A nanoporous gold-based electrochemical aptasensor for sensitive detection of cocaine

Abstract

The increasing application of aptamers in bioassays has triggered a lot of research interest for development of highly sensitive and selective sensing platforms. Herein, we report on the design of a sensitive cocaine biosensor by immobilizing the 5'-disulfide-functionalized end of an aptamer sequence on a nanoporous gold (NPG) electrode followed by the conjugation of its 3'-amino-functionalized end to 2,5-dihydroxybenzoic acid (DHBA) as the redox probe. In the presence of cocaine, the aptamer undergoes a conformational change from an open unfolded state to a closed conformation, which reduces the distance between DHBA and the electrode surface, resulting in the enhanced electron-transfer efficiency. Using square wave voltammetric method and under the optimal conditions, the cocaine aptasensor presented two linear responses in the concentration ranges between 0.05-1 and 1-35 μM, with an excellent detection limit of 21 nM. The proposed aptasensor provides a simple and low-cost method for cocaine detection with good reproducibility and accuracy. Furthermore, it could be regarded as a general model to investigate the unique function of aptamer-functionalized nanostructured electrodes to stablish highly advanced electrochemical biosensors for various target analytes of diagnostic importance.

Scheme 1. Schematic illustration of the fabrication process of the nanoporous gold-based cocaine aptasensor.

Fig. 1. Cyclic voltammograms of the flat gold electrode (a) and NPG electrode (b) at a scan rate of 50 mV s⁻¹ in 0.5 M H₂SO₄.

Fig. 2. SEM images of the flat (a) and NPG electrode at different magnifications (b and c).

Fig. 3. Cyclic voltammograms of 0.5 mM of [Fe(CN)₆]^3−/4− in 0.1 M PBS, pH 5.5, containing 0.1 M KCl at a scan rate of 50 mV s⁻¹ obtained for electrodes at different assembly steps: flat gold (a), NPG (b) and aptamer functionalized NPG electrode before (c) and after immobilization with DHBA redox probe.

Fig. 4. (a) Cyclic voltammograms of the aptamer functionalized NPG electrode in 0.02 M PBS (pH 5.5) at a scan rate of 50 mV s⁻¹ before (dashed, black line) and after DHBA immobilization (solid, red line), (b) SWV curves obtained for the proposed aptasensor in 0.02 M PBS, pH 5.5, before (dashed, red line) and after challenging with 1 μM cocaine (solid, blue line), the effect of incubation time (c), frequency (d), pulse width (e) and amplitude (f) on the SWV response of the aptasensor toward cocaine.

Fig. 5. (a) The SWV curves of the aptasensor obtained for different concentrations of cocaine in 0.02 M PBS, pH 5.5, and (b) the corresponding calibration graph of SWV response versus cocaine concentration. Inset shows the calibration graph in the concentration range of 0.05–1.00 μM (c) the selectivity of electrochemical aptasensor incubated with 10 μM cocaine in the absence and presence of 100 μM caffeine, heroine and morphine–codeine mixture.