Fluorescence Quenching Research Articles

Development of an assay to quantify tranexamic acid levels in plasma

Dysregulations of blood clot breakdown (fibrinolysis) during vascular trauma can lead to excessive blood loss. Tranexamic acid (TXA) is an inhibitor of fibrinolysis that works by blocking the interaction between plasminogen and fibrin degradation products (FDPs) – a key step in fibrinolysis. Despite the widespread usage, there are no tests available in a clinical setting to monitor TXA levels. We developed a fluorescence resonance energy transfer (FRET)-based assay to quantify TXA concentrations in plasma by using 1) fluorescently labeled plasminogen, and 2) FDPs labeled with a fluorescence quencher. Once plasminogen binds the FDPs, the fluorescent signal is quenched. TXA causes plasminogen to dissociate from the FDPs, thus increasing fluorescence signal in a dose-dependent manner. The dose response was sensitive between 1 and 100 μM (0.16 and 15.7 mg/L). The intraassay and interassay variabilities were determined to be 5.7 % and 3.0 %, respectively. Limit of detection was estimated to be 0.28 μM (0.044 mg/L). When tested for measuring known levels of TXA added to plasma samples, the ratio between measured and expected TXA concentration was 1.0151. Our study demonstrates a novel assay that can rapidly quantify TXA concentrations in plasma samples, thus demonstrating its potential as an in-hospital tool.

Fluorescent primers amplification refractory mutation system qPCR (FP ARMS-qPCR) for MTHFR C677T SNP genotyping.

The currently used methods for the detection of methylene tetrahydrofolic acid reductase (MTHFR) C677T single nucleotide polymorphism (SNP) are either time-consuming or expensive. In this study, we devised an accurate, rapid and easy-to-use SNP detection system based on fluorescent primers amplification refractory mutation system qPCR, known as FP ARMS-qPCR. Fluorescent primers (FPs) modified by fluorescent dye or quencher near the 3' terminal thymine were designed. The reaction conditions were optimized and the performance was evaluated. Using commercial kits as controls, 242 samples were tested in parallel to verify the feasibility of the FP ARMS-qPCR assay. We demonstrated the good sensitivity and specificity of the FP ARMS-qPCR with optimized conditions. The assay was able to accurately distinguish between different SNP sites of MTHFR C677T in less than 2h using as low as 50 pg of template genomic DNA. Completely consistent genotyping results reveal that FP ARMS-qPCR is concordant with commercial kits. We established a specific, sensitive, and rapid FP ARMS-qPCR method for the detection of MTHFR C677T genotype. This could also serve as a potential diagnostic tool for a variety of diseases.

Design of a Duplex-to-Complex Structure-Switching Approach for the Homogeneous Determination of Marine Biotoxins in Water

In this report, we describe a fluorescent assay for the detection of six marine toxins in water. The mechanism of detection is based on a duplex-to-complex structure-switching approach. The six aptamers specific to the targeted cyanotoxins were conjugated to a fluorescent dye, carboxyfluorescein (FAM). In parallel, complementary DNA (cDNA) sequences specific to each aptamer were conjugated to a fluorescence quencher BHQ1. In the absence of the target, an aptamer–cDNA duplex structure is formed, and the fluorescence is quenched. By adding the toxin, the aptamer tends to bind to its target and releases the cDNA. The fluorescence intensity is consequently restored after the formation of the complex aptamer–toxin, where the fluorescence recovery is directly correlated with the analyte concentration. Based on this principle, a highly sensitive detection of the six marine toxins was achieved, with the limits of detection of 0.15, 0.06, 0.075, 0.027, 0.041, and 0.026 nM for microcystin-LR, anatoxin-α, saxitoxin, cylindrospermopsin, okadaic acid, and brevetoxin, respectively. Moreover, each aptameric assay showed a very good selectivity towards the other five marine toxins. Finally, the developed technique was applied for the detection of the six toxins in spiked water samples with excellent recoveries.

