Multiplexed Digital Assays Using Color Combinatorics and Low Occupancy
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Solution Overview
Problem
Current multiplexed detection technologies are limited by the number of targets that can be simultaneously detected, the accuracy of differential detection, and the complexity of instrumentation, particularly in 3D partitioning systems and high-partition regimes, leading to reduced precision and increased costs.
Innovation Solution
The use of color combinatorics, stimulus-responsive probes, tandem probes, and conjugated polymer probes enables the detection of a large number of targets with high accuracy and efficiency in a digital assay, utilizing a high number of partitions to achieve low occupancy and minimize signal overlap, thereby expanding the dynamic range and improving signal-to-noise ratios.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional PCR multiplexing reactions use one probe per target conjugated with dyes of different excitation and emission spectra, then multiple targets can be differentiated, but the system is restricted to detecting only as many targets as there are available emission spectra
Solution Approach 1:
The patent uses fluorophores with different emission spectra (colors) to label different targets. By detecting multiple emission spectra simultaneously, the system can differentiate and quantify multiple targets in parallel, overcoming the limitation of traditional single-color detection methods.
Solution Approach 2:
The patent employs a universal detection platform that can handle multiple fluorophores and emission spectra through a single imaging subsystem. This multi-functional approach allows the same system to detect various targets labeled with different colored probes without requiring separate detection channels for each target.
2Measurement precision
If partition-based systems use a high number of partitions for digital analyses, then detection accuracy improves, but apparatus costs and material costs increase significantly
Solution Approach 1:
The patent divides the sample into multiple partitions (e.g., droplets or wells) to enable digital PCR analysis. Each partition independently contains target molecules, allowing statistical quantitation and high-precision detection. The segmentation approach transforms a continuous sample into discrete units that can be individually analyzed.
Solution Approach 2:
The patent varies partition size, number of partitions, and occupancy rates as adjustable parameters to optimize detection accuracy while controlling costs. By tuning these parameters, the system achieves high measurement precision without requiring excessively complex apparatus configurations.
3Productivity
If the system operates in a high-occupancy regime with many targets per partition, then throughput increases, but signal overlap occurs resulting in reduced precision in quantitation
Solution Approach 1:
The patent deliberately operates in a low-occupancy regime where only a fraction of partitions contain target molecules, rather than maximizing occupancy. This partial action approach prevents signal overlap between multiple targets in the same partition, maintaining quantitation precision while still achieving adequate throughput through statistical sampling of many partitions.
Solution Approach 2:
The patent replaces physical separation of all targets with optical detection methods that can distinguish multiple fluorophores simultaneously. By using spectral unmixing and multi-color imaging, the system resolves signals from different targets even when they coexist in the same partition, substituting mechanical separation with optical discrimination.
4Quantity of substance
If multiple emission spectra are used for detection of fluorescence from different probe dyes, then more targets can be differentiated, but the detection system becomes more complex and costly
Solution Approach 1:
The patent utilizes multiple emission spectra (colors) from different fluorophores to differentiate multiple targets. The imaging subsystem captures fluorescence signals across multiple spectral channels, enabling simultaneous detection and differentiation of numerous targets based on their distinct color signatures.
Solution Approach 2:
The patent employs composite probe designs that combine multiple fluorophores or use fluorophore pairs with distinct emission spectra. These composite labeling strategies enable multiplexed detection by encoding target identity through color combinations, increasing the number of distinguishable targets without proportionally increasing system 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 allows for the detection of over 100 targets in a single assay run with high accuracy and reduced background noise, achieving a dynamic range of several orders of magnitude and significantly improving the signal-to-noise ratio, enabling efficient and cost-effective multiplexed analyses.
Implementation Method 1
detected signals correspond to a set of color combinatorics of a set of color combinatorics paired with targets of a set of targets
Data Source
AI summary
The disclosure provides compositions, methods, and systems for implementation of highly multiplexed molecular diagnostic assays involving color combinatorics, stimulus-responsive probes, tandem probes, conjugated polymer probes, and other mechanisms for increasing the number of targets that can be simultaneously detected in a digital assay. Multiplexed detection of targets is achieved in a rapid manner, with respect to sample partitioning and target detection using multiple color channels for detection. Implementation of methods described also achieve detection with significantly improved signal-to-noise ratio (SNR) values.