Quantitative Study of DNA Probe Density for Enhanced Biosensing Applications

The purpose of this study is to explore a biosensing method based on DNA probe quantification and its applications. A single-stranded DNA probe sequence was selected and immobilized on the surface of a screen-printed electrode through Au-S self-assembly. The experimental conditions including probe concentration, incubation time, and detection time were initially optimized. Differential pulse voltammetry (DPV) was employed to study the binding efficiency of the target sequence. Subsequently, chronocoulometry (CC) was used to investigate the probe density after incubating the probe sequence on the electrode. The CC results together with the corresponding DPV response signals showed that the highest hybridization efficiency of 22-mer DNA probe was obtained when the probe density reached 5.57×l0<sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">12</sup> molecule/cm<sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup>. In addition, it was found that the electrostatic repulsion force caused by the high-density probe was higher than that of the low-density probe on the detection signal. The proposed hypothesis was further verified by applying this quantitative method to determine the hybridization efficiency using a long DNA probe (73-mer) and a short one (20-mer). This study provides an important theoretical and experimental basis for the development of advanced biosensing methods.