Steric Factors Controlling the Surface Hybridization of PCR Amplified Sequences Maria Lisa Del Giallo, † Fausto Lucarelli, † Elisabetta Cosulich, ‡ Erika Pistarino, ‡ Barbara Santamaria, ‡ Giovanna Marrazza, † and Marco Mascini* ,† Department of Chemistry, University of Florence, via della Lastruccia 3, 50019 Sesto F.no, Florence, Italy, and Laboratorio di Sicurezza Alimentare ed Ambientale, Dipartimento di Chimica e Chimica Industriale, University of Genova, via Dodecanneso 31, 16100 Genova, Italy This study elucidated the hybridization behavior of surface- bound oligonucleotides to their longer PCR-amplified targets. The screen-printed gold surface of disposable electrodes was the platform onto which thiol-tethered oligonucleotides (21-mer) were immobilized by chemi- sorption. As a model case, ∼600-bp amplicons were studied. Surface hybridization was monitored by means of an enzyme-linked assay with electrochemical detection. Use of different surface-tethered probe sequences over a wide range of surface densities was explored to achieve the highest duplex yield. Both the surface coverage by the probe and its relative position on the target strand were found to control the efficiency of capture of the target sequence. Interfacial hybridization occurred with the highest efficiency for a probe coverage of ∼2.9 × 10 12 molecules/cm 2 and when the 3′ end of the amplicon was involved. An unusual (bell-shaped) response/amplicon concentration profile was additionally found. It was hy- pothesised that when the amount of solution-phase target is relatively high, random collisions make reannealing of the ∼600-bp strands favored over formation of the surface- tethered probe-amplicon complex. This paper also de- scribes a strategy to enhance the sensitivity of enzyme- linked hybridization assays. Such a strategy relies on formation, around the long target sequence, of dendritic- like structures, which could offer multiple anchoring points for the enzyme conjugate. The results shown in this work might have great significance for the practical application of hybridization to oligonucleotide chips. In the past few years, an increasing number of researchers have exploited surface-immobilized oligonucleotides 1 for a variety of applications, including drug discovery, 2 study of gene expres- sion, 3 screening of genetic material for mutations, 4 investigation of the molecular basis of infectious diseases, 5 and sequencing of particular genes of interest among complex DNA samples. 6 In contrast to solution-phase hybridization, in which the concentra- tion and the diffusion of all reactants affect the thermodynamics and the kinetics of the process, chip-based hybridization is heavily dependent on the construction of the probe layer at the surface. During hybridization, immobilized oligomers experience environ- mental conditions which significantly differ from those of analo- gous solution-phase reactions. Therefore, to elucidate how hy- bridization of such probes is influenced by nearest-neighbor interactions between immobilized strands and between immobi- lized strands and the solid surface, several investigations have been performed. Shchepinov et al. explored the use of different spacer arms to mitigate the influence of the solid support (amino-modified polypropylene) on the hybridization behavior of immobilized 12- mer probes. 7 The optimal spacer length was determined to be at least 40 atoms in length, giving an up to 150-fold increase in the yield of hybridization, as compared to nontethered probes. Surface coverage was modulated using a combination of stable and cleavable linkers, giving the highest hybridization yields for surfaces containing ∼50% of the maximum concentration of oligonucleotides. Chemisorption of 25-mer thiol-derivatized oligonucleotides onto gold substrates was extensively characterized using a number of methods, including XPS, ellipsometry, 32 P-radiolabeling, 8 neutron reflectivity, 9 and electrochemical methods. 10 The ionic strength of thiolated probe solutions was found to have a profound effect on surface coverage, with chemisorption greatly enhanced at high salt concentrations. The authors attributed this trend to minimiza- tion of intermolecular electrostatic repulsion between neighboring strands, which were efficiently shielded under the high ionic strength conditions. Precise control over surface coverage and * Corresponding author. Tel. +39 055 4573283. Fax: +39 055 4573384. E-mail: marco.mascini@unifi.it. † University of Florence. ‡ University of Genova. (1) Southern E.; Mir K.; Shchepinov M. S. Nat. Genet. 1999, 21,5-9. (2) Debouck, C.; Goodfellow, P. N. Nat. Genet. 1999, 21, 48-50. (3) Bertucci, F.; Houlgatte, R.; Nguyen, C.; Viens, P.; Jordan, B. R.; Birnbaum, D. Lancet Oncol. 2001, 2 (11), 674-682. (4) Pusztai, L.; Ayers, M.; Stec, J.; Hortoba ´gyi, G. N. Oncologist 2003, 8, 252- 258. (5) Bryant, P. A.; Venter, D.; Robins-Browne, R.; Curtis, N. Lancet Infect. Dis. 2004, 4 (2), 100-111. (6) Chee, M.; Yang, R.; Hubbell, E.; Berno, A.; Huang, X. C.; Stern, D.; Winkler, J.; Lockhart, D. J.; Morris, M. S.; Fodor, S. P. A. Science 1996, 274, 610- 614. (7) Shchepinov, M. S.; Case-Green, S. C.; Southern, E. M. Nucleic Acids Res. 1997, 25 (6), 1155-1161. (8) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916-8920. (9) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787-9792. (10) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 4670-4677. Anal. Chem. 2005, 77, 6324-6330 6324 Analytical Chemistry, Vol. 77, No. 19, October 1, 2005 10.1021/ac0506175 CCC: $30.25 © 2005 American Chemical Society Published on Web 08/27/2005