Applications of 3D organoids in toxicological studies: a comprehensive analysis based on bibliometrics and advances in toxicological mechanisms.

A mechanism exploration is an important part of toxicological studies. However, traditional cell and animal models can no longer meet the current needs for in-depth studies of toxicological mechanisms. The three-dimensional (3D) organoid derived from human embryonic stem cells (hESC) or induced pluripotent stem cells (hiPSC) is an ideal experimental model for the study of toxicological effects and mechanisms, which further recapitulates the human tissue microenvironment and provides a reliable method for studying complex cell-cell interactions. This article provides a comprehensive overview of the state of the 3D organoid technology in toxicological studies, including a bibliometric analysis of the existing literature and an exploration of the latest advances in toxicological mechanisms. The use of 3D organoids in toxicology research is growing rapidly, with applications in disease modeling, organ-on-chips, and drug toxicity screening being emphasized, but academic communications among countries/regions, institutions, and research scholars need to be further strengthened. Attempts to study the toxicological mechanisms of exogenous chemicals such as heavy metals, nanoparticles, drugs and organic pollutants are also increasing. It can be expected that 3D organoids can be better applied to the safety evaluation of exogenous chemicals by establishing a standardized methodology.

Mitophagy and its regulatory mechanisms in the biological effects of nanomaterials.

Mitophagy is a selective cellular process critical for the removal of damaged mitochondria. It is essential in regulating mitochondrial number, ensuring mitochondrial functionality, and maintaining cellular equilibrium, ultimately influencing cell destiny. Numerous pathologies, such as neurodegenerative diseases, cardiovascular disorders, cancers, and various other conditions, are associated with mitochondrial dysfunctions. Thus, a detailed exploration of the regulatory mechanisms of mitophagy is pivotal for enhancing our understanding and for the discovery of novel preventive and therapeutic options for these diseases. Nanomaterials have become integral in biomedicine and various other sectors, offering advanced solutions for medical uses including biological imaging, drug delivery, and disease diagnostics and therapy. Mitophagy is vital in managing the cellular effects elicited by nanomaterials. This review provides a comprehensive analysis of the molecular mechanisms underpinning mitophagy, underscoring its significant influence on the biological responses of cells to nanomaterials. Nanoparticles can initiate mitophagy via various pathways, among which the PINK1-Parkin pathway is critical for cellular defense against nanomaterial-induced damage by promoting mitophagy. The role of mitophagy in biological effects was induced by nanomaterials, which are associated with alterations in Ca2+ levels, the production of reactive oxygen species, endoplasmic reticulum stress, and lysosomal damage.

Recombinant human metallothionein-III alleviates oxidative damage induced by copper and cadmium in Caenorhabditis elegans.

Recombinant human metallothionein III (rh-MT-III) is a genetically engineered product produced by Escherichia coli fermentation technology. Its molecules contain abundant reducing sulfhydryl groups, which possess the ability to bind heavy metal ions. The present study was to evaluate the binding effects of rh-MT-III against copper and cadmium in vitro and to investigate the antioxidant activity of rh-MT-III using Caenorhabditis elegans in vivo. For in vitro experiments, the binding rates of copper and cadmium were 91.4% and 97.3% for rh-MT-III at a dosage of 200 µg/mL at 10 h, respectively. For in vivo assays, the oxidative stress induced by copper (CuSO4 , 10 µg/mL) and cadmium (CdCl2 , 10 µg/mL) was significantly reduced after 72 h of exposure to different doses of rh-MT-III (5-500 µg/mL), indicated by restoring locomotion behavior and growth, and reducing malondialdehyde and reactive oxygen species levels in C. elegans. Moreover, rh-MT-III decreased the deposition of lipofuscin and fat content, which could delay the progression of aging. In addition, rh-MT-III (500 µg/mL) promoted the up-regulation of Mtl-1 and Mtl-2 gene expression in C. elegans, which could enhance the resistance to oxidative stress by increasing the enzymatic activity of antioxidant defense system and scavenging free radicals. The results indicated that supplemental rh-MT-III could effectively protect C. elegans from heavy metal stress, providing an experimental basis for the future application and development of rh-MT-III.

