Immobilization Of Horseradish Peroxidase Research Articles

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.

Support Materials of Organic and Inorganic Origin as Platforms for Horseradish Peroxidase Immobilization: Comparison Study for High Stability and Activity Recovery.

In the presented study, a variety of hybrid and single nanomaterials of various origins were tested as novel platforms for horseradish peroxidase immobilization. A thorough characterization was performed to establish the suitability of the support materials for immobilization, as well as the activity and stability retention of the biocatalysts, which were analyzed and discussed. The physicochemical characterization of the obtained systems proved successful enzyme deposition on all the presented materials. The immobilization of horseradish peroxidase on all the tested supports occurred with an efficiency above 70%. However, for multi-walled carbon nanotubes and hybrids made of chitosan, magnetic nanoparticles, and selenium ions, it reached up to 90%. For these materials, the immobilization yield exceeded 80%, resulting in high amounts of immobilized enzymes. The produced system showed the same optimal pH and temperature conditions as free enzymes; however, over a wider range of conditions, the immobilized enzymes showed activity of over 50%. Finally, a reusability study and storage stability tests showed that horseradish peroxidase immobilized on a hybrid made of chitosan, magnetic nanoparticles, and selenium ions retained around 80% of its initial activity after 10 repeated catalytic cycles and after 20 days of storage. Of all the tested materials, the most favorable for immobilization was the above-mentioned chitosan-based hybrid material. The selenium additive present in the discussed material gives it supplementary properties that increase the immobilization yield of the enzyme and improve enzyme stability. The obtained results confirm the applicability of these nanomaterials as useful platforms for enzyme immobilization in the contemplation of the structural stability of an enzyme and the high catalytic activity of fabricated biocatalysts.

Development of chia gum/alginate-polymer support for horseradish peroxidase immobilization and its application in phenolic removal

Chia gum’s molecular structure with distinctive properties as well as the alginate-based hydrogel’s three-dimensionally cross-linked structure can provide a potent matrix for immobilization of enzyme. Herein, chia gum (CG)/alginate (A)-polymeric complex was synthesized and employed as a support material for the immobilization of horseradish peroxidase (HRP). HRP was successfully immobilized on the developed ACG-polymeric support, and the highest immobilization recovery (75%) was observed at 1.0% CG and 2% A, pH 7.0, and 50 units of the enzyme. The structure, morphology, and thermal properties of the prepared ACG-HRP were demonstrated using Fourier Transform Infrared (FTIR), Scanning Electron Microscope, and Thermogravimetric (TGA) analyses. ACG-HRP showed a good reusability (60%) over ten reuses. The immobilized ACG-HRP displayed an acidic pH optimum (6.0), a higher temperature optimum (50 °C), and improved thermal stability (30–50 °C) compared to the soluble HRP at pH 7.0, 40 °C and (30–40 °C), respectively. ACG-HRP has a lower affinity for hydrogen peroxide (H2O2) and guaiacol and a higher oxidizing affinity for a number of phenolic substrates. The ACG-HRP demonstrated greater resistance to heavy metals, isopropanol, urea, Triton X-100, and urea, as well as improved efficiency for eliminating phenol and p-chlorophenol. The developed ACG-polymeric support provided improved enzyme properties, allowed the reuse of the immobilized HRP in 10 cycles, and made it promising for several biotechnological applications.

Immobilization of Horseradish Peroxidase on Ca Alginate-Starch Hybrid Support: Biocatalytic Properties and Application in Biodegradation of Phenol Red Dye.

In this study, horseradish peroxidase was extracted, purified, and immobilized on a calcium alginate-starch hybrid support by covalent bonding and entrapment. The immobilized HRP was used for the biodegradation of phenol red dye. A 3.74-fold purification was observed after precipitation with ammonium sulfate and dialysis. An immobilization yield of 88.33%, efficiency of 56.89%, and activity recovery of 50.26% were found. The optimum pH and temperature values for immobilized and free HRP were 5.0 and 50°C and 6.5 and 60°C, respectively. The immobilized HRP showed better thermal stability than its free form, resulting in a considerable increase in half-life time (t1/2) and deactivation energy (Ed). The immobilized HRP maintained 93.71% of its initial activity after 45days of storage at 4°C. Regarding the biodegradation of phenol red, immobilized HRP resulted in 63.57% degradation after 90min. After 10 cycles of reuse, the immobilized HRP was able to maintain 43.06% of its initial biodegradative capacity and 42.36% of its enzymatic activity. At the end of 15 application cycles, a biodegradation rate of 8.34% was observed. In conclusion, the results demonstrate that the immobilized HRP is a promising option for use as an industrial biocatalyst in various biotechnological applications.

