Polymer Journal, Vol. 36, No. 12, pp. 937—942 (2004) Synthesis of Polythiophene Derivatives and Their Application for Electrochemical DNA Sensor Seung Ku KANG,1 Ji-Heung KIM,2 Jeongho AN,1 Eun Kyu LEE,3 Junhoe CHA,4 y Geunbae LIM,4 Yong Soon PARK,5 and Dong June CHUNG1; 1Department of Polymer Science and Engineering, Sungkyunkwan University, Suwon, 440-746, Korea 2Department of Chemical Engineering, Sungkyunkwan University, Suwon, 440-746, Korea 3Department of Chemical Engineering, Hanyang University, Ansan, 425-791, Korea 4Biochip Project Team, MEMS Lab., Samsung Advanced Institute of Technology, P.O. Box 111, Suwon, Korea 5Department of Medical Information Engineering, Kwangju Health College, Kwangju, 506-701, Korea (Received June 10, 2004; Accepted September 24, 2004; Published December 15, 2004) ABSTRACT: In this study, we synthesized thiophene derivatives by the protection of the carboxyl group of 3-thi- ophene acetic acid(monomer) with different substituted benzyl groups for hybridized electrochemical DNA sensors composed of a conductive polymer and oligo-DNA (ODN). In general, while 3-thiophene acetic acid is not suitable for direct electro-polymerization because of the electron withdrawing effect of the –COOH group in above monomer, our synthesized thiophene monomer derivatives are easily electro-oxidized to form stable electro-active polymer films by the protection of –COOH group. These polymer films showed the specific electrochemical characteristics of poly 3-alkylthiophenes with a reversible redox transition in the range of 0.8–1.6 V in CV (cyclic voltammogram) measure- ment. Biological recognition was monitored by comparison with the CV signal from the hybridization of single and double strands of ODN. The oxidation current of double strand ODN was less than that of the single strand, which cor- responds to a decrease in electrochemical activity of the conducting polymer with an increase in stiffness of the side group of the electroactive polymer. [DOI 10.1295/polymj.36.937] KEY WORDS Biosensor / DNA Chip / Electrochemical Polymerization / Conductive Polymer / Polythiophene / In recent years, some studies on biosensors used for containing a functional group on electrode, the func- monitoring and diagnostic tests of disease from trans- tional group on the monomer must be protected by migration, virus, and bacterial infections have focused an activating agent (pentafluoro phenol, 4-chloroben- on the detection of unique signals from body compo- zyl alcohol and N-hydroxy phthalimide), because nu- nents such as blood-corpuscles, blood plasma, tissue cleophilic attack by monomeric functional groups such cells, and metabolites. Particularly, there has been as hydroxyl (–OH) and carboxyl (–COOH) to radical concentrated interest in the development of sequential cations of monomeric intermediate inhibit the electro detection methods by electrochemical sensors due to polymerization reaction.6,7 Therefore, protecting and the potential for fabricating small and economical deprotecting the functional group is required in second sensing chips.1 For the detection of an electrochemical step for direct electro polymerization.8 In third step, signal, it is essential to measure different electrical after electro-polymerization, an active biomolecule signals from a conducting polymer with recognition (oligo-DNA) for target molecule recognition is immo- molecules immobilized on electroactive films before bilized on the conductive polymer surfaces. and after hybridization with target molecules. Until We also investigated biological recognition precur- recently, when fabricating biosensors through active sor behavior by the electrochemical signal change biomolecules such as DNA and enzymes, such biomo- evaluation originating from hybridization of the probe lecules were entrapped (rather than directly bonded) oligonucleotide and the target molecule. onto a conducting polymer.2,3 Recently, however, the novel tool of immobilization for active biomole- EXPERIMENT cules on a synthesized conducting polymer surface through covalent bond has been reported.4,5 On the ba- Materials sis of these previous studies, our devices were fabri- 3-Thiophene acetic acid, pentafluorophenol, 4- cated through three stages, monomer modification, chlorobenzyl alcohol, N-hydroxyphthalimide, N,N0- electro-polymerization, and oligo-DNA immobiliza- dicyclohexylcarbodiimide (DCC), ferrocenyl acid tion. First, for electro-polymerization of a monomer (FeCOOH), acetonitrile (99.8% anhydrous) and di- yTo whom correspondence should be addressed (Tel: 82-31-290-7286, Fax: 82-31-292-8790, E-mail: [email protected]). 