A spatially-controlled DNA triangular prism nanomachine for AND-gated intracellular imaging of ATP in acidic microenvironment.

A DNA triangular prism nanomachine (TPN)-based logic device for intracellular AND-gated imaging of adenosine triphosphate (ATP) has been constructed. By using i-motif sequences and ATP-binding aptamers as logic control units, the TPN logic device is qualified to respond to the acidic environment and ATP in cancer cell lysosomes. Once internalized into the lysosome, the specific acidic microenvironment in lysosome causes the i-motif sequence to fold into a tetramer, resulting in compression of DNA tri-prism. Subsequently, the split ATP aptamer located at the tip of the collapsed triangular prism binds stably to ATP, which results in the fluorescent dyes (Cy3 and Cy5) modified at the ends of the split aptamer being in close proximity to each other, allowing Förster Resonance Energy Transfer (FRET) to occur. The FRET signals are excited at a wavelength of 543 nm and can be collected within the emission range of 646-730 nm. This enables the precise imaging of ATP within a cell. We also dynamically operate AND logic gates in living cells by modulating intracellular pH and ATP levels with the help of external drugs. Owing to the AND logic unit on TPN it can simultaneously recognize two targets and give corresponding intelligent logic judgment via imaging signal output. The accuracy of molecular diagnosis of cancer can be improved thus eliminating the false positive signal of single target-based detection. Hence, this space-controlled TPN-based logical sensing platform greatly avoids sensitivity to extracellular targets during the cell entry process, providing a useful tool for high-precision imaging of the cancer cell's endogenous target ATP.

Recent Progress of Spectroscopic Probes for Peroxynitrite and Their Potential Medical Diagnostic Applications.

Peroxynitrite (ONOO-) is a crucial reactive oxygen species that plays a vital role in cellular signal transduction and homeostatic regulation. Determining and visualizing peroxynitrite accurately in biological systems is important for understanding its roles in physiological and pathological activity. Among the various detection methods, fluorescent probe-based spectroscopic detection offers real-time and minimally invasive detection, high sensitivity and selectivity, and easy structural and property modification. This review categorizes fluorescent probes by their fluorophore structures, highlighting their chemical structures, recognition mechanisms, and response behaviors in detail. We hope that this review could help trigger novel ideas for potential medical diagnostic applications of peroxynitrite-related molecular diseases.

A fluorescent probe for monitoring sulfite in living cells with large Stokes shift and rapid response.

Sulfite (SO32-) is considered as a monitor of a wide range of physiological processes. However, cells and tissues are adversely affected when the body ingests high level of sulfite. Here, we designed and synthesized a "turn on" fluorescent probe ImiSft-1 with 2-cyano-N-methylacetamide as the specific recognition site of SO32-. This probe predominantly achieved high response intensity to SO32- and desirable properties such as large Stokes shift (∼180 nm), fast response time (within 15 s), and high sensitivity (LOD = 0.12 µM). Importantly, the probe was highly selective for sulfite from other bio-species including biological thiols. Other functional properties included broad pH adaptability (5.0-10.0) and low cytotoxicity. Given these advantages and the fluorescence imaging in living MCF-7 cells, it was demonstrated that probe ImiSft-1 could monitor the changes of sulfite concentration in living cells.

Selective optosensing of iron(III) ions in HeLa cells using NaYF4:Yb3+/Tm3+ upconversion nanoparticles coated with polyepinephrine.

Novel polyepinephrine-modified NaYF4:Yb,Tm upconversion luminescent nanoparticles (UCNP@PEP) were prepared via the self-polymerization of epinephrine on the surfaces of the UCNPs for selective sensing of Fe3+ inside a cell and for intracellular imaging. The proposed UCNP@PEP probe is a strong blue light emitter (λmax = 474 nm) upon exposure to an excitation wavelength of 980 nm. The probe was used for detecting Fe3+ owing to the complexation reaction between UCNP@PEP and Fe3+, resulting in reduced upconversion luminescence (UCL) intensity. The proposed probe has a detection limit of 0.2 µM and a good linear range of 1-10 µM for sensing Fe3+ ions. Moreover, the UCNP@PEP probe displays high cell viability (90%) and is feasible for intracellular imaging. The ability of the probe to sense Fe3+ in a human serum sample was tested and shows promising output for diagnostic purposes. The prepared UCNP@PEP probe was characterized by using UV-visible (UV-Vis) absorption spectrometry, fluorescence (FL) spectrometry, field emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FT-IR).

