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Stereoscopic imaging of single molecules at the plasma membrane of single cell requires spatial resolutions in 3 dimensions (x-y-z) at 10-nm level, which is rarely achieved using most optical super-resolution microscopies. Here, electrochemical stereoscopic microscopy with a detection limit down to a single molecule is achieved using a photoreduction-assisted cycle inside a 20-nm gel electrolyte nanoball at the tip of a nanopipette. On the basis of the electrochemical oxidation of Ru(bpy)3 2+ into Ru(bpy)3 3+ followed by the reduction of Ru(bpy)3 3+ into Ru(bpy)3 2+ by photogenerated isopropanol radicals, a charge of 1.5 fC is obtained from the cycling electron transfers involving one Ru(bpy)3 2+/3+ molecule. By using the nanopipette to scan the cellular membrane modified with Ru(bpy)3 2+-tagged antibody, the morphology of the cell membrane and the distribution of carcinoembryonic antigen (CEA) on the membrane are electrochemically visualized with a spatial resolution of 14 nm. The resultant stereoscopic image reveals more CEA on membrane protrusions, providing direct evidence to support easy access of membrane CEA to intravenous antibodies. The breakthrough in single-molecule electrochemistry at the cellular level leads to the establishment of high-resolution 3-dimensional single-cell electrochemical microscopy, offering an alternative strategy to remedy the imperfection of stereoscopic visualization in optical microscopes.
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The traditional recognition of extracellular matrix (ECM) at tissue sections relies on the time-consuming immunofluorescence that could not meet the demand of rapid diagnosis. Herein, we introduce a thickness-resolved electrochemiluminescence (ECL) microscopy to image thin-layer ECM at tissue sections for fast histopathological analysis. The unique surface-confined ECL mechanism enables to unveil the diversity and complexity of multiple tissue structures with varying thicknesses. Notably, the short lifetimes and the limited diffusion of electrogenerated coreactant radicals combined with their chemical reactivity result in a 2-fold increase in ECL intensity on ECM structures compared to the remaining tissue, enabling ECM visualization without specific labeling. The further quantitation of the ECM localization within tissue sections furnishes crucial insights into tumor progression and, more importantly, differentiates carcinoma and paracancerous tissues from patients in less than 30 min. Moreover, the reported electrochemistry-based microscopy is a dynamic approach allowing to investigate the transport, tortuosity, and trafficking properties through the tissues. This thickness-resolved recognition strategy not only opens new avenues for imaging complex samples but also holds promise for expediting tissue pathologic diagnosis, offering a more automated protocol with enhanced quantitative data compared to current intraoperative pathology methods.
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Técnicas Eletroquímicas , Matriz Extracelular , Neoplasias , Humanos , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Técnicas Eletroquímicas/métodos , Neoplasias/diagnóstico , Neoplasias/patologia , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Medições Luminescentes/métodos , Microscopia/métodosRESUMO
This work proposes the concept of single-cell microRNA (miR) therapy and proof-of-concept by engineering a nanopipette for high-precision miR-21-targeted therapy in a single HeLa cell with sensitive photoelectrochemical (PEC) feedback. Targeting the representative oncogenic miR-21, the as-functionalized nanopipette permits direct intracellular drug administration with precisely controllable dosages, and the corresponding therapeutic effects can be sensitively transduced by a PEC sensing interface that selectively responds to the indicator level of cytosolic caspase-3. The experimental results reveal that injection of ca. 4.4 × 10-20 mol miR-21 inhibitor, i.e., 26488 copies, can cause the obvious therapeutic action in the targeted cell. This work features a solution to obtain the accurate knowledge of how a certain miR-drug with specific dosages treats the cells and thus provides an insight into futuristic high-precision clinical miR therapy using personalized medicine, provided that the prerequisite single-cell experiments are courses of personalized customization.
