Highly Selective and Sensitive DNA Assay Based on Electrocatalytic Oxidation of Ferrocene Bearing Zinc(II)-Cyclen Complexes with Diethylamine Muhammad J. A. Shiddiky, † Angel A. J. Torriero, †,‡ Zhanghua Zeng, †,§ Leone Spiccia,* ,†,§ and Alan M. Bond* ,†,‡ School of Chemistry, ARC Special Research Centre for Green Chemistry, and ARC Centre of Excellence for Electromaterials Science, Monash UniVersity, Clayton, Victoria 3800, Australia Received March 13, 2010; E-mail: alan.bond@sci.monash.edu.au; leone.spiccia@sci.monash.edu.au Abstract: A highly selective and sensitive electrochemical biosensor has been developed that detects DNA hybridization by employing the electrocatalytic activity of ferrocene (Fc) bearing cyclen complexes (cyclen ) 1,4,7,10-tetraazacyclododecane, Fc[Zn(cyclen)H 2 O] 2 (ClO 4 ) 4 (R1), Fc(cyclen) 2 (R2), Fc[Zn(cyclen)H 2 O](ClO 4 ) 2 (R3), and Fc(cyclen) (R4)). A sandwich-type approach, which involves hybridization of a target probe hybridized with the preimmobilized thiolated capture probe attached to a gold electrode, is employed to fabricate a DNA duplex layer. Electrochemical signals are generated by voltammetric interrogation of a Fc bearing Zn-cyclen complexes that selectively and quantitatively binds to the duplex layers through strong chelation between the cyclen complexes and particular nucleobases within the DNA sequence. Chelate formation between R1 or R3 and thymine bases leads to the perturbation of base-pair (A-T) stacking in the duplex structure, which greatly diminishes the yield of DNA-mediated charge transport and displays a marked selectivity to the presence of the target DNA sequence. Coupling the redox chemistry of the surface-bound Fc bearing Zn-cyclen complex and dimethylamine provides an electrocatalytic pathway that increases sensitivity of the assay and allows the 100 fM target DNA sequence to be detected. Excellent selectivity against even single-base sequence mismatches is achieved, and the DNA sensor is stable and reusable. Introduction The development of sensitive DNA biosensors is of critical importance in many aspects of biomedical research, including disease diagnosis, drug development, gene mapping, and DNA sequencing. 1 Such biosensors commonly rely on hybridization with DNA sequences providing a readout in the form of fluorescence, 2 chemiluminescence, 3 surface plasmon resonance, 4 quartz crystal microbalance, 5 or electrochemical 6 signals. Of these detection formats, electrochemical methods offer elegant ways for interfacing biorecognition and transduction events and represent a substantial driver to achieve selective and sensitive detection of DNA hybridization. 7 Moreover, electrochemical DNA biosensors are compatible with current miniaturization technologies employed to produce, for example, microchip- based DNA bioassays. 8 In a typical electrochemical DNA biosensor scheme, hybrid- ization of a DNA probe on a solid-phase support exploits the interactions between a target single-stranded (ss)-DNA and an immobilized complementary ss-DNA recognition element, which is often labeled with a redox molecule to produce an electrochemical signal. 1a Therefore, transduction of the DNA recognition element is of considerable interest to achieve sensitive electrochemical DNA detection. 1,6 Much attention has been focused on sensitivity enhancement based on biobarcode, 9 hydrazine label, 10 functionalized liposome, 11 arrays of gold, 12 metal/semiconductor nanoparticle label, 13 redox-active reporter † School of Chemistry. ‡ ARC Special Research Centre for Green Chemistry. § ARC Centre of Excellence for Electromaterials Science. (1) (a) Palecek, E.; Fojta, M. Anal. Chem. 2001, 73, 75A. (b) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042. (c) Park, S.-J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 1503. (d) Sassolas, A.; Leca-Bouvier, B. D.; Blum, L. J. Chem. ReV. 2008, 108, 109. (2) (a) Gerion, D.; Parak, W. J.; Williams, S. C.; Zanchet, D.; Micheel, C. M.; Alivisatos, A. P. J. Am. Chem. Soc. 2002, 124, 7070. (b) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013. (c) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016. (3) Miao, W.; Bard, A. J. Anal. Chem. 2004, 76, 5379. (4) (a) He, L.; Musick, M. D.; Nicewarner, S. R.; Salinas, F. G.; Benkovic, S. J.; Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000, 122, 9071. (b) Fang, S.; Lee, H. J.; Wark, A. W.; Corn, R. M. J. Am. Chem. Soc. 2006, 128, 14044. (5) Yao, C.; Zhu, T.; Tang, J.; Wu, R.; Chen, Q.; Chen, M.; Zhang, B.; Huang, J.; Fu, W. Biosens. Bioelectron. 2008, 23, 879. (6) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192. (7) (a) Kuhr, W. G. Nat. Biotechnol. 2000, 18, 1042. (b) Willner, I. Science 2002, 298, 2407. (8) (a) Liu, D.; Perdue, R. K.; Sun, L.; Crooks, R. M. Langmuir 2004, 20, 5905. (b) Khandurina, J.; Jacobson, S. C.; Waters, L. C.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1999, 71, 1815. (c) Shiddiky, M. J. A.; Shim, Y. -B. Anal. Chem. 2007, 79, 3724. (9) (a) Stoeva, S. K.; Lee, J. -S.; Thaxton, C. S.; Mirkin, C. A. Angew. Chem., Int. Ed. 2006, 45, 3303. (b) He, P.; Shen, L.; Cao, Y.; Li, D. Anal. Chem. 2007, 79, 8024. (10) (a) Shiddiky, M. J. A.; Rahman, M. A.; Shim, Y. -B. Anal. Chem. 2007, 79, 6886. (b) Shiddiky, M. J. A.; Rahman, M. A.; Cheol, C. S.; Shim, Y.-B. Anal. Biochem. 2008, 379, 170. (11) Patolsky, F.; Lichtenstein, A.; Willner, I. Angew. Chem., Int. Ed. 2000, 39, 940. Published on Web 07/02/2010 10.1021/ja1021365  2010 American Chemical Society J. AM. CHEM. SOC. 2010, 132, 10053–10063 9 10053