Digital Multi-Temperature Fluorometric Detection for High Multiplexing
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Solution Overview
Problem
Current multiplex PCR-based detection methods are limited by the number of available color channels and the need for large, complex, and costly fluidic handling devices, restricting the ability to detect multiple analytes simultaneously.
Innovation Solution
A 3-dimensional melting curve labeling scheme combined with digital microfluidics to achieve high-level multiplexed detection, using a three-color detection system to generate up to 10,000 plex analysis, with at least 140 unique color-temperature codes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If additional fluorescence channels are used to detect more targets labeled with distinct fluorophores, then the number of detectable analytes increases, but the selection of commercially available fluorophores is limited
Solution Approach 1:
The patent transitions from detecting multiple analytes using multiple fluorescence channels (2D space) to using a single fluorescence channel combined with multiple melting temperatures (adding temperature dimension). This allows encoding multiple targets in one channel by measuring distinct Tm values, effectively moving from a limited color-channel-based approach to a temperature-dimension-based approach that overcomes fluorophore selection limitations.
Solution Approach 2:
The invention changes the detection parameter from fluorescence color (wavelength) to melting temperature. By using probes with different Tm values that all emit in the same fluorescence channel, the system can distinguish multiple targets without requiring multiple fluorophores, thus resolving the contradiction between detecting more analytes and fluorophore availability.
2Adaptability or versatility
If fluidic control is used to divide a sample into multiple reactions such that a single reaction contains a single analyte, then simultaneous measurement of multiple analytes is enabled, but large, complex, or expensive instruments are required
Solution Approach 1:
The patent merges multiple detection functions into a single reaction well by using probes with different melting temperatures that all respond in the same fluorescence channel. Instead of physically separating samples into multiple reactions (requiring complex fluidic systems), the invention combines multiple targets in one reaction and distinguishes them through temperature-dependent melting behavior, eliminating the need for expensive digital microfluidics instruments.
Solution Approach 2:
A single fluorescence channel performs the function of multiple channels by detecting probes with different Tm values. The system achieves multi-plexing capability using universal detection hardware, making the technique accessible without requiring specialized expensive instrumentation.
3Adaptability or versatility
If traditional melting curve analysis with individual melting curves for each target is used, then up to 6 targets can be distinguished using 2 fluorescence channels and 3 melting temperatures, but the level of multiplexing is limited
Solution Approach 1:
The patent creates a 3-dimensional encoding space by combining fluorescence channel (x-axis), melting temperature (y-axis), and probe design (z-axis). This allows exponential expansion of multiplexing capacity - with 3 color channels and multiple Tm values per channel, the system can distinguish far more than 6 targets, achieving ultra-high multiplexing without increasing instrument 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
Enables the simultaneous detection of multiple nucleic acids and proteins by generating distinct melt peaks in a single reaction, overcoming the limitations of traditional methods and expanding multiplexing capabilities.
Implementation Method 1
detecting targeted analytes by measuring the melting temperature hybridization with a fluorophore-labeled molecule probe... observing a decrease in the fluorescent signal
Implementation Method 2
measuring the melting temperature hybridization... the transition from hybridization to dissociation of the analyte and molecule probe
Data Source
AI summary
Provided herein are methods of performing a high-level of multiplexed analyte detection, including analytes such as nucleic acids and proteins. The methods include performing a melting curve analysis using multiple probe sets under conditions sufficient to generate a melting temperature (Tm) detection barcode data set from partitioned sample aliquots. Related systems and computer program products are also provided.