The ability to sense and digitally count individual RNA and mRNA biomarkers holds a great potential for early diagnosis of a wide range of diseases from cancer to infectious diseases, including the recent SARS-CoV-2. The ‘gold-standard’ method for RNA molecules quantification in clinical samples relies on reverse transcription, followed by a massive non-linear amplification of the DNA molecules using Reverse Transcription quantitative Real-Time Polymerase Chain Reaction (RT qRT-PCR). However, low abundance of the target RNA in the clinical sample has been shown to evade sensing, leading to false negative diagnosis, whereas excessive PCR amplification may deteriorate its specificity.
Solid-state Nanopores (ss-NPs) have recently emerged as label-free single-molecule sensing platforms for nucleic acids and proteins. To date however, the quantification of ultra-low RNAs from biological or clinical samples, have been challenging. This is attributed to the complexity associated with interpreting the signals obtained from highly heterogenous biological specimen. To address this limitation, we introduce a bio-chemical method for mRNA (or RNA) quantification using a ss-NP, coupled to a Gaussian Mixture Model data analysis.
This novel method was validated against RT qRT-PCR demonstrating that the nanopore method precedes, by roughly 5 orders of magnitude, the sensitivity of RT qRT-PCR technique. In further clinical testing, this method successfully identified plasma levels of known cancer biomarkers in pre-metastatic colorectal cancer patients.
- Fast detection of ultra-low RNA levels
- 5-times higher sensitivity relative to qRT-PCR
Applications and Opportunities
- In Vitro Diagnostics (IVD)
- Screening and early detection (e.g. through liquid biopsy)
- Molecular Diagnosis