Qin Ran

Covalent immobilization of horseradish peroxidase via click chemistry and its direct electrochemistry

A simple and versatile approach for covalent immobilization of redox protein on solid surface via self-assembled technique and click chemistry is reported. The alkynyl-terminated monolayers are obtained by self-assembled technique, then, azido-horseradish peroxidase (azido-HRP) was covalent immobilized onto the formed monolayers by click reaction. The modified process is characterized by reflection absorption infrared spectroscopy (RAIR), surface-enhanced Raman scattering spectroscopy (SERS) and electrochemical methods. All the experimental results suggest that HRP is immobilized onto the electrode surface successfully without denaturation. Furthermore, the immobilized HRP shows electrocatalytic reduction for H(2)O(2), and the linear range is from 5.0 to 700 μM. The heterogeneous electron transfer rate constant k(s) is 1.11 s(-1) and the apparent Michaelis-Menten constant is calculated to be 0.196 mM.

Direct electrochemistry of horseradish peroxidase immobilized on electrografted 4-ethynylphenyl film via click chemistry.

In this paper, a simple two-step approach for redox protein immobilization was introduced. Firstly, alkynyl-terminated film was formed on electrode surface by electrochemical reduction of 4-ethylnylphenyl (4-EP) diazonium compound. Then, horseradish peroxidase (HRP) modified with azido group was covalently immobilized onto the electrografted film via click reaction. Reflection absorption infrared (RAIR) spectroscopy and electrochemical methods were used to characterize the modification process. The results indicate that HRP retains its native structure and shows fast direct electron transfer. Moreover, the immobilized HRP shows excellent electrocatalytic reduction activity toward H(2)O(2) with a linear range of 5.0×10(-6) to 9.3×10(-4) mol L(-1).

A highly soluble poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid)/Au nanocomposite for horseradish peroxidase immobilization and biosensing

A highly soluble poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid)/Au (PEDOT-PSS/Au) nanocomposite was prepared via one-step chemical synthesis and the matrix was characterized by UV-vis spectroscopy (UV-vis), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM) and transmission electron microscope (TEM). Due to the excellent aqueous compatibility and biocompatibility, the PEDOT/PSS-Au nanocomposite can be used as biomaterial for enzymes immobilization. In this system, redox enzyme, horseradish peroxidase (HRP) was integrated with PEDOT/PSS-Au nanocomposite and the direct electron transfer of HRP was observed. Moreover, we find that the HRP/PEDOT-PSS/Au modified electrode shows excellent electrocatalytic ability for H(2)O(2) and the formal Michaelis-Menten constant (K(m)(app)) was 0.78 mmol/L. The response currents have good linear relation with the concentrations of H(2)O(2) with a linear range from 2.0 x 10(-7) to 3.8 x 10(-4)mol/L.

Covalent immobilization of redox protein via click chemistry and carbodiimide reaction: Direct electron transfer and biocatalysis

In this paper, a simple, facile and general strategy was proposed for the covalent biofunctionalization of Au surface with hemoglobin (Hb). Structurally well-defined azide-terminated organic self-assembled monolayers (SAMs) were formed on Au surface from a mixture solution of azidoundecanethiol and dilute thiol. 4-Pentynoic acid was used as a bifunctional linker to immobilized Hb via a selective, reliable, robust click reaction and a widely used carbodiimide reaction. The surface modification was characterized by reflectance absorption infrared (RAIR) spectroscopy, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The experimental data indicate that the proposed methodology is a highly versatile strategy for quantitative, stable and covalent attachment of biomacromolecules onto solid interface. The Hb functionalized Au electrode shows a pair of well-defined and quasi-reversible peaks at about -0.210 V (vs. SCE) in 0.1 mol/L pH 5.0 buffers, which stands for the reduction and oxidation of Hb Fe(III)/Fe(II) redox couple. The electron transfer rate constant (k(s)) was estimated to be 0.78 s(-1). The maximal surface coverage (Gamma) of the immobilized Hb was 8.3 x 10(-12) mol cm(-2), indicating the formation of a monolayer. Moreover, the biocatalytic activity of the Hb biofunctonalized electrode was investigated and an excellent electrocatalytic reduction toward O(2) and H(2)O(2) was observed.

High-Quality Covalently Grafting Hemoglobin on Gold Electrodes: Characterization, Redox Thermodynamics and Bio-electrocatalysis

Herein, we report a versatile surface chemistry methodology to covalently immobilize ligands and proteins to self-assembled monolayers (SAMs) on gold electrode. The strategy is based on two steps: 1) the coupling of soluble azido-PEG-amimo ligand with an alkynyl-terminated monolayer via click reaction and 2) covalent immobilization hemoglobin (Hb) to the amine-terminated ligand via carbodiimide reaction. Surface-enhanced Raman scattering spectroscopy (SERS), atomic force microscopy (AFM), reflection absorption infrared spectroscopy (RAIR) and cyclic voltammetry are used to characterize the model interfacial reactions. We also demonstrate the excellent biocompatibility of the interface for Hb immobilization and reliable application of the proposed method for H(2)O(2) biosensing. Moreover, the redox thermodynamics of the Fe(3+)/Fe(2+) couple in Hb is also investigated.