Development of Platforms for Sensing Plasma Low-Molecular-Weight Biothiols Using Microbial Redox Regulators

Abstract

Low-molecular-weight (LMW) biothiols, such as cysteine, homocysteine, and glutathione, play crucial roles in regulating redox homeostasis in living organisms. Due to their abilities to detoxify oxidative stress in intracellular and extracellular environments, LMW biothiols in blood serves as a potential indicator to aging and various diseases including cancer, dementia, atherosclerosis, and myocardial infarction. However, rapidly and effectively analyzing them remains challenging under the extracellular oxidative condition due to technical difficulties. Here I demonstrate newly designed platforms for rapid and cost-effective detection of plasma LMW biothiols using a Bacillus subtilis-derived organic hydroperoxide resistance regulatory (OhrRBS) protein and its operator dsDNA. Dimeric OhrR strongly binds to the dsDNA fragment with high affinity (Kd = 5 nM), whereas the OhrRBS-dsDNA complex rapidly dissociates by free LMW biothiols under the oxidation condition in the presence of organic hydroperoxide. Using this principle, three types of assay platforms were designed; affinity pulldown assay platform, DNA hairpin-based homogenous assay platform, and ion concentration polarization (ICP)-combined microfluidic assay platform. Affinity pulldown method enabled rapid determination of plasma LMW biothiols using FAM-labeled dsDNA and FLAG-tagged OhrRBS on anti-FLAG antibody-coated beads. The dsDNA element, engineered in the form of hairpin was able to more easily detect LMW biothiols without a pulldown process. ICP-coupled microfluidic assay allowed simple electrokinetic analysis based on the LMW biothiol-dependent separation of free FAM-dsDNA and complex FAM-dsDNA/OhrRB without purification process. Taking into consideration oxidized and reduced concentrations of LMW biothiols in plasma [1] [2], these methods were performed within the clinical dynamic range (≥ mM) of LMW biothiols. Compared to classical chemical probes, these methods were not perturbed by dithiothreitol (DTT) or high-molecular-weight biothiols. As a result, the concentrations of free (DTT-untreated sample) and total LMW biothiols (DTT-treated sample) were simultaneously measured in a single plasma sample. Furthermore, total plasma LMW biothiols (primarily Cys and Hcy) were relatively elevated in DLDLR mice or disease patient samples, compared to normal mice or normal human plasma samples. Owing to the rapid dissociation of OhrR/dsDNA complexes in response to LMW biothiols, those platforms are immediately suitable for monitoring rapid redox changes in plasma LMW biothiols as well as studying oxidative stress and diseases in blood.