent. Following this, the efficacy of immobilized HRP and Mb in removing aniline, phenol, and p-nitrophenol was assessed. Both immobilized enzymes removed all three organic pollutants from an aqueous solution, but Mb was more effective than HRP. After being immobilized, both enzymes became more resilient to changes in temperature and pH. Both immobilized enzymes retained their ability to eliminate organic pollutants through eight treatment cycles. Our study uncovered novel immobilized enzyme microspheres and demonstrated their successful application in wastewater treatment, paving the way for future research.

Highly Light-Harvesting MOF-on-MOF Heterostructure: Cascading Functionality to Flexible Photogating of Organic Photoelectrochemical Transistor and Bienzyme Cascade Detection.

Recently, organic photoelectrochemical transistor (OPECT) bioanalysis has become a prominent technique for the high-performance detection of biomolecules. However, as a sensitive index of the OPECT, the dynamic regulation transconductance (gm) is still severely deficient. Herein, this work reports a new photosensitive metal-organic framework (MOF-on-MOF) heterostructure for the effective modulation of maximum gm and natural bienzyme interfacing toward choline detection. Specifically, the bidentate ligand MOF (b-MOF) was assembled onto the UiO-66 MOF (u-MOF) by a modular assembly method, which could facilitate the charge separation and generate enhanced photocurrents and offer a biophilic environment for the immobilization of choline oxidase (ChOx) and horseradish peroxidase (HRP) through hydrogen-bonded bridges. The transconductance of the OPECT could be flexibly altered by increased light intensity to maximal value at zero gate bias, and sensitive choline detection was achieved with a detection limit of 0.2 μM. This work reveals the potential of MOF-on-MOF heterostructures for futuristic optobioelectronics.