A Reaction- Diffusion-Based Approach for Nucleic Acid Quantification

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Abstract Quantitative detection of nucleic acid sequences (DNA or RNA) is important in many biomedical applications including disease diagnosis, gene expression profiling. Nucleic acid amplification testing (NAAT), which takes advantage of enzymatic polymerization reaction to amplify specific nucleic acid sequences, provides high sensitivity and specificity and has become the gold standard for many infectious disease diagnostics. However, the NAAT has been severely hindered by the cost and complexity of the instrumentation for applications to point-of-care (POC) diagnostics, especially for use in resource-limited settings. The objective of the proposed research is to bridge this gap by developing a simple, affordable approach to quantify nucleic acids undergoing enzymatic polymerization reaction. To this end, we propose to study a new, reaction-diffusion-based, microfluidic method (dubbed the “nuclemeter”) for quantifying target nucleic acid molecules. The amount of target analytes in raw clinical samples can be quantitatively read out through the position of polymerization reaction-diffusion front in the nuclemeter at the endpoint, nearly as simply as reading temperature in a “mercury in glass” thermometer. As an example application, viral load testing in HIV infection will be used to evaluate and validate its clinical application. The hypothesis behind the proposed research is that the position of the enzymatic polymerization reaction-diffusion front indicates target nucleic acid concentration in samples. To test our hypothesis and demonstrate its suitability as a new, affordable, nucleic acid-based, molecular diagnostics approach, we assemble a multidisciplinary research team and propose the following specific aims: i) study a reaction-diffusion-based microfluidic device and method for endpoint quantification of nucleic acids, ii) develop a cellphone-based, label-free, bioluminescent detection platform; and iii) evaluate and validate the feasibility of clinical application of the nuclemeter for viral load testing. The proposed work is both innovative and immediately useful because it: i) introduces a novel, simple, affordable approach for nucleic acid endpoint quantification, ii) develops a minimally-instrumented, cellphone-based detection platform, and iii) proposes a new two-stage isothermal amplification assay for highly sensitive, specific, multiplex nucleic acid testing. If successful, it would open the door to affordable, mobile, personalized, molecular diagnosis and treatment. Beyond disease diagnostics, our nuclemeter system, as a technology platform of nucleic acid quantification, would also have broad applicability for many other biomedical research, such as high throughput DNA screening.