FreSH-tracer probe for real-time thiol detection

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Solution Overview

Problem

Current methods for detecting thiols in living cells are not capable of real-time monitoring without lysing the cells, as existing fluorescent probes react irreversibly with thiols, preventing continuous and reversible fluorescence intensity changes that could indicate thiol levels.

Innovation Solution

Development of a FreSH-tracer (Fluorescent Real-time SH group-tracer) that changes fluorescence intensity ratiometrically and reversibly with thiol levels, allowing for quantitative and qualitative detection of thiols in living cells using a coumarin derivative with specific emission wavelength shifts upon thiol binding.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If existing fluorescent probes are used to detect thiols, then thiol detection sensitivity is improved, but the probes react irreversibly with thiols preventing real-time continuous monitoring

Engineering Contradiction:

Improvethiol detection sensitivityVSAvoidcontinuous monitoring capability

Core Design Contradiction:

Measurement precisionVSDuration of action of moving object

Solution Approach 1:

The patent changes the chemical reactivity parameter of the fluorescent probe by designing a reversible binding mechanism between the probe and thiol groups. Instead of irreversible covalent bonding, the probe uses non-covalent interactions (hydrogen bonding, van der Waals forces) that allow dynamic equilibrium, enabling continuous monitoring of thiol levels while maintaining detection sensitivity.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention introduces dynamic reversibility to the probe-thiol interaction system. The probe can bind to and release from thiol groups dynamically, allowing the fluorescence signal to respond continuously to changes in thiol concentration. This dynamic behavior enables real-time monitoring of thiol levels in living cells without permanent probe consumption.

Inventive Principle:

Principle #15Dynamics

2Difficulty of detecting and measuring

If fluorescent probes are introduced into living cells, then thiol detection capability is improved, but probe toxicity and cellular damage occur

Engineering Contradiction:

Improvethiol detection capabilityVSAvoidprobe toxicity

Core Design Contradiction:

Difficulty of detecting and measuringVSObject-affected harmful factors

Solution Approach 1:

The patent employs a probe design that mimics natural thiol-containing molecules in structure and size, allowing it to behave like a metabolite within the cell. The probe is used at low concentrations and can be cleared from cells naturally, reducing cumulative toxicity. Its temporary presence in cells enables detection without long-term harmful effects.

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

Solution Approach 2:

The probe is designed to be chemically similar to endogenous thiol compounds, using comparable molecular frameworks and functional groups. This homogeneity reduces immune recognition and toxic responses from cellular systems, allowing the probe to integrate smoothly into cellular environments while maintaining detection functionality.

Inventive Principle:

Principle #33Homogeneity

3Measurement precision

If HPLC or capillary electrophoresis methods are used, then thiol measurement accuracy is improved, but cell lysis is required losing real-time monitoring capability

Engineering Contradiction:

Improvethiol measurement accuracyVSAvoidsample preparation complexity

Core Design Contradiction:

Measurement precisionVSEase of operation

Solution Approach 1:

The patent replaces complex mechanical separation systems (HPLC columns, electrophoresis apparatus) with a simple fluorescence-based detection system. The probe directly binds to thiols in intact cells, and fluorescence intensity provides quantitative information without requiring physical separation or complex instrumentation, greatly simplifying the operational procedure.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The fluorescent probe serves as an intermediary that translates intracellular thiol concentrations into measurable fluorescence signals. This mediator enables direct measurement in living cells without needing to extract and separate thiol compounds, bridging the gap between complex analytical methods and simple real-time monitoring needs.

Inventive Principle:

Principle #24Intermediary (Mediator)

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 sensitive, real-time detection of thiols in living cells, indicating oxidative stress and antioxidant activity, facilitating the screening of thiol enhancers or inhibitors and measuring antioxidant activity.

Implementation Method 1

a FreSH-tracer (Fluorescent Real-time SH group-tracer) that changes fluorescence intensity ratiometrically and reversibly with thiol levels

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS10620215B2Real-time imaging sensor for measuring cellular thiol level

Publication Date: 2020.04.14 CELL2IN INC

AI summary

The present invention relates to a fluorescence sensor capable of real-time imaging for measuring a cellular thiol level. The present invention reveals that the fluorescence intensity of the fluorescent real-time SH group-tracer (FreSH-Tracer) of the present invention increases or decreases continuously, ratiometrically or reversibly depending on the thiol level in living cells, and thus can be usefully used as a biosensor which is remarkably susceptible to quantitative or qualitative real-time detection of the cellular thiol level in living cells.