Quantitative Evaluation of DNA Probe Density by Electrochemical Surface Plasmon Resonance Measurement

Electrochemical surface plasmon resonance (EC-SPR) measurement enables changes in surface refractive index induced by an electrochemical reaction to be detected. Here, we carried out EC-SPR measurement to quantitatively evaluate a DNA sensor surface, which was constructed by immobilizing thiolated DNA probes on a gold film surface by a self-assembledmonolayer technique. This is because the performance of the sensor significantly depends on the probe density. Although the probe density can be evaluated quantitatively by measuring the current (charge) induced by electrochemical reductive desorption, this method removes all DNA probes on the sensor surface, meaning that the sensor cannot be used for further DNA sensing. In contrast, we can evaluate the DNA probe density more quantitatively by SPR measurement after obtaining the relationship between the charge induced by electrochemical desorption and the SPR angle shift by EC-SPR measurement. Both the amount of charge induced by reductive desorption and the SPR angle shift increased with increasing immobilization time from 0 to 24 h, and a strong relationship between the amount of charge and the SPR angle shift was observed. Therefore, the density of immobilized DNA probes can be evaluated without destroying the surface. We used EC-SPR results to evaluate the hybridization efficiency of target DNA by comparing the angle shifts observed by probe immobilization and target hybridization.