Optimizing Tris(2-Carboxyethyl)phosphine and Mercaptohexanol Concentrations for Thiolated Oligonucleotide Immobilization on Platinum Electrodes in Microfluidic Platforms

In this study, we propose a strategy to explore the impact of the proportion of tris(2-carboxyethyl)phosphine (TCEP) and 6-mercaptohexanol (MCH) on the efficiency of oligonucleotide functionalization on PDMS microfluidic channels equipped with pairs of homemade microfabricated platinum microelectrodes. We identified an optimal concentration of these compounds that enables the effective orientation and distribution of probes, thereby facilitating subsequent target hybridization. The experiment included optimizing sample injection into microfluidic channels. We used TCEP as a reducing agent to help the DNA probes adhere to the channel electrode better. This stopped the formation of disulfide bonds during the probe immobilization step. We found the optimal TCEP/MCH mixture ratio (5 mM TCEP and 50 mM MCH), which led to a more uniform distribution and orientation of the DNA probes on the platinum electrode. These optimized conditions resulted in a more compact DNA monolayer and enhanced detection capabilities. The biosensor's performance was evaluated by the detection of the hybridization of complementary DNA sequences in the presence of equimolar Fe(CN)₆^3-/Fe(CN)₆^4-. The detection of the synthetic GP8 resistance gene is facilitated by a measurable decrease in the electron transfer rate, which is directly proportional to its concentration. Under the optimized conditions, the DNA biosensor showed excellent sensitivity (with a detection limit of 10^-17 M) and high specificity when tested against noncomplementary DNA strands.