Cardiolipin Binding Assay Using a Stable Acridinium Fluorescent Probe

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Solution Overview

Problem

Current methods for evaluating the binding affinity of compounds with cardiolipin are time-consuming, semi-quantitative, require large amounts of materials, and suffer from non-specific interactions, making them unsuitable for rapid compound screening and quantitative characterization.

Innovation Solution

The use of 3,6-di(azetidin-1-yl)-10-(3-(trimethylsilyl)propyl)acridin-10-ium iodide as a fluorescent probe for determining the binding affinity of organic and inorganic substances with cardiolipin, allowing for quantitative characterization in a membrane model that resembles natural membranes, with flexible reaction conditions and suitable for high-throughput screening.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If 10-N-nonyl acridine orange (NAO) is used as a fluorescent probe, then fluorescence detection of cardiolipin binding is possible, but fluorescence intensity is low and stability is poor due to low solubility in aqueous medium

Engineering Contradiction:

Improvefluorescence stabilityVSAvoidsolubility in aqueous medium

Core Design Contradiction:

ReliabilityVSQuantity of substance

Solution Approach 1:

The patent modifies the chemical structure of acridine orange by replacing the nonyl chain with an azetidin-1-yl group at positions 3 and 6, and adding a trimethylsilylpropyl chain at position 10. This structural parameter change improves both aqueous solubility and fluorescence stability while maintaining cardiolipin binding affinity

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent creates a composite fluorescent probe combining azetidine fragments with acridinium salt structure and trimethylsilylpropyl substituent. This composite structure integrates the benefits of improved solubility, stability, and photoluminescence quantum yield while retaining specific binding to cardiolipin

Inventive Principle:

Principle #40Composite materials

2Measurement precision

If NMR methods are used to detect compounds binding with cardiolipin, then binding detection is possible, but the method is time-consuming and requires large amounts of both compound of interest and cardiolipin

Engineering Contradiction:

Improvebinding detection capabilityVSAvoidanalysis time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The patent replaces NMR (nuclear magnetic resonance) methodology with fluorescence spectroscopy using the acridinium salt probe. This substitution enables rapid binding detection with high sensitivity, requiring minimal amounts of both compound and cardiolipin, while dramatically reducing analysis time compared to NMR methods

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Measurement precision

If Ca2+ is used as a probe for evaluating compounds binding with anionic lipids, then binding evaluation is possible, but the method shows non-specific Ca2+ binding to cardiolipin and lacks hydrophobic interaction with cardiolipin

Engineering Contradiction:

Improvebinding evaluation capabilityVSAvoidbinding specificity

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent uses the acridinium salt probe as an intermediary molecule that specifically binds to cardiolipin through both electrostatic interactions (with the anionic phospholipid headgroups) and hydrophobic interactions (with the lipid acyl chains). This intermediary probe provides specific and reliable binding evaluation without the non-specific binding issues of Ca2+

Inventive Principle:

Principle #24Intermediary (Mediator)

4Productivity

If rapid compound screening for cardiolipin targeting is required, then productivity is improved, but measurement precision and binding affinity quantification may be compromised

Engineering Contradiction:

Improvescreening speedVSAvoidbinding affinity quantification

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The fluorescent probe automatically reports binding affinity through its photoluminescence quantum yield, which changes in response to cardiolipin binding. This self-reporting mechanism enables rapid screening of multiple compounds while maintaining precise binding affinity quantification, as each compound's affinity is directly reflected in the fluorescence signal intensity

Inventive Principle:

Principle #25Self-service

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 rapid, quantitative assessment of compound binding to cardiolipin, suitable for screening large numbers of compounds, predicting mitochondrial toxicity, and evaluating drug interactions, with improved sensitivity and stability compared to previous probes.

Implementation Method 1

utilization of 3,6-di(azetidin-1-yl)-10-(3-(trimethylsilyl)propyl)acridin-10-ium iodide (I) as a fluorescent probe for the determination of binding affinity of organic and inorganic compounds for CL

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS12625142B2Assay for measuring binding affinity for cardiolipin of biologically active compounds

Publication Date: 2026.05.12 LATVIAN INST OF ORGANIC SYNTHESIS

AI summary

The present invention relates to a method for the evaluation of binding affinity of biologically active substances for cardiolipin based on acridinium salt utilization as a fluorescent probe.