An investigation into the influence of secondary structures on DNA hybridization using surface plasmon resonance biosensing Fan-Ching Chien a , Jih-Shiou Liu b , Huang-Ju Su c , Li-An Kao b , Chung-Fan Chiou d , Wen-Yih Chen b , Shean-Jen Chen e, * a Institute of Optical Sciences, National Central University, Chung-Li 320, Taiwan b Department of Chemical and Material Engineering, National Central University, Chung-Li 320, Taiwan c Biomedical Engineering Center, Industrial Technology Research Institute, Hsinchu 300, Taiwan d Phalanx Biotech Inc., Hsinchu 300, Taiwan e Department of Engineering Science, National Cheng Kung University, Tainan 701, Taiwan Received 6 May 2004; in final form 12 August 2004 Abstract This study utilizes a surface plasmon resonance (SPR) biosensing and a theoretical secondary structure calculation to investigate the influence of secondary structures on the DNA hybridization. It is found that the SPR angular shifts associated with the three pairs of 60mer oligonucleotides with prominent secondary structures are lower than those observed for the two pairs of oligonucle- otides with no obvious secondary structures. It is also determined that increasing the DNA hybridization temperature from 35 to 45 °C reduces secondary structure effects. On the hybridization with mixture target oligonucleotides, the SPR results demonstrate that secondary structures interfere significantly. Ó 2004 Elsevier B.V. All rights reserved. 1. Introduction The pairing of complementary DNA sequences is commonly applied in a variety of biological techniques. Methods such as Southern hybridization and polymerase chain reaction are based on the pairing rela- tionships of DNA sequences. Recently, a simple high- throughput DNA hybridization method has been developed in which the reaction occurs on a solid sup- port surface containing the immobilized probe se- quences. This so-called microarray process [1] enables the simultaneous capture of a large quantity of informa- tion and is commonly applied in the detection of gene expressions [2,3]. In an ideal hybridization process, the single-stranded DNAs (ssDNA) will pair with the com- plementary probe sequences on the solid support. Under these conditions, the intensity of the measurement signal can then be translated directly into the relative quanti- ties of paired DNA. However, in practice, ssDNA olig- omers possessing self-complementary sequences may form stem-loop (i.e., hairpin) or duplex secondary struc- tures. DNA hairpin or duplex structures have been found to exhibit a relative stability and to interfere with the normal hybridization process [4,5]. Specifically, the kinetics of the normal duplex (probe–target) formation are influenced by these secondary structures since they reduce the overall pairing opportunities of the probe and target sequences. Therefore, it is necessary to take secondary structures in DNA targets or probe sequences into account when interpreting hybridization results. The potential for applying optical biosensing technol- ogy based on surface plasmon resonance (SPR) to the analysis of biomolecular interactions has attracted con- siderable attention [6]. In most cases, macromolecules 0009-2614/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2004.09.007 * Corresponding author. Fax: +886 6 276 6549. E-mail address: sheanjen@mail.ncku.edu.tw (S.-J. Chen). www.elsevier.com/locate/cplett Chemical Physics Letters 397 (2004) 429–434