High-Precision Single-Cell microRNA Therapy by a Functional Nanopipette with Sensitive Photoelectrochemical Feedback.

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.

Visual electrochemiluminescence from an all-solid-state electrochemical cell.

Visual electrochemiluminescence (ECL) emission from L012 and hydrogen peroxide is generated from an all-solid-state electrochemical cell with a polyacrylamide hydrogel as the solid electrolyte. The emission is strong enough to be visualized with the naked eye, which offers a new idea for the design of an all-solid-state ECL based sensor in air.

Observation of anodic electrochemiluminescence from silicon quantum dots for the detection of hydrogen peroxide.

Silicon quantum dots (QDs) with stable positively charged intermediates are prepared using chemical etching to generate strong anodic electrochemiluminescence (ECL) under a positive potential. Their surfaces could be passivated in the presence of strong oxidants, leading to enhanced ECL and offering the ability to carry out analysis for hydrogen peroxide.

Electrochemiluminescence Microscopy.

Electrochemiluminescence (ECL) is rapidly evolving from an analytical method into an optical microscopy. The orthogonality of the electrochemical trigger and the optical readout distinguishes it from classic microscopy and electrochemical techniques, owing to its near-zero background, remarkable sensitivity, and absence of photobleaching and phototoxicity. In this minireview, we summarize the recent advances in ECL imaging technology, emphasizing original configurations which enable the imaging of biological entities and the improvement of the analytical properties by increasing the complexity and multiplexing of bioassays. Additionally, mapping the (electro)chemical reactivity in space provides valuable information on nanomaterials and facilitates deciphering ECL mechanisms for improving their performances in diagnostics and (electro)catalysis. Finally, we highlight the recent achievements in imaging at the ultimate limits of single molecules, single photons or single chemical reactions, and the current challenges to translate the ECL imaging advances to other fields such as material science, catalysis and biology.

Thermoelectric Nanofluidics Probing Thermal Heterogeneity inside Single Cells.

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.

In Situ Spatial Analysis of Metabolic Heterogeneity in Single Living Tumor Spheroids Using Nanocapillary-Based Electrospray Ionization Mass Spectroscopy.

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.

Electrochemical Visualization of Membrane Proteins in Single Cells at a Nanoscale Using Scanning Electrochemical Cell Microscopy.

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.

Self-Enhanced Electrochemiluminescence Imaging System Based on the Accelerated Generation of ROS under Ultrasound.

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 ∼20-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.

Dynamic Mapping of Electrochemiluminescence Reactivity in Space: Application to Bead-Based Assays.

As an electrochemical technique offering an optical readout, electrochemiluminescence (ECL) evolved recently into a powerful microscopy technique with the visualization of a wide range of microscopic entities. However, the dynamic imaging of transient ECL events did not receive intensive attention due to the limited number of electrogenerated photons. Here, the reaction kinetics of the model ECL bioassay system was revealed by dynamic imaging of single [Ru(bpy)3]2+-functionalized beads in the presence of the efficient tripropylamine coreactant. The time profile behavior of ECL emission, the variations of the ECL layer thickness, and the position of maximum ECL intensity over time were investigated, which were not achieved by static imaging in previous studies. Moreover, the dynamics of the ECL emission were confronted with the simulation. The reported dynamic ECL imaging allows the investigation of the ECL kinetics and mechanisms operating in bioassays and cell microscopy.

Electrochemiluminescence imaging of a membrane carcinoembryonic antigen at single tissue sections.

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.

Nanokit coupled electrospray ionization mass spectrometry for analysis of angiotensin converting enzyme 2 activity in single living cell.