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MicroRNAs , Humanos , Células HeLa , Retroalimentação , Medicina de PrecisãoRESUMO
In the contemporary era of scientific and medical advancements, the accurate and ultra-sensitive detection of proteins, nucleic acids and metabolites plays a pivotal role in disease diagnosis and treatment monitoring. Single-molecule detection technologies play a great role in achieving this goal. In recent years, digital detection methods based on single molecule arrays (SIMOA) have brought groundbreaking contributions to the field of single-molecule detection. By confining the target molecules to femtoliter-sized containers, the SIMOA technology achieves detection sensitivity of attomolar. This review delves into the historical evolution and fundamentals of SIMOA technology, summarizes various approaches to optimize its performance, and describes the applications of SIMOA for the ultrasensitive detection of biomarkers for diseases such as cancer, COVID-19, and neurological disorders, as well as in DNA detection. Currently, some SIMOA technologies have been realized for high-throughput and multiplexed detection. It is believed that SIMOA technology will play a significant role in medical monitoring and disease prevention in the future.
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Ácidos Nucleicos , Proteínas , Técnicas Imunoenzimáticas , Ensaio de Imunoadsorção Enzimática/métodos , DNARESUMO
Accurate temperature measurement in one living cell is of great significance for understanding biological functions and regulation. Here, a nanopipet electric thermometer (NET) is established for real-time intracellular temperature measurement. Based on the temperature-controlled ion migration, the temperature change in solution results in altered ion mobilities and ion distributions, which can be converted to the thermoelectric responses of NET in a galvanostatic configuration. The exponential relationship between the voltage and the temperature promises highly sensitive thermoelectric responses up to 11.1 mV K-1, which is over an order of magnitude higher than previous thermoelectric thermometry. Moreover, the NET exhibits superior thermal resolution of 25 mK and spatiotemporal resolution of 100 nm and 0.9 ms as well as excellent stability and reproducibility. Benefiting from these unique features, both thermal fluctuations in steady-state cells and heat generation and dissipation upon drug administration can be successfully monitored, which are hardly achieved by current methods. By using NET, thermal heterogeneities of single cancer cells during immunotherapy were reported first in this work, in which the increased intracellular temperature was demonstrated to be associated with the survival benefit and resistance of cancer cells in immunotherapy. This work not only provides a reliable method for microscopic temperature monitoring but also gains new insights to elucidate the mechanism of immune evasion and therapeutic resistance.
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Eletricidade , Termômetros , Reprodutibilidade dos Testes , TemperaturaRESUMO
Biological channels can rapidly and continuously modulate ion transport behaviors in response to external stimuli, which play essential roles in manipulating physiological and pathological processes in cells. Here, to mimic the biological channels, a bionic nanochannel is developed by synergizing a cationic silicon-substituted rhodamine (SiRh) with a glass nanopipette for transmembrane single-cell quantification. Taking the fast and reversible nucleophilic addition reaction between glutathione (GSH) and SiRh, the bionic nanochannel shows a fast and reversible response to GSH, with its inner-surface charges changing between positive and negative charges, leading to a distinct and reversible switch in ionic current rectification (ICR). With the bionic nanochannel, spatiotemporal-resolved operation is performed to quantify endogenous GSH in a single cell, allowing for monitoring of intracellular GSH fluctuation in tumor cells upon photodynamic therapy and ferroptosis. Our results demonstrate that it is a feasible tool for in situ quantification of the endogenous GSH in single cells, which may be adapted to addressing other endogenous biomolecules in single cells by usage of other stimuli-responsive probes.
