(596c) Amplification-Free, Nucleic Acid-Based Detection of Pathogens Using the Lateral Flow Assay Format

An amplification-free, nanopore-based nucleic acid detection platform has been demonstrated for rapid, sensitive 16S rRNA sequence-specific detection of pathogenic bacteria. This new technology exhibits an ultralow RNA detection limit of ~100 zM (~10^-19 M) and has been used to assay Neisseria gonorrhoeae over a clinically important range of 10-100 CFU/mL in human urine with no false positives and with sensitivity and specificity of ~98% and ~100%, respectively. The nanopore detector has been integrated into the lateral flow assay (LFA) format for frontend sample processing, and the overall system can detect 10 fM Escherichia coli 16S rRNA in <15 minutes. In comparison, current point-of-care (POC) nucleic amplification tests (NAATs) exhibit assay times of 15-30 minutes while studies have shown that a substantial portion of the patient population is unwilling to wait for more than 20 minutes for a test result. In our system, a peptide nucleic acid (i.e., an uncharged polyamide analog to DNA) probe complementary to N. gonorrhoeae 16S rRNA, is bound covalently to microbeads. When the charge neutral bead-probe conjugates hybridize with target rRNA, a negatively charged complex is formed that exhibits mobility in an electric field. If the complex is directed to a smaller pore in a glass membrane chip, it will at least partially block it, resulting in a sustained drop in ionic current thereby signaling the presence of the target rRNA and N. gonorrhoeae. The single-use component of the system is composed of a lateral flow membrane strip with an integrated, glass chip-based nanopore detector that is preloaded with polystyrene microbeads conjugated with peptide nucleic acid (PNA) probe complementary to the target 16S rRNA. A ~250 µL sample deposited on the test strip quickly flows along the LFA membrane due to capillary action, and target rRNA hybridizes with the complementary PNA probe conjugated to the preloaded microbeads. Subsequent electromechanical detection events measured by conductimetry require low battery power and inexpensive electronics. Further, the strong electric field and an opposing electroosmotic flow at the glass pore mouth together serve as an active mechanism to avert false positives. This simple, rapid, low power and inexpensive amplification-free technology may prove promising for widespread POC use.