mBFP NADP(H) Detection via Fluorescence Binding
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Solution Overview
Problem
Current methods for detecting NADP(H) are limited by requiring complex pre-treatment, high enzyme costs, and low sensitivity, especially in biological and environmental samples, due to interference from other substances and the instability of NADPH under oxygen exposure.
Innovation Solution
A novel detection method using metagenome-derived blue fluorescent protein (mBFP) that directly binds to NADPH, increasing fluorescence intensity up to 20 times, allowing for rapid and accurate measurement of NADP(H) concentrations without additional substrates or enzymes, even under oxygen-free conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods for NADP(H) are used, then detection can be performed, but complex pre-treatment is required and sensitivity is low due to interference from other substances
Solution Approach 1:
The invention extracts and utilizes the specific binding property of mBFP for NADPH, separating the detection function from complex enzyme systems. By using only mBFP protein without requiring additional enzymes or substrates, the method eliminates complex pre-treatment steps while maintaining high detection sensitivity through the unique fluorescence enhancement when mBFP binds to NADPH.
Solution Approach 2:
The invention introduces mBFP as an intermediary mediator between NADPH and the detection system. mBFP acts as a bridge that specifically binds to NADPH and converts the binding event into a measurable fluorescence signal, enabling sensitive detection without requiring complex enzyme-catalyzed reactions or multiple reagents.
2Measurement precision
If conventional enzyme-based detection methods are used, then NADP(H) can be detected, but enzyme costs are high and measurement time is increased
Solution Approach 1:
The invention replaces expensive, unstable enzymes with a more stable and cost-effective mBFP protein that can be produced recombinantly. mBFP serves as a disposable detection reagent that maintains stability under various conditions and does not require the complex maintenance, storage, and handling that enzyme-based systems demand, thereby reducing both cost and measurement time.
Solution Approach 2:
The invention enables mBFP to self-perform the detection function without requiring additional enzymes or substrates. The mBFP protein inherently possesses the ability to bind NADPH and generate a fluorescence signal, eliminating the need for complex enzyme cascades or multiple reagent additions, thus simplifying the procedure and reducing measurement time.
3Reliability
If conventional detection methods are used, then NADP(H) concentration can be measured, but stability is poor under oxygen exposure
Solution Approach 1:
The invention creates an oxygen-insensitive detection environment by using mBFP, which does not rely on oxygen-dependent enzymatic reactions. The fluorescence-based detection method using mBFP remains stable under varying oxygen conditions, effectively protecting the measurement process from oxygen interference and enabling reliable measurements in both aerobic and anaerobic conditions.
4Measurement precision
If high sensitivity detection is achieved using conventional methods, then detection limit is reduced, but additional substrates and enzymes are required
Solution Approach 1:
The invention makes mBFP a universal detection reagent that performs multiple functions: it specifically binds to NADPH, generates a fluorescence signal, and does so without requiring additional substrates or enzymes. This multi-functionality eliminates the need for multiple reagents while maintaining high detection sensitivity, simplifying the detection system to require only mBFP and the sample.
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 method provides high sensitivity and accuracy for NADP(H) detection in various samples, reducing measurement time and eliminating the need for complex pre-treatment, while maintaining stability and sensitivity across a wide concentration range.
Implementation Method 1
detection method using metagenome-derived blue fluorescent protein (mBFP) that directly binds to NADPH, increasing fluorescence intensity up to 20 times
Data Source
AI summary
Provided is a detection method of NADP(H) from the change of a fluorescence intensity by a reaction between metagenome-derived blue fluorescent protein (mBFP) and NADPH. More particularly, the present invention relates to methods for detecting NADP(H) using mBFP or his-mBFP, or methods for detecting NADP(H) for measuring an activity of NADP(H) dependent dehydrogenase or oxidoreductase.