Genosensor on gold films with enzymatic electrochemical detection of a SARS virus sequence

Abstract

A hybridisation-based genosensor was designed on a 100 nm sputtered gold film. This material worked as an immobilisation and transduction surface. A 30-mer sequence that encodes a short lysine-rich region, unique to SARS (severe acute respiratory syndrome) virus, was chosen as target. A complementary strand (probe), labelled with a thiol group at the 3'-end, was immobilised on the film. After blocking the surface, hybridisation with the biotin-conjugated SARS strand (at the 3'-end) took place. Interaction with alkaline phosphatase-labelled streptavidin permits amplified indirect electrochemical detection. The analytical signal is constituted by an electrochemical process of indigo carmine, the soluble product of the enzymatic hydrolysis of 3-indoxyl phosphate. The use of a sensitive electrochemical technique such as square wave voltammetry allowed a detection limit of 6 pM to be obtained for this DNA sequence, lower than any other found in the bibliography. The parameters affecting the methodology were studied, with special attention being placed on selectivity. Specificity was clearly enhanced when interaction time and stringency (in the form of formamide percentage) were increased. With 1h of strand interaction and employing 50% of formamide in the hybridisation buffer, a 3-base mismatch strand was perfectly distinguished from the complementary.

Fig. 1

Schematic diagram of the three-electrode potentiostatic system.

Fig. 2

Assay procedures carried out for: (a) immobilisation and (b) hybridisation studies.

Fig. 3

Influence of double-labelled DNA concentration on the analytical signal when: (a) 10 μL of DNA are immobilised for 30 min at room temperature and (b) 5 μL of DNA are immobilised for 20 min at 37 °C.

Fig. 4

Influence of temperature on DNA immobilisation: C = 1 μM, V_drop = 5 μL.

Fig. 5

Signals and backgrounds obtained when: (a) albumin and (b) 1-hexanethiol are employed as blocking agents: C = 1 μM, V_drop = 5 μL, T_immob = 4 °C, t_immob = 12 h.

Fig. 6

Signals recorded for biotinylated SH-DNA, biotinylated DNA and background: C = 1 μM, V_drop = 5 μL, T_immob = 4 °C, t_immob = 12 h.

Fig. 7

Influence of temperature on the hybridisation step: C = 1 μM, V_drop SH-DNA = 5 μL, V_drop DNA = 20 μL, T_immob = 4 °C, t_immob = 12 h.

Fig. 8

Response curves for: (a) background; (b) 0.01 nM; (c) 0.10 nM; (d) 0.51 nM; (e) 1.01 nM c-DNA concentration. V_drop DNA = 20 μL, t_hybrid = 60 min, hybridisation buffer = 2× SSC + 50% formamide.