Mutant Blue Fluorescent Protein NADP(H) Quantification
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Solution Overview
Problem
Current methods for measuring NADP(H) and NAD(H) concentrations in cells face challenges such as low sensitivity, difficulty in distinguishing between NADP+/NADPH and NAD+/NADH, and require multi-step pretreatments, making real-time analysis and accurate quantification in natural samples difficult.
Innovation Solution
A mutant blue fluorescent protein (mBFP) with specific amino acid sequences is used, which is expressed in cells and measures fluorescence changes when added to NADP(H)-dependent oxidase/reductase, allowing for real-time quantification of NADP(H) with high sensitivity and minimal cell stress.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If intrinsic fluorescence measurement of NADP(H) is performed, then real-time measurement is possible, but sensitivity is too low for accurate quantification
Solution Approach 1:
The patent introduces a fluorescent probe as an intermediary substance that specifically binds to NADP(H). This probe acts as a mediator between the measurement system and the target coenzyme, amplifying the weak intrinsic fluorescence signal through its own fluorescent properties while maintaining real-time measurement capability. The probe's fluorescence signal is significantly stronger than the intrinsic signal, enabling accurate quantification.
2Measurement precision
If HPLC or coupling reaction methods are used, then measurement precision is improved, but device complexity and pretreatment steps increase
Solution Approach 1:
The patent extracts and utilizes the specific binding interaction between the fluorescent probe and NADP(H) as the core measurement mechanism. By isolating this specific molecular recognition event, the method eliminates the need for complex sample preparation steps such as extraction, separation, and derivatization required by HPLC and coupling reaction methods, achieving direct measurement in natural samples.
Solution Approach 2:
The patent replaces the mechanical and chemical complexity of HPLC separation systems and enzymatic coupling reactions with a simpler fluorescent detection system. The measurement is based on direct fluorescent signal detection upon probe-coenzyme binding, substituting complex mechanical separation and multiple chemical reactions with a single optical measurement step.
3Quantity of substance
If NADP(H) concentration is very low, then physiological relevance is maintained, but signal to noise ratio decreases making measurement difficult
Solution Approach 1:
The patent changes the detection parameter from relying on the weak intrinsic fluorescence of NADP(H) directly to measuring the enhanced fluorescence signal of the probe-NADP(H) complex. This parameter change in the detection mechanism allows sensitive detection of low concentrations by amplifying the signal through the probe's fluorescent properties while maintaining the physiological relevance of measuring actual endogenous coenzyme levels.
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 rapid, accurate, and sensitive measurement of NADP(H) concentrations in seconds, even at low concentrations, under various conditions, without the need for additional substrates or oxygen, and does not interfere with other coenzymes, facilitating real-time analysis and disease diagnosis.
Implementation Method 1
measuring concentration gradient and change in an intracellular coenzyme using a metagenome-derived blue fluorescent protein (mBFP) in real-time
Data Source
AI summary
Provided is a real-time imaging method of NAPDH-dependent metabolic activity in a cell and a use thereof, and more particularly, an imaging method capable of measuring concentration gradient and change in an intracellular coenzyme using a mutant of metagenome-derived blue fluorescent protein (mBFP) in real-time and an application method thereof. In particular, the method of imaging and quantifying the coenzyme contained in various biological samples may be performed by improving fluorescence in a short time even under anaerobic conditions, without adding a substrate required for coenzyme measurement or conversion without destroying cells, and without consuming the time required for formation of a special structure (fluorophore) for generating fluorescence unlike the existing quantitative systems. In particular, provided is a stable quantitative/imaging technique that is directly applicable to biological samples having various complex compositions without signal interference caused by impurities by removing an inherent enzymatic activity of mBFP.