Surface Dynamics Study of DNA Loop Formation Using Surface Plasmon Resonance

Thiol-terminated single-stranded DNA (SH ss-DNA) monolayer assembly on gold as a probe is a significant model for various biosensors. In electrochemical proximity assay (ECPA), SH ss-DNA with complementary DNA loop moves a methylene blue (MB)-conjugated oligonucleotide (MB ss-DNA) close to a gold surface, which forms a DNA loop like structure,  for transferring electron when target protein is present. After treatment of urea in DI water, the MB ss-DNA and DNA loop dissociate from the DNA loop like structure for reusability. Surface plasmon resonance (SPR) is a powerful optical technique for studying dynamic matrix of affinity biosensors.

In this study, we first confirmed DNA complementarities in flow mode with surface plasmon resonance detection. SPR results showed discriminative refractive angle change (DRA) in each DNA hybridization. However, the loss in DNA loop structure (SH-ssDNA or DNA loop) showed inconspicuous sensogram. Furthermore, we studied the effects of free thiols, which is a result of cleavage of disulfide precursors by reductant, in SH ss-DNA monolayer assembly on gold surface. Comparison of DRA in pure SH-ss DNA and non-purified SH-DNA in several concentrations showed free thiols can compete with SH-ss DNA on gold surface. DNA loop and MB ss-DNA added to two solutions is used for double confirmation of the recognition event. Mixture of different ratios of SH-ss DNA and free thiols were used to further study the competition between SH-ss DNA and free thiols. Moreover, we investigated reusable DNA Loop formation capable of several uses in simple matrix. Urea dissolved in DI water was utilized to dissociate DNA loop and MB ss-DNA from DNA loop-like structure. Optimization of concentration and pH of urea were performed. Study dynamics of this reusable DNA loop structure can improve sensitivity and feasibility of ECPA-based marker.