A Solid-State Nanopore miRNA Quantification Technology

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ABSTRACT Micro-RNAs are key gene expression regulators with great potential as biomarkers for cancer diagnosis, prognosis, and therapeutics. There is a great demand for accurate miRNA profiling in cancer research and in the clinics in the near future. However, the current technologies cannot perform this very challenging task with precision, ease, low cost, and high throughput. All main detection technologies, real-time reverse transcription PCR (qPCR), microarray hybridization, and next-generation sequencing (NGS) face challenges in mRNA profiling. Since different detection technologies, protocols and miRNA extraction and purification methods can lead to different results, interpretation of differential expression data and comparisons across different studies is tenuous. A technology capable of counting individual molecules of native miRNA in a complex sample would be much preferred. The overarching goal of this research is to develop a novel set of solid-state polymeric nanopore technologies for fast (1 hour), sensitive (100 molecules/sample) and quantitative measurement of a small set of miRNAs differentially expressed in cancer patients. This proposal will focus on its preliminary development and testing with non-clinical samples. Raw biological samples will be loaded in an integrated platform, which will perform sample cleanup and miRNA extraction and detection all in one-chip, for minimization of sample loss and protocol-dependent biases. In Specific Aim 1, we will combine multi- and single- pore solid-state technologies with dielectric thin film nanopore coatings and a newly discovered nanopore Ohmic heating phenomenon, to develop a miRNA “capture, release and count” strategy for concentration of a specific miRNA from a complex sample. In Specific Aim 2, we will integrate nanopore and ionic-membrane technologies into a platform for rapid detection of a small panel of miRNAS in raw cell-culture and/or tissue lysate samples. This design will then be expanded to oral cancer- related miRNAs by integrating several nanopore units in a multitarget detection platform. In Specific Aim 3 we will compare results of miRNA measurements in cell culture and/or tissue lysate using different sample preparation methods (the ionic-membrane chip of SA3a, and commercially available kits for miRNA extraction) in conjunction with the proposed nanopore platform and other reference methods (qPCR and NGS). Our long-term goal is to develop a robust, low cost, portable multi-disease profiling platform, capable of accurately quantifying a large miRNAs in complex samples, for research and clinical applications in cancer. Such platform would significantly speed up the process from biomarker discovery, validation, and regulatory approval, to translation into clinical setting.