Ligustilide improves functional constipation by non-covalently activating TRPA1 in colon tissue

Ethnopharmacological relevanceAngelica sinensis (Oliv.) Diels (AS), a medicinal plant renowned for its constipation-relieving properties, lacks comprehensive studies on its active pharmaceutical ingredients (APIs) and underlying mechanisms. In the gastrointestinal tract, TRP channels enhance colonic mucus secretion, expedite intestinal motility, and regulate gastrointestinal hormones; however, few reports have systematically established the relationship between TRPs and ligustilide (Lig), a key API of AS. Aim of the studyThis study aimed to explore the pharmacodynamic properties of AS in alleviating functional constipation, assess the potential of Lig for activating TRPs, and elucidate its mechanism of action. MethodsThe therapeutic efficacy of AS was assessed in a mouse model of loperamide hydrochloride-induced functional constipation. The APIs were screened via integrated activity-based UPLC profiling through periodic acid-Schiff (PAS) staining of the colon and immunofluorescence staining of HT-29 cells. The potential target was identified via target fishing and colocalization imaging via an alkynyl-modified Lig probe (AM-Lig). Molecular docking, microscale thermophoresis (MST), fluorescence quenching (FQ), and Fluo-4/Ca2+ influx assays were employed to reveal the interaction mode between Lig and the target protein. Finally, we assessed the efficacy of Lig in alleviating constipation in an animal model. ResultsThe efficacy of AS in improving functional constipation was demonstrated in a mouse constipation model, with Lig identified as the primary constituent responsible for inducing colon mucus secretion. Lig specifically targets TRPA1 in the colon, leading to calcium influx and subsequent mucus secretion, ultimately ameliorating functional constipation. Furthermore, a binding mode study revealed that Lig attaches to Thr684, located in the pre-S1 region, triggering TRPA1 channel activation. ConclusionsOur findings demonstrate that Lig, the API in AS for constipation treatment, activates TRPA1 through non-covalent interactions, increasing mucus secretion and improving functional constipation. These findings advance our understanding of the therapeutic mechanism of AS and Lig on constipation and suggest a new approach for developing TRPA1 agonists.

Validated Spectrofluorometric Assay for Folic Acid Determination in Pure and Pharmaceutical Forms via Basic Fuchsin Fluorescence Quenching.

A simple, specific, sensitive, rapid, and cost-effective spectrofluorometric method for quantifying folic acid has been developed using basic fuchsin as a fluorescence quenching agent. This method relies on the quenching effect of folic acid on the inherent fluorescence of basic fuchsin, facilitated by their interaction in Britton-Robinson buffer solution at pH 9, leading to the formation of an ion-associated complex. The decrease in fluorescence intensity of basic fuchsin was measured at 730 nm, with excitation at 365 nm. The method demonstrated a linear relationship (R2 = 0.9977) between the quenching fluorescence intensity (ΔF) and folic acid concentration over the range of 4-20 μg/mL, with a detection limit of 0.17 μg/mL. The method showed no significant interference from common excipients found in pharmaceutical tablets. The results obtained were in good agreement with those from high-performance liquid chromatography, with no significant differences in precision or accuracy. This spectrofluorimetric method was successfully applied to determine the folic acid content in various commercial pharmaceutical tablets.

A DNA Aptamer for 2-Aminopurine: Binding-Induced Fluorescence Quenching.

2-Aminopurine (2AP) is a fluorescent analog of adenine, and its unique properties make it valuable in various biochemical and biotechnological applications. Its fluorescence property probes local dynamics in DNA and RNA because stacking with the surrounding bases quench its fluorescence. 2AP-labeled DNA or RNA sequences have been used for the detection of genetic mutations, viral RNA, or other nucleic acid-based markers associated with diseases like cancer and infectious diseases. In this study, we isolated aptamers for 2AP using the library immobilization capture-SELEX technique. A dominating aptamer family was isolated after 15 rounds of selection. The Kd values for the most abundant 2AP1 aptamer are 209 nM in a fluorescence assay and 72 nM in an isothermal titration calorimetry test. A 32 nM 2AP limit of detection was tested based on its intrinsic fluorescence change upon aptamer binding. Additionally, we conducted some mutation analysis. Furthermore, we tested the selectivity of this aptamer and discovered that it can bind adenine and adenosine with approximately 100-fold lower affinity than 2AP.

