Fluorescent Probe Enzyme Activity Detection
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Solution Overview
Problem
Current methods for detecting enzyme activity, particularly for enzymes with high substrate specificity like acetylcholinesterase, face challenges in sensitivity and specificity, especially at the single molecule level.
Innovation Solution
A coupled assay system using a microdevice that generates thiol, selenol, or poly sulfur atoms through a reaction between an enzyme and a substrate analog, followed by the use of a fluorescent probe that selectively reacts with these groups to detect enzyme activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If a fluorescent substrate analog is used to detect enzyme activity, then fluorescence changes can be observed by metabolic reaction, but the types of detectable enzymes are limited and development is difficult for enzymes with strict substrate recognition
Solution Approach 1:
The patent introduces a substrate analog as an intermediary that the target enzyme (e.g., AChE) acts upon to generate a thiol group. This thiol group then reacts with the fluorescent probe (containing maleimide or nitroolefin groups) to produce a fluorescent signal. This two-step intermediary mechanism allows detection of enzymes with strict substrate specificity that cannot be directly detected by conventional fluorescent substrates.
2Measurement precision
If a fluorescent probe with relatively large molecular weight is used to bind to substrate, then enzyme activity can be detected, but binding is less likely for enzymes with strict substrate recognition
Solution Approach 1:
The detection system is segmented into three distinct components: (1) a small substrate analog that the target enzyme recognizes and acts upon, (2) a fluorescent probe containing reactive groups (maleimide or nitroolefin), and (3) the enzyme itself. This segmentation allows the small substrate analog to successfully bind to the enzyme's active site while the fluorescent probe separately reacts with the generated thiol group, avoiding the problem of large molecular weight probes interfering with substrate binding.
3Quantity of substance
If conventional protein analysis techniques are used, then enzyme presence can be detected, but sensitivity is insufficient for enzymes present in small amounts in blood
Solution Approach 1:
The patent replaces conventional mechanical/protein analysis techniques with a fluorescence-based optical detection system. The fluorescent probe generates a strong optical signal when reacting with the thiol group produced by enzyme action, enabling detection of extremely low enzyme concentrations in blood samples that would be undetectable by conventional methods.
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 enables high sensitivity and specificity in detecting enzyme activity, particularly for enzymes with high substrate specificity, at the single molecule level, facilitating early disease diagnosis.
Implementation Method 1
a substrate analog or a natural substrate generates a compound A having a thiol group (R—SH), a selenol group (R—SeH), or a poly sulfur atom (R—(S)n-H) by a reaction with the enzyme
Implementation Method 2
fluorescence is observed or measured, the fluorescence being emitted or enhanced from a fluorescent product generated by the reaction of the compound represented by General Formula (I) or the salt thereof with the thiol group, the selenol group, or the poly sulfur atom
Data Source
AI summary
An object of the present invention is to provide a method capable of detecting activity of an enzyme that strictly recognizes a substrate, such as acetylcholinesterase (AChE). There is provided a method for detecting activity of an enzyme in a biological sample using a microdevice, the method including a step of bringing the biological sample into contact with a substrate analog or a natural substrate, and a compound represented by General Formula (I) below or a salt thereof, wherein the substrate analog or the natural substrate generates a compound A having a thiol group (R—SH), a selenol group (R—SeH), or a poly sulfur atom (R—(S)n-H) (R is a hydrogen atom or an alkyl group, and n is 1 to 5) by a reaction with the enzyme, and fluorescence is observed or measured, the fluorescence being emitted or enhanced from a fluorescent product generated by the reaction of the compound represented by General Formula (I) or the salt thereof with the thiol group, the selenol group, or the poly sulfur atom.