RCA-Coupled Nanopore Counting for Salivary miRNA Detection
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Solution Overview
Problem
Current methods for diagnosing mild traumatic brain injury (mTBI) are inadequate due to delayed symptom onset, underdiagnosis, and the limitations of conventional subjective assessment techniques, as well as the challenges in detecting and differentiating salivary microRNAs (miRNAs) which are promising biomarkers, given their short length and high homogeneity.
Innovation Solution
A method using rolling circle amplification (RCA)-coupled resistive pulse counting with large solid-state pore structures to count mTBI-related salivary miRNAs, employing padlock probes for specific targeting and elongation of miRNAs into long ssDNA products that can be digitally counted with high signal-to-noise ratio, enabling rapid and accurate diagnosis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional miRNA detection methods (northern blotting, RT-PCR, microarrays, NGS) are used, then miRNA profiling can be achieved, but the methods suffer from complex workflows, long turnaround time, high cost, or limited sensitivity due to short miRNA length
Solution Approach 1:
The detection process is segmented into two distinct stages: (1) Rolling Circle Amplification (RCA) to exponentially amplify the short miRNA targets into long ssDNA products, and (2) Nanopore detection to measure the amplified products. This segmentation allows each stage to be optimized independently - RCA provides sensitivity through exponential amplification while nanopore provides simple, direct detection without complex workflows.
Solution Approach 2:
The patent introduces padlock probes as intermediary molecules that specifically hybridize to the target miRNA and serve as templates for RCA. These probes act as mediators that convert the short, difficult-to-detect miRNA into long, easily-detectable ssDNA products through the RCA process, enabling sensitive detection while maintaining simplicity.
2Productivity
If rapid and accurate mTBI diagnosis is achieved using salivary miRNAs, then diagnostic speed and accuracy improve, but the short length and high homogeneity of miRNAs make detection and differentiation challenging
Solution Approach 1:
The patent fundamentally changes the physical parameter of the target molecule by converting short miRNA (20-25 nucleotides) into long ssDNA products (kilobases) through RCA. This parameter change from short to long molecules makes the targets easily detectable by nanopore sensors while preserving the original miRNA sequence information for specific differentiation.
Solution Approach 2:
The padlock probes are designed with predetermined sequences that complementarily bind to specific miRNA targets before the RCA process. This preliminary specific binding ensures that only the correct miRNA targets are amplified, enabling accurate differentiation of highly homologous miRNAs before the amplification process begins.
3Reliability
If blood biomarkers are used for mTBI diagnosis, then objective diagnosis can be achieved, but the biomarkers are present at low concentrations, susceptible to degradation, and may have difficulty crossing the blood-brain barrier
Solution Approach 1:
The RCA process is self-amplifying and exponentially generates millions of copies from a single miRNA template molecule. This self-service amplification mechanism enables detection of extremely low concentrations of miRNA in saliva without requiring large initial amounts of biomarker, overcoming the low concentration problem of blood-based biomarkers.
4Reliability
If neuroimaging and electrophysiology are used for mTBI diagnosis, then objective diagnosis can be achieved, but expensive equipment and specialist interpretation are required, precluding point-of-care testing
Solution Approach 1:
The patent replaces complex mechanical and interpretive systems (neuroimaging machines requiring specialist interpretation) with a simpler biochemical system based on RCA and nanopore detection. The nanopore device provides direct electrical measurement of miRNA molecules, eliminating the need for expensive equipment and expert interpretation while maintaining diagnostic objectivity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for rapid, accurate, and cost-effective mTBI diagnosis by effectively differentiating miRNA concentrations and enabling multiplexed profiling, overcoming the limitations of existing technologies in sensitivity and specificity.
Implementation Method 1
The method relies on the linear and specific elongation of the miRNA to a much larger RCA product
Implementation Method 2
RCA-coupled resistive pulse counting platform for profiling mTBI-related miRNAs
Data Source
AI summary
A method of counting target salivary miRNAs related to mild traumatic brain injury (mTBI) includes the steps of binding the miRNAs to padlock probes specific to each miRNA forming a hybridized complex for each miRNA, ligating the hybridized complex forming a closed circular structure, elongating the hybridized complex producing an elongated ssDNA amplicon via rolling circle amplification (RCA) elongation, measuring the concentration of elongated ssDNA amplicon according to a translocation event rate using a solid-state pore structure with a diameter greater than 10 nm, and determining initial concentrations of miRNAs based on the quantity of the initial miRNA molecule which is linear with the concentration of elongated ssDNA amplicon.