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.

A novel immobilized horseradish peroxidase platform driven by visible light for the complete mineralization of sulfadiazine in water

A novel immobilized enzyme driven by visible light was prepared and used for complete mineralization of antibiotics in water bodies. The immobilized enzyme was composed of carbon nitride modified by biochar (C/CN) and horseradish peroxidase (HRP), establishing the photo-enzyme coupling system with synergistic effect. Among them, the introduction of biochar not only improves the stability and loading capacity of the enzyme, but also improves the light absorption capacity and carrier separation efficiency of the photocatalyst. After the optimization of immobilization process, the solid load of HRP could reach 251.03 mg/g, and 85.03 % enzyme activity was retained after 18 days of storage at 4 °C. In the sulfadiazine (SDZ) degradation experiment, the degradation rate of HRP/C3/CN reached 71.21 % within 60 min, which was much higher than that of HRP (2.33 %), CN (49.78 %) and C3/CN (58.85 %). In addition, under the degradation of HRP/C/CN, the total organic carbon (TOC) removal rate of SDZ reached 53.14 %, which was 6.47 and 1.74 times that of CN and C3/CN, respectively. This study shows that the introduction of biochar is of great significance to the photo-enzyme cascade coupling system and provides a new strategy for the application of HRP&g-C3N4 system in wastewater treatment.

Immobilization of horseradish peroxidase on metal-organic framework to improve enzyme activity for enhanced chemodynamic therapy

Bio-enzymes have the advantages of strong substrate specificity, high catalytic efficiency, and minimal toxic side effects, making them promising drugs in cancer therapy. However, the poor stability and cellular penetrability of uncoated protein in the physiological environment severely restricts the direct application of Bio-enzyme. To address it, we report a metal-organic framework (MOF), Hf-DBA (H2DBA, biphenyl carboxylic acid ligands). The morphology of the Hf-DBA was revealed by TEM and the diameter was in the range of 200 to 350 nm. Hf-DBA acted a carrier for intracellular delivery and protection of horseradish peroxidase (HRP). The prepared HRP@Hf-DBA can catalyze the excess H2O2 in the tumor cells to generation of •OH for chemodynamic therapy (CDT). Compared with free HRP, the catalytic activity of HRP@Hf-DBA is significantly improved, and the optimal catalytic conditions are explored. The catalytic stability of HRP@Hf-DBA remained above 70% after 12 cycles of catalysis. After treatment with HRP@Hf-DBA, the apoptosis rates of A549 and Hela cells was 71.64%, and 76.86%. The results in vitro show that HRP@Hf-DBA can effectively inhibit the growth of tumor cells through enhanced CDT.

Enzyme-immobilized hierarchically porous covalent organic framework biocomposite for catalytic degradation of broad-range emerging pollutants in water

Efficient enzyme immobilization is crucial for the successful commercialization of large-scale enzymatic water treatment. However, issues such as lack of high enzyme loading coupled with enzyme leaching present challenges for the widespread adoption of immobilized enzyme systems. The present study describes the development and bioremediation application of an enzyme biocomposite employing a cationic macrocycle-based covalent organic framework (COF) with hierarchical porosity for the immobilization of horseradish peroxidase (HRP). The intrinsic hierarchical porous features of the azacalix[4]arene-based COF (ACA-COF) allowed for a maximum HRP loading capacity of 0.76 mg/mg COF with low enzyme leaching (<5.0 %). The biocomposite, HRP@ACA-COF, exhibited exceptional thermal stability (∼200 % higher relative activity than the free enzyme), and maintained ∼60 % enzyme activity after five cycles. LCMSMS analyses confirmed that the HRP@ACA-COF system was able to achieve > 99 % degradation of seven diverse types of emerging pollutants (2-mercaptobenzothiazole, paracetamol, caffeic acid, methylparaben, furosemide, sulfamethoxazole, and salicylic acid)in under an hour. The described enzyme-COF system offers promise for efficient wastewater bioremediation applications.

