Multifunctional near-infrared fluorescent probe for sensing of lysine and Cu2+/Fe3+ and relay detection of biothiols.

Lysine (Lys), Cu2+ and Fe3+ ions and biothiols are essential to a myriad of biological and pathological pathways, and their dysregulation is implicated in a variety of diseases. Development of fluorescent probes capable of detecting multiple analytes may be of great significance for early and accurate diagnosis of diseases and remains a huge challenge. In this context, a novel coumarin-dicyanoisophorone-based probe, engineered for the concurrent sensing of Lys, Cu2+, Fe3+ and biothiols was developed. The probe exhibited turn-on response to Lys, colorimetric and turn-off response to Cu2+ by formation of the probe-Cu2+ complex, and ratiometric sensing of Fe3+. In addition, the probe-Cu2+ complex served colorimetric and fluorescence turn-on sensor for biothiols. The limit of detection (LOD) values for the analytes were in the range of 0.30-4.40 µM. Sensing mechanisms based on intramolecular charge transfer (ICT) and iron-mediated hydrolysis of Schiff base were proposed and substantiated through density functional theory (DFT) calculations. Application of the probe for living cell bioimaging was demonstrated.

Ultrasmall CuMn-His Nanozymes with Multienzyme Activity at Neutral pH: Construction of a Colorimetric Sensing Array for Biothiol Detection and Disease Identification.

Biothiol assays offer vital insights into health assessment and facilitate the early detection of potential health issues, thereby enabling timely and effective interventions. In this study, we developed ultrasmall CuMn-Histidine (His) nanozymes with multiple enzymatic activities. CuMn-His enhanced peroxidase (POD)-like activity at neutral pH was achieved through hydrogen bonding and electrostatic effects. In addition, CuMn-His possesses laccase (LAC)-like and superoxide dismutase (SOD)-like activities at neutral pH. Based on three different enzyme mimetic activities of CuMn-His at neutral pH, the colorimetric sensing array without changing the buffer solution was successfully constructed. The array was successfully used for the identification of three biothiols, glutathione (GSH), cysteine (Cys), and homocysteine (Hcy). Subsequently, excellent application results were shown in complex serum and cellular level analyses. This study provides an innovative strategy for the development of ultrasmall bimetallic nanozymes with multiple enzymatic activities and the construction of colorimetric sensing arrays.

A Red-Emission Fluorescent Probe for Intracellular Biothiols and Hydrogen Sulfide Imaging in Living Cells.

This research centers on the development and synthesis of a longwave fluorescence probe, labeled as 60T, designed for the simultaneous detection of hydrogen sulfide, cysteine/homocysteine, and glutathione. The probe showcases a swift response, good linearity range, and heightened sensitivity, boasting that the detection limits of the probe for Cys, Hcy, GSH and H2S were 0.140, 0.202, 0.259 and 0.396 µM, respectively. Notably, its efficacy in monitoring thiol status changes in live MCF-7 cells is underscored by a substantial decrease in fluorescence intensity upon exposure to the thiol trapping reagent, N-ethyl maleimide (NEM). With an impressive red emission signal at 630 nm and a substantial Stokes shift of 80 nm, this probe exhibits remarkable sensitivity and selectivity for biothiols and H2S, indicating promising applications in the diagnosis and surgical navigation of relevant cancers.

Theory and experiment: The synthesis and drug application of "ON-OFF-ON" fluorescent probes for copper and biothiols detection.

Abnormal copper ions (Cu2+) and biothiols have potential impacts on environmental pollution and human health, so the detection of these substances with high selectivity and sensitivity has become an important research topic. In this study, we designed and synthesized two fluorescent probes (L1 and L2) based on naphthalene and anthracene derivatives that could specifically detect Cu2+ and biothiols. Owing to the paramagnetic effect of Cu2+, the strong fluorescent intensity was quenched after the addition of Cu2+. When biothiols were added to the solution (L-Cu2+), the fluorescence intensity was significantly enhanced and recovered. So, the interaction process was accompanied with "ON-OFF-ON" phenomenon in fluorescent intensity. Two complexes (L-Cu2+) showed low limit of detection for biothiols (Cys was 3.4 ×10-5 M and GSH was 2.0 ×10-5 M) and weak cytotoxicity (< 150 µg/mL). Theoretical investigation analysis revealed that the intramolecular hydrogen bond existed in the structure of probes and the roles of molecular frontier orbitals in molecular interplay. In addition, two probes also showed good applicability in actual drug Atomolan. The GSH content in the tested Atomolan reached over 99.9% of the labeling which was accord with the percentage of pharmacopoeia. Therefore, two probes have the real application value in the detection of Cu2+, biothiols and drug efficacy in various environments.

