Small-Molecule Detection Reagent Kit Using FRET to Cut Background Fluorescence
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Solution Overview
Problem
The existing chemiluminescence detection method for small molecule substances is hindered by background fluorescence from single fluorescent groups, leading to measurement errors.
Innovation Solution
A reagent combination using a first conjugate formed by coupling a first nucleic acid molecule to an antibody, a second conjugate with a substrate protein and a second fluorescent group, and a third conjugate with a first fluorescent group, along with an oxidation inhibiting reagent, to minimize background fluorescence through fluorescence resonance energy transfer and competitive binding, allowing for accurate detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If a single fluorescent group is used to emit fluorescence for detection, then the detection method is simple, but background fluorescence is produced causing measurement errors
Solution Approach 1:
The single fluorescent group is segmented into two distinct fluorescent groups: a first fluorescent group that emits background fluorescence and a second fluorescent group that emits signal fluorescence. By separating the fluorescent functions into different groups, the patent eliminates the background fluorescence interference while maintaining detection capability, thus resolving the contradiction between method simplicity and measurement precision.
Solution Approach 2:
A nucleic acid molecule acts as an intermediary to connect the first fluorescent group and the second fluorescent group through hybridization. This intermediary mechanism allows the system to distinguish between background fluorescence from the first group and signal fluorescence from the second group, enabling precise measurement without the complexity of multiple separate detection systems.
2Measurement precision
If background fluorescence is eliminated by using a second fluorescent group, then measurement precision is improved, but the reagent combination complexity increases
Solution Approach 1:
The patent merges the first fluorescent group, second fluorescent group, and nucleic acid molecule into a single integrated reagent combination. By combining these elements in one mixture, the patent achieves precise measurement through the second fluorescent group's signal while avoiding the complexity of separate reagent systems, thus resolving the contradiction between precision improvement and reagent complexity.
3Ease of operation
If the first fluorescent group is used for detection, then the detection process is straightforward, but background fluorescence interferes with detection accuracy
Solution Approach 1:
Instead of using the first fluorescent group directly for detection (which causes background interference), the patent inverts the approach by using the second fluorescent group as the primary detection signal. The first fluorescent group's background fluorescence is effectively ignored or subtracted, allowing straightforward detection operations while achieving high measurement precision through the inverted detection strategy.
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
The method reduces background fluorescence interference, enhancing detection sensitivity and accuracy by using the second fluorescent group's fluorescence as the measurement signal, enabling both qualitative and quantitative analysis of small molecule substances.
Implementation Method 1
allowing for accurate detection by using the second fluorescent group's fluorescence as the measurement signal
Implementation Method 2
an oxidation inhibiting reagent, to minimize background fluorescence through fluorescence resonance energy transfer and competitive binding
Implementation Method 3
allowing for accurate detection by using the second fluorescent group's fluorescence as the measurement signal, enabling both qualitative and quantitative analysis of small molecule substances
Data Source
AI summary
A reagent combination, a kit, a detection system and a detection method for detecting a small molecule substance. In the condition that a solution to be tested does not contain or contains a small amount of the small molecule substance, an antibody binds to a substrate protein conjugate to form an immune complex, a stem-loop structure is generated by means of a complementary pairing among a plurality of nucleic acid molecules, a first fluorescent group excites a second fluorescent group to emit a second fluorescence on the basis of fluorescence resonance energy transfer, and the content of the small molecule substance is calculated. In the condition that the solution to be tested contains a large amount of the small molecule substance, the immune complex and the stem-loop structure cannot be formed, and the second fluorescent group cannot emit the second fluorescent.