Zinc-Finger-Protein-Based Microfluidic Electrophoretic Mobility Reversal Assay for Quantitative Double-Stranded DNA Analysis

Abstract

We report for the first time a microfluidic electrophoretic mobility reversal assay (MEMRA) for double-stranded DNA (dsDNA) detection using zinc-finger proteins (ZFPs) and a polyacrylamide-gel (PAG) sieving matrix. Microfluidic DNA analysis was actively studied because of its importance in biology and medicine. Most microfluidic DNA detection techniques rely on time-consuming denaturation and hybridization processes. To address this limitation, ZFP was employed as a novel affinity probe, which directly binds to a specific sequence of dsDNA without denaturation and renaturation. A mildly alkaline electrophoresis buffer (pH 8.6) was used for our MEMRA, instead of a strongly alkaline buffer (pH 10.75) for separating the ZFP-dsDNA complex from interfering species. At pH 8.6, the mobility of ZFP was reversed upon binding with dsDNA (complex pI = similar to 5.33), and unbound ZFP (pI = similar to 9.3) was excluded from loading. Therefore, the ZFP-dsDNA complex was detected without zone interferences. Furthermore, nonspecific interactions and band dispersion, observed in strongly alkaline buffer, were effectively mitigated in the MEMRA. The ZFP-dsDNA complex was fully separated (separation resolution >= 2.0) and detected rapidly (12-15 s at a separation distance of 160-240 mu m) using on-chip photopatterned 3-16%T discontinuous PAG. The MEMRA performance was excellent, providing a detection limit of 50 pM and a detection range of 100 pM-500 nM for seb (Staphylococcus enterotoxin B) gene dsDNA oligonucleotides. We expect that our ZFP-based MEMRA will find broad utility in biology and medicine where the rapid, specific, and quantitative detection of dsDNA is of paramount importance.