Full Paper Attachment of Ferrocene-Capped Gold Nanoparticle/Streptavidin Conjugates onto Electrode Surfaces Covered with Biotinylated Biomolecules for Enhanced Voltammetric Analysis Alfred J. Baca, a Feimeng Zhou, a,b * Jun Wang, a Jingbo Hu, c Jinhua Li, c Jianxiu Wang, b Zarui S. Chikneyan a a Department of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, California 90032; *e-mail: fzhou@calstatela.edu b Department of Chemistry, Graduate School, Chinese Academy of Sciences, Beijing 100039, P.R. China c Department of Chemistry, Beijing Normal University, Beijing 100875, P.R. China Received: June 26, 2003 Final version: August 13, 2003 Abstract Ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates were attached onto electrodes covered with either biotinylated oligodeoxynucleotides (ODNs) duplexes or N-biotinoyl-N'-(6-maleimidedohexanoyl)hydrazide that had been preadsorbed onto the reduced form of glutathione (GSH) immobilized onto a dithiobissuccinmidyl propionate self-assembled monolayer (SAM). Effects of the DNA probe and target strand lengths on the voltammetric behavior of the Fc groups residing at the nanoparticle conjugates were studied. The voltammetric behavior was also compared to that arising from the Fc groups present at the end of the ODN duplex in a sandwich complex (Wang et al. Anal. Chem. 2003, 75, 3941). It was found that shorter duplexes would lead to a more reversible voltammetric wave. However, the formation of duplexes with a greater number of base pairs will result in a lower detection level. For the analysis of a biotinylated 30mer target at a mixed SAM of a 30mer probe and hexanethiol, a concentration level as low as 0.25 pM could be detected (which is even lower than our previously reported detection level of 2 pM for an ODN sandwich complex). The detection level for GSH (1 nM) was found to be comparable to those of many other analytical techniques. The capability of our method for trace analysis of biotinylated biomolecules can be attributed to the enhanced voltammetric detection by the large number of Fc moieties present at each conjugate. The method described herein is simple, reproducible, versatile, and selective, and does not require sophisticated analytical instrumentation. Keywords: Ferrocene-capped gold nanoparticles, DNA analysis, Voltammetry 1. Introduction The incorporation of nanoparticles in nucleic acid analyses has resulted in improvements of the analytical ™figures of merit∫ofanumberoftechniques[1±14].Forexample,gold nanoparticles capped with thiol-modified oligodeoxynu- cleotides (ODNs) allowed Mirkin and coworkers to detect single-base mismatch at a sensitive level [15]. Willner and co-workers have recently showed that gold nanoparticles can serve as tiny electronic plugs to connect enzymes to the electrode surface for biocatalytical assays [16]. Gold nano- particles as tags have been employed to enhance the small mass increase associated with the adsorption of DNA molecules at a quartz crystal surface [8, 11, 12] and to amplify the weak signals of surface plasmon resonance [13] given rise by DNA hybridization at the metal/solution interface. Recently Wang and co-workers have amplified the voltammetric detection of nucleic acids by employing polystyrene microspheres loaded with ferrocene (Fc) for chronopotentiometric DNA assays [4]. Silver staining (deposition) onto thiolated ODN-capped gold nanoparti- cles or gold nanoparticle/streptavidin conjugates has im- proved the scanometric [9], calorimetric [10], and scanning electrochemical microscopic [17] imaging of DNA hybrid- ization reactions. We recently reported the synthesis and use of Fc-capped gold nanoparticle/streptavidin conjugates for the analysis of ODN and polynucleotide targets in the form of sandwich complexes[18].Theremarkabledetectionlimit(10 amolfor the analysis of a 39mer DNA) has been attributed to the amplification of the voltammetric signal by the large number of Fc moieties present on the conjugates (ca. 127 Fc moieties per gold nanoparticle). However, the factors affecting the voltammetric behavior of faradaic currents from the oxidation of the Fc groups on the conjugates were not elucidated. In this work, we carried out the direct hybridization between a surface-confined ODN probe and a biotinylated target and detected such a surface reaction with the aid of the Fc-capped gold nano- particle/streptavidin conjugates. Because non-sandwich DNA complexes are used, the procedure is simpler and can provide more direct means to probe the relationship between the surface parameters (e.g., probe length and surface density) and the observed voltammetric behavior. By varying the probe and target lengths and the number of basepairsintheduplexesandcomparingtheresultstothose 73 Electroanalysis 2004, 16, No. 1-2 ¹ 2004 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim DOI: 10.1002/elan.200302920