Multicolor Two-Photon Nanosystem for Multiplexed Intracellular Imaging and Targeted Cancer Therapy.

The novel theranostic nanosystems based on two-photon fluorescence can achieve higher spatial resolution of deep tissue imaging for simultaneous diagnosis and therapy of a variety of cancers. Herein, we have designed and prepared FRET-based two-photon mesoporous silica nanoparticles (MTP-MSNs) for single-excitation multiplexed intracellular imaging and targeted cancer therapy for the first time. This nanosystem includes two constituents, containing (1) multicolor two-photon mesoporous silica nanoparticles and (2) cancer cell-targeting aptamers that act as gatekeepers for MTP-MSNs. After incubation with cancer cells, the Dox-loaded and aptamer-capped MTP-MSNs could be internalized into the cells, opening the pores and releasing the drug. Furthermore, using two-photon multicolor fluorescence, MTP-MSNs could serve as good contrast agents for multicolor two-photon intracellular imaging with increased imaging depth and improved spatial localization of tissue. In sum, these multicolor MTP-MSNs provide a promising system for traceable targeted cancer therapy with further applications in multiplex intracellular imaging and the screening of drug.

A nanoprobe for ratiometric imaging of glutathione in living cells based on the use of a nanocomposite prepared from dual-emission carbon dots and manganese dioxide nanosheets.

A ratiometric fluorescence assay for glutathione (GSH) was developed. The novel assay is based on a nanoprobe composed of manganese dioxide nanosheets (MnO2 NS) and dual-emission carbon dots (de-CDs) with intrinsic GSH-response property. After construction of the nanoprobe, two emission peaks of de-CDs were suppressed to varying degrees by MnO2 NS. The suppression was relieved and the two emission peaks recovered proportionally when MnO2 NS was decomposed by GSH, thus realizing the ratiometric assay for micromolar GSH. The intrinsic responsiveness of de-CDs to millimolar GSH broadens the analytical range of the nanoprobe. An appropriate precursor, calcon-carboxylic acid, was screened out to synthesize de-CDs via one-step hydrothermal treatment. The de-CD@MnO2 NS nanoprobe can measure GSH concentrations through the fluorescence intensity ratio between 435 and 516 nm excited at 365 nm. The range of response was from 1 µM to 10 mM and the detection limit reached 0.6 µM (3σ criterion). Benefiting from its good biocompatibility, the proposed nanoprobe has excellent applicability for intracellular GSH imaging.Graphical abstract Schematic representation of glutathione (GSH) ratiometric detection. The nanoprobe is prepared from dual-emission carbon dots (de-CDs) and manganese dioxide nanosheets (MnO2 NS). GSH removes quenching effect by decomposing MnO2 NS and induces intrinsic response of de-CDs, which realizes ratiometric detection.

Dual-emissive carbon dots for dual-channel ratiometric fluorometric determination of pH and mercury ion and intracellular imaging.

Dual-emissive carbon dots (CDs) were fabricated for dual-channel ratiometric fluorometric determination of pH and mercury ion (Hg2+) and intracellular imaging. Dual-emissive CDs were synthesized by one-pot solvothermal treatment of cabbage. The CDs exhibited two distinctive fluorescence emissions at 500 and 678 nm under single excitation at 410 nm. The green emission (500 nm) had reversible linear response to pH (7.0-12.0) due to deprotonation and protonation of surface functional groups and their non-covalent interactions. On the other hand, the red emission (678 nm) had efficient and selective fluorescence response to Hg2+ by formation of non-emission complex between CDs and Hg2+. The limit of detection (LOD) and limit of quantification (LOQ) for Hg2+ were 6.25 and 20.63 nM, respectively. The CDs have been successfully applied for label-free ratiometric fluorometric determination of pH and Hg2+ in fish and human serum samples with good recoveries (92.0-108.3%). In addition, the CDs had excellent photostability, low cytotoxicity, and good biocompatibility for intracellular imaging. All in all, the system was multi-functional in determination, high in sensitivity, and excellent in selectivity, which demonstrated wide and promising applicability for biosensing and bioimaging in the future. Graphical abstract Schematic presentation of dual-emission carbon dots (CDs) synthesized by solvothermal treatment of cabbage for dual-channel determination of pH and Hg2+.