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Biônica , Ferroptose , Vidro , Glutationa , Transporte de Íons , RodaminasRESUMO
Electrochemiluminescence (ECL) imaging, as an optical technology, has been developed at full tilt in the field of life science and nanomaterials. However, the relatively low ECL intensity or the high co-reactant concentration needed in the electrochemical reaction blocks its practical application. Here, we developed an ECL imaging system based on the rGO-TiO2-x composite material, where the co-reactant, reactive oxygen species (ROS), is generated in situ under the synergetic effect of of ultrasound (US) and electric irradiation. The rGO-TiO2-x composites facilitate the separation of electron (e-) and hole (h+) pairs and inhibit recombination triggered by external US irradiation due to the high electroconductivity of rGO and oxygen-deficient structures of TiO2, thus significantly boosting ROS generation. Furthermore, the increased defects on rGO accelerate the electron transfer rate, improving the electrocatalytic performance of the composite and forming more ROS. This high ultrasonic-electric synergistic efficacy is demonstrated through the enhancement of photon emission. Compared with the luminescence intensity triggered by US irradiation and electric field, an enhancement of â¼20-fold and 10-fold of the US combined with electric field-triggered emission is observed from this composite. Under the optimized conditions, using dopamine (DA) as a model target, the sensitivity of the US combined ECL strategy for detection of DA is two orders of magnitude higher than that of the ECL method. The successful detection of DA at low concentrations makes us believe that this strategy provides the possibility of applying ECL imaging for cellular single-molecule analysis and cancer therapy.
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Spatial metabolomic analysis of individual tumor spheroids can help investigate metabolic rearrangements in different cellular regions of a spheroid. In this work, a nanocapillary-based electrospray ionization mass spectroscopy (ESI-MS) method is established that could realize the spatial sampling of cellular components in different regions of a single living tumor spheroid and the subsequent MS analysis for a metabolic study. During the penetration of the nanocapillary into the spheroid for sampling, this "wound surface" at the outer layer of the spheroid takes only 0.1% of the whole area that maximally maintains the cellular activity inside the spheroid for the metabolic analysis. Using the ESI-MS analysis, different metabolic activities in the inner and outer (upper and lower) layers of a single spheroid are revealed, giving a full investigation of the metabolic heterogeneity inside one living tumor spheroid for the first time. In addition, the metabolic activities between the outer layer of the spheroid and two-dimensional (2D)-cultured cells show obvious differences, which suggests more frequent cell-cell and cell-extracellular environment interactions during the culture of the spheroid. This observation not only establishes a powerful tool for the in situ spatial analysis of the metabolic heterogeneity in single living tumor spheroids but also provides molecular information to elucidate the metabolic heterogeneity in this three-dimensional (3D)-cultured cell model.
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Neoplasias , Esferoides Celulares , Humanos , Esferoides Celulares/patologia , Espectrometria de Massas por Ionização por Electrospray , Neoplasias/patologiaRESUMO
The electrochemical visualization of proteins in the plasma membrane of single fixed cells was achieved with a spatial resolution of 160 nm using scanning electrochemical cell microscopy. The model protein, the carcinoembryonic antigen (CEA), is linked with a ruthenium complex (Ru(bpy)32+)-tagged antibody, which exhibits redox peaks in its cyclic voltammetry curves after a nanopipette tip contacts the cellular membrane. Based on the potential-resolved oxidation or reduction currents, an uneven distribution of membrane CEAs on the cells is electrochemically visualized, which could only be achieved previously using super-resolution optical microscopy. Compared with current electrochemical microscopy, the single-cell scanning electrochemical cell microscopy (SECCM) strategy not only improves the spatial resolution but also utilizes the potential-resolved current from the antibody-antigen complex to increase electrochemical imaging accuracy. Eventually, the electrochemical visualization of cellular proteins at the nanoscale enables the super-resolution study of cells to provide more biological information.
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Proteínas de Membrana , Microscopia , Microscopia/métodos , Oxirredução , Membrana Celular , Microscopia Eletroquímica de VarreduraRESUMO
Histopathological molecular testing of tissue sections is an essential step in tumor diagnosis; however, the commonly used immunohistochemical methods have problems such as low specificity and the subjective bias of the observer. Here, we report an electrochemiluminescence (ECL) imaging method to detect a membrane carcinoembryonic antigen (CEA) at the single tissue sections of cancer patients. By permeabilizing the tissue attached to a glassy carbon electrode, Ru(bpy)32+ tagged at the membrane CEA of the tissue could electrochemically react with TPrA in solution to emit ECL that has near-zero background and an extremely high signal-to-background ratio. Using the established ECL method, the expression differences and distribution characteristics of the CEA protein in the carcinoma and paracancerous tissues of pancreatic ductal carcinoma (PDAC) and lung adenocarcinoma (LUAD) patients are investigated. The images reveal that CEA proteins are mostly distributed in the acini and surrounding areas both in PDAC and LUAD tissues. Therefore, the presented approach could be able to provide a new molecular recognition method for the diagnosis of adenocarcinoma and other tumors.