937 S. K. KANG et al. OH O R DCC/PY HO R O CH2Cl2 O arrS +- --arrS O R Electropolymerization O S n F F O H2 F -C-0-Cl -N?) F F O Pentafluorophenol 4-chlorobenzylalcohol N-hydroxy phthalimide Scheme 1. Synthesis of thiophene derivatives with different protecting groups and their electro polymerization. methyl sulfoxide (DMSO) were purchased from Al- drich Chem. Co. (Milwaukee, USA). Electrochemi- cal grade tetrabutylammonium hexafluorophosphate (TBAPF6) was purchased from Fluka Chim. Gmbh. (Buchs, Switzerland). Protease free phosphate buffer (pH 7.4), DNase, RNase and sodium chloride were purchased from Sigma Chemical Co. (St. Louis, USA). Target ODNs and amino-modified oligonucleo- 0 tide (probe-ODN; 5 -NH2-(CH2)6-(GTTCTTCTCAT- CATC)-30) were purchased from Genotech Co., Ltd. (Taejeon, Korea). All chemicals were used without any purification. To characterize the conducting polymer films, po- tentiostat (EG&G 273A, Gaithersburg, USA), FT IR (Mattson 5000, Wisconsin, USA) and 1H/13C NMR (Varian Unity Inova, 500MH, Germany) measure- Figure 1. Photograph of Pt chip electrode. ments were performed. Modification of the Monomers solvent removal under reduced pressure, the monomer The thiophene derivatives were synthesized by re- was purified by a silicagel column chromatogram action of 3-thiophene acetic acid with three kinds of using an MC or MC/Hexane mixture for eluent. benzyl alcohols using DCC as a coupling reagent ac- cording to Scheme 1.9 A reaction mixture of 3-thio- Preparation of Chip Electrodes phene acetic acid (10 mmole), benzyl alcohols (10 Pt was used for working, counter, and internal pseu- mmole) and pyridine (20 mmole) was stirred with a do-reference electrode material on a chip (Figure 1).10 magnetic bar in 50 mL methylene chloride (MC) for The area of the working electrode was 2 mm2 and that 20 min. Excess DCC was then added at 0 C. After of the counter and reference electrodes was 8 mm2 re- the reaction, the reaction mixture was kept for 20 h at spectively. The distance between the working and ref- the same temperature. The product was filtered for erence electrode was 1 mm. The overall glass chip elimination of N,N0-dicyclohexylcarbourea (DCU, by size was 2 Â 3 cm in width and length. The Pt refer- product) and the filtrate was washed with 4% HCl, ence electrode was calibrated with an Ag/AgCl stand- 8% NaHCO3, and distilled water several times respec- ard reference electrode in 0.1 M acetonitrile/FeCOOH tively, and then dehydrated using MgSO4. After the mixed solution. 938 Polym. J., Vol. 36, No. 12, 2004 Polythiophene Derivatives and Application for DNA Sensor HN GTT CTT CTC ATC ATC O R O O NH2 -ODN S n S n Scheme 2. Modification of polythiophene film surface with probe-ODN. Electro-polymerization (probe and target ODN) DNA on a hybrid chip, the The polymer films were prepared in the Pt coated change of CV current was measured before and after glass chip described above by electro polymeriza- hybridization of the target ODN. First, the Pt chip tion.11 The Pt chip was pre-cleaned in piranha solution was rinsed in acetonitrile several times to remove un- (H2O2/H2SO4, 1/3 in volume) for 10 min to remove binded target ODN. The reaction medium of the CV organic impurities, rinsed with distilled water several measurement was prepared from TBAPF6 (0.1 mol/ times, and dried in air. The reaction medium of electro L) in acetonitrile as an electrolyte (monomer free polymerization was prepared from synthesized mono- electrolyte solution). For measurement, the hybridized mer (0.1 mol/L) and TBAPF6 (0.1 mol/L) in acetoni- chip was monitored by the three electrode system un- trile as an electrolyte. During polymerization, the der a potential range of 0–1.2 V (vs. Pt electrode) and monomers were electro-polymerized by the three a scanning rate of 50 mV/s per cycle. electrode system under a potential range of 0–1.7 V (vs. Pt electrode) and a scanning rate of 50 mV/s at RESULTS AND DISCUSSION one cycle. After polymerization, the modified chip was rinsed with acetonitrile and then electro activities Chemical Modification of the Monomers were assessed in a monomer free electrolyte solution. FT IR spectroscopy clearly revealed that all modi- In this case, a potential range of 0–1.2 V (vs. Pt) and fied monomers showed disappearance of the –OH scan rate of 50 mV/s were applied. peak at 3200–3600 cmÀ1 originating from the –COOH group in the original monomers and also showed two Removal of Protecting Group and Oligo DNA Immo- new characteristic ester peaks at 1736 and 1751 cmÀ1 bilization on Film Surface from the R–COO–R0 group in the modified mono- Scheme 2 showed simultaneous chemical reactions mers. These were well defined C=O stretch peaks in for removal of the protecting group on the polymer the range of 1735–1750 cmÀ1 of normal esters instead film in the chip and introduction of ODN to the same of a C=O stretch in the range of 1700–1710 cmÀ1 polymer film. A coupling reaction between –NH2 in arising from normal carboxyl acid in the original ODN and –COOH in polymer film occurred in a so- monomer. dium bicarbonate buffer solution (pH 9.6) containing 1H NMR data of the modified monomers were as a small amount of DMSO for 1 d at room temperature. follows; in the case of pentafluorophenyl thiophene- 1 After the coupling reaction, the DNA immobilized 3-acetate ( H NMR (CDCl3): 7.42 (d, 1H, –SCHCH–), chip was rinsed with same buffer several times.