Rapid Electrokinetic Detection of Low-Molecular-Weight Thiols by Redox Regulatory Protein-DNA Interaction in Microfluidics

Abstract

Despite the diagnostic potential of low-molecular-weight (LMW) thiols in human diseases, their rapid and simple detection is challenging due to their reversible redox changes. Herein, we report a rapid electrokinetic detection of LMW thiols using the interaction between Bacillus subtilis-derived organic hydroperoxide resistance regulatory protein (OhrRBS) and its operator DNA element in ion concentration polarization (ICP)-coupled microfluidic multiple channels. The dimeric OhrRBS was tightly bound to the dye-labeled dsDNA element with high binding affinity (KD ≈ 4 nM) under the reduced conditions. The presence of organic hydroperoxide (OHP) and LMW thiol (X–SH) caused rapid oxidation of the reduced Cys residue (OhrRBS–SH) of the protein into the sulfenic acid form (OhrRBS–SOH) by OHP and subsequently into the disulfide form (OhrRBS–S–S–X) by LMW thiol via S-thiolation, leading to a rapid release of OhrRBS from the fluorescent dsDNA element. Based on this principle, the fluorescence zones of the dsDNA-protein complex in response to LMW thiols were rapidly separated in the microfluidic channels by electrokinetic mobility. Owing to the ability of ICP-coupled microfluidics to concentrate charged samples, this electrokinetic method enabled the rapid determination (<35 min) and improved sensitivity (>2 μM) of LMW thiols, depending on their molecular weights and concentrations. Additionally, this strategy enabled the simultaneous detection of free and total LMW thiols in mouse serum. These results suggest that the ICP-coupled microfluidic platform in combination with the OhrRBS–DNA complex will be useful for monitoring rapid redox changes of LMW thiols in real samples.