Flow-through colorimetric assay for detection of nucleic acids in plasma

Abstract

A flow-through colorimetric assay for detection of nucleic acids in plasma is reported. The proposed assay features an array of four polyvinylidene fluoride (PVDF) membranes impregnated with cationic poly (3-alkoxy-4-methylthiophene) (PT) as an optical reporter. The sensing strategy is based on monitoring the changes in optical properties of PT, upon complexation with target nucleic acids in the presence and in the absence of their corresponding complementary peptide nucleic acids (PNAs). As a proof of concept, the proposed methodology is validated using two biomarkers; lung cancer associated microRNA (mir21) and hepatitis B virus DNA (HBV-DNA). The flow-through colorimetric assay enabled detection of mir21 and HBV-DNA in plasma without requiring tedious sample pre-treatment and clean up protocols. Colorimetric responses for mir21 and HBV-DNA were obtained at nanomolar concentrations over five orders of magnitudes (from 1 nM to 10 μM), with a limit of detection of ∼0.6 nM and ∼2 nM in DI water and plasma, respectively. A logic gate system was developed to utilize the colorimetric assay responses as inputs for discrimination of mir21 and HBV-DNA and subsequently to obtain a profile of nucleic acids in samples that exceed respective clinical threshold limits, thereby enabling rapid and point of care (POC) disease diagnosis. Furthermore, the proposed methodology can be utilized for detection of a large number of nucleic acids in plasma by extending the array of PT impregnated membranes incorporated with their corresponding complementary PNAs.