Anti-Fouling Magnetic Beads Combined with Signal Amplification Strategies for Ultra-Sensitive and Selective Electrochemiluminescence Detection of MicroRNAs in Complex Biological Media.

Herein, an electrochemiluminescence (ECL) microRNA biosensor based on anti-fouling magnetic beads (MBs) and two signal amplification strategies was developed. The newly designed anti-fouling dendritic peptide was wrapped on the surfaces of MBs to make them resistant to nonspecific adsorption of biomolecules in complex biological samples so as to realize accurate and selective target recognition. One of the amplification strategies was achieved through nucleic acid cycle amplification based on the DNAzyme on the surfaces of MBs. Then, the output DNA generated by the nucleic acid cycle amplification program stimulated the hybrid chain reaction (HCR) process on the modified electrode surface to generate the other amplification of the ECL response. Titanium dioxide nanoneedles (TiO2 NNs), as a co-reaction accelerator of the Ru(bpy)2(cpaphen)2+ and tripropylamine (TPrA) system, were wrapped with the electrodeposited polyaniline (PANI) on the electrode surface to enhance the ECL intensity of Ru(bpy)2(cpaphen)2+. The conducting polymer PANI can not only immobilize the TiO2 NNs but also improve the conductivity of the modified electrodes. The biosensor exhibited ultra-high sensitivity and excellent selectivity toward the detection of miRNA 21, with a detection limit of 0.13 fM. More importantly, with the anti-fouling MBs as a unique separation tool, this ECL biosensor was capable of assaying targets in complex biological media such as serum and cell lysate.

From Passive Signal Output to Intelligent Response: "On-Demand" Precise Imaging Controlled by Near-Infrared Light.

"On-demand" accurate imaging of multiple intracellular miRNAs will significantly improve the detection reliability and accuracy. However, the "always-active" design of traditional multicomponent detection probes enables them to passively recognize and output signals as soon as they encounter targets, which will inevitably impair the detection accuracy and, inevitably, result in false-positive signals. To address this scientific problem, in this work, we developed a near-infrared (NIR) light-activated multicomponent detection intelligent nanoprobe for spatially and temporally controlled on-demand accurate imaging of multiple intracellular miRNAs. The proposed intelligent nanoprobe is composed of a rationally designed UV light-responsive triangular DNA nano sucker (TDS) and upconversion nanoparticles (UCNPs), named UCNPs@TDS (UTDS), which can enter cells autonomously through endocytosis and enable remote regulation of on-demand accurate imaging for multiple intracellular miRNAs using NIR light illumination at a chosen time and place. It is worth noting that the most important highlight of the UTDS we designed in this work is that it can resist nonspecific activation as well as effectively avoid false-positive signals and improve the accuracy of imaging of multiple intracellular miRNAs. Moreover, distinguishing different kinds of cell lines with different miRNA expressions levels can be also achieved through this NIR light-activated intelligent UTDS, showing feasible prospects in precise imaging and disease diagnosis.

Neurotoxicity of metal-containing nanoparticles and implications in glial cells.

With the development of nanotechnology, metal-containing nanoparticles are used widely in the diagnosis, monitoring and treatment of central nervous system (CNS) diseases. The neurotoxicity of these nanoparticles has drawn attention. Glial cells (particularly microglial cells and astrocytes) have important functions in the CNS. Neural disorders are related to functional/histologic damage to glial cells. Dysfunctions of microglial cells or astrocytes injure the brain, and cause the neurodegeneration seen in Alzheimer's disease and Parkinson's disease. We have summarized the route of access of metal-containing nanoparticles to the CNS, as well as their neurotoxicity and potential molecular mechanisms involved in glial cells. Metal-containing nanoparticles cross or bypass the blood-brain barrier, access the CNS and cause neurotoxicity. The potential mechanisms are related to inflammation, oxidative stress, DNA and/or mitochondrial damage and cell death, all of which are mediated by microglial cell activation, inflammatory factor release, generation of reactive oxygen species, apoptosis and/or autophagy in glial cells. Moreover, these processes increase the burden of the CNS and even accelerate the occurrence or development of neurodegenerative diseases. Some important signaling pathways involved in the mechanism of neurotoxicity in glial cells caused by nanoparticles are also discussed.

