Holistic Prediction of AuNP Aggregation in Diverse Aqueous Suspensions Based on Machine Vision and Dark-Field Scattering Imaging.

The localized surface-plasmon resonance of the AuNP in aqueous media is extremely sensitive to environmental changes. By measuring the signal of plasmon scattering light, the dark-field microscopic (DFM) imaging technique has been used to monitor the aggregation of AuNPs, which has attracted great attention because of its simplicity, low cost, high sensitivity, and universal applicability. However, it is still challenging to interpret DFM images of AuNP aggregation due to the heterogeneous characteristics of the isolated and discontinuous color distribution. Herein, we introduce machine vision algorithms for the training of DFM images of AuNPs in different saline aqueous media. A visual deep learning framework based on AlexNet is constructed for studying the aggregation patterns of AuNPs in aqueous suspensions, which allows for rapid and accurate identification of the aggregation extent of AuNPs, with a prediction accuracy higher than 0.96. With the aid of machine learning analysis, we further demonstrate the prediction ability of various aggregation phenomena induced by both cation species and the concentration of the external saline solution. Our results suggest the great potential of machine vision frameworks in the accurate recognition of subtle pattern changes in DFM images, which can help researchers build predictive analytics based on DFM imaging data.

DNAzyme-Powered DNA Walker for Cooperative Expression Imaging of Mutant p53 and Telomerase in Cancer Cells.

Cooperative expression of multiple cancer biomarkers is of great significance in influencing cell pathways and drug treatment. However, the simultaneous analysis of low-abundance biomarkers in living cells remains a challenge. Here, we report a DNAzyme-powered DNA walker to visualize the cooperative expression of mutant p53 and telomerase in living cells. The activation of the DNA walker is orthogonally powered by mutated p53 and telomerase, which enables the unlocking of the walking strand and the subsequently repeated substrate cleavage, producing fluorescence recovery for the imaging of the two target molecules in living cells. The DNA walker allows for real-time monitoring of the expression profile of mutant p53 and active telomerase in cancer cells under various antitumor drug treatments, and the results demonstrate the cooperative expression of mutant p53 and telomerase via the Akt pathway, which may bring new insights into the study of cancer pathway-relevant biomarkers.

Amperometric Identification of Single Exosomes and Their Dopamine Contents Secreted by Living Cells.

Dopamine (DA) is an important neurotransmitter, which not only participates in the regulation of neural processes but also plays critical roles in tumor progression and immunity. However, direct identification of DA-containing exosomes, as well as quantification of DA in single vesicles, is still challenging. Here, we report a nanopipette-assisted method to detect single exosomes and their dopamine contents via amperometric measurement. The resistive-pulse current measured can simultaneously provide accurate information of vesicle translocation and DA contents in single exosomes. Accordingly, DA-containing exosomes secreted from HeLa and PC12 cells under different treatment modes successfully detected the DA encapsulation efficiency and the amount of exosome secretion that distinguish between cell types. Furthermore, a custom machine learning model was constructed to classify the exosome signals from different sources, with an accuracy of more than 99%. Our strategy offers a useful tool for investigating single exosomes and their DA contents, which facilitates the analysis of DA-containing exosomes derived from other untreated or stimulated cells and may open up a new insight to the research of DA biology.

Dual-Modal Apoptosis Assay Enabling Dynamic Visualization of ATP and Reactive Oxygen Species in Living Cells.

ATP and reactive oxygen species (ROS) are considered significant indicators of cell apoptosis. However, visualizing the interplay between apoptosis-related ATP and ROS is challenging. Herein, we developed a metal-organic framework (MOF)-based nanoprobe for an apoptosis assay using duplex imaging of cellular ATP and ROS. The nanoprobe was fabricated through controlled encapsulation of gold nanorods with a thin zirconium-based MOF layer, followed by modification of the ROS-responsive molecules 2-mercaptohydroquinone and 6-carboxyfluorescein-labeled ATP aptamer. The nanoprobe enables ATP and ROS visualization via fluorescence and surface-enhanced Raman spectroscopy, respectively, avoiding the mutual interference that often occurs in single-mode methods. Moreover, the dual-modal assay effectively showed dynamic imaging of ATP and ROS in cancer cells treated with various drugs, revealing their apoptosis-related pathways and interactions that differ from those under normal conditions. This study provides a method for studying the relationship between energy metabolism and redox homeostasis in cell apoptosis processes.

Carbon dots with hydrogen bond-controlled aggregation behavior.

