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

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 p I = ~ 5.33), and unbound ZFP (p I = ~ 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 μm) 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.