Neurobehavior and neuron damage following prolonged exposure of silver nanoparticles with/without polyvinylpyrrolidone coating in Caenorhabditis elegans.

Silver nanoparticles (AgNPs) have become widespread in the environment with increasing industrial applications. But the studies about their potential health risks are far from enough, especially in neurotoxic effects. This study aimed to investigate the neurotoxic effects of longer-term exposure (prolonged exposure for 48 h and chronic exposure for 6 days) of 20nm AgNPs with/without polyvinylpyrrolidone (PVP) coating at low concentrations (0.01-10 mg·L-1 ) to Caenorhabditis elegans. The results suggested that exposure to AgNPs induced damage to nematode survival, with the longest and relative average life span reduced. Exposure to AgNPs caused neurotoxicity on locomotion behaviors (head thrashes, body bends, pharyngeal pumping frequency, and defecation interval) and sensory perception behaviors (chemotaxis assay and thermotaxis assay), as well as impaired dopaminergic, GABAergic, and cholinergic neurons, except for glutamatergic, based on the alters fluorescence intensity, in a dose- and time-dependent manner. Further investigations suggested that the low-dose AgNPs (0.01-0.1 mg·L-1 ) exposure raises receptors of GABAergic and dopamine in C. elegans at the genetic level, whereas opposite results were observed at higher doses (1-10 mg·L-1 ), which implied that AgNPs could cause neurotoxicity by impairing neurotransmitter delivery. The PVP-AgNPs could cause a higher fatality rate and neurotoxicity at the same dose. Notably, AgNPs did not cause any deleterious effect on nematodes at the lowest dose of 0.01 mg·L-1 . In general, these results suggested that AgNPs possess the neurotoxic potential in C. elegans and provided useful information to understand the neurotoxicity of AgNPs, which would offer an inspiring perspective on the safe application.

Mitophagy-lysosomal pathway is involved in silver nanoparticle-induced apoptosis in A549 cells.

With the increasing use of silver nanoparticles (AgNPs) in biological materials, the cytotoxicity caused by these particles has attracted much attention. However, the molecular mechanism underlying AgNP cytotoxicity remains unclear. In this study, we aimed to systematically investigate the toxicity induced by AgNP exposure to the lung adenocarcinoma A549 cell line at the subcellular and signaling pathway levels and elucidate the related molecular mechanism. The survival rate of cells exposed to AgNPs at 0, 20, 40, 80, and 160 µg/mL for 24 or 48 h decreased in a dose- and time-dependent manner. AgNPs induced autophagy and mitophagy, determined by the transmission electron microscopy investigation and upregulation of LC3 II/I, p62, PINK1, and Parkin expression levels. AgNP treatment induced lysosomal injury, including the decline of lysosomal membrane integrity and increase in cathepsin B level. The decreased in mitochondrial membrane potential, along with upregulation of cytochrome c, caspases 9 and 3, and BAX/BCL2, further suggested that mitochondrial injury were involved in AgNP-induced apoptosis. In addition, mitochondrial injury may further lead to excessive production of reactive oxygen species and oxidative/ antioxidant imbalance. The results suggested that AgNPs could regulate autophagy via mitochondrial and lysosome injury in A549 cells. The information of the molecular mechanism will provide an experimental basis for the safe application of nanomaterials.

Silver nanoparticles induced cytotoxicity in HT22 cells through autophagy and apoptosis via PI3K/AKT/mTOR signaling pathway.

