Friday, November 22, 2024
11/22/2024
Project Summary The goal of this project is to demonstrate feasibility of a system for the point-of-care (POC) detection of Chlamydia trachomatis (CT) in human urine samples that integrates a patented detector of pathogens based on identification of species-specific, nucleic acid (NA) sequences. The detector relies on a novel electromechanical signal transduction mechanism that enables the low-cost, optics-free and amplification-free (e.g., no PCR) detection of DNA/RNA at ultralow concentration (~10-19 M). A compelling need persists for rapid (minutes), cost effective, POC NA detection devices for infectious disease diagnostics so as to facilitate prompt, well-informed therapies and counseling and to avoid potentially harmful actions including the inappropriate prescription of antibiotics. In earlier work, we demonstrated the detection of Neisseria gonorrhoeae (NG), a CDC top-three public health threat, with sensitivity of ~98% and specificity of ~100%. Chlamydia, the most common notifiable disease in the US, is most often tested for in parallel with NG and is therefore the next most logical target for us to address. A key feature of the patented detector is the use of peptide nucleic acid (PNA) capture probes, which are uncharged polyamide analogs to NAs that share the same base chemistry. Since bead-PNA conjugates are designed to be charge neutral, they do not exhibit electrophoretic movement in the presence of a DC electric field. However, the substantial negative charge acquired upon capture of a target NA sequence makes the hybridized conjugate mobile. Electrophoresis of the bead-PNA conjugate with hybridized target NA to the mouth of a smaller diameter glass pore causes a significant increase in pore resistance, thereby resulting in a strong, sustained drop in measured ionic current. Nonspecifically bound NA is removed from the bead conjugate in the strong electric field in the pore mouth resulting in no sustained signal. Further, the opposing electroosmotic flow through the glass pore sweeps PNA-bead conjugates without hybridized target away from the pore mouth. In such a way, this simple conductometric device gives a highly selective (no observed false positives), binary response signaling the presence or absence of the target NA (and associated pathogen). This project focuses on two Aims: 1) Demonstration of the detection of CT in remnant urine samples from the UCLA Clinical Microbiology Laboratory with competitively high sensitivity (≥95%) and specificity (≥95%) and 2) Development and feasibility testing of key steps in an integrated and robust prototype device for rapid (<5 mins) bacterial detection based on the use of magnetic beads that minimizes the need for multiple chambers, pumps and valves. Successful achievement of these Aims will result in a device concept ready for prototype development that is capable of NG and CT testing with competitive accuracy and likely with significant advantages of cost, rapidity, power and robustness. Further research and development work is envisioned to expand sampling capabilities to other pathogens, both microbial and viral, and to other complex sample media including blood and sputum.
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