A Fluorescent Probe for Investigating the Role of Biothiols in Signaling Pathways Associated with Cerebral Ischemia-Reperfusion Injury.

Cerebral ischemia-reperfusion injury (CIRI) is intimately associated with the redox regulation of biothiol, a crucial antioxidant marker that precludes the onset of ROS. We designed a novel fluorescent probe, DCI-Ac-Py, showing various physicochemical properties, such as high selectivity, exceptional signal-to-noise ratio, near-infrared (NIR) optical window, and blood-brain barrier (BBB) penetrability, for detecting biothiols in the brain. The picolinate serves as a specific recognition group that is rapidly activated by biothiol and undergoes nucleophilic substitution with the adjacent acrylic ester to yield the desired NIR probe. Additionally, the probe's lipid solubility is improved through the inclusion of halogen atoms, which aids in penetrating the BBB. Using DCI-Ac-Py, we investigated changes of biothiols in vivo in the brains of mice during CIRI. We found that biothiol-mediated NF-kB classical (P65-related) and nonclassical (RelB-related) pathways contribute to abundant ROS production induced by CIRI and that biothiols are involved in redox regulation. These findings provide new insights into the study of CIRI and shed light on the physiological and pathological mechanisms of biothiols in the brain.

All-Nontoxic Fluorescent Probe for Biothiols and Its Clinical Applications for Real-Time Glioblastoma Visualization.

Fluorescence-guided surgery (FSG) is a surgical method to selectively visualize the tumor site using fluorescent materials with instrumental setups in the operation rooms. It has been widely used in the surgery of brain tumors, such as glioblastoma (GBM), which is difficult to distinguish from normal tissue. Although FSG is crucial for GBM surgery, the commercially available fluorescent materials for FSG have shown serious adverse effects. To satisfy the clinical demand, we recently reported reaction-based fluorescent probes based on a 4-chloro-7-nitrobenzofurazan (NBD) fluorophore that can detect cysteine (Cys) and homocysteine (Hcy), a biomarker of GBM, and their applications for the GBM diagnosis and FSG. However, our probes have cellular toxicity issues arising from the leaving group (LG) that is generated after the reaction of the fluorescent probe and the analytes. In this study, we disclosed a nontoxic fluorescent probe for sensing biothiols and their clinical applications for real-time human glioblastoma visualization. Systematic toxicity analysis of several LGs was conducted on several cell lines. Among the LGs, 2-hydroxy-pyridine showed negligible toxicity, and its fluorescent probe derivative (named NPO-o-Pyr) showed high specificity and sensitivity (LOD: 0.071 ppm for Cys; 0.189 ppm for Hcy), a fast response time (<5 min) to Cys and Hcy, and high biocompatibility. In addition, NPO-o-Pyr can significantly detect the GBM site both in actual clinical samples as well as in the GBM-xenografted mouse model. We are confident that NPO-o-Pyr will become a new substitute in FSG due to its capability to overcome the limitations of the current fluorescent probes.

A Cerium Organic Framework with {Cu2I2} Cluster and {Cu2I2}n Chain Modules: Structure and Fluorescence Sensing Properties.

In this work, a copper iodine module bearing a coordination polymer (CP) with a formula of [(Cu2I2)2Ce2(INA)6(DMF)3]·DMF (1, HINA = isonicotinic acid, DMF = N,N'-dimethyl formamide) is presented. The title compound features a three dimensional (3D) structure, in which the {Cu2I2} cluster and {Cu2I2}n chain modules are coordinated by N atoms from a pyridine ring in INA- ligands, while the Ce3+ ions are bridged by the carboxylic groups of INA- ligands. More importantly, compound 1 exhibits an uncommon red fluorescence (FL) with a single emission band maximized at 650 nm belonging to near infrared (NIR) luminescence. The temperature dependent FL measurement was applied to investigate the FL mechanism. Remarkably, 1 could be used as a FL sensor to cysteine and the nitro-bearing explosive molecule of trinitropheno (TNP) with high sensitivity, demonstrating its potential FL sensing applications for biothiol and explosive molecules.

