PCR-free nucleic acid detector based on nanopore sensing

The development of a rapid, sensitive, and cost-effective nucleic acid (NA) detection platform is highly desired for a range of diverse applications. We designed and developed an optical label free, PCR independent and potentially low-cost device for sequence-specific nucleic acid detection. The detection is based on conductance change measurement of a pore blocked by electrophoretically mobilized bead-(peptide nucleic acid (PNA) probe) conjugates upon hybridization with target nucleic acid. As the target NAs hybridize to the complementary PNA-beads, the beads acquire negative charge and become electrophoretically mobile. An applied electric field guides these NA-PNA-beads toward the pore, which they obstruct, leading to an indefinite, electrically detectable blockade of the pore. In the present of noncomplementary NA, even to the level of single base mismatch, permanent pore blockade was not seen. We show application of this platform to detection of the anthrax lethal factor sequence. Next, we demonstrated the operation of our device with longer DNA targets, and we described the resulting improvement in the limit of detection (LOD). We investigated the detection of DNA oligomers of 110, 235, 419, and 1613 nucleotides at 1 pM to 1 fM and found that the LOD decreased as DNA length increased, with 419 and 1613 nucleotide oligomers detectable down to 10 fM. Also, target DNA fragments at 10 fM concentration (approximately 6 105 molecules) were detected against a DNA background simulating the non-complementary genomic DNA present in real sample. In addition, no false positive responses were obtained with non-complementary, control DNA fragments of similar length. The 1613-base DNA oligomer is similar in size to 16S rRNA, which suggests that our device may be useful for detection of pathogenic bacteria at clinically relevant concentration based on recognition of species-specific 16S rRNA sequences. To investigate that we detected the specific sequence of 16S rRNA of non pathogenic E.Coli K-12 at 10fM detection limit. Two different non-pathogenic bacteria were used as negative experimental control and the universal PNA probe complementary to all three bacteria was used as positive experimental control. We could successfully detect E.Coli K-12 16S rRNA with no false negative and only one false positive at 3.5 x 104 colony forming units (CFU). This detection limit is below the threshold concentration limit for detecting the pathogenic E.Coli in urine and therefore our device has a potential to be used for detecting the clinically significant pathogens