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A simple surface modification procedure is utilized to construct a multispot DNA chip for the surface plasmon resonance (SPR) imaging measurements in this investigation to observe the influence of DNA secondary structures on hybridization. To prepare a multispot DNA chip, thiolated single-stranded oligonucleotides (1μM) mixed with oligo (ethylene glycol) (OEG) alkanethiol (50μM) were immobilized onto a gold surface. The composition of mixed ssDNA/OEG has been reported. In investigating the effect of secondary structures on hybridization, complementary DNA sequences were injected into the SPR system to interact with the immobilized ssDNA/OEG spots at two different temperatures. The experimental results demonstrated the method is applicable to provide an easy and rapid way of making multispot DNA chips for the detection of DNA hybridization. The results also showed that increasing the DNA …