Amplification-Free Detection of 16S rRNA for Next Generation Point-of-Care Diagnostics

The ongoing COVID-19 global pandemic has highlighted the need for point-of-care (POC) testing to monitor public health threats and to provide timely healthcare to patients. POC testing involves performing a diagnostic test that produces a rapid and reliable result outside the laboratory at the point of first contact between patients and healthcare professionals. In previous work, our group successfully demonstrated a novel nucleic acid (NA) sensing method based on simple pore blockage. In our approach, uncharged peptide nucleic acid (PNA) probes are conjugated to carboxyl-functionalized microspheres to form nearly neutral complexes that do not exhibit electrophoretic movement in an electric field. When the probe-bead conjugates capture the target NA, they gain negative charge and therefore become mobile in the presence of an electric field. If the probe-bead conjugate with hybridized target NA is directed to a smaller diameter pore in a thin glass membrane, it will at least partially block it resulting in a sustained drop in ionic current, which serves as the detection signal. We achieved a limit of detection of 1 aM (10-18 M) E. coli 16S rRNA with this simple scheme, yet this approach required laboratory-based manipulations and a turnaround time of 10 hours. In this dissertation, we present our work to reduce the total assay time and complexity so that this technology can meet the criteria for POC testing.To reduce the lengthy sampling time, alkaline lysis followed by simple filtration was explored to accelerate the NA isolation process. In addition, kinetically enhanced hybridization was accomplished by passing NA samples through a compact bed of charge neutral peptide nucleic acid (PNA) capture probes conjugated to submicron polystyrene beads. With these two improvements, we shortened the lab-based process time to 30 minutes and achieved a limit of detection (LOD) of 100 zM (10-19 M) E. coli 16S rRNA. However, work with E. coli spiked in sterile, pooled human urine suggested that a subsequent cleanup is needed for alkaline extraction in complex media. Rapid commercial RNA extraction kits therefore were used to achieve more consistent results in later work. To evaluate the capability of our technology to detect an important pathogen in complex media, kinetically enhanced hybridization was used to capture the 16S rRNA of Neisseria gonorrhoeae spiked in human urine. Based on 44 test runs, the ability to detect N. gonorrhoeae over the range of 10 to 100 CFU/mL spiked in human urine was demonstrated successfully with sensitivity and specificity of ~98% and ~100%, respectively. No false positives were observed for the control group of representative background flora at 1000 CFU/mL. To further improve our technology for POC applications, we integrated the nanopore detector with the lateral flow assay (LFA) format. In this approach, an extracted NA sample quickly flows along the LFA membrane by capillary action and hybridizes with preloaded PNA-bead conjugates. The resulting conjugates that are hybridized with negatively charged target RNA therefore move toward and block the smaller glass nanopore under the influence of an external electric field. The detection of 10 aM E. coli 16S rRNA against 10 fM P. putida 16S rRNA within 15 minutes has been successfully demonstrated. Finally, our LFA format device rapidly detected E. coli at 10 CFU/mL against a one-million-fold background of viable P. putida. With further improvements in sensitivity and reliability, this simple, rapid, and inexpensive amplification-free technology may be promising for widespread diagnostic usage in defense of public health