Harnessing Contact-Quenched, Profluorescent Chemical Probes for Sensitive Determination and High-Throughput Measurements of Enzyme Activity

Sep 29, 2026

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Dual-labeled, profluorescent chemical probes are engineered to quantify and visualize a specific enzyme’s activity in complex biological media. Intact probes show low fluorescence due to fluorescence quenching, yet intrinsic fluorescence is restored through a favorable enzyme-catalyzed reaction. Contact quenching is used via a fluorophore–quencher ground-state complex, enabling dual-labeled probes as intramolecular dimers. A mono-exo-BCN-cystamine scaffold is sequentially coupled with two FAM derivatives to create four bis-FAM probes and to enable kinetic characterization, serum biomarker assays (BChE, PON1), and high-throughput detection in human serum.

LanguageEnglish

CategoryResearch & Report

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Pages11

Reading Time28 min

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Abstract

Introduction

Profluorescent probes and fluorescence quenching

Contact quenching constructs and ground-state dimers

Received/Revised/Accepted/Published

Figures and experimental characterization

This article is licensed under CC-BY 4.0 [http://pubs.acs.org/journal/acsodf](http://pubs.acs.org/journal/acsodf) Article Harnessing Contact-Quenched, Profluorescent Chemical Probes for Sensitive Determination and High-Throughput Measurements of Enzyme Activity Chien-Hui Huang,○ Chia-Yen Dai,○ Su-Hung Wang,○ Scott Severance, Chi-Ching Hwang, Yu-Chen Liu, Bao-Lin Yeh, Yung-Chieh Weng, Siao-Lei Yu, Hsing-Tao Kuo, Li-Fang Wang, Jeh-Jeng Wang, and Tzu-Pin Wang * Cite This: ACS Omega 2025, 10, 58566−58576 Read Online | | | | | | | | --- | --- | --- | --- | --- | --- | | ACCESS | Metrics & More | | Article Recommendations | | *sı Supporting Information | ABSTRACT: Dual-labeled, profluorescent chemical probes have been developed to quantify and visualize a specific enzyme’s activity in complex biological media. The intact chemical probes often exhibit minimal fluorescence due to fluorescence quenching, but their intrinsic fluorescence can be released by a favorable enzymecatalyzed reaction. Contact quenching represents one of several fluorescence quenching mechanisms. However, it is seldom intentionally implemented in the synthesis of dual-labeled, profluorescent chemical probes, because the structure of a contact quenching construct requires the formation of a ground-state fluorophore-quencher/-fluorophore complex. The ability of such duallabeled molecular probes to act as intramolecular dimers cannot be predicted. We previously revealed that a mono exobicyclo[6.1.0]nonyne (exo-BCN)-derivatized cystamine framework was critical to synthesizing sensitive, dual-labeled, profluorescent chemical probes capable of contact quenching. Here, we exploited the nonsymmetrical mono-exo-BCN-cystamine backbone by sequentially coupling it with two different carboxyfluorescein (FAM) derivatives and subsequently synthesizing four nonsymmetrical bis-FAM chemical probes. The fluorescence turn-on properties of the bis-FAM chemical probe with the lowest background FAM fluorescence were characterized by kinetic studies. Moreover, the release of two FAM equivalents from the fluorescence turn-on chemical probe during reactions was utilized to develop sensitive assays for measuring the activity and inhibition of two serum biomarkers, butyrylcholinesterase (BChE) and paraoxonase 1 (PON1) lactonase. We also developed an efficient, high-throughput assay for detecting BChE activity based on the chemical probe. Finally, the fluorescence assays successfully quantified the activities of BChE and PON1 lactonase in human serum. ■ INTRODUCTION Fluorophore-labeled probes are used in a wide variety of chemical and biochemical assays. Many of these assays exploit profluorescent probes in biochemical processes to increase the fluorescence signal in order to facilitate the detection and quantification of intrinsic activities in biological systems.1,2 Profluorescent probes are typically synthesized by incorporating one or two fluorophores into the fluorescence-quenched constructs.1,2 Dual-labeled, profluorescent probes can contain fluorophore-quencher or fluorophore−fluorophore pairs and have background fluorescence intensity intricately controlled by the fluorophore-quencher or fluorophore−fluorophore distance. Molecular beacons are prominent examples of duallabeled profluorescent probes used in DNA/RNA analysis.3,4 Close contact of the reporter and quencher moieties in molecular beacons ensures effective fluorescence quenching contributed by contact quenching effect.5,6 Effective contact quenching requires the formation of an intramolecular, nonfluorescent ground-state dimer between the moiety of a reporter fluorophore and the moiety of an acceptor, which can be either a fluorophore or a quencher.5,6 The close contact of dye−dye-paired moieties in contact quenching constructs promotes strong, intramolecular, dipole−dipole coupling of the dye components and perturbs the absorbance of intramolecular ground-state, reporter-acceptor comp

FAQs

How do the dual-labeled profluorescent probes generate a fluorescence signal during enzyme activity measurements?

The probes are designed so quenching keeps fluorescence low in the intact state. Upon a favorable enzyme-catalyzed reaction, intrinsic fluorescence is released and fluorescence turns on to report activity.

Why is contact quenching important for the synthesized chemical probes?

Effective contact quenching requires forming an intramolecular, nonfluorescent ground-state dimer between a reporter fluorophore and an acceptor (fluorophore or quencher). This close-contact arrangement enables strong intramolecular dipole–dipole coupling and reduced background fluorescence.

What assays are developed using the fluorescence turn-on chemical probe?

Fluorescence turn-on is used to develop sensitive assays for measuring activity and inhibition of serum biomarkers BChE and PON1 lactonase. An efficient high-throughput assay for BChE activity is also established, with fluorescence assays used to quantify activities in human serum.

Sep 29, 2026

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