Digital Real-Time PCR with CMOS Imaging for Wide-Range Quantification
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Solution Overview
Problem
Existing digital PCR technologies face challenges in accurately distinguishing between positive and negative partitions, particularly in low-concentration samples, leading to false positives and negatives, and have limited dynamic ranges, making it difficult to quantify nucleic acids across a wide concentration range.
Innovation Solution
A digital real-time PCR method using a cartridge with a microfluidic chamber, well array, and CMOS photosensor array to capture reaction images in real-time, allowing for the extraction of cycle threshold values and target numbers in individual partitions, thereby eliminating false positives and negatives and expanding the measurable concentration range.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If end-point digital PCR is used to simplify the measurement process, then the measurement precision deteriorates because it cannot provide real-time curves for accurate quantification
Solution Approach 1:
The system divides the measurement process into discrete PCR cycles with real-time fluorescence detection at each cycle, segmenting the continuous amplification process into measurable stages that provide both operational simplicity and quantification accuracy through cycle threshold determination
Solution Approach 2:
The system implements real-time feedback by continuously monitoring fluorescence intensity during each PCR cycle and using this feedback to determine cycle threshold values, enabling accurate quantification while maintaining automated operation
2Measurement precision
If the number of partitions is increased to improve the limit of detection for low-concentration samples, then the measurement precision deteriorates because intermediate intensity partitions increase proportionally
Solution Approach 1:
The system changes the parameter used for partition classification from arbitrary user-defined fluorescence intensity thresholds to objectively determined cycle threshold values derived from real-time fluorescence curves, maintaining reliability while improving detection sensitivity through increased partition count
Solution Approach 2:
The system replaces the mechanical/manual threshold-setting process with an automated algorithmic determination of cycle thresholds based on fluorescence curve analysis, eliminating user bias and improving consistency in partition classification
3Measurement precision
If droplet digital PCR is used to achieve high measurement precision, then the device complexity increases due to requiring multiple specialized equipment
Solution Approach 1:
The system merges the droplet generation function, PCR amplification, and fluorescence detection into a single integrated microfluidic device, eliminating the need for separate droplet generators, transfer equipment, and analysis instruments while maintaining high measurement precision
Solution Approach 2:
The microfluidic chip performs multiple functions including sample partitioning, thermal cycling for PCR amplification, and real-time fluorescence detection, allowing a single device to replace multiple specialized equipment while achieving the same measurement precision
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
The method provides real-time graphs for individual partitions, enabling accurate quantification of nucleic acids across a wide concentration range, from high to low, and reduces errors by repeatedly evaluating PCR reactions thousands of times with one cartridge.
Implementation Method 1
a CMOS photosensor array located on a bottom surface of the well array and configured to capture a reaction image of the analysis target sample filled in a plurality of partitions provided in the well array in real-time
Data Source
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AI summary
The present invention relates to a digital real-time PCR analysis method and aims to provide a digital real-time PCR method that enables digital real-time PCR analysis for samples across a wide concentration range, including both high and low concentration samples that exceed the measurement limits of conventional methods.