Portable intelligent paper-based sensors for rapid colorimetric and smartphone-assisted analysis of hydrogen peroxide for food, environmental and medical detection applications

One of the effective approaches to safeguard human health is the rapid and sensitive identification of food additives, environmental pollutants, and disease markers. This study developed a portable intelligent paper-based sensor for point-of-care detection of hydrogen peroxide (H2O2) and sarcosine (SA) using smartphone colorimetric analysis, supplemented by UV-Vis verification. The paper-based sensor utilizes a chemical modification of conventional filter paper by immobilizing horseradish peroxidase (HRP) from the horseradish plant, achieved through boronate affinity and metal chelation techniques. For H2O2 detection, the sensor exhibited a linear range of 2–100 µmol L−1 and a limit of detection (LOD) of 1.0 µmol L−1. Similarly, a range of 2–400 μmol L−1 for SA detection and an LOD of 1.0 μmol L−1. The sensors were successfully employed for determining the H2O2 concentration in liquor samples, environmental water, and the amount of SA in urine. Significantly, the detection ranges of these sensors surpassed those of commercially available test strips. In summary, we first proposed enzyme-immobilized paper-based sensors for immobilizing HRP from plants to detect H2O2 and SA, which are intelligent, portable, fast, green, low-cost, highly sensitive, and selective, with broad application prospects in food analysis, environmental monitoring, and medical diagnostics.

Immobilization of Horseradish Peroxidase for Phenol Degradation.

The use of enzymes to degrade environmental pollutants has received wide attention as an emerging green approach. Horseradish peroxidase (HRP) can efficiently catalyze the degradation of phenol in the environment; however, free HRP exhibits poor stability and temperature sensitivity and is easily deactivated, which limit its practical applications. In this study, to improve their thermal stability, HRP enzymes were immobilized on mesoporous molecular sieves (Al-MCM-41). Specifically, Al-MCM-41(W) and Al-MCM-41(H) were prepared by modifying the mesoporous molecular sieve Al-MCM-41 with glutaraldehyde and epichlorohydrin, respectively, and used as carriers to immobilize HRP on their surface, by covalent linkage, to form the immobilized enzymes HRP@Al-MCM-41(W) and HRP@Al-MCM-41(H). Notably, the maximum reaction rate of HRP@Al-MCM-41(H) was increased from 2.886 × 105 (free enzyme) to 5.896 × 105 U/min-1, and its half-life at 50 °C was increased from 745.17 to 1968.02 min; the thermal stability of the immobilized enzyme was also significantly improved. In addition, we elucidated the mechanism of phenol degradation by HRP, which provides a basis for the application of this enzyme to phenol degradation.

Immobilization of commercial horseradish peroxidase in calcium alginate-starch hybrid support and its application in the biodegradation of phenol red dye

In this study, horseradish peroxidase (HRP) was immobilized for the first time on Ca alginate-starch hybrid beads and employed for the biodegradation of phenol red dye. The optimal protein loading was 50 mg/g of support. Immobilized HRP demonstrated improved thermal stability and maximum catalytic activity at 50 °C and pH 6.0, with an increase in half-life (t1/2) and enzymatic deactivation energy (Ed) compared to free HRP. After 30 days of storage at 4 °C, immobilized HRP retained 109% of its initial activity. Compared to free HRP, the immobilized enzyme exhibited higher potential for phenol red dye degradation, as evidenced by the removal of 55.87% of initial phenol red after 90 min, which was 11.5 times greater than free HRP. In sequential batch reactions, the immobilized HRP demonstrated good potential efficiency for the biodegradation of phenol red dye. The immobilized HRP was used for a total of 15 cycles, degrading 18.99% after 10 cycles and 11.69% after 15 cycles, with a residual enzymatic activity of 19.40% and 12.34%, respectively. Overall, the results suggest that HRP immobilized on Ca alginate-starch hybrid supports shows promise as a biocatalyst for industrial and biotechnological applications, particularly for the biodegradation of recalcitrant compounds such as phenol red dye.