Novel metal–organic framework biosensing platform for detection of COVID-19 RNA

The latest pandemic resulting from the novel SARS-CoV-2 coronavirus has significantly affected public health, the worldwide economy, and social life. Metal organic frameworks (MOFs) are currently being implemented in biosensors for rapid and accurate detection of viruses thanks to their exceptional properties. This research aims to develop a Zeolitic Imidazolate Framework-8 (ZIF-8) based fluorescent biosensor for facile and rapid COVID-19 RNA sequence detection. ZIF-8 was characterized using several tests, such as FT-IR, TGA, and PXRD, to examine the MOF’s crystalline structure and thermal stability. The results demonstrated high crystallinity and thermal stability up to a temperature of 550 °C. The experimental study showed that ZIF-8 is an excellent fluorescence quencher, with 78.39% quenching efficiency. Analyzing the adsorption mechanism of probe DNA into ZIF-8 revealed that they can form electrostatic and π–π stacking interactions, forming a P-DNA@ZIF-8 complex and that PET is more dominant than FRET in the quenching mechanism. This ZIF-8 biosensing platform showed high sensitivity towards COVID-19 RNA with an ultra-low limit of detection of 6.24 pM, a rapid detection time of 8 min, and high selectivity to COVID-19 RNA. Indeed, ZIF-8 experienced much lower fluorescence recovery when tested on two mismatched RNAs. The experimental results show the potential use of ZIF-8 as a novel biosensor for a rapid and sensitive COVID-19 diagnosis.

Evaluating the Fluorescence Quenching of Troxerutin for Commercial UV Sunscreen Filters

Evaluating the Fluorescence Quenching of Troxerutin for Commercial UV Sunscreen Filters

Induction of Ca2+ signaling and cytotoxic responses of human lung fibroblasts upon an antihistamine drug oxatomide treatment and evaluating the protective effects of Ca2+ chelating.

Oxatomide, an antihistamine drug of the diphenylmethylpiperazine family, has anti-inflammatory effects in airway disease. Because oxatomide was shown to cause diverse physiological responses in several cell models, the impact of oxatomide on Ca2+ signaling and its related physiological effects has not been explored in IMR-90 human fetal lung fibroblasts. This study assessed the effect of oxatomide on cell viability and intracellular free Ca2+ concentrations ([Ca2+]i) and examined whether oxatomide-induced cytotoxicity through Ca2+ signaling in IMR-90 cells. Cell viability was measured by the cell proliferation reagent (WST-1). [Ca2+]i was measured by the Ca2+-sensitive fluorescent dye fura-2. Oxatomide (10-40 μM) concentration dependently reduced cell viability and induced [Ca2+]i rises in IMR-90 cells. This cytotoxic effect was reversed by chelation of cytosolic Ca2+ with BAPTA-AM. In terms of Ca2+ signaling, oxatomide-caused Ca2+ entry was inhibited by modulators of store-operated Ca2+ channels (2-APB and SKF96365) and protein kinase C (PKC) inhibitor (GF109203X). Furthermore, oxatomide-induced Ca2+ influx was confirmed by Mn2+-induced quench of fura-2 fluorescence. In a Ca2+-free medium, preincubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin inhibited oxatomide-evoked [Ca2+]i rises. Conversely, treatment with oxatomide abolished thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 also inhibited oxatomide-caused [Ca2+]i rises. In IMR-90 cells, oxatomide-induced cytotoxicity by preceding [Ca2+]i rises involving PKC-sensitive store-operated Ca2+ entry and PLC-dependent Ca2+ release from the endoplasmic reticulum. BAPTA-AM, with its Ca2+ chelating effects, may be a potential compound for preventing oxatomide-induced cytotoxicity.

Multifunctional Au–Ag NCs for luminescence and colorimetric double signal sensing of H2S and catalytic reduction of nitrophenol

In this study, the N-Acetyl-l-Cysteine (NAC)-capped gold and silver bimetallic nanoclusters (NAC@Au–Ag NCs) was synthesized by reflux method. Due to the silver effect, the NAC@Au–Ag NCs exhibited strong photoluminescence at far-red/near-infrared regions and better catalytic performance than Au or Ag NCs. Upon addition of H2S, the Au–Ag NCs exhibited obvious fluorescence quench and color changes through the generation of metal sulfides and a static quenching process. The Au–Ag NCs displayed a wide linear luminescence response for H2S (18.5–217 μmol/L) with a detection limit of 0.269 μmol/L. Moreover, the visible color of Au–Ag NCs changed from white to brown-yellow along with increased H2S, the corresponding RGB values also displayed good linearity with the concentration of H2S (90.1–678 μmol/L). Notably, the fabrication of test strips provided a convenient and intuitive tool to screen the freshness of eggs by the color change of test strips. Au–Ag NCs could be used for living HeLa cells bioimaging and recognition of H2S abnormalities. Furthermore, it can be used as a catalyst to reduction of nitrophenols (NPhs), specific included 2NP, 3NP and 4NP. The reaction was regarded as a pseudo-first-order kinetic reaction due to the presence of excess NaBH4. At 298K, the catalytic rate constants(k) of 2NP, 3NP and 4NP were 0.1754 min−1, 0.1734 min−1 and 0.2782 min−1, respectively. The NAC@Au–Ag NCs catalyst still showed good catalytic activity and reusability after five cycles. Therefore, this study developed a H2S sensor for food samples and biological systems. And this nanocatalyst had great application potential for removed the nitrophenol pollutants in water.