Here, hydrophilic carbon dots (H-CDs) are prepared by a facile room temperature method. The strength of hydrogen bonds can be controlled by introducing proton and aprotic solvents, respectively, so as to realize the tunable aggregation state of H-CDs. Because of the ultrasensitive response to dimethyl sulfoxide (DMSO), H-CDs can serve as optical probes for detecting DMSO in a linear range of 0.005% to 0.75% and with a detection limit of 0.001%.

Schiff Base Reaction in a Living Cell: In Situ Synthesis of a Hollow Covalent Organic Polymer To Regulate Biological Functions.

Artificially performing chemical reactions in living biosystems to attain various physiological aims remains an intriguing but very challenging task. In this study, the Schiff base reaction was conducted in cells using Sc(OTf)3 as a catalyst, enabling the in situ synthesis of a hollow covalent organic polymer (HCOP) without external stimuli. The reversible Schiff base reaction mediated intracellular Oswald ripening endows the HCOP with a spherical, hollow porous structure and a large specific surface area. The intracellularly generated HCOP reduced cellular motility by restraining actin polymerization, which consequently induced mitochondrial deactivation, apoptosis, and necroptosis. The presented intracellular synthesis system inspired by the Schiff base reaction has strong potential to regulate cell fate and biological functions, opening up a new strategic possibility for intervening in cellular behavior.

Specially Resolved Single Living Cell Perfusion and Targeted Fluorescence Labeling Based on Nanopipettes.

Targeted delivery and labeling of single living cells in heterogeneous cell populations are of great importance to understand the molecular biology and physiological functions of individual cells. However, it remains challenging to perfuse fluorescence markers into single living cells with high spatial and temporal resolution without interfering neighboring cells. Here, we report a single cell perfusion and fluorescence labeling strategy based on nanoscale glass nanopipettes. With the nanoscale tip hole of 100 nm, the use of nanopipettes allows special perfusion and high-resolution fluorescence labeling of different subcellular regions in single cells of interest. The dynamic of various fluorescent probes has been studied to exemplify the feasibility of nanopipette-dependent targeted delivery. According to experimental results, the cytoplasm labeling of Sulfo-Cyanine5 and fluorescein isothiocyanate is mainly based on the Brownian movement due to the dyes themselves and does not have a targeting ability, while the nucleus labeling of 4',6-diamidino-2-phenylindole (DAPI) is originated from the adsorption between DAPI and DNA in the nucleus. From the finite element simulation, the precise manipulation of intracellular delivery is realized by controlling the electro-osmotic flow inside the nanopipettes, and the different delivery modes between nontargeting dyes and nucleus-targeting dyes were compared, showcasing the valuable ability of nanopipette-based method for the analysis of specially defined subcellular regions and the potential applications for single cell surgery, subcellular manipulation, and gene delivery.

Dynamic Visualization of Endoplasmic Reticulum Stress in Living Cells via a Two-Stage Cascade Recognition Process.

The endoplasmic reticulum (ER) is crucial for the regulation of multiple cellular processes, such as cellular responses to stress and protein synthesis, folding, and posttranslational modification. Nevertheless, monitoring ER physiological activity remains challenging due to the lack of powerful detection methods. Herein, we built a two-stage cascade recognition process to achieve dynamic visualization of ER stress in living cells based on a fluorescent carbon dot (CD) probe, which is synthesized by a facile one-pot hydrothermal method without additional modification. The fluorescent CD probe enables two-stage cascade ER recognition by first accumulating in the ER as the positively charged and lipophilic surface of the CD probe allows its fast crossing of multiple membrane barriers. Next, the CD probe can specifically anchor on the ER membrane via recognition between boronic acids and o-dihydroxy groups of mannose in the ER lumen. The two-stage cascade recognition process significantly increases the ER affinity of the CD probe, thus allowing the following evaluation of ER stress by tracking autophagy-induced mannose transfer from the ER to the cytoplasm. Thus, the boronic acid-functionalized cationic CD probe represents an attractive tool for targeted ER imaging and dynamic tracking of ER stress in living cells.

AuNPs-COFs Core-Shell Reversible SERS Nanosensor for Monitoring Intracellular Redox Dynamics.

