A Study on DNA Loop Formation Dynamics Using Surface Plasmon Resonance (SPR)

Surface plasmon resonance (SPR) is a well-established optical technique for studying DNA-DNA binding (hybridization) in the fields of molecular biology and biomedical sensing. With the emergence of high performance protein-to-DNA switches as electrochemical biosensors, the importance of understanding DNA binding dynamics at gold surfaces has significantly increased. One such method, the electrochemical proximity assay (ECPA), has been shown capable of direct protein quantitation as low as 20 fM. We report here the SPR study of a DNA loop model of ECPA that mimics probe/target binding with a DNA loop. In the assay, the loop-like structure holds methylene blue (MB) in close proximity with the gold surface when target is present, ensuring efficient electron transfer. Investigations on hybridization dynamics were carried out by immobilizing thiolated capture DNA onto the gold surface of the SPR sensor chip to form self-assembled monolayer. DNA loop oligonucleotides and MB-DNA sequences were alternately hybridized to the complementary strands to form a loop-like structure. Sensogram results showed increase in refractive angle (RA) in real time (flow-mode) analysis of binding sequence of the DNA complementarities. In order to verify this binding recognition event, one of the counterpart DNA probe sequences (DNA loop or SH-DNA) were eliminated which showed no discernable RA signal, suggesting loss in loop structure. Further investigations on analysis of time required for thiolated DNA to self-assemble onto the gold surface and optimization of concentrations under stopped-flow (batch mode) conditions were carried out. With this proof-of-concept dynamics of the DNA loop model system, we anticipate that this study can significantly improve the speed and sensitivity of ECPA-based biomarker recognition.