Construction of fluorescent aptasensor based on graphene oxide for the detection of metronidazole in food

Aiming at the detection of metronidazole (MNZ) residues in food, a high sensitivity fluorescent aptasensor was designed, which carboxyfluorescein (FAM)-labeled nucleic acid aptamer (FAM-MNZ) as the core probe and graphene oxide (GO) as an efficient fluorescence quencher. The principle is that GO adsorbs the aptamer onto the surface, causing short-range contact between the fluorescent group of the aptamer and GO. Due to its sp2 hybridized domains, GO undergoes fluorescence resonance energy transfer (FRET). Thus, the fluorescence is quenched. When MNZ is added, it preferentially forms a stable complex with the aptamer without FRET. Under optimized conditions, milk samples can be accurately detected with a detection limit of 0.033 μM. In addition, molecular docking, lumo electron orbital simulations, and experimental methods were used to explore the effect of different average radial sizes of single-layer GO on sensing performance. It is shown that GO with a larger average size makes it easy for aptamers to be regularly arranged on its surface. This also provides a basis for electron transfer, resulting in higher fluorescence quenching ability through the FRET mechanism. Therefore, the constructed fluorescent aptamer sensor has a significant application value in food detection.

A near-infrared fluorescent probe with a large Stokes shift for the detection and imaging of biothiols in vitro and in vivo.

In this study, a new near-infrared (NIR) fluorescent turn-on probe featuring a large Stokes shift (198nm) was developed for the detection of biothiols. The probe was based on a dicyanoisophorone derivative serving as the fluorophore and a 2,4-dinitrobenzenesulfonyl (DNBS) group functioning as both a recognition site and a fluorescence quencher. In the absence of biothiols, the fluorescence of the probe was low due to the photoinduced electron transfer (PET) effect between the fluorophore and DNBS. Upon the presence of biothiols, the DNBS group underwent a nucleophilic aromatic substitution reaction with the sulfhydryl group of biothiols, leading to the release of the fluorophore and a notable emission peak at 668nm. This developed probe exhibited exceptional selectivity and sensitivity to biothiols in solution, with an impressive detection limit of 28nM for cysteine (Cys), 22nM for homocysteine (Hcy), and 24nM for glutathione (GSH). Furthermore, the probe demonstrated its applicability by successfully visualizing both endogenous and exogenous biothiols in living systems.

Bilayer MOF nanomachine for precision breast cancer cell fluorescent imaging and therapy.

Abilayer MOF reporter (ZIF-67@FAM-mRNA@ZIF-8) was synthesized, and the ZIF-67 was used as a carrier and fluorescent quencher to connect the FAM reporter through electrostatic adsorption and coordination effect. The ZIF-8 covering the outer layer can improve the stability of the probe and cell permeability, which helps the FAM reporter effectively release. After entering the cancer cells, the acidic environment in the cells induced the decomposition of ZIF-8. The excess ATP in the tumor cells competitively binds ZIF-67, causing the FAM reporter to shed and restore fluorescence. The shed FAM reporter was specifically bound to the overexpressed miRNA-21 in breast cancer cells to achieve fluorescence imaging and therapy of breast cancer. The results of specific imaging and apoptosis experiments of breast cancer cells indicate that bilayer MOF nanomachine provides an effective nanotherapy platform for accurate fluorescence imaging.

