Multiplex Assay Signal Separation Using Thermally Distinct Probes

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Solution Overview

Problem

Challenges arise in multiplex nucleic acid detection assays due to spectral similarity of detectable labels, making it difficult to resolve and quantify individual target nucleic acids, limiting the number of targets that can be detected in a single reaction mixture.

Innovation Solution

Utilizing cleavable and non-cleavable probes with different reaction condition responses, allowing separation of detectable signals with spectral similarity within the same detection channel by varying reaction conditions and measuring signal responses under different sets of conditions.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If multiple detectable labels with spectral similarity are used in a multiplex assay, then the number of detectable targets increases, but the ability to resolve and quantify individual targets deteriorates

Engineering Contradiction:

Improvenumber of detectable targetsVSAvoidability to resolve individual targets

Core Design Contradiction:

Quantity of substanceVSMeasurement precision

Solution Approach 1:

The patent segments the detection process by using two different probe types (first and second probe types) that differ in thermal and temporal properties. Each probe type responds differently to varying reaction conditions, allowing the composite signal to be mathematically separated into individual contributions from each target, thus resolving the spectral overlap problem

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent changes reaction conditions (temperature, time) to exploit the different thermal and temporal properties of the two probe types. By measuring signals under different conditions and using the known differential responses, the system can deconvolute the composite signal to determine individual target amounts, maintaining measurement precision while detecting multiple targets

Inventive Principle:

Principle #35Parameter changes

2Quantity of substance

If detectable labels with spectral similarity are used, then the number of targets detectable in a single channel increases, but the complexity of signal resolution and data analysis increases

Engineering Contradiction:

Improvenumber of targets in single channelVSAvoidcomplexity of signal resolution

Core Design Contradiction:

Quantity of substanceVSDevice complexity

Solution Approach 1:

The patent incorporates known differential thermal and temporal properties into the probe design beforehand. This preliminary characterization allows the system to use predetermined mathematical models for signal deconvolution, reducing the complexity of real-time signal resolution while enabling multiplexed detection in a single channel

Inventive Principle:

Principle #10Preliminary action

3Device complexity

If the same detectable label is used for different target nucleic acids, then the number of channels required decreases, but the ability to distinguish between targets deteriorates

Engineering Contradiction:

Improvenumber of detection channelsVSAvoidability to distinguish targets

Core Design Contradiction:

Device complexityVSMeasurement precision

Solution Approach 1:

The patent makes the detection system dynamic by varying reaction conditions (temperature, time) and exploiting the different responses of the two probe types to these changes. This dynamic approach allows the same detectable label to be used for multiple targets while maintaining the ability to distinguish between them through differential signal responses under varying conditions

Inventive Principle:

Principle #15Dynamics

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 quantification of multiple targets using the same detectable label without additional channels, increasing the 'plexy' of multiplex assays and allowing the same label to be used for different target nucleic acids.

Implementation Method 1

The reaction mixture can include one or more detectable labels each designed to associate with a different target nucleic acid and generate a signal that corresponds to the amount of the associated target nucleic acid

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS20250391509A1Systems and methods for analyte detection from multiplexed assays

Publication Date: 2025.12.25 LIFE TECHNOLOGIES CORP

AI summary

Systems and methods that enable analyte detection in a multiplexed amplification process can include obtaining, at multiple time points during the amplification process, composite emission signal data associated with a composite emission signal from at least a first probe type comprising a first label configured to generate a first emission signal and a second probe type comprising a second label configured to generate a second emission signal which has spectrally similar characteristics as said first emission signal. the first probe type and the second probe type differing in thermal and/or temporal properties; and determining, based at least partially on the composite emission signal data, emission signal data associated with a emission signal from a given probe type of the first probe type or the second probe type during the amplification process.