Amplified FRET Nanoflares: An Endogenous mRNA-Powered Nanomachine for Intracellular MicroRNA Imaging.

It is of great value to detect biological molecules in live cells. However, probes for imaging low-abundance targets in live cells are limited by the one-to-one signal-triggered model. Here, we introduce the concept of the amplified FRET nanoflare, which employs high-abundance endogenous mRNA as fuel strands to amplify the detection of low abundance intracellular miRNA. As far as we know, this is the first report of an endogenous mRNA-powered nanomachine for intracellular molecular detection. We experimentally prove the mechanism of the nanomachine and demonstrate its specificity and sensitivity. The proposed amplified FRET nanoflare can act as an excellent intracellular molecular detection strategy that is promising for biological and medical applications.

A nanohybrid composed of MoS2 quantum dots and MnO2 nanosheets with dual-emission and peroxidase mimicking properties for use in ratiometric fluorometric detection and cellular imaging of glutathione.

A nanohybrid probe was fabricated from manganese dioxide nanosheets (MnO2 NSs), molybdenum disulfide quantum dots (MoS2 QDs) and o-phenylenediamine (OPD) for ratiometric detection of glutathione (GSH) in aqueous solutions and living cells. The MoS2 QDs act as the fluorescent "turn off-on" units. The MnO2 NSs have 3 functions, viz. (a) as fluorescence quencher, (b) as fluorescence initiator for oxidized OPD (ox OPD) and (c) as selective recognizer of GSH. The quenched blue fluorescence of the MoS2 QDs can be restored by introducing GSH that reduces the MnO2 NSs. However, the green fluorescence of ox OPD is decreased through the loss of peroxidase activity of MnO2 NSs in the presence of GSH. Therefore, the ratio of the fluorescence intensities at 560 and 400 nm (from ox OPD and MoS2 QDs, respectively) linearly decreases with increasing concentrations of GSH. Under the optimal conditions, the detection limit for GSH is as low as 90 nM. The method was successfully applied to the determination of GSH in human serum samples. This nanohybrid also is shown to be membrane-permeable and to have low cytotoxicity. This paved the way to intracellular sensing of GSH in living normal HFF and cancerous HeLa cells. Additionally, by combining with logic gate, this assay was successfully applied to visually discriminate changes in the intracellular GSH. The combination of ratiometric fluorometry and peroxidase mimicking can provide a wide range of application in bioanalysis and intracellular imaging. Graphical abstract Schematic representation of the ratiometric fluorometric detection and cellular imaging of glutathione using a nanohybrid composed of MoS2 quantum dots and MnO2 nanosheets with dual (blue and green emission and peroxidase mimicking properties.

In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing.

Over the past two decades, enormous progress has been made in designing fluorescent sensors or probes for divalent metal ions. In contrast, the development of fluorescent sensors for monovalent metal ions, such as sodium (Na(+)), has remained underdeveloped, even though Na(+) is one the most abundant metal ions in biological systems and plays a critical role in many biological processes. Here, we report the in vitro selection of the first (to our knowledge) Na(+)-specific, RNA-cleaving deoxyribozyme (DNAzyme) with a fast catalytic rate [observed rate constant (ko(bs)) ∼ 0.1 min(-1)], and the transformation of this DNAzyme into a fluorescent sensor for Na(+) by labeling the enzyme strand with a quencher at the 3' end, and the DNA substrate strand with a fluorophore and a quencher at the 5' and 3' ends, respectively. The presence of Na(+) catalyzed cleavage of the substrate strand at an internal ribonucleotide adenosine (rA) site, resulting in release of the fluorophore from its quenchers and thus a significant increase in fluorescence signal. The sensor displays a remarkable selectivity (>10,000-fold) for Na(+) over competing metal ions and has a detection limit of 135 µM (3.1 ppm). Furthermore, we demonstrate that this DNAzyme-based sensor can readily enter cells with the aid of α-helical cationic polypeptides. Finally, by protecting the cleavage site of the Na(+)-specific DNAzyme with a photolabile o-nitrobenzyl group, we achieved controlled activation of the sensor after DNAzyme delivery into cells. Together, these results demonstrate that such a DNAzyme-based sensor provides a promising platform for detection and quantification of Na(+) in living cells.

Carbon dots doped with nitrogen and boron as ultrasensitive fluorescent probes for determination of α-glucosidase activity and its inhibitors in water samples and living cells.