Identification of S-phenylmercapturic acid using heterometallic Zn–Eu MOF as a fluorescence sensor

S-phenylmercapturic acid (S-PMA) can be served as an important biomarker for detecting benzene metabolites in clinical diagnosis. In this work, a 3D luminescent heterometal-organic framework (HMOF) with two kinds of pores was designed and synthesized by choosing d/f heterometals salts and two ligands with abundant coordination sites, {[EuZn2(imdc)2(bpe)(H2O)3(OH)]·bpe}n (H3imdc = 1H-imidazole-4,5-dicarboxylic acid; bpe = 1,2-bis (4-pyridyl)-ethylene), namely, MOF 1, which could be utilized to selectively recognize S-PMA by the fluorescence quench. It demonstrated that an efficient recoverable luminescent probe MOF 1 had determined S-PMA with excellent sensitivity (the limit of detection = 3.0 × 10–8 M), rapid response time (less than 20 s), and a wide linear concentration range (3.70 ∼ 180 µM). More interestingly, this is the first instance of utilizing a luminescent MOF for the detection of S-PMA. A low-cost portable composite film for the convenient detection of S-PMA had been designed to recognize S-PMA by visual changes in luminescent color. In addition, the fluorescence sensing mechanism of S-PMA by MOF 1 was elaborated.

Portable multimodal platform with carbon nano-onions as colorimetric and fluorescent signal output for trypsin detection

Multimodal biosensors with independent signaling pathways can self-calibrate and improve the reliability of disease biomarker detection. Herein, a colorimetric-fluorescent dual-mode paper-based biosensor with PAN/Fe(III)–CNOs (FPCs) as core components has been developed, which information is recognized by smartphone and naked eye. Using 1-(2-pyridylazo)-2-naphthol (PAN) as a mediator, Fe(III) is enriched on the surface of carbon nano-onions (CNOs), endowing FPCs with excellent mimetic enzyme activity and photothermal conversion ability, which allows it to output amplified colorimetric signals under laser irradiation. In addition, the complexation of PAN with Fe(III) broadens its absorption spectrum, which makes FPCs more suitable to be energy acceptors to quench fluorescence of polymer dots (Pdots), resulting in the changes of output fluorescent signal. Based on the above design, a portable colorimetric-fluorescent dual-mode biosensor is proposed for trypsin detection with Pdots as fluorescence sources and FPCs as fluorescence quenchers and nanoenzymes. This work provides a convenient way for constructing portable visual multimodal biosensors, which is expected to applied in various disease diagnosis.

The effect of metal-to-ligand charge transfer on linear and nonlinear optical properties of hexaphyrin and metallo-hexaphyrins

Charge transfer from metal to ligand has implications in various fields of chemistry, physics and biology. The manipulation of spin properties would be necessary for controlling charge transfer in spintronics and biological applications. Upon binding to the metal atoms (Ni, Cu, Au, etc.), the spin properties would be modulated in organic conjugated molecules of less intrinsic spin–orbit coupling (SOC) due to SOC coming from the heavy elements. Emission spectra calculation is necessary to validate them as suitable fluorescence quenchers. The nonlinear optical properties like first-order hyperpolarisability β and second-order hyperpolarisability γ calculations are important to find out their relation between fluorescence quenching and higher order nonlinear optical (NLO) properties. From the second-order NLO properties, we computed degenerate four-wave mixing (DFWM) component ( γ ( 2 ) ( − ω ; ω , ω , − ω )) and finally quadratic nonlinear refractive indices of the expanded porphyrins (EPs). How the linear and nonlinear optical properties are dependent on the SOC coming from the heavy materials (Ni, Cu, Au), and charge transfer process is the most interesting question to be addressed for their potential uses for various optoelectronic and spintronic applications.

Interplay between the Glass Transition Temperature, Analyte Diffusion, and Fluorescence Quenching for Detection of Nitro-Group Containing Explosives Using Organic Semiconducting Films.

