Sub-femtomolar detection of DNA and discrimination of mutant strands using microwell-array assisted digital enzyme-linked oligonucleotide assay

Detection methods that do not rely on the amplification of DNA but can reach sensitivity, specificity and throughput of gold standard methods, such as qPCR, have been extensively explored in recent years. Here, we present a hydrophilic-in-hydrophobic (HIH)-microwell array platform that empowers a panel of different amplification-free DNA bioassays: digital enzyme-linked oligonucleotide assay (ELONA), ligation-assisted (LA) digital ELONA and so-called 'analog' bioassays. We developed all three bioassays by using magnetic beads for capturing DNA target, followed by hybridization of enzyme-labelled detection probes and sealing of the built complexes into the femtoliter HIH microwells to achieve the fluorescent readout of single DNA molecules. With the optimized digital ELONA bioassay, we successfully detected 97 and 200 nt-long ssDNA molecules down to 68 and 92 aM, respectively, demonstrating extremely high sensitivity of the bioassay and its flexibility towards targets of different lengths. Importantly, we also proved that the same bioassay concept was suited to detect substantially higher concentrations of ssDNA (up to picomolar levels) by quantifying the total fluorescent intensity rather than counting fluorescent events for digital quantification. Finally, we advanced this concept towards LA digital ELONA capable of differentiating wildtype strands from those carrying single-point mutations even when the former were constituting only 1% of the DNA mixture and were present at 2 fM concentration. In conclusion, the developed platform showed remarkably high sensitivity, specificity and versatility for amplification-free detection of DNA and as such can be valuable for numerous applications in medical diagnostics, gene analysis, food safety and environmental monitoring.