Multiplexed Digital Target Quantitation With Color-Combinatorial Probes
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Solution Overview
Problem
Existing systems for multiplexed detection and quantitation of targets are limited by the number of targets that can be detected simultaneously, the mechanism of differential detection, the accuracy of partitioning technologies, and the high costs and complexity of instrumentation.
Innovation Solution
The use of color combinatorics, stimulus-responsive probes, tandem probes, and conjugated polymer probes to increase the number of targets that can be simultaneously detected in a digital assay, achieving high multiplexing capability with low occupancy of partitions and high dynamic range.
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 detected, but the system is limited by the number of colors available for detection and signal overlap occurs
Solution Approach 1:
The invention segments the detection space by partitioning the sample into many individual partitions (droplets or wells), with each partition containing at most one target molecule. This segmentation eliminates signal overlap between different targets and enables precise quantitation by counting positive partitions, resolving the contradiction between detecting multiple targets and maintaining measurement precision.
Solution Approach 2:
The invention transitions from detecting multiple targets in a single bulk reaction (one-dimensional detection) to distributing targets across many individual partitions (adding the dimension of partition index). This dimensional expansion allows simultaneous detection of multiple targets without signal overlap, as each partition's signal is independently measured and counted.
2Quantity of substance
If partition-based systems use a high number of partitions for digital analyses, then more targets can be detected, but the device complexity and instrumentation costs increase
Solution Approach 1:
The invention uses simple, identical partition templates (droplets or wells) that can be mass-produced through straightforward microfluidic generation or plate fabrication. Each partition is a copy of the same basic structure, eliminating the need for complex, customized instrumentation and reducing device complexity while enabling large numbers of partitions.
Solution Approach 2:
The invention employs universal reagents and detection protocols that work across all partitions simultaneously. The same primers, probes, and thermal cycling conditions apply to every partition, eliminating the need for target-specific customization and reducing instrumentation complexity while supporting high-throughput multiplexed detection.
3Productivity
If traditional approaches operate in a high-occupancy regime, then fewer partitions are needed, but statistical error correction factors are required and quantitation precision is reduced
Solution Approach 1:
The invention ensures that each individual partition has low occupancy (containing zero or one target molecule with high probability), creating locally optimal detection conditions in each partition. This local quality control eliminates the need for statistical error correction and enables direct, precise quantitation by simply counting positive partitions, while the aggregate data from many partitions maintains high productivity.
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 the detection and quantitation of a large number of targets with high accuracy and efficiency, reducing signal overlap and improving signal-to-noise ratio, while minimizing instrumentation complexity and cost.
Implementation Method 1
a first fluorophore-labeled oligonucleotide corresponding to the first flanking sequence, the first fluorophore-labeled oligonucleotide comprising a first fluorophore configured to transmit a first target signal if the target region is amplified
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.