Electrochemical Coding of Single-Nucleotide Polymorphisms By Monobase-Modified Gold Nanoparticles Kagan Kerman, †,‡ Masato Saito, † Yasutaka Morita, † Yuzuru Takamura, † Mehmet Ozsoz, ‡ and Eiichi Tamiya* ,† School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan, and Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, Bornova, Izmir, 35100 Turkey Rapidly increasing information about the human genome requires a fast and simple method for the detection of single-nucleotide polymorphisms (SNPs). To date, the conventional SNP detection technologies have been un- able to identify all possible SNPs and needed further development in cost, speed, and sensitivity. Here we describe a novel method to discriminate and code all possible combinations. SNPs were coded by monitoring the changes in the electrochemical signal of the mono- base-modified colloidal gold (Au) nanoparticles. First, a chitosan layer was formed on the alkanethiol self-as- sembled monolayer-modified Au nanoparticle. The mono- bases were then attached onto the chitosan-coated Au nanoparticles through their 5 ′ phosphate group via the formation of a phosphoramidate bond with the free amino groups of chitosan. The size of the surface-modified Au nanoparticle was found to be 8 .4 6 ( 1.53 nm by using atomic force microscopy. If there is a SNP in DNA and the mismatched bases are complementary to the mono- base, Au nanoparticles accumulate on the electrode surface in the presence of DNA polymerase I (Klenow fragment), thus resulting in a significant change in the Au oxide wave. In this report, monobase-modified Au nano- particles show not only the presence of a SNP, but also identify which bases are involved within the pair. Espe- cially, the identification of a transversion SNP, which contains a couple of the same pyrimidine or purine bases, is greatly simplified. A model study was performed by using a synthetic 21-base DNA probe related to tumor necrosis factor (TNF-r) along with its all possible mutant combinations. This versatile nanoparticle-based electro- chemical protocol is a promising candidate for coding all mutational changes. Current efforts in DNA-based research are focused on making use of the extensive library of the Human Genome Project to reveal the secrets of biological events. The biggest challenge in these efforts is certainly the detection of a single nucleotide polymorphism (SNP). A base change in the somatic cells may lead to an inherited or a noninherited genetic disease. The transmission of the genetic code depends entirely on the specific pairings of adenine (A) with thymine (T), and cytosine (C) with guanine (G), as first described by Watson and Crick five decades ago. 1 The base-pair mismatch types can be grouped into transition SNPs, which pair a purine with the wrong pyrimidine; and transversion SNPs, which pair either two purines or two pyrim- idines. The main drawback in SNP identification protocols is that many combinations can be formed even with the alteration of a single base. As a matter of fact, eight possible SNPs can be foreseen; such as A-C, A-A, A-G, C-C, C-T, T-T, T-G, and G-G. In the past decade, several important protocols for detecting SNPs have been described; however, these protocols cannot be applied to detect all possible SNPs listed above. Cleaving the region of the DNA, which contained SNP with a single-strand- specific DNase, such as S1 nuclease 2 or T4 endonucleoase VII, 3 was a direct approach; however, many different combinations for enzyme and reaction conditions made this protocol a difficult task. Additionally, A-A and T-T transversion SNPs were not cleaved in any mismatched strands. 3 Protein recognition of SNPs has been an attractive research field, because no enzyme cleavage is required. A protein, which had the ability to recognize and bind to a SNP was isolated from E. coli, and referred to as mutS. 4 Once the mutS bound to the SNP, the detection of this complex could be carried out by using several different analytical methods. 5 The main disadvantage of this protocol was that not all mismatches could be detected. For instance, C-C mismatches could not be recognized by mutS. 4,5 Bi et al. 6 has recently reported a protein chip based on a genetically modified mutS. The genetically modified mutS could successfully detect SNPs in PCR amplified samples. * To whom correspondence should be addressed. E-mail: tamiya@ jaist.ac.jp. † Japan Advanced Institute of Science and Technology. ‡ Ege University. (1) Watson, J. D.; Crick, F. H. C. Nature 1953 , 171, 737-738. (2) Dodgson, J. B.; Wells, R. D. Biochemistry 1977 , 16, 2374-2379. (3) Youil, R.; Kemper, B. W.; Cotton, R. G. H. Proc. Natl. Acad. Sci. U.S.A. 1995 , 92, 87-91. (4) Lishanski, A.; Ostrander, E. A.; Rine, J. Proc. Natl. Acad. Sci. U.S.A. 1994 , 91, 2674-2678. (5) Smith, J.; Modrich, P. Proc. Natl. Acad. Sci. U.S.A. 1996 , 93, 4374-4379. (6) Bi, L.-J.; Zhou, Y. F.; Zhang, J.-Y.; Zhang, Z.-P.; Xie, B.; Zhang, C.-G. Anal. Chem. 2003 , 75, 4113-4119. Anal. Chem. 2004, 76, 1877-1884 10.1021/ac0351872 CCC: $27.50 © 2004 American Chemical Society Analytical Chemistry, Vol. 76, No. 7, April 1, 2004 1877 Published on Web 03/06/2004