A coumarin-based fluorescent probe: single-wavelength excitation, discrimination of Cys/Hcy and GSH by naked eyes.

Biothiols mainly include cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), which play an important role in life activities and abnormal changes in their concentrations are closely related to certain diseases. Therefore, the quantitative tracking and analysis of biothiols in living organisms has become a hot research topic in recent years. In this work, a coumarin-based fluorescent probe COUN was designed and synthesized for the comparable color recognition of Cys/Hcy and GSH by introducing the phenylethynyl group as the recognition site of biothiols, which showed significant fluorescence enhancement and green fluorescence under the UV light at 365 nm. The probe specifically recognized Hcy, showing 40-fold fluorescence enhancement and strong green fluorescence at 492 nm. Moreover, there was a good linear relationship between the fluorescence intensity of the probe and certain concentrations of Cys/Hcy and GSH, with detection limits of 36.6 nM, 86.4 nM, and 174 nM, respectively. The recognition mechanism of COUN to distinguish Cys/Hcy and GSH was studied by TDDFT calculations. More importantly, COUN was successfully used for imaging biothiols in living cells. The results showed that this probe could provide an effective contribution to the understanding of the role of biothiols, especially Hcy.

Colorimetric determination of biothiols based on peroxidase-mimicking Ag nanoparticles decorated Ti3C2 nanosheets.

Ag nanoparticle-decorated Ti3C2 nanosheets (AgNPs@Ti3C2 NSs) were facilely synthesized via a self-reduction approach, in which Ti3C2 NSs acted as both reductant and supporter. The AgNPs@Ti3C2 NS nanocomposite exhibited excellent peroxidase-like activity with o-phenylenediamine (OPD) and H2O2 as substrates. The catalytic behavior followed the typical Michaelis-Menten kinetics; Michaelis constant (Km) and maximum initial velocity (Vmax) for OPD were 0.263 mM and 43.2 × 10-8 M-1 s, indicating high affinity and high catalytic efficiency towards OPD. The catalytic mechanism was revealed to be an accelerated electron transfer process. Based on the inhibition effect on the peroxidase-like activity of AgNPs@Ti3C2 NSs, a simple, fast, and sensitive colorimetric method for detection of low-weight biothiols (cysteine (Cys), homocysteine (Hcy), and glutathione (GSH)) was developed by measuring the absorbance at 425 nm. The colorimetric method displayed wide linear range (50 nM to 50 µM for Cys, 10 nM to 250 µM for Hcy, 10 nM to 50 µM for GSH), low limit of detection (48.5 nM for Cys, 5.5 nM for Hcy, 7.0 nM for GSH), and good selectivity and short assay time (3 min). Moreover, the feasibility of this colorimetric sensor was demonstrated by accurately determining Cys in diluted human serum samples; good recovery (95.9-101.0%) and low relative standard deviations (2.8-4.9%) were obtained, showing great promise for point-of-care test in clinical samples.

A paper-based optical sensor for the screening of viruses through the cysteine residues of their surface proteins: A proof of concept on the detection of coronavirus infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to human health. Current methods such as reverse transcription polymerase chain reaction (qRT-PCR) are complex, expensive, and time-consuming. Rapid, and simple screening methods for the detection of SARS-CoV-2 are critically required to fight the current pandemic. In this work we present a proof of concept for, a simple optical sensing method for the screening of SARS-CoV-2 through its spike protein subunit S1. The method utilizes a target-specific extractor chip to bind the protein from the biological specimens. The disulfide bonds of the protein are then reduced into a biothiol with sulfhydryl (SH) groups that react with a blue-colored benzothiazole azo dye-Hg complex (BAN-Hg) and causes the spontaneous change of its blue color to pink which is observable by the naked eye. A linear relationship between the intensity of the pink color and the logarithm of reduced S1 protein concentration was found within the working range 130 ng.mL-1-1.3 pg mL-1. The lowest limit of detection (LOD) of the assay was 130 fg mL-1. A paper based optical sensor was fabricated by loading the BAN-Hg sensor onto filter paper and used to screen the S1 protein in spiked saliva and patients' nasopharyngeal swabs. The results obtained by the paper sensor corroborated with those obtained by qRT-PCR. The new paper-based sensing method can be extended to the screening of many viruses (e.g. the human immunodeficiency virus, the human polyomavirus, the human papilloma virus, the adeno associated viruses, the enteroviruses) through the cysteine residues of their capsid proteins. The new method has strong potential for screening viruses at pathology labs and in remote areas that lacks advanced scientific infrastructure. Further clinical studies are warranted to validate the new sensing method.