The redox homeostasis in living cells is greatly crucial for maintaining the redox biological function, whereas accurate and dynamic detection of intracellular redox states still remains challenging. Herein, a reversible surface-enhanced Raman scattering (SERS) nanosensor based on covalent organic frameworks (COFs) was prepared to dynamically monitor the redox processes in living cells. The nanosensor was fabricated by modifying the redox-responsive Raman reporter molecule, 2-Mercaptobenzoquione (2-MBQ), on the surface of gold nanoparticles (AuNPs), followed by the in situ coating of COFs shell. 2-MBQ molecules can repeatedly and quickly undergo reduction and oxidation when successively treated with ascorbic acid (AA) and hypochlorite (ClO-) (as models of reductive and oxidative species, respectively), which resulted in the reciprocating changes of SERS spectra at 900 cm-1. The construction of the COFs shell provided the nanosensor with great stability and anti-interference capability, thus reliably visualizing the dynamics of intracellular redox species like AA and ClO- by SERS nanosensor. Taken together, the proposed SERS strategy opens up the prospects to investigate the signal transduction pathways and pathological processes related with redox dynamics.

Combination Cancer Treatment: Using Engineered DNAzyme Molecular Machines for Dynamic Inter- and Intracellular Regulation.

Despite the promise of combination cancer therapy, it remains challenging to develop targeted strategies that are nontoxic to normal cells. Here we report a combination therapeutic strategy based on engineered DNAzyme molecular machines that can promote cancer apoptosis via dynamic inter- and intracellular regulation. To achieve external regulation of T-cell/cancer cell interactions, we designed a DNAzyme-based molecular machine with an aptamer and an i-motif, as the MUC-1-selective aptamer allows the specific recognition of cancer cells. The i-motif is folded under the tumor acidic microenvironment, shortening the intercellular distance. As a result, T-cells are released by metal ion activated DNAzyme cleavage. To achieve internal regulation of mitochondria, we delivered another DNAzyme-based molecular machine with mitochondria-targeted peptides into cancer cells to induce mitochondria aggregation. Our strategy achieved an enhanced killing effect in zinc deficient cancer cells.

Cell Surface Engineering Using DNAzymes: Metal Ion Mediated Control of Cell-Cell Interactions.

Regulating cell-cell interactions and cell behaviors via cell surface engineering is of significance for biological research such as cell fate control and cell therapy. While extensive efforts have been made to induce cell-cell assembly via various cell surface modifications triggered by macromolecules or organic metabolites, controllable cell-cell interactions that include both assembly and disassembly triggered by metal ions remain a challenge. Herein, we report a strategy based on DNAzymes to realize controllable cell-cell interactions, triggered by metal ions. The metal-dependent DNAzyme-based cleavage can effectively manipulate cell behaviors, including cell-cell conjunctions and disaggregation. Using a Zn2+-specific DNAzyme, a Mg2+-specific DNAzyme, and their respective substrate strands as the building blocks, the corresponding DNA double-chain switches enabling two-factor disassembly are demonstrated. Moreover, the method has been applied to control the assembly and disassembly between two cell spheroids. Since a wide variety of metal-specific DNAzymes are available, this method can be readily applied to construct cell dynamic systems controlled by other metal ions, providing a smart and versatile platform to regulate dynamic cell behavior.

Nanopipette-Based Nanosensor for Label-Free Electrochemical Monitoring of Cell Membrane Rupture under H2O2 Treatment.

H2O2 is an essential signaling molecule in living cells that can cause direct damage to lipids, proteins, and DNA, resulting in cell membrane rupture. However, current studies mostly focus on probe-based sensing of intracellular H2O2, and these methods usually require sophisticated probe synthesis and instruments. In particular, local H2O2 treatment induces cell membrane rupture, but the level of cell membrane destruction is unknown because the mechanical properties of the cell membrane are difficult to accurately determine. Therefore, highly sensitive and label-free methods are required to measure and reflect mechanical changes in the cell membrane. Here, using an ultrasmall quartz nanopipette with a tip diameter less than 90 nm as a nanosensor, label-free and noninvasive electrochemical single-cell measurement is achieved for real-time monitoring of cell membrane rupture under H2O2 treatment. By spatially controlling the nanopipette tip to precisely approach a specific location on the membrane of a single living cell, stable cyclic membrane oscillations are observed under a constant direct current voltage. Specifically, upon nanopipette advancement, the mechanical status of the cell membrane can be sensibly displayed by continuous current versus time traces. The electrical signals are collected and processed, ultimately revealing the mechanical properties of the cell membrane and the degree of cell apoptosis. This nanopipette-based nanosensor paves the way for developing a facile, label-free, and noninvasive strategy to assay the mechanical properties of the cell membrane during external stimulation at the single-cell level.

In Situ Monitoring of Hydrogen Peroxide Released from Living Cells Using a ZIF-8-Based Surface-Enhanced Raman Scattering Sensor.

