Enzyme biosensor based on the immobilization of HRP on SiO2/BSA/Au composite nanoparticles

Abstract

In this work, an enzyme biosensor based on the immobilization of horseradish peroxidase (HRP) on SiO₂/BSA/Au/thionine/nafion-modified gold electrode was fabricated successfully. Firstly, nafion was dropped on the surface of the gold electrode to form a nafion film followed by chemisorption of thionine (Thi) as an electron mediator via the ion-exchange interaction between the Thi and nafion. Subsequently, the SiO₂/BSA/Au composite nanoparticles were assembled onto Thi film through the covalent bounding with the amino groups of Thi. Finally, HRP was immobilized on the SiO₂/BSA/Au composite nanoparticles due to the covalent conjugation to construct an enzyme biosensor. The surface topographies of the SiO₂/BSA/Au composite nanoparticles were investigated by using scanning electronic microscopy. The stepwise self-assemble procedure of the biosensor was further characterized by means of cyclic voltammetry and chronoamperometry. The enzyme biosensor showed high sensitivity, good stability and selectivity, a wide linear response to hydrogen peroxide (H₂O₂) in the range of 8.0 x 10⁻⁶ ~3.72 x 10⁻³ mol/L, with a detection limit of 2.0 x 10⁻⁶ mol/L. The Michaelies-Menten constant K(app)(M) value was estimated to be 2.3 mM.

Versatile Matrix for Constructing Enzyme-Based Biosensors

“…The value of K M app was calculated to 0.59 mM. The comparison of these results with that published − in Table proves that such a matrix is a sensitive platform for constructing different enzyme-based biosensors for the detection of hydrogen peroxide.…”

“…For example, a pretty wide linear working range (from 20 μM to 13.7 mM) was reported with a detection limit of 3 μM on a HRP-Au NPs/ALG/Au electrode, although the sensitivity (40.1 μA mM –1 cm –2 ) is lower than those on the CAT/nanoNiO/MWCNTs/GCE matrix and the HRP/DANN-Ag NPs/PDDA-Au NP matrix . On our matrix, the K M app value is much smaller than those reported; ,,− the linearity obtained is relatively wide (3 μM to 5.86 mM); the detection limit (LOD) is as low as 2 μM, which is lower than most of the reported results. ,, Although smaller K M app value (in other words high enzymatic affinities) was obtained, the sensitivity of our CAT/Au NPs/DMSA/Au matrix is required to be improved. In conclusion, the hydrogen peroxide sensor using the CAT/Au NPs/DMSA/Au matrix shows relatively better sensing performance than most developed sensors.…”

“…The best fitting line gives a slope of 0.0067, resulting in the value of K M app of 0.57 mM. This value is much lower than those reported, − indicating high catalytic activity of CAT on such a matrix toward the reduction of hydrogen peroxide.…”

“…Table compares the achieved results on such a matrix with those published on other matrixes regarding the linearity, limit of detection (LOD), the sensitivity for the monitoring of hydrogen peroxide, and the value of K M app . − In the literature, various types of electrodes (e.g., gold electrode, glassy carbon electrode, and platinum electrode) have been used. To immobilize enzymes for the catalytic reduction of hydrogen peroxide, those electrodes were modified or functionalized with different nanomaterials (e.g., gold nanoparticles, nano NiO, carbon nanotubes, nano Fe 3 O 4 particle, graphene, and DNA) through numerous approaches (e.g., direct coating/casting, self-assembling, and sol–gel methods).…”

“…Various nanomaterials such as metal nanoparticles, carbon nanotubes, graphene, silica nanoparticles, and other semiconductor nanoparticles (NPs) have been extensively used. − Among them, gold nanoparticles have been paid intensive attention − because of the advantages of facile control of their sizes and surfaces, easy synthesis protocols, excellent catalytic ability, and their high biocompatibility. − Different from flat planar electrodes, those matrixes constructed with self-assembled monolayers and nanomaterials always show three-dimensional structures. On them direct electron-transfer processes of redox enzymes as well as highly efficient catalytic abilities of enzymes (e.g., for electrocatalytic monitoring of small-sized biomolecules like hydrogen peroxide, and glucose) have been achieved. − Because of deeply buried redox centers inside enzymes, those activities are difficult to be achieved on flat planar electrodes. However, those three-dimensional matrixes make the orientation of enzymes possible and the denaturation of enzymes difficult, resulting in the realization of fast electron transfer of enzymes and their high electrocatalytic activities.…”