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Técnicas Eletroquímicas , Medições Luminescentes , Humanos , Técnicas Eletroquímicas/métodos , Medições Luminescentes/métodos , Antígeno Carcinoembrionário/análise , Antígeno Carcinoembrionário/metabolismo , Adenocarcinoma/química , Adenocarcinoma/metabolismo , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/metabolismo , Membrana Celular/química , Membrana Celular/metabolismoRESUMO
The simultaneous and accurate detection of intracellular pH (pHi) and extracellular pH (pHe) is essential for studying the complex physiological activities of cancer cells and exploring pH-related therapeutic mechanisms. Here, we developed a super-long silver nanowire-based surface-enhanced Raman scattering (SERS) detection strategy for simultaneous sensing of pHi and pHe. A surface-roughened silver nanowire (AgNW) with a high aspect ratio is prepared at a nanoelectrode tip using a Cu-mediated oxidation process, which is then modified by pH-sensitive 4-mercaptobenzoic acid (4-MBA) to form 4-MBA@AgNW as a pH sensing probe. With the assistance of a 4D microcontroller, 4-MBA@AgNW is efficient in simultaneously detecting pHi and pHe in both 2D and 3D culture cancer cells by SERS, with minimal invasiveness, high sensitivity, and spatial resolution. Further investigation proves that the surface-roughened single AgNW can also be used in monitoring the dynamic variation of pHi and pHe of cancer cells upon stimulation with anticancer drugs or under a hypoxic environment.
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Nanopartículas Metálicas , Nanofios , Prata , Análise Espectral Raman/métodos , Compostos de SulfidrilaRESUMO
Single-cell protein therapeutics is expected to promote our in-depth understanding of how a specific protein with a therapeutic dosage treats the cell without population averaging. However, it has not yet been tackled by current single-cell nanotools. We address this challenge by the use of a double-barrel nanopipette, in which one lumen was used for electroosmotic cytosolic protein delivery and the other was customized for ionic evaluation of the consequence. Upon injection of protein DJ-1 through the delivery lumen, upregulation of the antioxidant protein could protect neural PC-12 cells against oxidative stress from phorbol myristate acetate exposure, as deduced by targeting of the cytosolic hydrogen peroxide by the detecting lumen. The nanotool developed in this study for single-cell protein therapeutics provides a perspective for future single-cell therapeutics involving different therapeutic modalities, such as peptides, enzymes and nucleic acids.
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Terapia Baseada em Transplante de Células e Tecidos , Proteína Desglicase DJ-1 , Íons , Peptídeos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Sistemas de Liberação de Fármacos por Nanopartículas , Proteína Desglicase DJ-1/farmacologia , Proteína Desglicase DJ-1/uso terapêutico , Estresse Oxidativo , Acetato de TetradecanoilforbolRESUMO
Single-cell interrogation with the solid-state nanoprobes enables understanding of the linkage between cellular behavior and heterogeneity. Herein, inspired by the charge property of the organic molecular probe (OMP), a generic ionic current rectification (ICR) single-cell methodology is established, exemplified by subcellular detection of glutathione (GSH) with high selectivity, sensitivity, and recyclability. The as-developed nanosensor can transduce the subcellular OMP-GSH interaction via a sensitive ionic response, which stems from the superior specificity of OMP and its essential charge property. In addition, the nanosensor exhibits good reversibility, since the subsequent tandem reaction after the recognition can well recover the sensing surface. Given the diverse structures and tailorable charge properties of OMP, this work underpins a new and general method of OMP-based ICR single-cell analysis.