Immobilization of horseradish peroxidase with zwitterionic polymer material for industrial phenolic removal.

Horseradish peroxidase (HRP) is a hemoglobin composed of a single peptide chain that catalyzes the oxidation of various substrates such as phenol and aniline in the presence of hydrogen peroxide via its iron-porphyrin catalytic center. This enzyme is widely used in industrial phenol removal, food additives, biomedicine, and clinical test reagents due to its rapid reaction rate and obvious reaction outcomes. However, the large-scale use of HRP in industrial applications still faces numerous challenges, including activity, stability, and sustainability. This study demonstrates that when peroxidase is immobilized in zwitterionic polymer hydrogels, polycarboxybetaine (PCB) and polysulfobetaine (PSB), the properties of the enzyme are improved. PCB and PSB-embedded HRP exhibit a 6.11 and 1.53 times increase in Kcat/Km value, respectively, compared to the free enzyme. The immobilized enzyme also experiences increased activity over a range of temperatures and better tolerance to extreme pH and organic solvents, including formaldehyde. In addition, immobilized HRP exhibits excellent performance in storage and reproducibility. Remarkably, PCB-HRP still retains 80% of the initial activity after a 6-week storage period and can still attain the initial catalytic level of the free enzyme after six repeated cycles. It also removes 90% of phenol within 12 min, surpassing the current pharmacy on the market. These experimental results indicated that we have successfully designed a set of stable and efficient support substrates for horseradish peroxidase, which enhances its suitability for deployment in industrial applications.

Preparation of multifunctional hydrogels with accessible isothiouronium groups via radical cross-linking copolymerization

Hydrogels can be equipped with functional groups for specific purposes. Isothiouronium groups can enhance adsorptivity, or allow coupling of other functional groups through mild reactions after transformation to thiol groups. Here we present a method to prepare multifunctional hydrogels by introducing isothiouronium groups into poly(ethylene glycol) diacrylate (PEGDA) hydrogels, and convert them into thiol-functionalized hydrogels by the reduction of the isothiouronium groups. For this purpose, the amphiphilic monomer 2-(11-(acryloyloxy)-undecyl)isothiouronium bromide (AUITB), containing an isothiouronium group, was synthesized and copolymerized with PEGDA. In this convenient way, it was possible to incorporate up to 3 wt% AUITB into the hydrogels without changing their equilibrium swelling degree. The successful functionalization was demonstrated by surface analysis of the hydrogels with water contact angle measurements and increased isoelectric points of the hydrogel surfaces from 4.5 to 9.0 due to the presence of the isothiouronium groups. The hydrogels showed a suitability as an adsorbent, as exemplified by the pronounced adsorption of the anionic drug diclofenac. The potential of the functionalization for (bio)conjugation reactions was demonstrated by the reduction of isothiouronium groups to thiols and subsequent immobilization of the functional enzyme horseradish peroxidase on the hydrogels. The results show that fully accessible isothiouronium groups can be introduced into radically cross-linked hydrogels.

Horseradish Peroxidase Immobilized in Metal Aerogels Boosts Electron Transfer and an Interfacial Reaction for Photoelectrochemical Immunoassay.

Photoelectrochemical (PEC) enzymatic biosensors have attracted widespread attention for their specificity and sensitivity, but the charge migration between an enzyme and a semiconductor remains uncertain. In this work, horseradish peroxidase (HRP) was successfully immobilized on ionic liquid-functioned Cu@Cu2O (IL-Cu@Cu2O) aerogels to boost charge transfer and an interfacial redox reaction. The photogenerated electrons flow from the conduction band of Cu2O to HRP under the assistance of Cu and are subsequently captured by [Fe(CN)6]3- in the electrolyte, which boosts the PEC response. The improved interfacial catalytic ability after the immobilization of HRP is proved by the enhanced redox ability under light irradiation. Benefiting from the excellent PEC activity and catalysis reaction of IL-Cu@Cu2O@HRP, an immunoassay platform was constructed for sensing prostate-specific antigens, which presents a wide detection range and a low limit of detection. An in-depth understanding of the direct electronic communication between a photoactive material and an enzyme for boosted charge transfer and interfacial catalysis provides a new view for the design of advanced PEC sensing platforms.

