Fluorescent Probe for Real-Time BCAA Detection in Cells
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Solution Overview
Problem
Existing methods for detecting branched chain amino acids (BCAAs) are time-consuming, require cell fragmentation, and are not suitable for high-throughput, real-time, or in situ analysis with high spatial-temporal resolution.
Innovation Solution
A fluorescent sensor is developed by fusing a leucine responsive polypeptide with an optically active polypeptide, such as green fluorescent protein, to visualize the binding of BCAAs or leucine in real-time, allowing for high-throughput and quantitative detection inside or outside the cell.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional detection methods (mass spectrometry, chromatography, capillary electrophoresis) are used, then detection accuracy is achieved, but sample processing is time-consuming and requires cell fragmentation
Solution Approach 1:
The patent replaces mechanical/chemical separation systems (chromatography columns, electrophoresis apparatus) with a fluorescent sensing system. The fluorescent probe directly binds to BCAAs and emits optical signals, eliminating the need for complex mechanical separation and sample processing equipment while maintaining detection accuracy.
Solution Approach 2:
The fluorescent probe acts as an intermediary between the BCAA analyte and the detection system. Instead of directly measuring BCAA through complex instrumentation, the probe converts BCAA binding events into fluorescent signals that can be easily detected and quantified, simplifying the detection pathway.
2Measurement precision
If traditional detection methods are used, then quantitative analysis is achieved, but separation, extraction and purification of amino acid are difficult
Solution Approach 1:
The patent extracts only the essential detection function from complex separation and purification systems. The fluorescent probe specifically binds to BCAAs in complex biological matrices, selectively extracting the signal from the target analyte without requiring physical separation of other components.
Solution Approach 2:
The fluorescent probe system serves multiple functions simultaneously: it detects BCAA presence, provides quantitative measurement, and operates in complex biological environments without requiring separate purification steps. This multi-functionality eliminates the need for multiple dedicated devices for separation, extraction, and purification.
3Measurement precision
If fluorescent spectrometry with Ni (II) tetraazamacrocyclic complex is used, then fluorescence detection is achieved, but temperature related spin exchange equilibrium is required
Solution Approach 1:
The patent uses a stable fluorescent probe that does not require temperature-dependent equilibrium processes. The probe provides reliable fluorescence signals under physiological conditions without needing controlled temperature environments, making the detection system simpler and more adaptable to living systems.
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 fluorescent sensor enables real-time localization and quantitative detection of BCAAs with a large fluorescence dynamic range, eliminating time-consuming sample processing steps and enabling detection in subcellular structures and high-throughput screening.
Implementation Method 1
an optically active polypeptide, such as green fluorescent protein
Data Source
Figure 1A~1B
Figure 2A
Figure 2B
AI summary
The disclosure provides a fluorescent sensor, comprising a) a responsive polypeptide, and b) an optically active polypeptide, wherein the optically active polypeptide is inserted into the responsive polypeptide. The present disclosure also relates to a nucleic acid sequence encoding the fluorescent sensor according to any embodiment, or a complementary sequence thereof. The disclosure also provides an expression vector comprising the nucleic acid sequence or a complementary sequence thereof of the disclosure operably linked to an expression control sequence. The disclosure also provides a cell containing the expression vector of the disclosure. The present disclosure also provides a method for preparing the fluorescent sensor of this disclosure, comprising the following steps: providing a host cell comprising an expression vector that expresses the fluorescent sensor of the disclosure, culturing the host cell under conditions suitable for the expression of the host cell, and separating the fluorescent sensor. The disclosure also provides uses of the fluorescent sensor described in the disclosure or the fluorescent sensor prepared according to the method of the disclosure in detecting BCAAs. In one embodiment, the branched chain amino acids are selected from leucine, isoleucine and valine. The detection can be conducted in vitro, in vivo, in situ, or at subcellular level. The disclosure also provides a kit, comprising the fluorescent sensor described in the disclosure or the fluorescent sensor prepared according to the method of the disclosure.