Ratiometric Fluorescence Coding for Multiplex Nucleic Acid Detection
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Solution Overview
Problem
Current fluorescence-based nucleic acid amplification testing (NAAT) methods are limited in multiplexing capacity due to spectral overlaps between fluorophores, restricting the number of targets that can be detected in a single assay.
Innovation Solution
The method employs a ratiometric fluorescence coding approach, using a combination of multiple fluorophores to encode each target sequence. This involves designing oligonucleotide target-specific probes (TSPs) with pre-defined ratios of fluorescent probe binding regions, allowing for the detection of multiple targets using a limited number of fluorophores.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple fluorophores are used to detect multiple targets, then the multiplexing capacity increases, but spectral overlaps occur between fluorophores
Solution Approach 1:
The patent changes the detection parameter from absolute fluorescence intensity to fluorescence ratio. By measuring the ratio of fluorescence intensities between two channels, the system can distinguish multiple targets even when using fluorophores with overlapping spectra, as each target produces a characteristic ratio signature
Solution Approach 2:
The patent makes the TSPs universal by incorporating common primer-binding regions that are recognized by universal primers. This allows a single set of TSPs to be used for detecting multiple different nucleic acid targets through ratio-based fluorescence coding, eliminating the need for target-specific primer sets for each fluorophore
2Device complexity
If the number of fluorophores is limited, then the cost and complexity are reduced, but the number of detectable targets is restricted
Solution Approach 1:
The patent transitions from one-dimensional detection (single fluorescence intensity per target) to two-dimensional detection (fluorescence intensity ratio between two channels). This dimensional change allows encoding of multiple targets using only two fluorophores, as each target can be assigned a unique ratio signature
Solution Approach 2:
The patent changes from using multiple fluorophores (increasing complexity) to using fluorophore ratios (maintaining simplicity). By measuring the ratio of intensities from two fluorophores, the system can distinguish up to multiple targets without requiring an equal number of distinct fluorophores
3Adaptability or versatility
If target-specific probes with multiple fluorescent probe binding regions are designed, then the multiplexing capacity is expanded, but the probe design complexity increases
Solution Approach 1:
The patent segments the TSP structure into distinct functional modules: target-binding regions, common primer-binding regions, and fluorescent probe binding regions. This modular segmentation simplifies probe design by allowing independent optimization of each function while maintaining overall probe effectiveness
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 significantly expands the multiplexing capacity of NAAT, enabling the detection of multiple nucleic acid targets in a single assay without the limitations of spectral overlaps, while maintaining high sensitivity and specificity.
Implementation Method 1
contacting the TSSs with a plurality of differently labeled fluorescent probes (FPs) such that a TSS-FP complex comprising the TSS and at least one of the first FP and the second FP is generated
Implementation Method 2
each of the TSPs includes: at least one target-binding region configured to bind to at least a portion of the distinct target nucleic acid sequence
Data Source
Figure 1A
Figure 1A
Figure 1B~1C
AI summary
Methods for multiplexed detection of a nucleic acid sequence in a sample including the use of a plurality of oligonucleotide target-specific probes (TSPs) configured to bind to a distinct target nucleic acid sequence, where each of the TSPs includes one or more copies of a first fluorescent probe (FP) binding region and one or more copies of a second FP binding region, and where a predetermined ratio of the one or more copies of the first FP binding region to the one or more copies of the second FP binding region is indicative of the distinct target nucleic acid sequence the TSP is configured to bind to.