Immobilization Of Horseradish Peroxidase Enzyme Research Articles

Efficient immobilization of horseradish peroxidase enzyme on transition metal carbides

In this study, the molecular dynamics simulation is performed to investigate the adsorption of horseradish peroxidase (HRP, a model enzyme) on Ti3C2 and Ti3C2OH (as templates for enzyme immobilization). It has been found that van der Waals and electrostatic interactions regulate enzyme immobilization, whereas the hydrogen bonding between enzyme and MXene is involved in the adsorption process. The simulation outcomes indicate that various MXene can effectually adsorb HRP, and the adsorption strength is more for the Ti3C2@HRP complex, as revealed by energy analysis (interaction energy = −710.92 vs −449.02 kJ mol−1). Moreover, the root means square deviation, root means square fluctuation and secondary structure analysis indicate that Ti3C2 protects the conformation of enzymes more than Ti3C2OH. Hence, HRP can be effectually immobilized after the Ti3C2 in comparison to the Ti3C2OH. In the following, the evaluation of 2-thiouracil (2-Thi) ability to modulate HRP activity is investigated. The obtained results show that 2-Thi inhibitor has a great capacity to inhibit HRP enzyme. Eventually, the stability of immobilized HRP at higher temperatures (330 and 363 K) and acidic pH is examined. The present work suggests theoretical insights intorealizing the biocompatibility of MXenes and instructs for modeling efficacious synthetic inhibitors.

Mg and Ni nano-hydrotalcites modified with gold nanoparticles as platform of enzymatic electrochemical sensors for H2O2 detection

The immobilization of Horseradish Peroxidase enzyme over Magnesium (Mg-nHT) and Nickel (Ni-nHT) nano Hydrotalcite clays were synthesized by the co-precipitation method, and later modified with gold nanoparticles through in-situ synthesis and delamination-restructuration, is reported. The synthesized materials were characterized by Fourier-Transform Infrared Spectroscopy, X-ray diffraction, Scanning Electronic Microscopy, Flame Atomic Absorption Spectroscopy and Cyclic Voltammetry. Results show that the size of the synthetized materials is 26 nm for the gold nanoparticles, and 38.92 nm and 8.44 nm for Mg-AlnHT and Ni-AlnHT, respectively. The clays were used to modify the surface of graphite electrodes (GE); the inclusion of Au nanoparticles in the Hydrotalcites structure improved the enzyme-electrode electric communication. Figures of merit for detection of H2O2 by Differential-Pulse Voltammetry at HRP/Mg-AlnHT/GE, HRP/Ni-AlnHT/GE, HRP-AuNps/Mg-AlnHT/GE and HRP-AuNps/Ni-AlnHT/GE modified electrodes were determined, and respective detection limits of 0.012 μmolL−1, 0.014 μmolL−1, 0.009 μmolL−1, and 0.010 μmolL− 1 were obtained.

On the Use of a TiO2 Sol-Gel for Horseradish Peroxidase Enzyme Immobilization for Development of an Amperometric Biosensor

The sol-gel technique has drawn considerable attention in the area of biomolecules immobilization by virtue of its intrinsic properties including chemical inertness, thermal stability, and good biocompatibility. The present work reports the development of a bio-sensing methodology which involves the immobilization of horseradish peroxidase enzyme (HRP) on pencil graphite electrode (PGE) using TiO2 sol-gel and its application for 5-aminosalicylic acid (5-ASA) determination in pharmaceutical formulations. TiO2 sol-gel film was deposited on the PGE surface by dip coating, and the HRP enzyme was immobilized on the film by adsorption and cross-linked binding using glutaraldehyde. The analytical performance of the methodology was investigated through amperometric measurements at –0.08 V in the presence of 5-ASA (electron mediator) and hydrogen peroxide (substrate). The biosensor was successfully applied for 5-ASA determination, where limits of detection and quantification of 3.3 and 10.0 μmol L-1, respectively, were obtained. The application of the methodology for 5-ASA determination in pharmaceutical formulations led to satisfactory recovery rates ranging from 80 to 98%. The biosensor developed in this work is simple, inexpensive and has the potential to be applied for the determination of other pharmaceutical compounds, which also act as electron mediators in the catalytic cycle of HRP.

Immobilization of horseradish peroxidase enzyme on nanoporous titanium dioxide electrodes and its structural and electrochemical characterizations.

Hierarchically ordered, honeycomb-like nanoporous TiO2 electrodes are prepared by a simple electrochemical anodization process using ammonium fluoride dissolved in ethylene glycol as an electrolytic medium. Formation of hexagonally arranged nanopores along with the tubular structure and anatase crystalline phase of TiO2 is confirmed by field emission scanning electron microscope (FESEM) and X-ray diffraction (XRD) studies. Further, these nanoporous TiO2 electrodes are employed as a substrate for enzyme (horseradish peroxidase, HRP) immobilization in an attempt to enhance the electron transport across the semiconductor electrode-electrolyte interface. Two different strategies, namely, physical entrapment and covalent linking, are used for anchoring the enzyme. Various parameters such as conductivity, stability, enzyme loading, enzymatic activity, sensitivity, linear range, etc., are investigated by using electrochemical techniques. Structural and morphological analyses of enzyme-modified electrodes are carried out using spectroscopic (UV - vis) and microscopic (AFM) methods. In the case of physical entrapment, a simple drop casting method of HRP solution on the nanoporous TiO2 electrodes is used in contrast to chemical linking method where a monolayer of 3-aminopropyltrimethoxy silane (APTMS) is formed initially on TiO2 followed by HRP immobilization using an amide coupling reaction. Interestingly, both of these methods result in anchoring of HRP enzyme, but the amount of enzyme loading and the stability are found to be higher in the covalent linking method. Cyclic voltammetric studies reveal the formation of a well-defined reversible peak for HRP enzyme. Dependence of peak current with the scan rate suggests that HRP enzyme is immobilized and stable and that the overall electron transfer process is predominantly controlled by a diffusion process. Enzymatic activity of HRP is investigated by monitoring the reduction process of hydrogen peroxide by incremental addition using cyclic voltammetry and amperometry techniques, from which several kinetic parameters are determined.

Immobilization of HRP Enzyme on Layered Double Hydroxides for Biosensor Application

We present a new biosensor for hydrogen peroxide (H2O2) detection. The biosensor was based on the immobilization of horseradish peroxidase (HRP) enzyme on layered double hydroxides- (LDH-) modified gold surface. The hydrotalcite LDH (Mg2Al) was prepared by coprecipitation in constant pH and in ambient temperature. The immobilization of the peroxidase on layered hybrid materials was realized via electrostatic adsorption autoassembly process. The detection of hydrogen peroxide was successfully observed in PBS buffer with cyclic voltammetry and the chronoamperometry techniques. A limit detection of 9 μM of H2O2was obtained with a good reproducibility. We investigate the sensitivity of our developed biosensor for H2O2detection in raw milk.