Electrochemical Biosensor for Acetamiprid Based on Aptamer As Captured Probe and Horseradish Peroxidase for Signal Amplification

Abstract

Nowadays, people pay more attention to the environmental protection and food safety problems caused by pesticide residues. In this paper, a biosensor for sensitive detection of acetamiprid, a kind of insecticide, was fabricated based on aptamer as a captured probe of acetamiprid and horseradish peroxidase (HRP) as a biocatalyst for signal amplification. The schematic diagram of fabrication of the biosensor was shown in Fig. 1. Fig. 1 The preparation of biosensor and the detection of acetamiprid Labeled aptamer of acetamiprid and a complementary DNA strand (cDNA) of the aptamer were synthesized by Shanghai Sangon Biological Engineering Technological Co. Ltd. (China). The base sequences are as-follows:[1] aptamer: 5′-NH2-(CH2)6-TGT AAT TTG TCT GCA GCG GTT CTT GAT CGC TGA CAC CAT ATT ATG AAG A-3′; cDNA: 5′- (SH) - (CH2)6 - T CTT CAT AAT ATG GTG TCA GCG ATC AAG AAC CGC TGC AGA CAA ATT ACA -3′. A 10 mL graphene oxide (GNO) suspension of 1.0 mg/mL was uniformly coated on the surface of a glassy carbon electrode (GCE) by drop-casting and allowed it to dry naturally in the air. The modified electrode (GNO/GCE) was immersed in 0.1 mol/L PBS solution containing of 0.4 mM xanthurenic acid (Xa) and was performed cyclic voltammetry scan from 0.65 V to -1.70 V (vs.SCE) for 10 cycles. GNO as the starting material was electrochemical reduction to ERGO at cathodic potential and the electropolymerization of xanthurenic acid (PXa) was achieved at anodic potential in a simply one-step process by cyclic voltammetry (CV), simultaneously. Because of the π–π* interaction between the conjugated GNO layers and aromatic rings of Xa, GNO can serve as a strong adsorbing platform for Xa monomer and promote the electropolymerization of Xa effectively. [2] After the PXa-ERGO composite film was synthesized, the PXa-ERGO/GCE electrode was immersed in 0.1 M phosphate buffer solution (PBS) of pH 7.0 containing 5.0 mM ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and 8.0 mM N-hydroxysuccinimide (NHS) to convert the terminal carboxylic group to active NHS ester. Double-helix DNA (dsDNA) was prepared by the hybridization between aptamer and cDNA. Then the dsDNA solution was coated on the surface of the PXa-ERGO/GCE electrode and incubated for 2 hours. The rich carboxylic groups of PXa-ERGO film were applied to immobilize the dsDNA with amino groups at 5’ end of cDNA by covalent bonding. The biosensor was denoted as dsDNA/PXa-ERGO/GCE. Gold nanoparticles (AuNPs) and HRP–AuNPs conjugates were prepared. Transmission electron microscope (TEM) images and ultraviolet-visible absorption spectra were used to characterize the AuNPs. AuNPs-HRP conjugates were modified onto the dsDNA/PXa-ERGO/GCE though Au–thiol interactions. Hydroquinone (HQ) and H2O2 in solution were catalyzed by HRP to generate an anodic voltammetric peak. The aptamer biosensor was successfully applied to determine acetamiprid by differential pulse voltammetry (DPV). The analysis was based on the change in the anodic peak current (i p) before and after sample solutions were applied to the surface of the working electrode at room temperature. When the background current stabilized, the anodic peak current response was recorded as i p0. In the presence of acetamiprid, aptamers bind with acetamiprid to produce a complex on the electrode surface. The HRP–AuNP–cDNA conjugates therefore fall off from the electrode surface. The anodic peak current decreased and was recorded as i p. The change in current was given by Δi p = i p0 - i p. A 25 mL aliquot of 0.1 M PBS (pH 7.4) was placed in a voltammetric cell and the required volumes of 3.0 mM HQ and 1.5 mM H2O2 solution were added. The DPV potential scan was performed from -0.4 V to 0.6 V (vs.SCE) at pulse amplitude of 20 mV, pulse frequency of 25 Hz and increment potential of 4 mV. Under the optimal conditions, the Δi p value highly and linearly increased with the increase in the logarithm of the acetamiprid concentration (c) over a range from 0.010 to 20 μg/L, with a detection limit of 0.005 μg/L. This aptamer sensor was applied in the determination of acetamiprid in river water with high specificity, sensitivity and selectivity.