Enzyme-Specific Fluorescent Compound for Single-Cell Imaging
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Solution Overview
Problem
Conventional fluorescent probes for imaging β-galactosidase activity in living cells face challenges such as leakage of enzyme reaction products and the need for cytotoxic ultraviolet light, making it difficult to visualize cells at a single-cell level effectively, especially for cancer diagnosis.
Innovation Solution
Development of an enzyme-specific retainable fluorescent compound that changes fluorescence properties upon reaction with β-galactosidase, covalently bonding to proteins within cells, allowing for specific visualization of cells expressing the enzyme without leakage, using a compound structure optimized to retain fluorescence in cells and emit light in the visible spectrum.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescent probes (HMDER-βGal) are used for imaging β-galactosidase activity, then enzyme-specific fluorescence can be achieved, but the fluorescent product leaks from cells making single-cell visualization difficult
Solution Approach 1:
The patent uses a spiro-indoxyl derivative as an intermediate compound that reacts with β-galactosidase to form a fluorescent product. This intermediate acts as a mediator that enables specific fluorescence generation only at the target site (cells expressing β-galactosidase) while the reaction product remains trapped within cells due to its molecular structure, preventing leakage and enabling clear single-cell visualization.
Solution Approach 2:
The patent modifies the molecular parameters of the fluorescent probe by using a spiro-indoxyl derivative with specific structural characteristics. This parameter change in the probe structure results in a fluorescent product that is retained within cells, fundamentally changing the leakage behavior from the conventional HMDER-βGal probe and enabling stable single-cell imaging.
2Measurement precision
If ultraviolet light is used for excitation to achieve fluorescence imaging, then enzyme activity can be detected, but cytotoxic effects prevent clear imaging of living cells
Solution Approach 1:
The patent changes the excitation wavelength parameter from ultraviolet to visible light range. The spiro-indoxyl derivative probe is designed to be excited by visible light, which eliminates the cytotoxic effects of UV radiation while maintaining the ability to detect β-galactosidase activity through fluorescence emission, enabling imaging of living cells without harm.
3Measurement precision
If X-Gal substrate is used for imaging, then β-galactosidase activity can be detected, but it cannot be applied to living cells
Solution Approach 1:
The patent fundamentally changes the chemical and physical parameters of the imaging substrate from X-Gal (which requires cell fixation and permeabilization) to a spiro-indoxyl derivative that can penetrate living cell membranes and function in living cells. This parameter change enables the probe to detect β-galactosidase activity in living cells while maintaining detection precision.
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 clear, single-cell level visualization of cells expressing β-galactosidase, both in living and fixed states, with no leakage of fluorescent dye, suitable for cancer cell imaging and diagnostic applications, using normal microscopy and without the need for special devices.
Implementation Method 1
A relationship between aging and expression of β-galactosidase in cells has recently been suggested
Implementation Method 2
HMDER-βGal and the like have been developed as β-galactosidase fluorescent probes which can be applied to living cells
Data Source
Figure 1(a)~1(c)
Figure 2(a)~2(c)
Figure 3(a)~4(b)
AI summary
Provided is an enzyme-specific fluorescent compound capable of being retained in cells, which can emit fluorescence specifically in a target cell, particularly a cell capable of expressing a reporter enzyme such as β-galactosidase, and can covalently bind to a protein in the cell to exhibit an excellent property of being retained in the cell. The fluorescent compound comprises a compound represented by formula (I') or a salt thereof. In formula (I'), A, X, Y and R1 to R9 are as described in claim 1.