Multiplexed Digital Assays Using Color Combinatorics for Clearer Quantitation
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Solution Overview
Problem
Existing multiplexed detection technologies are limited by the number of partitions available, partition format, number of colors for analysis, high apparatus costs, and signal overlap, leading to reduced precision in quantitation and sample characterization.
Innovation Solution
Implementing systems and methods that utilize color combinatorics, stimulus-responsive probes, tandem probes, and conjugated polymer probes to increase the number of targets detected in a digital assay, achieving low occupancy of partitions and high partition numbers, enabling differential detection of up to 100 targets with improved signal-to-noise ratio and dynamic range.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional PCR multiplexing reactions use one probe per target conjugated with dyes of different excitation and emission spectra, then targets can be differentiated, but multiplexing is restricted to systems that can cope with multiple emission spectra for detection of fluorescence
Solution Approach 1:
The patent uses probes with different emission spectra (colors) to label different targets. By detecting fluorescence at multiple emission wavelengths, the system can differentiate multiple targets simultaneously. This approach enables multiplexing by varying the color emission of probes rather than requiring complex spatial or temporal separation of signals.
2Measurement precision
If partition-based systems are used for digital quantitation of multiple targets, then detection capability is improved, but the number of partitions available, partition format, and number of colors available limit the detection capacity
Solution Approach 1:
The patent extends multiplexing from a single dimension (number of partitions) to multiple dimensions by combining partitioning with multi-color fluorescence detection. Each partition can contain multiple targets labeled with different colored probes, and the system detects them by measuring fluorescence at multiple emission wavelengths. This dimensional expansion allows the number of detectable targets to exceed the number of physical partitions.
Solution Approach 2:
The patent makes each partition multi-functional by enabling it to detect multiple targets simultaneously through the use of multiple colored probes. Instead of requiring one partition per target, a single partition can serve multiple detection functions by containing targets labeled with probes of different emission spectra, which are then distinguished by wavelength-resolved fluorescence detection.
3Adaptability or versatility
If signal amplitude-based differentiation is used to achieve unique endpoints for target detection, then targets can be differentiated, but signal overlap results in reduced precision in quantitation
Solution Approach 1:
Instead of relying on signal amplitude differentiation which suffers from overlap and reduced precision, the patent uses color (emission wavelength) differentiation. Each target is labeled with a probe that emits at a distinct wavelength, allowing clear spectral separation of signals. This wavelength-based differentiation eliminates the signal overlap problems inherent in amplitude-based methods and provides superior quantitation 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 system achieves accurate and efficient multiplexed detection of up to 100 targets with a dynamic range of over 7 orders of magnitude, significantly reducing signal overlap and background noise, and allowing for high-throughput analysis with reduced infrastructure costs.
Implementation Method 1
The set of color combinatorics involves combinations of up to 3 colors, up to 4 colors, up to 5 colors, up to 6 colors, up to 7 colors, or greater (from each of the set of partitions), where each combination of colors has a corresponding target associated with the respective combination
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.