Sequence-Specific DNA Detection at 10 fM by Electromechanical Signal Transduction

Abstract

Target DNA fragments at 10 fM concentration (approximately 6 × 105 molecules) were detected against a DNA background simulating the noncomplementary genomic DNA present in real samples using a simple, PCR-free, optics-free approach based on electromechanical signal transduction. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is highly desired for a range of diverse applications. We previously described a potentially low-cost device for sequence-specific nucleic acid detection based on conductance change measurement of a pore blocked by electrophoretically mobilized bead-(peptide nucleic acid probe) conjugates upon hybridization with target nucleic acid. Here, we demonstrate the operation of our device with longer DNA targets, and we describe 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. In addition, no false positive responses were obtained with noncomplementary, 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 concentrations based on recognition of species-specific 16S rRNA sequences.

PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor

“…We concluded that the concentration detection limit of our sensor for 16S rRNA is ~10 fM. This limit is consistent with previous work with our sensor where we found a 10 fM LOD for 1613-base ssDNA, which corresponds to ~100–1000 CFU/mL (depending on the growth rate of the bacterium at the time of harvest and the corresponding rRNA levels within the cells) [ 24 , 25 ]. In the experiments reported here, we observed fewer transient blockades compared to previous studies with ssDNA detection.…”

“…Our technology is based on the observed step change reduction in ionic current through a pore when blocked by a peptide nucleic acid-functionalized bead that has acquired electrophoretic mobility upon target NA hybridization [ 24 ]. We demonstrated the operation of our device for PCR-free detection of longer DNA targets (1613 bases) with limit of detection (LOD) down to 10 fM in the presence of a high concentration of non-specific DNA to simulate the genomic background of a realistic biological application [ 25 ]. Since 16S rRNA is close in size (~1500 bases) to that detected in our previous work and it has high copy number (6700–71,000 per viable cell) [ 26 ], it may prove possible to detect 16S rRNA at a level corresponding to a low viable bacterial cell concentration (colony forming units/mL (CFU/mL)) with our device.…”

Multi-frequency impedance sensing for detection and sizing of DNA fragments

“…While highly sensitive, the precise manufacturing of nanoscale pores with high-yield and robust operation resilient to clogging and device failure still remains a challenge 38 . Instead of using nanopores, Esfandiari et al achieved sequence-specific DNA detection by investigating the blocked current of the bead hybridizing with a target DNA 39 . Previously, Sohn et al reported the use of resistive pulse sensing across microscale pores for protein detection 40 , where quantification relied on the use of target antigen binding to antibody coated colloids, and detecting changes in DC current across pores as a result of binding.…”

Sequence-Specific Detection of DNA Strands Using a Solid-State Nanopore Assisted by Microbeads

“…In this way, they could discriminate between the ‘‘probe’’ and ‘‘target’’ bound beads. Schmidt’s group [ 38 , 39 ] proposed to sequence-specifically capture DNA by PNA probes conjugated beads, which leads to the neutral beads become negatively charged. Then the target attached beads were able to be electrically driven to the sensing zone of the nanopore and cause signature pulse signals.…”