TRAINING & CAREERS
2017 BESIP Project
Microfabrication and Microfluidics Unit, Biomedical Engineering and Physical Science Shared Resource
Nicole Morgan, Ph.D.
Laboratory and Project Description
Microfluidic PNA-based HIV RNA Detection System
Commercially available rapid tests for HIV-1 can designate an individual as "preliminarily reactive," but positive results from these initial tests must be confirmed with additional assays that are based on the detection of HIV RNA using PCR. The Appella lab has identified a new method to directly detect and quantify HIV RNA using Peptide Nucleic Acids (PNAs). PNAs are stable, synthetic molecules that can bind DNA and RNA with high sequence specificity, which in this application allows for highly sensitive detection of HIV RNA in patient plasma. Using an ELISA-based experimental protocol in a 96-well plate format, the optimized set of PNA probes is competitive with a standard PCR assay for HIV RNA.
Current research efforts are focused on moving the PNA-based HIV assay to a microfluidic platform, with the aim of simplifying the assay and, in the long run, developing a portable instrument to enable routine testing and immediate results in the clinic. This transition requires a collaborative interdisciplinary effort, which includes: design and fabrication of the microfluidic devices; refinement of existing protocols for the assay using the device; modification of the protocols, including the use of different reporter molecules to facilitate low-cost optical detection; and the development of a simple detector with user-friendly image-based quantification of the results.
To date, we have preliminary results showing feasibility for direct detection of RNA at the desired sensitivity levels (about 50 molecules of target RNA) using a simple PDMS-based microchannel and a commercially available inspection microscope system. Based on these results, we are working to optimize the microfluidic design, evaluate and improve the assay sensitivity and reproducibility, reduce the number of steps requiring operator intervention, refine the detection system, and optimize the image analysis algorithm. Our long term goal is to develop a low-cost, user-friendly PNA-based HIV RNA detection system that can be deployed to areas with limited access to the advanced medical centers typically found in larger urban areas.
A BESIP student working on this project should have an interest in microfluidics, molecular assays, image analysis, and biomedical instrumentation. Working closely with the interdisciplinary team, the intern will help construct microfluidic devices, create testing assays to evaluate reproducibility and sensitivity requirements, and evaluate the ability to use low cost cameras (such as a cell phone camera) as an imaging source. Based on these efforts, the student will work with the team to modify the microfluidic devices and imaging systems to fit the needs of the project.
Appella lab: Expertise in chemical synthesis of PNA, and optimization of PNA for diagnostic applications to detect RNA. Current focus is to make PNA probes to signal the presence of HIV RNA and provide quantitative read-out as an alternative to PCR-based assays for HIV viral load testing in patients.
Morgan lab: Expertise in and facilities for microfabrication, device characterization and device operation. Other collaborations include a number of devices aimed at controlling cellular environments in culture, including a 3D chemotaxis model and a bioreactor for controlled oxygen delivery to 3D cultures.
Pohida lab: Provides electrical, electronic, electro-optical, mechanical, computer, and software engineering expertise to NIH projects that require in-house technology development. Collaborations involve advanced signal transduction and data acquisition; real-time signal and image processing; control and monitoring systems (e.g., robotics and process automation); and rapid prototype development. Collaborations result in the design of first-of-a-kind biomedical/clinical research systems, instrumentation, and methodologies.