Fluorescent Probe Platform for Rapid ccf-DNA Detection
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Solution Overview
Problem
Current methods for detecting and quantifying circulating cell-free DNA (ccf-DNA) in liquid biopsies are labor-intensive, costly, and not suitable for rapid, point-of-care diagnostics, particularly for monitoring tissue/organ injury and disease progression.
Innovation Solution
A novel platform using DNA-specific fluorescent probes, such as PicoGreen, for rapid detection and quantification of ccf-DNA within extracellular vesicles, including mitochondrial-derived vesicles, without the need for mtDNA-specific primers, enabling detection in emergency or field settings.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If real-time quantitative PCR (qPCR) is used to detect ccf-DNA, then detection accuracy is improved, but labor intensity and cost increase significantly
Solution Approach 1:
The patent replaces the mechanical/chemical qPCR system with a fluorescence-based detection system. The fluorescent probe binds to ccf-DNA and emits detectable signal, eliminating the need for complex thermal cycling, primers, and extensive laboratory processing while maintaining detection accuracy
Solution Approach 2:
The fluorescent probe system is designed to automatically detect ccf-DNA without requiring manual intervention for each step of the qPCR process. The probe self-bind to target DNA and generate detectable signal, reducing labor intensity while preserving measurement precision
2Measurement precision
If real-time quantitative PCR (qPCR) is used to detect ccf-DNA, then detection accuracy is improved, but cost efficiency deteriorates
Solution Approach 1:
The patent employs disposable fluorescent probes that are inexpensive to manufacture and use. These probes can be discarded after single use, eliminating the need for expensive qPCR reagents, primers, and specialized equipment while maintaining adequate detection accuracy for clinical applications
Solution Approach 2:
By replacing the expensive qPCR mechanical system with a simpler fluorescence detection approach, the patent significantly reduces equipment costs, reagent costs, and operational expenses while preserving the essential function of accurate ccf-DNA detection
3Measurement precision
If qPCR is used for ccf-DNA detection, then primer-specific detection is achieved, but speed and scalability deteriorate
Solution Approach 1:
The fluorescent probe is pre-designed and pre-bound to the detection system, ready to immediately detect ccf-DNA upon sample introduction. This eliminates the time-consuming thermal cycling and primer annealing steps of qPCR, achieving rapid detection without sacrificing specificity
Solution Approach 2:
The patent replaces the slow, multi-step qPCR mechanical process with a rapid fluorescence-based detection system that provides real-time or near-real-time results, dramatically improving detection speed and throughput while maintaining analytical specificity through probe design
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 provides a fast, scalable, and cost-effective method for assessing tissue/organ injury severity and monitoring disease progression, with results comparable to conventional qPCR, suitable for point-of-care diagnostics.
Implementation Method 1
adding a fluorescent probe such as PicoGreen to the isolated EVs or to the liquid biopsy sample, and measuring any increase in fluorescence intensity
Data Source
AI summary
The present invention relates in general to the field of rapid detection and quantification of tissue/organ injury, and more particularly, to a novel platform for monitoring and quantification of circulating cell-free DNA including within extracellular vesicles, together with extracellular vesicles specific markers, and extracellular vesicles sizes also in combination with PCR-related technologies in biospecimens, and liquid biopsies for the assessment and prediction of severity of tissue/organ injury, monitoring of disease progression as well as the assessment of the response to therapeutic interventions.