Single-Molecule Dehydrogenase Assay Using a Universal Fluorescent Probe
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Solution Overview
Problem
Existing methods fail to detect oxidative activity of dehydrogenases at the single-molecule level and require multiple fluorescent probes for each dehydrogenase, which is difficult due to high substrate specificity.
Innovation Solution
A coupled assay system using a single fluorescent probe, dehydrogenase, nucleoside diphosphate derivative, and DT-diaphorase in an aqueous solution allows for oxidative activity evaluation at the single-molecule level, enabling differentiation of multiple dehydrogenases using various substrates and derivatives.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a standard substrate evaluation method is used, then the evaluation process is simple, but the activity of low-expression enzyme subtypes is buried and cannot be detected
Solution Approach 1:
The invention segments the enzyme population into individual single-molecule units by diluting the sample to achieve an average of less than 0.01 enzyme molecules per reaction chamber. This segmentation allows detection of low-expression subtypes that would be buried in bulk measurements, directly resolving the contradiction between detection sensitivity and system complexity.
Solution Approach 2:
The invention introduces a coupled assay system as an intermediary mechanism, where the enzyme of interest catalyzes conversion of substrate A to substrate B, and a second enzyme converts substrate B to a fluorescent product. This intermediary coupling amplifies the signal from single enzyme molecules, enabling detection of low-expression subtypes without requiring complex direct detection methods.
2Measurement precision
If multiple fluorescent probes are developed for each dehydrogenase, then detection specificity is improved, but the development difficulty increases due to high substrate specificity
Solution Approach 1:
The invention makes the fluorescent probe universal by using a common probe design that detects the reduced form of nucleoside diphosphate derivatives produced by various dehydrogenases. Instead of developing specific probes for each dehydrogenase substrate, the system uses a single fluorescent probe that can detect the coenzyme product across multiple dehydrogenase types, eliminating the need for multiple probe developments while maintaining detection specificity through the coupled assay design.
Solution Approach 2:
The invention introduces DT-diaphorase and nucleoside diphosphate derivatives as intermediaries between the dehydrogenase and the fluorescent probe. The dehydrogenase produces reduced nucleoside diphosphate, which then serves as a substrate for DT-diaphorase to reduce the fluorescent probe. This intermediary mechanism allows a single fluorescent probe to detect multiple dehydrogenases, resolving the contradiction between detection specificity and probe development difficulty.
3Device complexity
If a single fluorescent probe is used for multiple dehydrogenases, then the system complexity is reduced, but the ability to distinguish different dehydrogenase activities is lost
Solution Approach 1:
The invention applies local quality by assigning specific substrates to detect specific dehydrogenase activities. Each dehydrogenase subtype has its characteristic substrate (e.g., lactate for LDH, pyruvate for PDK), and the fluorescent signal intensity in each reaction chamber reflects the activity of that specific enzyme subtype. This allows a single fluorescent probe to differentiate multiple dehydrogenases based on their unique substrate-specific catalytic activities.
Solution Approach 2:
The invention uses parameter changes by varying the substrate type to detect different dehydrogenase activities. By changing the substrate parameter (lactate, pyruvate, alanine, etc.) in different reaction chambers, the system can selectively detect different dehydrogenase subtypes using the same fluorescent probe. The fluorescent signal intensity serves as the measurable parameter that reflects the activity level of each enzyme subtype.
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 detection of oxidative activities of multiple dehydrogenases with a single fluorescent probe, facilitating the evaluation of enzyme activity in specific conditions and identifying samples with enhanced activity in diseases like cancer.
Implementation Method 1
A dehydrogenase is an oxidase that oxidizes a substrate by using, as a coenzyme, an oxidized nucleoside diphosphate derivative such as an oxidized nicotinamide adenine dinucleotide derivative or an oxidized flavin adenine dinucleotide derivative
Implementation Method 2
the DT-diaphorase is capable of using a reduced form of the nucleoside diphosphate derivative as a coenzyme, and wherein the fluorescent probe is a compound whose fluorescence intensity increases upon reduction by the DT-diaphorase
Implementation Method 3
the fluorescent probe is a compound whose fluorescence intensity increases upon reduction by the DT-diaphorase
Data Source
Figure 1(A)~1(B)
Figure 2
Figure 3
AI summary
Disclosed is a method for evaluating the oxidation activity of a dehydrogenase, the method comprising causing the dehydrogenase and DT-diaphorase to catalyze an oxidation-reduction reaction in an aqueous solution containing one molecule of the dehydrogenase, a substrate of the dehydrogenase, a nucleoside diphosphate derivative, the DT-diaphorase, and a fluorescent probe, and measuring the fluorescence after the reaction.