Angiotensin-converting enzyme 2 (ACE2) is not only an enzyme but also a functional receptor on cell membrane for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, the activity of ACE2 in single living cell is firstly determined using a nanokit coupled electrospray ionization mass spectrometry (nanokit-ESI-MS). Upon the insertion of a micro-capillary into the living hACE2-CHO cell and the electrochemical sorting of the cytosol, the target ACE2 enzyme hydrolyses angiotensin II inside the capillary to generate angiotensin 1-7. After the electrospray of the mixture at the tip of the capillary, the product is differentiated from the substrate in molecular weight to achieve the detection of ACE2 activity in single cells. The further measurement illustrates that the inflammatory state of cells does not lead to the significant change of ACE2 catalytic activity, which elucidates the relationship between intracellular ACE2 activity and inflammation at single cell level. The established strategy will provide a specific analytical method for further studying the role of ACE2 in the process of virus infection, and extend the application of nanokit based single cell analysis.

Click-Chemistry-Enabled Nanopipettes for the Capture and Dynamic Analysis of a Single Mitochondrion inside One Living Cell.

The in-depth study of single cells requires the dynamically molecular information in one particular nanometer-sized organelle in a living cell, which is difficult to achieve using current methods. Due to high efficiency of click chemistry, a new nanoelectrode-based pipette architecture with dibenzocyclooctyne at the tip is designed to realize fast conjugation with azide group-containing triphenylphosphine, which targets mitochondrial membranes. The covalent binding of one mitochondrion at the tip of the nanopipette allows a small region of the membrane to be isolated on the Pt surface inside the nanopipette. Therefore, the release of reactive oxygen species (ROS) from the mitochondrion is monitored, which is not interfered by the species present in the cytosol. The dynamic tracking of ROS release from one mitochondrion reveals the distinctive "ROS-induced ROS release" within the mitochondria. Further study of RSL3-induced ferroptosis using nanopipettes provides direct evidence for supporting the noninvolvement of glutathione peroxidase 4 in the mitochondria during RSL3-induced ROS generation, which has not previously been observed at the single-mitochondrion level. Eventually, this established strategy should overcome the existing challenge of the dynamic measurement of one special organelle in the complicated intracellular environment, which opens a new direction for electroanalysis in subcellular analysis.

Direct Visualization of Nanoconfinement Effect on Nanoreactor via Electrochemiluminescence Microscopy.

Nanoconfinement in mesoporous nanoarchitectures could dramatically change molecular transport and reaction kinetics during electrochemical process. A molecular-level understanding of nanoconfinement and mass transport is critical for the applications, but a proper route to study it is lacking. Herein, we develop a single nanoreactor electrochemiluminescence (SNECL) microscopy based on Ru(bpy)3 2+ -loaded mesoporous silica nanoparticle to directly visualize in situ nanoconfinement-enhanced electrochemical reactions at the single molecule level. Meanwhile, mass transport capability of single nanoreactor, reflected as long decay time and recovery ability, is monitored and simulated with a high spatial resolution. The nanoconfinement effects in our system also enable imaging single proteins on cellular membrane. Our SNECL approach may pave the way to decipher the nanoconfinement effects during electrochemical process, and build bridges between mesoporous nanoarchitectures and potential electrochemical applications.

Electrochemical Single-Cell Protein Therapeutics Using a Double-Barrel Nanopipette.

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.

An Integrated Dual-Functional Nanotool Capable of Studying Single-Cell Epigenetics and Programmable Gene Regulation.

Single-cell epigenetics is envisioned to decipher manifold epigenetic phenomena and to contribute to our accurate knowledge about basic epigenetic mechanisms. Engineered nanopipette technology has gained momentum in single-cell studies; however, solutions to epigenetic questions remain unachieved. This study addresses the challenge by exploring N6-methyladenine (m6 A)-bearing deoxyribozyme (DNAzyme) confined within a nanopipette for profiling a representative m6 A-modifying enzyme, fat mass and obesity-associated protein (FTO). Electroosmotic intracellular extraction of FTO could remove the m6 A and cause DNAzyme cleavage, leading to the altered ionic current signal. Because the cleavage can release a DNA sequence, we simultaneously program it as an antisense strand against FTO-mRNA, intracellular injection of which has been shown to induce early stage apoptosis. This nanotool thus features the dual functions of studying single-cell epigenetics and programmable gene regulation.