Hydrogen peroxide (H2O2) widely involves in intracellular and intercellular redox signaling pathways, playing a vital role in regulating various physiological events. Nevertheless, current analytical methods for the H2O2 assay are often hindered by relatively long response time, low sensitivity, or self-interference. Herein, a zeolitic imidazolate framework-8 (ZIF-8)-based surface-enhanced Raman scattering (SERS) sensor has been developed to detect H2O2 released from living cells by depositing ZIF-8 over SERS active gold nanoparticles (AuNPs) grafted with H2O2-responsive probe molecules, 2-mercaptohydroquinone. Combining the superior fingerprint identification of SERS and the highly efficient enrichment and selective response of H2O2 by ZIF, the ZIF-8-based SERS sensor exhibits a high anti-interference ability for H2O2 detection, with a limit of detection as low as 0.357 nM. Satisfyingly, owing to the enhanced catalytic activity derived from the successful integration of AuNPs and ZIF, the response time as short as 1 min can be obtained, demonstrating the effectiveness of the SERS sensor for rapid H2O2 detection. Furthermore, the developed SERS sensor enables real-time detection of H2O2 secreted from living cells under phorbol myristate acetate stimulation, as cells can be cultured on-chip. This study will pave the way toward the development of a metal-organic framework-based SERS platform for application in the fields of biosensing and early disease diagnosis associated with H2O2 secretion, thus exhibiting promising potential for future therapies.

Probing the Membrane Vibration of Single Living Cells by Using Nanopipettes.

The vibration of a cell membrane plays a key role in the regulation of cell shape and the behavior of cells. However, most existing approaches for the measurement of cell vibration require either exogenous modification or sophisticated techniques, and the main challenge lies in developing methods that can monitor membrane vibration of living cells directly. Herein, a noninvasive strategy based on ultrasmall quartz nanopipettes is introduced. With a tip size of less than 100 nm, nanopipettes can be spatially controlled for precision targeting of a specific location on the membrane of single living cells. Surprisingly, by employing a constant voltage, stable cyclic oscillations are observed from the continuous current versus time traces. The time-domain current can be decomposed into two basic waves: the high-frequency one indicates the local membrane vibration driven by the electro-osmotic flow from the nanopipette, whereas the low-frequency one indicates the natural frequency of the whole cell. This provides a simple but reliable method to test local and global membrane vibration of single living cells simultaneously with little damage, which provides a tool for the quantification of drugs, disease, or mutations of the cell structure.

A polydopamine-based biomimetic multifunctional nanoplatform for multilayer imaging of cancer biomarkers carried by extracellular vesicles.

Cancer-derived extracellular vesicles (EVs) have attracted considerable attention for clinical diagnosis. However, a limiting factor in current EV assays is the ability to detect various EV cancer biomarkers expressed at different locations. Here, we report a biomimetic multifunctional nanoplatform for multilayer imaging of cancer biomarkers from the EV surface to the interior without complex pretreatment. Constructed from polydopamine-wrapped gold nanoparticles modified with multiple functional molecules, this nanoplatform can capture EVs from complex samples and target different EV cancer biomarkers for imaging analysis at the single-vesicle level. Combined with 96-well plates, this assay can distinguish cancer cell-derived EVs from normal ones in a high-throughput manner. Using serum samples, EVs from hepatocellular carcinoma (HCC) patients can be distinguished from healthy controls. This convenient workflow represents a promising tool for EV-based cancer diagnosis.

Label-free chlorine and nitrogen-doped fluorescent carbon dots for target imaging of lysosomes in living cells.

Lysosomes with a single-layered membrane structure are mainly involved in the scavenging of foreign substances and play an important role in maintaining normal physiological functions of living cells. In this work, near-neutrally charged fluorescent carbon dots (CDs) were prepared with lipophilicity through a facile one-pot hydrothermal carbonization of chloranil and triethylenetetramine at 160 °C for 3 h. The as-obtained CDs are proved to have good photostability, low cost, and excellent biocompatibility. Importantly, the as-prepared CDs with high quantum yield of 30.8% show excitation-dependent emission with great stability, and thus, they can be well used for the long-term target imaging of lysosomes in living cells without further modification. Meanwhile, the CDs can quickly enter into the lysosomes within 30 min, and the green fluorescence (FL) of CDs reaches the plateau when incubated for 60 min. By comparing the fluorescent intensity, the information about distribution and amount of lysosomes in different cells can be obtained. The proposed CD-based strategy demonstrates great promise for label-free target imaging of lysosomes in living cells. Graphical abstract The near-neutral carbon dots (CDs) with lipophilicity are used as label-free fluorescent nanoprobes for the long-term imaging of lysosomes in living cells.