With the widespread application and inevitable environmental exposure, silver nanoparticles (AgNPs) can be accumulated in various organs. More serious concerns are raised on the biological safety and potential toxicity of AgNPs in the central nervous system (CNS), especially in the hippocampus. This study aimed to investigate the biological effects and the role of PI3K/AKT/mTOR signaling pathway in AgNPs mediated cytotoxicity using the mouse hippocampal neuronal cell line (HT22 cells). AgNPs reduced cell viability and induced membrane leakage in a dose-dependent manner, determined by the MTT and LDH assay. In doses of 25, 50, 100 µg mL-1 for 24 h, AgNPs promoted the excessive production of reactive oxygen species (ROS) and caused the oxidative stress in HT22 cells. AgNPs induced autophagy, determined by the transmission electron microscopy observation, upregulation of LC3 II/I and downregulation of p62 expression levels. The mechanistic investigation showed that the PI3K/AKT/mTOR signaling pathway was activated by phosphorylation, which was enrolled in an AgNP-induced autophagy process. AgNPs could further trigger the apoptosis by upregulation of caspase-3 and Bax and downregulation of Bcl-2 in HT22 cells. These results revealed AgNP-induced cytotoxicity in HT22 cells, which was mediated by autophagy and apoptosis via the PI3K/AKT/mTOR signaling pathway. The study could provide the experimental evidence and explanation for the potential neurotoxicity triggered by AgNPs in vitro.

Detection of DNA methyltransferase activity using template-free DNA polymerization amplification based on aggregation-induced emission.

A sensitive and selective fluorescence assay for DNA methyltransferase (MTase) activity detection was designed based on aggregation-induced emission (AIE) and target initiated template-free DNA polymerization. Quaternized tetraphenylethene salt was synthesized as the AIE probe, which binds to single-stranded DNA by electrostatic interaction. A hairpin probe was designed with a specific sequence for DNA MTase. In the presence of DNA MTase, the methylation reaction initiated DNA polymerization with terminal deoxynucleotidyl transferase (TdT), which activated the fluorescence intensity through AIE. The designed DNA sensor displayed a linear response to concentrations of DNA adenine methyltransferase (Dam) MTase from 0.5 U·mL-1 to 100 U mL-1, with a limit of detection of 0.16 U mL-1. The assay was also effective for detection of DNA MTase activity in human serum and for showing the inhibitory effect of 5-fluorouracil on Dam MTase.

Biodistribution and organ oxidative damage following 28 days oral administration of nanosilver with/without coating in mice.

This study evaluated the biodistribution and organ oxidative effects of silver nanoparticles (AgNPs) coated with/without polyvinylpyrrolidone (PVP) (AgNP-20 and AgNP-PVP) in mice; these were administered by gavage at a dose of 10-250 mg/kg body weight per day for 28 days. The results showed that both the AgNPs could induce subacute toxicity and oxidative damage to mice and were mainly accumulated in the liver and spleen and excreted by feces. AgNPs could be absorbed into blood and might cross the blood-brain barrier, and be distributed extensively in mice. The malondialdehyde content in the liver, lungs and kidneys increased in both AgNP groups, while the content of glutathione decreased, and the activity of superoxide dismutase increased at first and then decreased along with the increased doses. Inflammatory pathological changes in the lung and liver at high dose of both AgNPs were consistent with increases in glutamate pyruvic transaminase, glutamate oxaloacetic transaminase and the total protein in serum detection. The Ag content was detected in organs, with the highest content in the liver, followed by spleen, while the Ag content in feces was about 500 times higher than that in urine. AgNP-PVP could induce higher oxidative stress and subacute toxicity than AgNP-20 at the same dose, which might be related to the higher concentrations and more Ag+ ions released in mice after AgNP-PVP exposure. The data from this research provided information on toxicity and biodistribution of AgNPs following gavage administration in mice, and might shed light for future application of AgNPs in daily life.

DNAzyme-functionalized porous carbon nanospheres serve as a fluorescent nanoprobe for imaging detection of microRNA-21 and zinc ion in living cells.

The present study shows that a dual-signal nanoprobe consisting of DNAzyme-functionalized porous carbon nanospheres (PCNs) responds to microRNA-21 and zinc ion (Zn2+). The fluorescent probe undergoes an increase in the fluorescence intensity of fluorescein isothiocyanate (FITC) (with excitation/emission wavelengths at 488/517 nm) and the fluorescence intensity of cyanine-5 (Cy5) (with excitation/emission wavelengths at 633/670 nm) in the presence of microRNA-21 and Zn2+. The recognition between microRNA-21 and its complementary strand in the PCNs induces the separation of Zn2+-specific DNAzyme from PCNs, thus resulting in the increase of green fluorescence, and the exogenous Zn2+ triggers the rupture of cleavage strand of DNAzyme and recovery of red fluorescence. This nanoprobe allows us to acquire in vitro the determination of microRNA-21 in the range of 2-300 nM with a detection limit of 0.57 nM and the determination of Zn2+ in the range 2-100 nM with a detection limit of 0.43 nM, and in situ simultaneous imaging in MCF-7 breast cancer cells. Therefore, this strategy permits to obtain the expression levels of different biomarkers in living cells, providing a useful tool for diagnosis of cancers and understanding their biological process. Graphical abstract Schematic representation of the DNAzyme-functionalized porous carbon nanospheres for the imaging analysis of microRNA-21 and Zn2+ in living cells.