Brønsted Acid-Functionalized Ionic Co(II) Framework: A Tailored Vessel for Electrocatalytic Oxygen Evolution and Size-Exclusive Optical Speciation of Biothiols.

Metal-organic frameworks (MOFs) not only combine globally demanded renewable energy generation and environmental remediation onto a single platform but also rationalize structure-performance synergies to devise smarter materials with remarkable performance. The robust and non-interpenetrated cationic MOF exemplifies a unique bifunctional scaffold for the efficient electrochemical oxygen evolution reaction (OER) and ultrasensitive monitoring of biohazards. The microporous framework containing Brønsted acid-functionalized [Co2(µ2-OH)(CO2)2] secondary building units (SBUs) exhibits remarkable OER performance in 1 M KOH, requiring 410 mV overpotential to obtain 10 mA cm-2 anodic current density, and a low Tafel slope of 55 mV/dec with 93.1% Faradaic efficiency. Apart from the high turnover frequency and electrochemically assessable surface area, steady OER performance over 500 cycles under potentiodynamic and potentiostatic conditions result in long-term catalyst durability. The highly emissive attribute from nitrogen-rich fluorescent struts benefits the MOF in recyclable and selective fluoro-detection of three biothiols (l-cysteine, homocysteine, and glutathione) in water with a fast response time. In addition to colorimetric monitoring in the solid and solution phases, control experiments validate size-exclusive biothiol speciation through molecular-dimension-mediated pore diffusion. The role of SBUs in the OER mechanism is detailed from density functional theory-derived free energy analysis, which also validates the importance of accessible N-sites in sensing via portraying framework-analyte supramolecular interactions.

Recent Progress of Glutathione (GSH) Specific Fluorescent Probes: Molecular Design, Photophysical Property, Recognition Mechanism and Bioimaging.

The selective detection of glutathione (GSH) in vitro and in vivo has attracted great attentions, credited to its important role in life activities and association with a series of diseases. Among all kinds of analytical techniques, the fluorescent probe for GSH detection become prevalent recently because of its ease of operation, high temporal-spatial resolution, visualization and noninvasiveness, etc. The special structural features of GSH, such as the nucleophilicity of sulfhydryl group, the concerted reaction ability of amino group, the negative charged nature, the latent hydrogen bonding ability along with its flexible molecular chain, are all potent factors to be employed to design the specific fluorescent probe for GSH and discriminate it from other bio-species including its analogues cysteine (Cys) and homocysteine (Hcy). This paper reviewed the studies in the last 3 years and was organized based on the reaction mechanism of each probe. According to the reactivity of GSH, various recognition mechanisms including Michael addition, nucleophilic aromatic substitution, ordinary nucleophilic substitution, multi-site reaction, and other unique reactions have been utilized to construct the GSH specific fluorescent probes, and the molecular design strategy, photophysical property, recognition mechanism, and bioimaging application of each reported probe were all discussed here systematically. Great progress has been made in this area, and we believe the analyses and summarization of these excellent studies would provide valuable message and inspiration to researchers to advance the research toward clinic applications.

A Facile Probe for Fluorescence Turn-on and Simultaneous Naked-Eyes Discrimination of H2S and biothiols (Cys and GSH) and Its Application.

Hydrogen sulfide and biothiol molecules such as Cys and GSH acted important roles in many physiological processes. To simultaneously detect and distinguish them was quite necessary by a suitable fluorescent probe. A novel chemosensor 4-(4-(benzo[d]thiazol-2-yl)-2-methoxyphenoxy)-7-nitrobenzo[c][1,2,5]oxadiazole (BMNO) was designed to detect H2S/Cys/GSH using the combination of nitrobenzofurazan (NBD) and benzothiazole fluorophores linked by a facile ether bond. The probe BMNO was developed for simultaneous identification of H2S, Cys and GSH. Noticeably, the color changes (from colorless to light purple, light orange and light yellow) of probe BMNO solutions for sensing H2S, Cys and GSH could be observed by naked eyes, respectively. The probe BMNO exhibited high selectivity and sensitivity for H2S, Cys and GSH showing distinct optical signal with detection limit as low as 0.15 µM, 0.03 µM and 0.14 µM, respectively. The sensing mechanism was clarified by spectrum analysis and some controlled experiments. In addition, these outstanding properties of probe BMNO enabled its practical applications in detection H2S in beer, and in cell imaging for Cys and GSH as well.