An ultrasensitive fluorometric assay is described for the determination of the activity of the enzyme α-glucosidase in waters and living cells. Carbon dots doped with nitrogen and boron (N,B-CDs) were prepared that have excitation/emission peaks at 400/510 nm and a fluorescence quantum yield of 47%. 4-Nitrophenylglucoside is added and then hydrolyzed by α-glucosidase to form yellow 4-nitrophenol which screens off fluorescence due to an inner filter effect. The method was applied to the determination of α-glucosidase activity and has a 3 mU mL-1 detection limit. It was subsequently applied to the determination of the α-glucosidase inhibitor acarbose which can be determined in a concentration as low as 10 nM (at three times the standard deviation versus slope). The method was also applied to the determination of α-glucosidase activity and acarbose in living HeLa cells and MCF-7 cells. The method is simple, sensitive, and excellently selective. Graphical abstract N,B-CDs as ultrasensitive fluorescence probe for α-glucosidase activity and its inhibitor in waters and living cells based on IFE.

A ratiometric fluorescent core-shell nanoprobe for sensing and imaging of zinc(II) in living cell and zebrafish.

A zinc(II)-responsive ratiometric fluorescent core-shell nanoprobe (referred to as QPNPs) is described. It consist of an optimized combination of an internal reference dye (TBAP) encapsulated in the core, and a Zn(II)-specific indicator dye (PEIQ) in the shell. The nanoprobe was synthesized via single-step graft copolymerization induced by tert-butyl hydroperoxide at 80 °C. QPNPs exhibit a well-defined core-shell nanostructure and well-resolved dual emissions after photoexcitation at 380 nm. After exposure to Zn(II), the QPNPs display a green fluorescence peaking at ~500 nm that increases with the concentration of Zn(II), while the pink fluorescence of the porphine-derived reference dye peaking at ~650 nm remains unchanged. This results in color change from pink to green and thus enables Zn(II) to be detected both spectroscopically and with bare eyes. Zn(II) can be quantified with a 3.1 nM detection limit. The core-shell structured nanoprobe was also applied to real-time imaging of Zn(II) in living HeLa cells and in zebrafish. This work establishes a reliable approach to synthesize ratiometric fluorescent nanoprobes. It enables such nanoprobes to be prepared also by those not skilled in nanomaterial synthesis. Graphical abstract A zinc(II)-responsive core-shell nanoprobe (referred to as QPNP) is synthesized via single-step graft copolymerization. Zn(II) can be quantitated with a 3.1 nM detection limit by the QPNPs through ratiometric fluorescence strategy (PEIQ as the Zn(II) indicator and TBAP as the reference dye).

Detection of Copper(II) Ions Using Glycine on Hydrazine-Adsorbed Gold Nanoparticles via Raman Spectroscopy.

A facile, selective, and sensitive detection method for the Cu2+ ions in environmental and biological solutions has been newly developed by observing the unique CN stretching peaks at ~2108 cm-1 upon the dissociative adsorption of glycine (GLY) in hydrazine buffer on gold nanoparticles (AuNPs). The relative abundance of Cu species on AuNPs was identified from X-ray photoelectron spectroscopy analysis. UV-Vis spectra also indicated that the Au particles aggregated to result in the color change owing to the destabilization induced by the GLY-Cu2+ complex. The CN stretching band at ~2108 cm-1 could be observed to indicate the formation of the CN species from GLY on the hydrazine-covered AuNP surfaces. The other ions of Fe3+, Fe2+, Hg2+, Mg2+, Mn2+, Ni2+, Zn2+, Cr3+, Co2+, Cd2+, Pb2+, Ca2+, NH4⁺, Na⁺, and K⁺ at high concentrations of 50 µM did not produce such spectral changes. The detection limit based on the CN band for the determination of the Cu2+ ion could be estimated to be as low as 500 nM in distilled water and 1 µM in river water, respectively. We attempted to apply our method to estimate intracellular ion detection in cancer cells for more practical purposes.

New Dual Fluorescent Probe for Simultaneous Biothiol and Phosphate Bioimaging.

