Arginine Fluorescent Probe for Real-Time Live-Cell Quantification
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Solution Overview
Problem
Existing methods for detecting arginine in live cells and subcellular organelles are time-consuming and cannot perform real-time, in-situ, high-throughput, and high spatiotemporal resolution detection.
Innovation Solution
Development of an arginine-sensitive polypeptide variant with specific mutations and a fluorescent protein insertion, allowing for real-time localization and quantitative detection of arginine inside and outside cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods (capillary electrophoresis, HPLC, enzymelinked immunosorbent assay, UV-visible spectrophotometry, fluorescence spectrometry) are used to detect arginine, then detection can be performed with established protocols, but time-consuming sample processing procedures (cell disruption, separation, extraction, purification) are required and real-time in-situ detection in live cells cannot be achieved
Solution Approach 1:
The patent replaces mechanical/chemical sample processing systems with an optical detection system. A fluorescent probe comprising a ligand-binding domain (specific to arginine) and a fluorescent protein domain detects arginine through fluorescence intensity changes, eliminating the need for cell disruption, separation, extraction, and purification steps while achieving real-time in-situ detection in live cells
Solution Approach 2:
The patent introduces a fluorescent probe as an intermediary molecule that binds to arginine and translates its presence into a measurable fluorescence signal. The probe consists of a ligand-binding domain that specifically recognizes arginine and a fluorescent protein domain that emits detectable signals, serving as a mediator between the target analyte and the detection system
2Loss of time
If conventional detection methods are used, then established protocols are available, but in-situ, real-time, dynamic, high-throughput, and high spatiotemporal resolution detection in live cells and subcellular organelles cannot be performed
Solution Approach 1:
The patent replaces complex sample processing mechanical systems with a simple optical detection system that can be performed directly in live cells. The fluorescent probe allows real-time monitoring of arginine dynamics in subcellular organelles without cell disruption or complex separation procedures
Solution Approach 2:
The fluorescent probe is designed to be expressed within the cell itself, allowing the cell to perform its own detection. The probe autonomously binds to arginine molecules in their natural cellular environment and generates fluorescent signals that can be detected and imaged in real-time
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 arginine fluorescent probe exhibits large fluorescence dynamic changes, high specificity, and can be expressed in cells for real-time localization and quantitative detection, eliminating the need for time-consuming sample processing steps, and enabling high-throughput detection in subcellular structures and compound screening.
Implementation Method 1
arginine fluorescent probe exhibits large fluorescence dynamic changes
Data Source
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AI summary
The present invention relates to an arginine fluorescent optical probe. Specifically, the present invention provides an arginine-sensitive polypeptide, wherein the arginine-sensitive polypeptide is a variant of an arginine binding protein, and formula (1) thereof has a sequence as shown in SEQ ID NO: 1 and has mutations at one, two, or three or more sites selected from the following. Compared with a probe formed by a wild arginine-sensitive polypeptide, the fluorescent probe of the present invention has greater dynamic changes in fluorescence and better specificity, and can be used for the high-throughput and quantitative detection of arginine inside and outside of cells.