Biothiol-Functionalized Cuprous Oxide Sensor for Dual-Mode Sensitive Hg2+ Detection.

Hg2+ ions are one of the highly poisonous heavy metal ions in the environment, so it is urgent to develop rapid and sensitive detection platforms for detecting Hg2+ ions. In this work, a novel electrochemical and photoelectrochemical dual-mode sensor (l-Cys-Cu2O) was successfully fabricated, and the sensor exhibits a satisfactory detection limit (0.2 and 0.01 nM) for the detection of Hg2+, which is far below the dangerous limit of the U.S. Environmental Protection Agency. The linear ranges of dual-mode Hg2+ detections were 0.33-3.3 and 0.17-1.33 µM, respectively. Moreover, the sensor shows desirable stability, selectivity, and reproducibility for detecting Hg2+ ions. For river water samples, the recoveries of 96.6-101.4% (electrochemical data) and 93.0-105.6% (photoelectrochemical data) were obtained, indicating that the sensor could be successfully applied in the determination of Hg2+ ions in environmental water. Therefore, the designed sensor has a potential in the trace-level detection of Hg2+ ions.

Substitution-rearrangement-cyclization strategy to construct fluorescent probe for multicolor discriminative analysis biothiols in cells and zebrafish.

Discriminative detection of biothiols (Cysteine, homocysteine and glutathione) is of great significance to clarificate their complex physiological processes, the occurrence and development of related diseases. However, similar structure and reactivity among such species pose huge challenges in developing fluorescent probes to distinguish among of them. In this work, a dual-site probe CTT reacted with the analytes to regulate molecular conjugation through substitution-rearrangement-cyclization strategy, utilizing a multi-channel signal combination mode to realize the distinguishing detection of the three biothiols. Cell and zebrafish imaging experiments sufficiently demonstrated that CTT could semiquantify biothiols, which will provide valuable chemical tool for elucidating the complex biological functions of biothiols.

Red-emitting fluorescent turn-on probe with specific isothiocyanate recognition site for cysteine imaging in living systems.

Cysteine (Cys) is an effective biomarker in life systems and is closely related to a variety of diseases, so developing a specific and efficient detection method for Cys is of great significance. To date, extensive work has been undertaken toward this goal. However, the differentiation of Cys from other biothiols still represents a challenge from an experimental point of view. Toward this end, a selective and sensitive red-emitting probe (TMN-NCS) with an isothiocyanate (ITC)-based structure was proposed in this paper. A large Stokes shift (210 nm) was observed upon addition of Cys to a solution of TMN-NCS. In addition, TMN-NCS showed low toxicity, a low detection limit (120 nM), and excellent cell permeability. The results suggested that TMN-NCS holds great promise for biological applications.

Rational design of a far-red fluorescent probe for endogenous biothiol imbalance induced by hydrogen peroxide in living cells and mice.

Intracellular biothiols are correlated with many diseases such as nerve disorder and Parkinson's disease likely due to a redox imbalance. In this work, we designed an ultrafast fluorescent probe (Cou-DNBS) for biothiols with a large Stokes shift (131 nm). The probe was constructed through linking the 2,4-dinitrobenzenesulfonyl moiety as the specially recognizing biothiols site to an iminocoumarin fluorophore Cou-NH obtained by fusing an additional benzene ring. The presence of biothiols could ultrafast perform a significant fluorescence emission at 617 nm upon the excitation of 480 with the low limits of detection (2.5 nM for Cys, 1.7 nM for Hcy and 0.84 nM for GSH). HRMS spectra as well as theoretical calculations further evidenced the rationale of recognition mechanism. Furthermore, the probe can successfully visualize endogenous biothiol recovery in living cells damaged by H2O2.

A vinyl sulfone clicked carbon dot-engineered microfluidic paper-based analytical device for fluorometric determination of biothiols.

