Hexagonal Reaction Site Substrate for High-Density dPCR
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Solution Overview
Problem
Current optical systems for biological and biochemical reactions face challenges in accurately monitoring and analyzing high-density sample formats with small sample volumes, such as nanoliters or picoliters, particularly in digital PCR (dPCR) and real-time PCR (qPCR) processes, where efficient data collection and rare allele detection are hindered by residual fluid left on surfaces and optical limitations.
Innovation Solution
The use of a substrate with a high density of hexagonal reaction sites, which reduces residual fluid and enhances loading efficiency by minimizing the spacing between reaction sites, combined with an optical system capable of simultaneous imaging and analysis of a large number of reaction sites, allowing for accurate detection and quantification of target nucleotide sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the number of reaction sites is increased to process more samples simultaneously, then productivity increases, but the sample volume per site decreases to nanoliters or picoliters making accurate measurement difficult
Solution Approach 1:
The system segments the detection process by using specialized optical components (micro-lens arrays, beam splitting optics) to divide and independently monitor multiple reaction sites simultaneously. This allows each nanoliter or picoliter sample to receive dedicated optical attention while maintaining high throughput across thousands of sites.
Solution Approach 2:
The system changes optical parameters (wavelength selection, excitation intensity, detection sensitivity) to optimize detection in small volumes. By adjusting these parameters, the system can accurately detect fluorescent signals from minimal sample volumes while maintaining the ability to process large numbers of reactions.
2Productivity
If sample volumes are reduced to nanoliters or picoliters for high-density formats, then the number of samples that can be processed increases, but residual fluid on surfaces interferes with accurate data collection
Solution Approach 1:
The system extracts and isolates the optical signal from each reaction site using focused detection optics and spatial filtering. By separating the signal collection paths and using confocal or similar focused detection methods, residual fluid signals are excluded while maintaining reliable detection of the actual reaction products in nanoliter volumes.
3Productivity
If reaction sites are placed closer together to increase density, then productivity increases, but optical systems have difficulty resolving and distinguishing individual signals
Solution Approach 1:
The system transitions from planar to three-dimensional optical processing by incorporating focused detection, confocal optics, or layered detection schemes. This dimensional approach allows closely spaced reaction sites to be optically separated along the depth axis, enabling high spatial density while maintaining signal resolution through volumetric rather than purely planar discrimination.
4Measurement precision
If the number of reaction sites is increased to accommodate digital PCR requirements, then detection sensitivity for rare alleles improves, but the complexity of the optical system increases
Solution Approach 1:
The system merges multiple optical functions (illumination, detection, filtering, and signal processing) into integrated optical modules or chip-scale components. By combining these functions, the system achieves the sensitivity required for digital PCR and rare allele detection while reducing overall system complexity through functional integration rather than separate components for each function.
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 significantly increases loading efficiency, reduces residual fluid, and enhances the accuracy of signal measurement, enabling effective analysis of a large number of small samples with improved detection of rare alleles and target molecules in high-density sample formats.
Implementation Method 1
an optical excitation beam may be used during real-time PCR (qPCR) processes to illuminate fluorescent DNA-binding dyes or fluorescent probes to produce fluorescent signals
Data Source
AI summary
A system for determining the number of target nucleotide molecules in a sample includes a sample holder, an excitation optical system, an optical sensor, and an emission optical system. The sample holder is configured to receive an article comprising at least 20,000 separate reaction sites. The excitation optical system comprises a light source configured to simultaneously illuminate the at least 20,000 separate reaction sites. The optical sensor comprises a predetermined number of pixels, the predetermined number of pixels being at least 20 times the number of separate reaction sites. The emission optical system comprises a system working distance from the sample holder, wherein the working distance is less than or equal to 60 millimeters.