Multiplex PCR Chip Spatial Isolation Probe Stability
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Solution Overview
Problem
Conventional multiplex PCR chips face challenges in forming and maintaining spatially isolated probe particles, leading to difficulties in simultaneous multiplex detection due to probe droplet spreading, particle instability, and limited detectable targets, as well as interference from overlapping fluorescent dye wavelengths.
Innovation Solution
A multiplex PCR chip with spatially separated particle-forming grooves on a transparent plastic substrate, where probes with different primers and fluorescent dyes are fixed using a supporting body that forms a three-dimensional structure, and particle holders with protrusions prevent particle separation, allowing for varied probe shapes and positions for simultaneous detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If probes are fixed in a single reaction chamber using conventional spatial isolation, then multiple target nucleic acids can be detected, but probe droplets spread and particles become unstable
Solution Approach 1:
The reaction chamber is segmented into multiple distinct reaction regions, each containing isolated probe particles. This spatial segmentation prevents probe droplet spreading while maintaining the ability to detect multiple target nucleic acids simultaneously through the use of different fluorescent dyes in each region.
Solution Approach 2:
A supporting body is introduced as an intermediary structure to hold probe particles in fixed positions within the reaction chamber. This mediator prevents particle instability and maintains spatial isolation of probes during the PCR process.
2Productivity
If various fluorescent dyes are used for multiplex detection, then multiple target genes can be detected simultaneously, but wavelength overlap causes interference
Solution Approach 1:
Different reaction regions are assigned different fluorescent dyes with distinct emission wavelengths. By localizing specific dye types to specific regions and using spectral unmixing algorithms, the system resolves wavelength overlap interference while maintaining high detection efficiency for multiple targets.
3Measurement precision
If probe particles are spatially separated to prevent interference, then detection accuracy improves, but the number of detectable targets is limited
Solution Approach 1:
The system transitions from two-dimensional spatial arrangement to three-dimensional positioning of probe particles using supporting bodies. This additional dimensional freedom allows denser packing of probe particles while maintaining spatial isolation, thereby increasing the number of detectable targets without sacrificing detection accuracy.
4Adaptability or versatility
If multiple primers and fluorescent dyes are combined in probes, then multiplex detection is enabled, but probe design becomes complex
Solution Approach 1:
The supporting body serves as a universal platform that can hold multiple types of probe particles with different primer-dye combinations. This multi-functional structure simplifies probe design by providing a standardized framework that accommodates various probe configurations without requiring complex custom designs for each multiplex assay.
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
Enables efficient simultaneous amplification and detection of multiple target nucleic acids by spatially isolating probe particles, reducing interference, and facilitating multiplex detection through distinct probe positions and shapes, while ensuring all reaction chambers are filled with a single sample injection and maintaining probe stability.
Implementation Method 1
a supporting body which forms a three-dimensional structure when a physical stimulus is applied so as to fix the primers
Implementation Method 2
a DNA denaturation step, an annealing step, and a DNA extension step are repeatedly performed, wherein the DNA denaturation step is a step of separating double-stranded DNA into single-stranded DNA by heating the sample solution
Implementation Method 3
the annealing step is a step of injecting primers having sequences complementary to specific nucleotide sequences to be amplified into the sample solution after the DNA denaturation step, and hybridizing the primers to the specific nucleotide sequences of the single-stranded DNA
Implementation Method 4
the target nucleic acids may be detected by way of detecting fluorescent light generated from a fluorescent dye combined with the target nucleic acids
Data Source
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Figure 3A~3D
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AI summary
A multiplex PCR chip capable of simultaneously detecting multiple target genes and a multiplex PCR method using the same are proposed. More specifically, in the multiplex PCR chip and multiplex PCR method, after a plurality of spatially separated particle-forming grooves is formed in one or more reaction chambers and a probe in a solution state is injected into the particle-forming grooves, planar shapes of the particle-forming grooves are varied or shapes and patterns of particle holders respectively formed on inner surfaces of the particle-forming grooves are varied, and the probe including primers specifically hybridizing with sequences of different nucleic acid molecules is injected into the particle-forming grooves, whereby simultaneous multiplex detection is possible by allowing multiple target genes to be detected on the basis of positions and shapes of the probe particles and the shapes and patterns of the particle holders respectively formed inside of the probe particles.