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Glutationa , Sondas MolecularesRESUMO
The analysis of biomolecules in a 3D cell model is crucial for the collection of spatial information close to the actual organ. In this work, a highly sensitive platinized open carbon nanocavity electrode is fabricated to investigate reactive oxygen species (ROS) in three regions (proliferating zone, quiescent zone, and necrotic core) of a 3D CT26 cell model. The presence of a nanocavity permits more frequent collisions of ROS on the Pt surface, accelerating electron transfer, and thus pushes the detection limit down to 1 nM. This improved detection sensitivity guarantees the spatial investigation of the ROS distribution in a 3D cell sphere, including a high concentration in the outer proliferating layer even without any external stimulus, a low concentration in the quiescent layer, and almost no ROS at the center. The observation of ROS in the cell sphere without the stimulus reveals the presence of oxygen stress in the 3D cancer cell model, which is obviously different from the previous observation in living cultured 2D cells. This discovery provides direct evidence about the discrepancy about the metabolism in 2D and 3D cells, which could also direct a new study in cell electroanalysis to achieve more actual molecular information in life study.
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Carbono , Oxigênio , Eletrodos , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Análise EspacialRESUMO
The recent surge of interest in metal-organic gels (MOGs) has emerged for their soft porous structure, large surface area, and abundant active metal sites, making them a promising candidate for building catalyst matrices. In this work, facilely synthesized Fe(III)-organic gel was directly used as a robust electrode matrix. Detailed studies illustrated that their Fe(III) centers can speed up the electro-oxidation/reduction of the H2O2 coreactant to produce reactive oxygen species for enhancing a potential-resolved dual electrochemiluminescence (ECL) emission. Among them, the anodic signal of luminol varied with the cell concentration based on the impedance ECL mechanism, while the cathodic signal of CdS quantum dots traced the VEGF165 subtype at cell surface by specific aptamer recognition. Based on this, a ratiometric strategy was proposed for accurate cytosensing by eliminating environmental interference. Moreover, by cooperating these two signals, a novel strategy was developed for direct evaluation of the VEGF165 subtype, further realizing rapid drug screening and subtype assessment on different cell lines. This work not only opens up the promising application of MOGs as an effective catalyst matrix but also develops reliable cell assays and protein subtype identification for clinical diagnosis and research.
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Técnicas Biossensoriais , Nanopartículas Metálicas , Catálise , Técnicas Eletroquímicas , Géis , Peróxido de Hidrogênio , Ferro , Medições Luminescentes , Luminol , Nanopartículas Metálicas/química , Fator A de Crescimento do Endotélio VascularRESUMO
The use of double-barreled nanopipette (θ-nanopipette) to electrically sample, manipulate, or detect biomaterials has recently seen strong growth in single-cell studies, driven by the potential of the nanodevices and applications that they may enable. Considering the pivotal roles of Na/K ratio (RNa/K) at cellular level, herein we describe an engineered θ-nanopipette for measuring single-cell RNa/K. The two independently addressable nanopores, located within one nanotip, allow respective customization of functional nucleic acids but simultaneous deciphering of Na and K levels inside a single cell of a non-Faradic manner. Two ionic current rectification signals, corresponding to the Na- and K-specific smart DNA responses, could be easily used to derive the RNa/K. The applicability of this nanotool is validated by practical probing intracellular RNa/K during the drug-induced primary stage of apoptotic volume decrease. Especially, the RNa/K has been shown by our nanotool to be different in cell lines with different metastatic potential. This work is expected to contribute to futuristic study of single-cell RNa/K in various physiological and pathological processes.
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Herein, a single biomolecule is imaged by electrochemiluminescence (ECL) using Ru(bpy)32+-doped silica/Au nanoparticles (RuDSNs/AuNPs) as the ECL nanoemitters. The ECL emission is confined to the local surface of RuDSNs leading to a significant enhancement in the intensity. To prove the concept, a single protein molecule at the electrode is initially visualized using the as-prepared RuDSN/AuNPs nanoemitters. Furthermore, the nanoemitter-labeled antibody is linked at the cellular membrane to image a single membrane protein at one cell, without the interference of current and optical background. The success in single-biomolecule ECL imaging solves the long-lasting task in the ultrasensitive ECL analysis, which should be able to provide more elegant information about the protein in cellular biology.