Ultrasensitive electrochemical DNA biosensor based on a tetrahedral structure and proximity-dependent surface hybridization.

The DNA tetrahedron has developed a broad spectrum of applications in biosensor construction thanks to its excellent mechanical rigidity and structural stability. However, how to construct a highly sensitive biosensor using a DNA tetrahedron is still a challenge. In this work, an ultrasensitive electrochemical biosensor based on a DNA tetrahedral nanostructure was developed with the help of synergy from proximity-dependent hybridization. To decrease the steric hindrance of DNA tetrahedra to proximity-dependent hybridization, the detection signal was set on the inclined side chain structure of a DNA tetrahedral sensing system. Additionally, when the target hybridized with the DNA probe, the ferrocene (Fc) labeled on the end of the DNA probe was driven close to the surface of the biosensor, providing a sensitive faradaic current. The experimental results exhibited a good linear relationship from 1 fM to 10 pM with a linear correlation coefficient of 0.9977, and a high sensitivity with a detection limit of 0.2 fM. Our DNA biosensor also showed good stability according to electrode characterization and target detection at different time scales and the anti-jamming capabilities in a complicated biological extraction environment were excellent. The electrochemical sensing system established here has greatly improved the detection sensitivity of a DNA biosensor based on a DNA tetrahedron, which will further promote its practical applications.

Rapid detection of foodborne pathogen Listeria monocytogenes by strand exchange amplification.

A strand exchange amplification (SEA) method to detect foodborne pathogen Listeria monocytogenes was developed. SEA is a novel nucleic acid amplification method that only requires one pair of primers. The specie-specific primers were designed by targeting the 16S rRNA gene and the amplification reaction was performed as short as 60 min at 61 °C. Notably, SEA method could not only detect genomic DNA but also detect RNA by one step without requiring extra reverse transcription. The result could be visualized by naked eyes so that water bath pot would be the only equipment needed. Moreover, culture fluids and bacteria colony could be successfully detected without any pretreatment and the method displayed good specificity and strong anti-jamming capacity. These features greatly simplified the operating procedure and made SEA method be potential for developing point-of-care testing (POCT) devices to detect viable L. monocytogenes.

Innate Reverse Transcriptase Activity of DNA Polymerase for Isothermal RNA Direct Detection.

RNA detection has become one of the most robust parts in molecular biology, medical diagnostics and drug discovery. Conventional RNA detection methods involve an extra reverse transcription step, which limits their further application for RNA rapid detection. We herein report a novel finding that Bst and Klenow DNA polymerases possess innate reverse transcriptase activities, so that the reverse transcription step and next amplification reaction can be combined to one step in isothermal RNA detection. We have demonstrated that Bst and Klenow DNA polymerases could be successfully used to reverse transcribe RNA within 125-nt length by real time RT-PCR and polyacrylamide gel electrophoresis (PAGE). Our findings will spur the development of a myriad of simple and easy to use RNA detection technologies for isothermal RNA direct detection. This will just meet the future needs of bioanalysis and clinical diagnosis to RNA rapid detection in POC settings and inspection and quarantine.

Detection of thrombin based on aptamer isothermal exponential signal amplification technique.

In this report, a strategy based on an aptameric molecule switch to realize homogeneous and isothermal signal exponential amplification and protein detection has been described. This method was simple and sensitive, allowing detection of as low as 2.6 × 10(-12) M thrombin.

Oxidative stress-activated Nrf2 remitted polystyrene nanoplastic-induced mitochondrial damage and inflammatory response in HepG2 cells.

