Digital PCR Ion Concentration Detection Method
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Solution Overview
Problem
Current digital PCR (dPCR) methods rely on fluorescent probes and light-based detection, which can lead to errors and biases, and lack sensitivity for accurate detection of nucleic acids.
Innovation Solution
A method involving fractionating a sample into multiple volumes, subjecting them to amplification conditions, detecting changes in ion concentration, and counting amplified target nucleic acids, using ion-sensitive detection and potentially combining with primers, probes, and beads on treated substrates or matrices, to determine nucleic acid quantities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent probes and light-based detection methods are used in dPCR, then nucleic acid amplification can be detected, but errors and biases increase and sensitivity decreases
Solution Approach 1:
The patent replaces the optical detection system (fluorescent probes and light-based detection) with an electrical detection system using ion-sensitive field-effect transistors (ISFETs). This substitution eliminates the errors and biases inherent in optical methods while maintaining detection capability, directly resolving the contradiction between measurement precision and reliability.
Solution Approach 2:
The patent changes the detection parameter from optical signal (fluorescence intensity) to electrical signal (ion concentration changes detected by ISFETs). This parameter change enables more reliable and accurate detection by measuring ionic changes during amplification rather than relying on fluorescent signal intensity, which is prone to errors and biases.
2Measurement precision
If sample is fractionated into many partitions for dPCR, then sensitivity of nucleic acid detection increases, but device complexity increases
Solution Approach 1:
The patent applies segmentation by dividing the sample into many small partitions (droplets or wells), each containing a limited number of target nucleic acid molecules. This segmentation enables sensitive detection through Poisson statistics while the use of ISFET sensors simplifies the overall detection system compared to traditional optical methods requiring complex imaging systems.
Solution Approach 2:
The patent uses multiple identical ISFET sensors to detect ion concentration changes in parallel across many partitions. This copying approach allows simultaneous detection in numerous partitions without requiring complex individualized detection systems for each partition, thereby maintaining sensitivity while controlling device complexity.
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 enhances the accuracy and precision of nucleic acid detection, offering improved sensitivity and reducing errors in dPCR-based analyses.
Implementation Method 1
Both PCR and dPCR carry out one reaction per single sample, dPCR also carries out a single reaction within a sample
Implementation Method 2
detecting a change in ion concentration in a sample volume wherein a target nucleic acid is present
Data Source
AI summary
A method of detection of a target nucleic acid is provided. The method includes fractionating a sample into a plurality of sample volumes wherein more than 50% of the fractions contain no more than 1 target nucleic acid molecule per sample volumes, and subjecting the plurality of sample volumes to conditions for amplification. The method further includes detecting a change in ion concentration in a sample volume wherein a target nucleic acid is present, counting the number of fractions with an amplified target nucleic acid, and determining the quantity of target nucleic acid in the sample.