The simultaneous detection of relevant metabolites in living organisms by using one molecule introduces an approach to understanding the relationships between these metabolites in healthy and deregulated cells. Fluorescent probes of low toxicity are remarkable tools for this type of analysis of biological systems in vivo. As a proof of concept, different naturally occurring compounds, such as biothiols and phosphate anions, were the focus for this work. The 2,4-dinitrobenzenesulfinate (DNBS) derivative of 9-[1-(4-tert-butyl-2-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (Granada Green; GG) were designed and synthesized. This new sulfinyl xanthene derivative can act as a dual sensor for the aforementioned analytes simultaneously. The mechanism of action of this derivative implies thiolysis of the sulfinyl group of the weakly fluorescent DNBS-GG by biological thiols at near-neutral pH values, thus releasing the fluorescent GG moiety, which simultaneously responds to phosphate anions through its fluorescence-decay time. The new dual probe was tested in solution by using steady-state and time-resolved fluorescence and intracellularly by using fluorescence-lifetime imaging microscopy (FLIM) in human epithelioid cervix carcinoma (HeLa) cells.

Gold nanoparticles explore cells: cellular uptake and their use as intracellular probes.

Understanding uptake of nanomaterials by cells and their use for intracellular sensing is important for studying their interaction and toxicology as well as for obtaining new biological insight. Here, we investigate cellular uptake and intracellular dynamics of gold nanoparticles and demonstrate their use in reporting chemical information from the endocytotic pathway and cytoplasm. The intracellular gold nanoparticles serve as probes for surface-enhanced Raman spectroscopy (SERS) allowing for biochemical characterisation of their local environment. In particular, in this work we compare intracellular SERS using non-functionalised and functionalised nanoparticles in their ability to segregate different but closely related cell phenotypes. The results indicate that functionalised gold nanoparticles are more efficient in distinguishing between different types of cells. Our studies pave the way for understanding the uptake of gold nanoparticles and their utilisation for SERS to give rise to a greater biochemical understanding in cell-based therapies.

Fluorescent Sensors for Tetracycline Detection in Aqueous Medium: A Mini-Review.

Tetracycline (TC) is a commonly used antibiotic in human therapy and animal husbandry. Public concerns about TC residues inflated due to their negative impact on the environment, food, and human health concerns. To ensure human health and safety, there is a need for fluorogenic chemosensors that can easily detect TC antibiotics with high selectivity and sensitivity in the aqueous medium. This mini-review discusses the progress and achievements in several fluorometric antibiotic tetracycline detection methods. Fluorogenic chemosensors for tetracycline antibiotics with easy-to-use, high selectivity, and sensitivity have been essentially required to regulate food safety and secure human health and safety. Moreover, we gave more attention to the practical applicability of chemosensors for tetracycline antibiotics in food and water quality assessment. This article starts with a section that constitutes an overview of the problems of antibiotics and the typical features of traditional techniques of antibiotic detection. It then goes on to describe up-to-date optical methods for the selective detection and efficient removal of TC. These methods involve a variety of platforms, like tetraphenylethylene polymers, metal complexes, self-assembled CuNCs, and hydrogel. The article also discusses the practical applicability of chemosensors for tetracycline antibiotics in food and water quality.

Dual-functionalization of fluorescent carbon dots via cyclodextrin and aminosilane for visual detection of ß-glucuronidase and bioimaging.

A sensitive method for the detection of ß-glucuronidase was established using functionalized carbon dots (ß-CD-SiCDs) as fluorescent probes. The ß-CD-SiCDs were found to be obtained through in situ autopolymerization by mixing the solutions of methyldopa, mono-6-ethylenediamine-ß-cyclodextrin and N-(ß-aminoethyl)-γ-aminopropyltrimethoxysilane at room temperature. The method has the characteristics of low energy consumption, simple and rapid. ß-CD-SiCDs exhibited green fluorescence at 515 nm emission with a quantum yield of 7.9 %. 4-nitrophenyl-ß-D-glucuronide was introduced as a substrate for ß-glucuronidase to generate p-nitrophenol. Subsequently, p-nitrophenol self-assembled with ß-CD-SiCDs through host-guest recognition to form a stable inclusion complex, resulting in the fluorescence quenching of ß-CD-SiCDs. The linear range of ß-CD-SiCDs for detecting ß-glucuronidase activity was 0.5-60 U L-1 with a detection limit of 0.14 U L-1. For on-site detection, gel reagents were prepared by a simple method and the images were visualized and quantified by taking advantage of smartphones, avoiding the use of large instrumentation. The constructed fluorescence sensing platform has the benefits of easy operation and time saving, and has been successfully used for the detection of ß-glucuronidase activity in serum and cell imaging.

