Multiplexed Nucleic Acid Detection via Microfluidic Segmentation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for high-throughput detection of nucleic acid and protein targets in samples face limitations in simultaneously detecting multiple targets across a broad range of concentrations, with existing multiplexing techniques suffering from high fluorescence background and limited dynamic range.
Innovation Solution
The method involves using target-specific probes with nucleic acid tag sequences, distributed across a microfluidic device, where each aliquot is queried for the presence of probe products using PCR primers and dual-labeled fluorogenic oligonucleotide probes, enabling the detection and quantification of multiple targets with a dynamic range of at least 4 orders of magnitude.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If multiplexing methods are used to detect multiple targets simultaneously, then the number of detectable targets increases, but the fluorescence background increases and dynamic range is limited
Solution Approach 1:
The patent segments the detection process by dividing the sample into multiple aliquots, each processed in separate microfluidic chambers. Each chamber contains specific probe sets for particular targets, allowing isolated detection reactions that minimize cross-contamination and fluorescence background interference while maintaining the ability to detect multiple targets across the entire sample set.
Solution Approach 2:
The patent introduces nucleic acid tag sequences as intermediaries between the targets and detection probes. These tags serve as amplifiable markers that can be specifically detected through PCR, separating the detection function from the target binding function and enabling sensitive detection without direct fluorescence from the targets themselves, thereby reducing background interference.
2Quantity of substance
If multiplexing methods are used to detect multiple targets simultaneously, then the number of detectable targets increases, but the dynamic range is limited
Solution Approach 1:
The patent implements dynamic detection capabilities through real-time PCR monitoring in the microfluidic chambers. The system can adaptively adjust detection parameters and process multiple aliquots with different probe sets, enabling dynamic range of at least 4 orders of magnitude while maintaining simultaneous detection of multiple targets through coordinated processing of sample aliquots.
Solution Approach 2:
The patent performs preliminary actions by pre-dividing the sample into aliquots and pre-configuring microfluidic chambers with specific probe sets before analysis. This preparation enables systematic detection across a broad dynamic range by ensuring optimal probe-target combinations are ready in advance, allowing accurate quantification from low to high concentrations without signal saturation or loss.
3Measurement precision
If target-specific probes with nucleic acid tags are used, then detection specificity improves, but assay complexity increases
Solution Approach 1:
The patent creates universal detection capability through the use of common nucleic acid tag sequences that can be amplified by standardized PCR primers across different probe sets. This universal tagging system allows the same detection machinery to identify multiple different targets, reducing the need for target-specific detection reagents and simplifying the overall assay workflow despite the multiplexed nature of the analysis.
Solution Approach 2:
The patent replaces complex mechanical separation and detection systems with a biochemical approach using nucleic acid hybridization and PCR amplification. The microfluidic device facilitates this substitution by providing controlled reaction environments where probe-target binding and subsequent PCR amplification occur automatically, reducing mechanical complexity while maintaining high specificity through molecular recognition mechanisms.
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 simultaneous detection and quantification of numerous targets with high specificity and accuracy, overcoming the limitations of existing multiplexing techniques by providing a broader dynamic range and improved sensitivity.
Implementation Method 1
combining target-specific probes with the sample under conditions in which the probe binds a target, if the target is present in the sample
Implementation Method 2
the tag sequence is amplified using PCR
Implementation Method 3
the amplified tag sequence is detected using a fluorogenic nuclease assay
Data Source
AI summary
The invention provides an assay method for detection and/or quantification of a plurality of nucleic acid or protein targets in a sample. In the method probes are used to associate a detectable tag sequence with each of the selected targets present in the sample. Probes or primers sufficient to identify at least 25, and preferably at least 500, different targets are used. The method involves segregating aliquots of the sample from each other and detecting the tag sequences in each aliquot.