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Queratina-19/química , Substâncias Luminescentes/química , Nanopartículas Metálicas/química , Imagem Individual de Molécula/métodos , Complexos de Coordenação/química , Técnicas Eletroquímicas/métodos , Ouro/química , Humanos , Luminescência , Medições Luminescentes/métodos , Células MCF-7 , Estudo de Prova de Conceito , Compostos de Rutênio/química , Dióxido de Silício/químicaRESUMO
Conventional photoelectrochemical (PEC) analysis mostly utilizes photoactive material modified planar indium tin oxides (ITOs) to obtain photocurrent responses for the measurement of analytes in solution. In this work, a CdS quantum dot (QD) modified nanopipette was prepared for the PEC analysis of the alkaline phosphatase (ALP) activity in single MCF-7 cells. The nanopipette was filled with ascorbic acid 2-phosphate (AAP) that was egressed outside the nanopipette by electrochemical pumping. Next, AAP was catalyzed by ALP to generate ascorbic acid (AA), which is an efficient electron donor for CdS QDs under illumination. Based on the result that the nanopipette showed a linear photocurrent response to AA, a nearly linear correlation between the photocurrent and the activity of ALP was established. Accordingly, using these CdS QD modified nanopipettes, the ALP activity in single MCF-7 cells was determined to be 0.12 U mL-1 by PEC analysis. This work does not expand the application of PEC bioanalysis, but offers a new strategy for single cell analysis.
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Técnicas Biossensoriais , Pontos Quânticos , Fosfatase Alcalina , Técnicas EletroquímicasRESUMO
Developing an electrochemiluminescence (ECL) coreactant to minimize the biotoxicity and to maximize the enhancement factor is critical to single-cell ECL microscopy. Here, we reported a guanine-rich single-stranded DNA (G-ssDNA)-loaded high-index faceted gold nanoflower (Hi-AuNF) as a synergistic coreactant of Ru(bpy)32+ for single-cell ECL imaging. Because of the excellent catalytic performance and huge specific surface area, Hi-AuNF serves as not only an ECL enhancer but also a carrier for G-ssDNA. Guanine in G-ssDNA specifically reacts with Ru(bpy)32+ through a so-called "catalytic route" and thus significantly enhances the ECL signal of Ru(bpy)32+. To endow targeting ability to the synergistic coreactant, an aptamer of carcinoembryonic antigen (CEA) is incorporated into the G-ssDNA to form G-ssDNA-Apt for the recognition of human breast adenocarcinoma cells, which overexpress CEA on the cytomembrane. Accordingly, the ECL imaging of CEA on the cytomembrane was realized by using the highly selective Hi-AuNF@G-ssDNA-Apt as the probe as well as the luminophore of Ru(bpy)32+. Compared with the common coreactant tripropylamine with high toxicity and volatility, the Hi-AuNF@G-ssDNA-Apt is considered as a high-performance and biocompatible coreactant, providing exciting opportunities in single-cell imaging and detection.
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DNA de Cadeia Simples , Ouro , Guanina , Humanos , Medições LuminescentesRESUMO
Emerging nanopipette tools have demonstrated substantial potential for advanced single-cell analysis, which plays vital roles from fundamental cellular biology to biomedical diagnostics. Highly recyclable nanopipettes with easy and quick regeneration are of special interest for precise and multiple measurements. However, existing recycle strategies are generally plagued by operational complexity and limited efficiency. Light, acting in a noncontact way, should be the ideal external stimulus to address this issue. Herein, we present the photocontrolled nanopipette capable of probing cellular adenosine triphosphate (ATP) gradient at single-cell level with good sensitivity, selectivity, and reversibility, which stems from the use of ATP-specific azobenzene (Azo)-incorporated DNA aptamer strands (AIDAS) and thereby the sensible transduction of variable nanopore size by the ionic currents passing through the aperture. Photoisomerized conformational change of the AIDAS by alternative UV/vis light stimulation ensures its noninvasive regeneration and repeated detection. Inducement and inhibition of the cellular ATP could also be probed by this nanosensor.