A microfluidic paper-based analytical device integrating carbon dot (CDs) is fabricated and used for a fluorometric off-on assay of biothiols. Vinyl sulfone (VS) click immobilization of carbon dots (CDs) on paper was accomplished by a one-pot simplified protocol that uses divinyl sulfone (DVS) as a homobifunctional reagent. This reagent mediated both the click oxa-Michael addition to the hydroxyl groups of cellulose and ulterior covalent grafting of the resulting VS paper to NH2-functionalized CDs by means of click aza-Michael addition. The resulting cellulose nanocomposite was used to engineer an inexpensive and robust microfluidic paper-based analytical device (µPAD) that is used for a reaction-based off-on fluorometric assay of biothiols (GSH, Cys, and Hcy). The intrinsic blue fluorescence of CDs (with excitation/emission maxima at 365/450 nm) is turned off via the heavy atom effect of an introduced iodo group. Fluorescence is turned on again due to the displacement of iodine by reaction with a biothiol. The increase in fluorescence is related to the concentration over a wide range (1 to 200 µM for GSH and 5-200 µM for Cys and Hcy, respectively), and the assay exhibits a low detection limit (0.3 µM for GSH and Cys and 0.4 µM for Hcy). The method allows for rapid screening and can also be used in combination with a digital camera readout. Graphical abstract Schematic representation of a µPAD based on click immobilized carbon dots and used for a reaction-based fluorometric off-on assay of biothiols. The intrinsic blue fluorescence of carbon dots is turned off via the heavy atom effect of an introduced iodo group and turned on by the displacement of this atom by reaction with a biothiol.

Didactic approach recounting advances and limitations in novel glutathione and cysteine detection (reduced GSH probe) with mixed coumarin, aldehyde, and phenyl-selenium chemistry.

We describe the pertinent research steps and analysis, many of which are chemical, to achieve a novel molecular probe for glutathione (GSH) which has been published and patented based on two recent articles: "Exceptional time response, stability and selectivity in doubly-activated phenyl selenium-based glutathione-selective platform" and "Enhanced Doubly Activated Dual Emission Fluorescent Probes for Selective Imaging of Glutathione or Cysteine in Living Systems" (Kim et al., 2015; Mulay et al., 2018). The papers involve coumarin probes. Reaction/detection unfolds with aminothiol attack at an electrophilic ring carbon position. An adjacent -CHO group is heavily involved in resonance aspects of the C-Se position, as well as the binding of the pendant N-group; the coumarin lactone carbonyl also allows for resonance to be achieved (vide infra). The leaving group, -SePh, while precedented in some systems, depends on electronic tuning (Fig. 1). For 1, the response times with GSH was ~100ms; a 100-fold fluorescence increase is observed (Compound 1). The probe also reacts with cysteine (Cys) and homocysteine (Hcy), albeit differently. For glutathione probing, the greater wavelength maxima (1: 550nm, DACP-1: 555nm, DACP-2: 590nm) enabled eventual cell studies (confocal microscopy) and animal studies. The limits of detection (LOD, 1: 270nM DACP-1: 10.1nM DACP-2: 17.0nM), as measured using the 3σ/k method. We provide a didactic presentation from probe conception to probe in vivo testing, etc., with additional considerations presented; a variety of factors/issues (2.1-2.28) help maintain a realistic sequence, a flow from wider to narrower, of the factors that go into developing medical, biological and neurodegenerative disease-related probes, meant to help other researchers follow our intention, gain perspective, and overcome current limitations.

A turn-on fluorescence strategy for biothiols determination by blocking Hg(II)-mediated fluorescence quenching of adenine-rich DNA-templated gold nanoclusters.

Fluorescent adenine (A)-rich DNA-templated gold nanoclusters were demonstrated to be a novel probe for determination of biothiols (including cysteine, glutathione, and homocysteine). Fluorescence intensity of adenine-rich DNA-templated gold nanoclusters could be greatly quenched by Hg(II) ions through the formation of a gold nanoclusters-Hg(II) system. When biothiols (cysteine as the model) were introduced into the system, the fluorescence intensity recovered due to the formation of a more stable Hg(II)-thiol coordination complex using Hg-S metal-ligand bonds, which inhibited the Hg(II)-mediated fluorescence quenching of adenine-rich DNA-templated gold nanoclusters. Based on this fluorescence phenomenon, an on-off-on fluorescence strategy was designed for the sensitive determination of biothiols. The method allowed sensitive detection of cysteine with a linear detection range from 100 nM to 5 µM and a limit of detection of 30 nM. Additionally, the assay can be applied for detection of biothiol levels in human plasma samples. Therefore, it can provide a simple and rapid fluorescent platform for biothiol detection.