Ultrafast Mapping of Subcellular Domains via Nanopipette-Based Electroosmotically Modulated Delivery into a Single Living Cell.

Recently, a variety of strategies have been developed for single-cell detection. However, the precise probing of the given area at single-cell level is still a challenge. Here, we put forward a rapid and targeted imaging approach for the mapping of subcelluar domains, which realizes the precise injection of multifluorescence into a single living cell via an ultrasmall quartz capillary nanopipette (∼100 nm) and can successfully transport different fluorescent probe molecules to the pointing subcellullar area around the tip in the cytoplasm within 20 s. This method is also applied for monitoring the loss of intracellular mitochondrial membrane potential under the treatment of metformin in a single MCF-7 breast cancer cell. The major driven force in the nanopipette, electroosmotic flow, is evaluated by a theory calculation method and finite element simulations, and the solution indicates a confined solute distribution profile around the tip within the working range. Overall, the nanopipette approach realizes the precise and simultaneous delivery of multiple probe molecules into the single living cell through the electroosmotically modulated, nondestructive, and one-step injection, which is especially powerful and convenient for multichannel single-cell imaging and monitoring, indicating favorable potential for understanding, monitoring, and controlling the biological processes from the single cell to subcellular organelles.

Controllable Aggregation-Induced Exocytosis Inhibition (CAIEI) of Plasmonic Nanoparticles in Cancer Cells Regulated by MicroRNA.

Recent advances in nanotechnology have produced plenty of intracellular drug delivery systems based on various functional nanoparticles. Although much progress has been achieved in improving cellular uptake efficiency, the retention time of these engineered nanoparticles in living cells has not yet received wide attention. Here, we report the controllable exocytosis of plasmonic gold nanoparticles (GNPs) based on a microRNA-21 (miRNA-21) targeted binary system. Rapid intracellular accumulation of GNPs was observed in miRNA-21 positive MCF-7 breast cancer cells, which blocked the exocytosis of the GNP aggregates. Under near-infrared (NIR) irradiation, MCF-7 cells were successfully killed due to the far-red and NIR absorption of the GNP aggregates. In contrast, in miRNA-21 negative cells, the dispersive GNPs escaped from the cells after 6 h. The traces of GNPs could be conveniently captured under the dark-field microscope. This work provides a promising platform for the study of controllable aggregation-induced exocytosis inhibition (CAIEI) of nanocarriers, which is inspiring for the design of more effective nanodrugs for the treatment of cancer.

Plasmon Resonance Energy Transfer: Coupling between Chromophore Molecules and Metallic Nanoparticles.

Plasmon resonance energy transfer (PRET) from a single metallic nanoparticle to the molecules adsorbed on its surface has attracted more and more attentions in recent years. Here, a molecular beacon (MB)-regulated PRET coupling system composed of gold nanoparticles (GNPs) and chromophore molecules has been designed to study the influence of PRET effect on the scattering spectra of GNPs. In this system, the chromophore molecules are tagged to the 5'-end of MB, which can form a hairpin structure and modified on the surface of GNPs by its thiol-labeled 3'-end. Therefore, the distance between GNPs and chromophore molecules can be adjusted through the open and close of the MB loop. From the peak shift, the PRET interactions of different GNPs-chromophore molecules coupling pairs have been calculated by discrete dipole approximation and the fitting results match well with the experimental data. Therefore, the proposed system has been successfully applied for the analysis of PRET situation between various metallic nanoparticles and chromophore molecules, and provides a useful tool for the potential application in screening the PRET-based nanoplasmonic sensors.

A Two-Stage Dissociation System for Multilayer Imaging of Cancer Biomarker-Synergic Networks in Single Cells.

The monitoring of cancer biomarkers is crucial to the early detection of cancer. However, a limiting factor in biomarker analysis is the ability to obtain the multilayered information of various biomarker molecules located at different parts of cells from the plasma membrane to the cytoplasm. A two-stage dissociation nanoparticle system based on multifunctionalized polydopamine-coated gold nanoparticles (Au@PDA NPs) is reported, which allows for the two-stage imaging of cancer biomarkers in single cells. We demonstrate the feasibility of this strategy on sialic acids (SAs), p53 protein, and microRNA-21 (miRNA-21) in MCF-7 breast cancer cells by two custom-built probes. Furthermore, the multicolor fluorescence information extracted is used for the monitoring of biomarker expression changes under different drug combinations, which allows us to investigate the complex interactions between various cancer biomarkers and to describe the cancer biomarker-synergic networks in single cells.