Elsevier

Biosensors and Bioelectronics

Volume 18, Issue 10, September 2003, Pages 1241-1247
Biosensors and Bioelectronics

DNA hybridization electrochemical sensor using conducting polymer

https://doi.org/10.1016/S0956-5663(03)00088-5Get rights and content

Abstract

We report the use of poly(thiophen-3-yl-acetic acid 1,3-dioxo-1,3-dihydro-isoindol-2-yl ester (PTAE) for application to electrochemical hybridization sensor. A synthetic route for the thiophen-3-yl-acetic acid 1,3-dioxo-1,3-dihydro-isoindol-2-yl ester (TAE) is described, which is used as a monomer of conducting polymer sensor. A direct chemical substitution of probe oligonucleotide to good leaving group site in the PTAE is carried out on the conducting polymer film. A biological recognition can be monitored by comparison with the electrochemical signal (cyclic voltammogram) of single and double strand state oligonucleotide. The sensitivity of the electrochemical sensor is 0.62 μA/nmole and the detection limit is 1 nmole. The oxidation current of double strand state oligonucleotide is a half of that of single strand, that is corresponding to the decrease of electrochemical activity of conducting polymer with increase of stiffness of side group of the polymer. The oxidation current decreasing ratios of perfect matched and single nucleotide mismatched samples are 52 and 25–30%, respectively. The more decreasing ratio is attributable to the more steric hindrance of single nucleotide mismatched sample.

Introduction

DNA diagnostics has become a focus of the biotechnology era (Pividori et al., 2000). Specially, DNA probe assay for the detection of specific base sequences of DNA have been enormous scope of application in biotechnology and medicine, ranging from agriculture and farming to detection of pathogens in foods to genetic diagnostics on human subjects (Hoch et al., 1996).

Currently, popular DNA sequence tests, microarrays for example, are based on fluorescence signals. The polymerase chain reaction (PCR) multiplies tiny amounts of DNA into readable quantities. Although these techniques are extremely sensitive and quantitative, they require time, sample preparation, and expansive equipment. Moreover the systems are so big that they can not be satisfied for the portable point-of-care-test (POCT) application up to now (Wilson, 1998).

DNA Lab-on-a-chips (LOC) are POCT applicable devices that comprise a biological recognition agent (probe single strand DNA, for instance) that confers selectivity, a transducer that provides sensitivity and converts the recognition event into a measurable electronic signal and a microfluidic channel that provides delivery and preparation of sample (Pividori et al., 2000, Lim and Cho, 2000, Service, 1998).

The sequence recognition events depend more on the biological components of sensors in LOC. Thus one key factor in sensor design and construction is the development of tethering technologies for stabilizing biomolecules to surfaces (Lin et al., 2000, Cho et al., 2001).

The immobilization of probe DNA molecules onto solid surfaces is enormous interest in studies of DNA diagnostics. Thus various protocols have been developed to confine oligonucleotides (ODN) and chromosomal DNA onto solid sensors (Wang and Jiang, 2000).

By virtue of possibility of miniaturization and direct detection of electrochemical signal on a noble metal electrode, electrochemical systems have been paid attention to the candidate of LOC sensors. In all electrochemical systems, DNA is bound onto a solid electrode to detect an electrochemical signal that inherently involves surfaces and interfaces of the electrodes. There are a number of examples related electrochemical sensors using conducting polymer (de Lumley-Woodyear et al., 1996, Campbell et al., 2002, Patolsky et al., 2001, Patolsky et al., 2002, Wang and Jiang, 1998, Korri-Youssoufi et al., 1997a, Korri-Youssoufi et al., 1997b, Bäuerle and Emge, 1998). de Lumley-Woodyear et al. introduced water soluble copolymer of acrylamide and vinylimidazole, modified with hydrazine and osmium complex to measure directly as an electrical current. The current flows as a result of continuous electroreduction of H2O2, electrocatalyzed by the horseradish peroxidase label of an oligonucleotide strand when the complementary strand was covalently bound to a hydrogel (de Lumley-Woodyear et al., 1996). Garnier et al. introduced functionalized polypyrrole derivatives that comprise pyrrole monomer for the electrochemical sensing and linker for the immobilization of oligonucleotide. They observed decrease of electrochemical activities due to the change of the size of side group between before and after hybridization (Livache et al., 1994, Godillot et al., 1996, Garnier et al., 1999, Korri-Youssoufi et al., 1997b). Bäuerle et al. introduced nucleobase functionalized polythiophenyl derivatives. They observed changes of absorption maximum and the decrease of the electrochemical activities as well due to the hydrogen bond formation with complementary nucleobases (Bäuerle and Emge, 1998, Emge and Bäuerle, 1997).

This paper shows the procedure used for the novel synthesis of thiophenyl monomer and electropolymerization, the specificity of the immobilization and the ability of the probe oligonucleotide linked to the polythiophenyl compound to be hybridized. We also demonstrate electrochemical phenomena on chip electrode system.

Section snippets

Materials

3-thiophene acetic acid, N-hydroxyphthalimide (NHP), N,N′-dicyclohexylcarbodiimide (DCC), ferrocenyl carboxylic acid (FeCOOH), acetonitrile (99.8%, anhydrous), chloroform, and DMSO were purchased from Aldrich. DNase, RNase, and protease none detected phosphate buffer (pH 7.4) and sodium chloride were purchased from Sigma. An electrochemical grade tetrabutylammonium hexafluorophosphate (TBAHFP) was purchased from Fluka. Target ODNs and amino-modified immobilization probes were purchased from

Characterization of patterned electrochemical chip and its electrochemical phenomena

To investigate performance and consistency of patterned chip, cyclic voltammetric (CV) measurements were performed with the chips in 0.1 M ferrocenyl carboxylic acid in acetonitrile solution (Fig. 1). Fig. 1(a) and (b) show the cyclic voltammogram of Ag/AgCl and Pt pseudo reference electrode system, respectively. It was found from the result that an electrode potential of the Pt pseudo reference electrode was 0.60 V vs. SHE.

Electropolymerization and characterization of PTAE film

The TAE can be electropolymerized in the electrolyte

Conclusion

In summary, the combination of target ODN and probe ODN with conjugated conducting polymers leads to sensitive sequence recognizable hybridization sensor. The next step, the physical and chemical conditions affecting on the sensor response, the combination of microfluidic devices for the precise control of the sample volume and the built-in hybridization sensor are under way in our laboratory. A further improvement in sensitivity and selectivity can also be expected from fully integrated

Acknowledgements

This work was supported by the Ministry of Commerce, Industry and Energy (MOCIE) of the Republic of Korea under the next generation new technology development project (00008069) through the Biochip Project Team at Samsung Advanced Institute of Technology (SAIT).

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