Generated from plastics, microplastics (MPs) and nanoplastics (NPs) are difficult to completely degrade in the natural environment, which can accumulate in almost all lives. Liver is one of the main target organs. In this study, HepG2 and L02 cells were exposed to 0-50 µg/mL polystyrene (PS)-NPs to investigate the mechanism of mitochondrial damage and inflammation. The results showed mitochondria damage and inflammatory caused by NPs, and it can be inhibited by N-acetyl-L-cysteine (NAC). In addition, reactive oxygen species (ROS) activated nuclear factor erythroid-derived factor 2-related factor (Nrf2) pathway. Nrf2 siRNA exacerbated the injury, suggesting Nrf2 plays a protective role. Moreover, p62 siRNA increased ROS and mitochondrial damage by inhibiting Nrf2, but didn't affect the inflammation. In conclusion, Nrf2 was activated by ROS and played a protective role in PS-NPs-mediated hepatotoxicity. This study supplemented the data of liver injury caused by PS-NPs, providing a basis for the safe disposal of plastics.

Silver nanoparticles induced synaptic degeneration via Ca2+/CaMKII signal and Drp1-dependent mitochondrial disorder in HT22 cells.

Silver nanoparticles (AgNPs) have been widely used in biomedicine and cosmetics, increasing their potential risks in neurotoxicity. But the involved molecular mechanism remains unclear. This study aims to explore molecular events related to AgNPs-induced neuronal damage by RNA-seq, and elucidate the role of Ca2+/CaMKII signal and Drp1-dependent mitochondrial disorder in HT22 cells synaptic degeneration induced by AgNPs. This study found that cell viabilities were decreased by AgNPs in a dose/time-dependent manner. AgNPs also increased protein expression of PINK1, Parkin, synaptophysin, and inhibited PGC-1α, MAP2 and APP protein expression, indicating AgNPs-induced synaptic degeneration involved in disturbance of mitophagy and mitochondrial biogenesis in HT22 cells. Moreover, inhibition of AgNPs-induced Ca2+/CaMKII activation and Drp1/ROS rescued mitophagy disturbance and synaptic degeneration in HT22 cells by reserving aforementioned protein express changes except for PGC-1α and APP protein. Thus, AgNPs-induced synaptic degeneration was mediated by Ca2+/CaMKII signal and Drp1-dependent mitochondrial disorder in HT22 cells, and mitophagy is the sensitive to the mechanism. Our study will provide in-depth molecular mechanism data for neurotoxic evaluation and biomedical application of AgNPs.

Comparative oxidative damages induced by silver nanoparticles with different sizes and coatings in Caenorhabditis elegans.

Silver nanoparticles (AgNPs) are widely used in many commercial and medical products. Serious concerns are paid on their adverse potentials to the environment and human health. In this study, toxic effects and oxidative stress induced by AgNPs with different sizes and coatings (20 nm AgNPs, 20 nm polyvinylpyrrolidone (PVP) -AgNPs and 50 nm AgNPs) in Caenorhabditis elegans (C. elegans) were investigated. The toxic effects including the shortened lifespan and decreased frequency of head thrashes and body bends of C. elegans were induced in a dose-dependent manner by AgNPs. The reactive oxygen species (ROS) production and the oxidative stress-related indicators including malondialdehyde (MDA) and glutathione (GSH) in nematodes were changed after exposure to three kinds of AgNPs. These effects were the most obvious in a 20 nm PVP-AgNPs exposure group. AgNPs could also induce the expression of genes related to oxidative stress in nematodes. In addition, the up-regulation of mtl-1 and mtl-2 in nematodes might reduce the oxidative damage caused by AgNPs, by using transgenic strains CF2222 and CL2120 nematodes. Metallothionein (MT), an antioxidant, could relieve the oxidative damage caused by AgNPs. These results suggested that 20 nm PVP-AgNPs with a smaller particle size and better dispersion have stronger toxic effects and the oxidative damage to nematodes. Mtl-1 and mtl-2 might be involved in alleviating the oxidative damage caused by AgNPs. Our findings provide clues for the safety evaluation and mechanism information of metal nanoparticles.

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.