Electrochemical Molecule Trap-Based Sensing of Low-Abundance Enzymes in One Living Cell.

Measuring the activity of low-abundance enzymes, down to a few molecules in one living cell, is important but challenging to elucidate their biological function. Here, an electrochemical molecule trap is established at the tip of a nanopipette with an electrochemical detector, in which the diffusion of the molecules away from the electrochemical detector is prevented by electro-osmotic flow (EOF). Accordingly, a limited amount of enzymes is trapped to continuously catalyze the conversion of the substrate to generate a sufficient amount of the byproduct hydrogen peroxide for electrochemical measurements. The resistive pulse sensing of the enzymes in single liposomes validates the detection sensitivity down to 15 molecules. Using this ultrasensitive electrochemical strategy, the activity of 60 sphingomyelinase molecules inside single unstimulated living J774 cells is measured, which was hardly detected by previous methods. The established electrochemical molecule trap-based sensing approach opens the door toward single-molecule electrochemical detection in one living cell. This success will solve the long-standing problem regarding the study of the activity of low-abundance proteins in cells in their native physiological state and greatly enhance the understanding of the roles of proteins in cellular behavior.

Fluorescent Polymerase Chain Reaction Nanokit for the Detection of DNA Sequence in Single Living Cells.

Here, a fluorescent polymerase chain reaction (PCR) nanokit is established to detect the specific DNA sequence in a single living cell. Different from well-developed protocols to load cell-permeable probes into single cell for recognition, the DNA sequence in a cellular nucleus is sorted into a nanopipette in our strategy. The target DNA sequence is reacted with the PCR kit components in the nanopipette to complete a PCR amplification reaction. SYBR Green prefilled in the nanopipette is intercalated into double-stranded DNA to induce fluorescence emission for real-time detection down to a single copy. An obvious increase in the fluorescence is observed that validates the detection of the target DNA sequence in single living cells. The established real-time fluorescent PCR nanokit could adapt the PCR kit for single cell analysis and thus offers an alternatively general and highly sensitive strategy for the detection of specific DNA sequences in single living cells.

An Iron(III)-Based Metal-Organic Gel-Catalyzed Dual Electrochemiluminescence System for Cytosensing and In Situ Evaluation of the VEGF165 Subtype.

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.

Spatial Analysis of Reactive Oxygen Species in a 3D Cell Model Using a Sensitive Nanocavity Electrode.

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.

Trihexyltetradecylphosphonium chloride based ratiometric fluorescent nanosensors for multiplex anion discrimination.

Compared with the well-studied cations, the development of methods for anion detection is relatively slow due to the anion characteristics such as a complex geometry, strong hydration and a low charge density. Herein, a colorimetric and ratiometric fluorescent anion sensing platform based on trihexyltetradecylphosphonium chloride ([THTP][Cl]) was developed for the first time. Such nanosensors exhibited a pH response of 5-7 as well as a high selectivity to perchlorate. The selectivity behavior followed the Hofmeister series in which lipophilic anions were more readily co-extracted. To deviate from the Hofmeister series, anion ionophores should be introduced for selective complexation of the target anions. As a proof of concept, the organomercury compounds ETH9033 and ETH9009 were employed as model ionophores. The obtained nitrate- and chloride-selective [THTP][Cl]-based nanosensors demonstrated prominent colorimetric and spectroscopic transformations specifically induced by the anion species. The fluorescence (I675/I600) and absorbance (A650/A500) intensities versus the logarithm values of anion concentrations proved a high selectivity towards the major anion. The excellent performance such as high selectivity, good sensitivity and fast response times enabled the accurate determination of nitrate in mineral water. More importantly, through simply altering the ionophores, a pool of [THTP][Cl]-based anion-selective nano-optodes for extended targets could be achieved. The nanosensor shows great potential for anion determination in the environmental and biomedical fields.