Development of a fluorometric and colorimetric dual-mode sensing platform for acid phosphatase assay based on Fe3+ functionalized CuInS2/ZnS quantum dots.

BACKGROUND: The spectral dual-mode response towards analyte has been attracted much attention, benefiting from the higher detection accuracy of such strategy in comparison to single signal readout. However, the currently reported dual-mode sensors for acid phosphatase (ACP) activity are still limited, and most of them more or less exist some deficiencies, such as complicated construction procedure, high-cost, poor biocompatibility, aggregation-caused quenching and limited emission capacity. RESULTS: Herein, we employed Fe3+ functionalized CuInS2/ZnS quantum dots (CIS/ZnS QDs) as nanosensor to develop a novel fluorometric and colorimetric dual-mode assay for ACP activity, combing with ACP-triggered hydrolysis of ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA). The Fe3+ binding to CIS/ZnS QDs can be reduced into Fe2+ during the determination, resulting in the dramatically weakened photoinduced electron transfer (PET) effect and the disappearance of competition absorption. Thus, a highly sensitive ACP assay in the range of 0.22-12.5 U L-1 through fluorescence "turn-on" mode has been achieved with a detection of limit (LOD) of 0.064 U L-1. Meanwhile, the ACP activity can also be quantified by spectrophotometry based on the chromogenic reaction of the formed Fe2+ with 1,10-phenanthroline (Phen). Moreover, the designed nanosensor with good biocompatibility was successfully applied to image and monitor the ACP levels in living cells. SIGNIFICANCE: We believe that the proposed method has remarkable advantages and potential application for ACP assay in terms of the high accuracy, simplicity, low cost, as well as its adequate sensitivity.

A selective fluorescent probe for hydrogen sulfide from a series of flavone derivatives and intracellular imaging.

In this work, through the orthogonal design of two fluorophores and two recognition groups, a series of fluorescent probes were developed from the flavone derivatives for hydrogen sulfide (H2S). The probe FlaN-DN stood out from the primarily screening on the selectivity and response intensities. It could respond to H2S with both the chromogenic and fluorescent signals. Among the recent reported probes for the H2S detection, FlaN-DN indicated the most highlighted advantages including the rapid response (within 200 s) and the high response multiplication (over 100 folds). FlaN-DN was sensitive to the pH condition, thus could be applied to distinguish the cancer micro-environment. Moreover, FlaN-DN suggested practical capabilities including a wide linear range (0-400 µM), a relatively high sensitivity (limit of detection 0.13 µM), and high selectivity towards H2S. As a low cytotoxic probe, FlaN-DN achieved the imaging in living HeLa cells. FlaN-DN could detect the endogenous generation H2S and visualize the dose-dependent responses to the exogenous H2S level. This work provided a typical case of natural-sourced derivatives as functional implements, which might inspire the future investigations.

Photophysical Study of Heteroatom-Doped Carbon Dots-MnO2-Based Nanosensor: Selective Detection of Glutathione in the Nanomolar Level.

Heteroatom doping on carbon dots (Cdots) has been developed as an efficient approach to modify its optical and electronic properties. The four different types of heteroatom-doped Cdots (undoped Cdots (u-Cdots, nitrogen-doped Cdots (N-Cdots), sulfur-doped Cdots (Cdots), nitrogen, sulfur codoped Cdots (N, S-Cdots)) have been synthesized through a simple heat treatment of 5 min. Among four different heteroatoms doped nanosensors, N, S-Cdots with MnO2 nanospheres (Mn NS) showed one of the best fluorescents "on-off-on" nanosensors for selective sensing of glutathione (GSH) and cell imaging. N, S-Cdots showed a high fluorescence quantum yield, good photostability, ionic strength, and pH stability. N, S-Cdots with Mn NS demonstrated extremely high fluorescence quenching efficiency and the maximum fluorescence recovery rate after adding GSH to the produced solution. The photophysical study of N, S-Cdots-Mn NS used as a sensor confirms the inner filter effect (IFE) quenching mechanism between them. The developed sensor has an 80 nM limit of detection (LOD) for GSH. The heteroatom-doped framework of Cdots plays a significant role in the sensitive detection of GSH. N, S-Cdots-Mn NS have good permeability, biocompatibility, and low toxicity, due to which it was used in the intracellular imaging of GSH in living cells. The prepared sensor is rapid, economical, less toxic, and highly applicable in diagnosing diseases.