Microfabricated Disposable DNA Sensors Based on Enzymatic Amplification Electrochemical Detection

Abstract

A new electrochemical biosensing method was developed based on the immobilization of DNA probes on photolithographic gold film electrodes by self‐assembled monolayer technique. The complementary sequence was hybridized to the modified electrode and the hybridization reaction was carried out at 37 °C for 1 h. Avidin‐akaline phosphatase was coupled to the hybrid through avidin‐biotin complex and hydrolyzed to produce an electroactive α‐naphthol, which could be detected by different pulse voltammetry (DPV). Numerous factors including the concentration of the substrate and incubation time are optimized to maximize the sensitivity and speed in the assay time. The sensors were employed to detect PCR products relative to the hepatitis B plasmid. The method has a linear calibration range of 1.5 nmol/L–50 nmol/L and the detection limit is 30 fmol.

Carbon and gold electrodes as electrochemical transducers for DNA hybridisation sensors

“…A large number of uniform gold electrodes is required for the practical evaluation of real samples. The availability of disposable printed (Umek et al, 2001) and photolithographed (Hashimoto et al, 1998;Xu et al, 2001) gold electrodes greatly facilitates biosensing protocols. As in the case of disposable carbon strips, disposable gold electrodes also obviate the need for a regeneration step so that their future use for decentralised genetic testing can be easily envisioned.…”

“…The most widely used is a method based on the cyclic voltammetric measurement of the [Fe(CN) 6 ] 3− /[Fe(CN) 6 ] 4− faradic response (Hashimoto et al, 1994b;Steel et al, 1998;Kelley and Barton, 1997;Xu et al, 2001;Nakayama et al, 2002). The peak currents of the redox couple decreased and the peak-to-peak separation ( E p ) increased when probe-modified electrodes were used and compared with bare electrodes.…”

“…However, the use of different hybridisation accelerators, covalently-modified oligonucleotides or new strategies to detect single-point mutations are highlighted below. Extreme hybridisation conditions were described by Xu et al (2001). Besides high ionic strength hybridisation buffer (e.g.…”

“…Several authors have reported the electrochemical analysis of PCR-amplified real samples (Hashimoto et al, 1998;Miyahara et al, 2002;Umek et al, 2001;Xu et al, 2001). Willner and co-workers even reported the direct detection of specific genomic sequences with no PCR pre-amplification (Patolsky et al, 2001).…”

“…A biosensing method, based on the enzyme-amplified detection of hybridisation reaction, has been reported by Xu et al (2001). Probe-modified gold electrodes were first hybridised to a biotinylated complementary sequence.…”