Efficient detection of chemical analytes using fluorescence-based sensors necessitates an in-depth understanding of the physical interaction between the analyte molecules and the sensor films. This study explores the interplay between the thermal properties of a series of triphenylamine-centered fluorescent dendrimers with different glass transition temperatures (Tg) for detecting nitroaromatic explosives. When exposed to 4-nitrotoluene (pNT) vapors, biphasic diffusion kinetics were observed for all the dendrimers, corresponding to Super Case II kinetics, suggesting rapid film swelling during initial analyte uptake. The diffusion kinetics were further analyzed using a diffusion-relaxation model, where a strong Tg dependence was observed for both the initial concentration-driven diffusion phase and the slower film relaxation phase. Additionally, a difference in kinetics between analyte uptake and release was observed. The photoluminescence (PL) kinetics also showed a Tg dependence, with more efficient PL recovery observed for films composed of dendrimers that had a lower Tg. Rapid quenching of over 40% with little PL recovery was seen in the dendrimer with the highest Tg (107 °C), while a smaller quench with efficient PL recovery was observed in the dendrimer that had a Tg close to room temperature. The results highlight the critical role of the thermal properties of sensor films in achieving rapid and sensitive detection.

Tannic acid/non-covalent interaction-mediated modification of hemp seed proteins: Focused on binding mechanisms, oil-water interface behavior, and rheological properties

In this study, hemp seed protein (HSP) was treated to prepare a hemp seed protein-tannic acid non-covalent complex (Nov-HSP). To obtain an emulsifier with excellent properties at the oil-water interface and to propose a potential mechanism of action. The results showed that the α-helix and β-sheet content of Nov-HSP decreased and the intensity of the UV absorption peak increased, respectively, compared with that of HSP. The positions of the maximum fluorescence emission peaks of the synchronized fluorescence spectra were red-shifted to 301.8 nm and 340.6 nm, respectively. Fluorescence quench analysis revealed that static quench occurred between tannins and HSP, with ΔH of −16.138 kJ/mol and ΔS of −0.1073 kJ/mol. The physicochemical and functional properties of HSP and Nov-HSP were analyzed, and it was found that the volume mean diameter of Nov-HSP was 449.28 nm, the absolute value of zeta potential was 20.52 mV, and the solubility was 33.42% at a tannic acid concentration of 1.0 mg/mL. Focusing on the oil-water interfacial properties of Nov-HSP as an emulsifier, it was found that having non-covalent binding effectively increased the diffusion rate of HSP at the interface, thus enhancing the rheological properties of the emulsion. And the emulsification activity index and emulsion stability index were improved to 71.67 m2/g and 32.01 min. These findings provide new perspectives for the phenolic compound-mediated structural remodeling of HSP, expanding its application in emulsion food production.

Fluorescence Quenching for Determination of Catalyst Concentration in the Parahydrogen‐Induced Polarization Method SABRE

AbstractIn recent years, parahydrogen‐induced hyperpolarization has become a focus for future medical applications. Similar to the established dynamic nuclear polarization method, a biocompatible bolus from a hyperpolarized sample can be produced for in vivo studies. However, this requires removing toxic hydrogenation catalysts, which inevitably must be used. Additionally, the ratio between the substrate to be hyperpolarized (e. g. pyruvate) and a mandatory catalyst must also be maintained. Even the smallest differences can lead to a reduction in generated signal amplification. In particular, weighing small amounts of catalysts leads to inaccuracies in sample preparations. Fluorescence spectroscopy provides a rapid and sensitive enough approach to determine catalyst concentration. The Ir‐IMes metal complexes used in SABRE lead to a quenching of the fluorescence of the solvent, dependent on its concentration. This can be used to quickly estimate the actual concentration in a solution with very small quantities of catalysts. Hence, fluorescence spectroscopy offers a rapid and reliable quality control method for the preparation of samples to be hyperpolarized. In addition, it can also be used as a quality control method to assess filtration efficacy before administration of hyperpolarized samples.

Fluorescence Quenching Effect of a Highly Active Nitroxyl Radical on 7-amino-4-methylcoumarin and Glutathione Sensing.

Nitroxyl radical compounds, such as 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), are stable radical compounds with a variety of unique characteristics, including fluorescence quenching. In this study, we investigated the fluorescence quenching effect of nortropine N-oxyl (NNO), which is a highly active nitroxyl radical that is more active than TEMPO in oxidation catalysis. The fluorescence intensity of 7-amino-4-methylcoumarin (AMC) was quenched by NNO and TEMPO to 5% and 95% of the initial fluorescence intensity, respectively, indicating highly efficient quenching by NNO. In addition, we used this reaction to measure glutathione concentration. The quenching effect of NNO was abrogated by the chemical reaction with glutathione, resulting in restoration of AMC fluorescence. This response was observed at glutathione concentrations from 10 µM to 1 mM, and good calibration curves were obtained from 10 to 250 µM.