Functionalized chitosan and sodium alginate for the effective removal of recalcitrant organic pollutants

In this work, the study of an oxidative-coupling reaction by immobilization of laccase or horseradish peroxidase (HRP) onto chitosan and sodium alginate was reported. The oxidative-coupling reaction of three recalcitrant organic pollutants (ROPs) such as chlorophenol compounds including 2,4-dichlorophenol (DCP), 2,4,6- trichlorophenol (TCP), pentachlorophenol (PCP) was studied. The results showed that the systems with immobilized laccase or horseradish peroxidase had broader range of optimum pH and temperature when compared to that of free laccase and horseradish peroxidase. The removal efficiencies of DCP, TCP and PCP within 6 h were found to be 77 %, 90 % and 83 %, respectively. The rate constants of the first order reactions for laccase were arranged as 0.30 h−1 (TCP) > 0.13 h−1 (DCP) > 0.11 h−1 (PCP) and the rate constants for HRP were arranged as 0.42 h−1 (TCP) > 0.32 h−1 (PCP) > 0.25 h−1 (DCP). The removal rate of TCP was found to be the highest among all and the removal efficiency of ROPs by HRP was always better than that of laccase. The major products of the reaction were identified by LC-MS and confirmed as humic-like polymers.

Immobilized horseradish peroxidase on boric acid modified polyoxometalate molecularly imprinted polymer for biocatalytic degradation of phenol in wastewater: Optimized immobilization, degradation and toxicity assessment

The degradation of phenol from wastewater is crucial for environmental protection. Biological enzymes, such as horseradish peroxidase (HRP), have shown great potential in the degradation of phenol. In this research, we prepared a hollow CuO/Cu2O octahedron adsorbent with a carambola matrix shape through the hydrothermal method. The surface of the adsorbent was modified by silane emulsion self-assembly, where 3-aminophenyl boric acid (APBA) and polyoxometalate (PW9) were combined with silanization reagents and grafted onto the surface. The adsorbent was then molecularly imprinted with dopamine to obtain boric acid modified polyoxometalate molecularly imprinted polymer (Cu@B@PW9@MIPs). This adsorbent was used to immobilize HRP, which served as a biological enzyme catalyst from horseradish. The adsorbent was characterized, and its synthetic conditions, experimental conditions, selectivity, reproducibility, and reusability were evaluated. The maximum adsorption amount of HRP under optimized conditions was 159.1 mg g−1, as determined using high-performance liquid chromatography (HPLC). At pH 7.0, the immobilized enzyme showed a high efficiency of up to 90.0% in removing phenol, after 20 min of reaction with 25 mmol L−1 H2O2 and 0.20 mg mL−1 Cu@B@PW9@HRP. Growth tests of aquatic plants confirmed that the adsorbent reduced harm. Gas chromatography-mass spectrometry (GC-MS) tests revealed that the degraded phenol solution contained about fifteen phenol derivatives intermediates. This adsorbent has the potential to become a promising biological enzyme catalyst for dephenolization.

Catalytic activity of glucose oxidase after dielectrophoretic immobilization on nanoelectrodes.

Dielectrophoresis (DEP) is an AC electrokinetic effect that is proven to be effective for the immobilization of not only cells, but also of macromolecules, e.g., antibodies and enzyme molecules. In our previous work, we have already demonstrated the high catalytic activity of immobilized horseradish peroxidase after DEP. To evaluate the suitability of the immobilization method for sensing or research in general, we want to test it for other enzymes, too. In this study, glucose oxidase (GOX) from Aspergillus niger was immobilized on TiN nanoelectrode arrays by DEP. Fluorescence microscopy showed the intrinsic fluorescence of the immobilized enzymes flavin cofactor on the electrodes. The catalytic activity of immobilized GOX was detectable, but a fraction of less than 1.3% of the maximum activity that was expected for a full monolayer of immobilized enzymes on all electrodes was stable for multiple measurement cycles. Therefore, the effect of DEP immobilization on the catalytic activity strongly depends on the used enzyme. This article is protected by copyright. All rights reserved.