ABSTRACT
Cell-substrate interaction plays a critical role in determining the mechanical status of living cell membrane. Changes of substrate surface properties can significantly alter the cell mechanical microenvironment, leading to mechanical changes of cell membrane. However, it is still difficult to accurately quantify the influence of the substrate surface properties on the mechanical status of living cell membrane without damage. This study addresses the challenge by using an electrochemical sensor made from an ultrasmall quartz nanopipette. With the tip diameter less than 100 nm, the nanopipette-based sensor achieves highly sensitive, noninvasive and label-free monitoring of the mechanical status of single living cells by collecting stable cyclic membrane oscillatory signals from continuous current versus time traces. The electrochemical signals collected from PC12 cells cultured on three different substrates (bare ITO (indium tin oxides) glass, hydroxyl modified ITO glass, amino modified ITO glass) indicate that the microenvironment more favorable for cell adhesion can increase the membrane stiffness. This work provides a label-free electrochemical approach to accurately quantify the mechanical status of single living cells in real-time, which may help to better understand the relationship between the cell membrane and the extra cellular matrix.
Subject(s)
Biosensing Techniques , Cell Membrane , Electrochemical Techniques , Tin Compounds , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , Animals , Rats , PC12 Cells , Tin Compounds/chemistry , Electrochemical Techniques/methods , Cell Membrane/chemistry , Cell Adhesion , Vibration , Surface Properties , Equipment DesignABSTRACT
Intracellular glucose detection is crucial due to its pivotal role in metabolism and various physiological processes. Precise glucose monitoring holds significance in diabetes management, metabolic studies, and biotechnological applications. In this study, we developed an innovative and expedient cell-permeable nanoreactor for intracellular glucose based on surface-enhanced Raman scattering (SERS). The nanoreactor was designed with gold nanoparticles (AuNPs), which were engineered with glucose oxide (GOx) and a H2O2-responsive Raman reporter 2-mercaptohydroquinone (2-MHQ). The interaction between 2-MHQ and H2O2 generated by glucose and GOx could simultaneously induce the appearance in the peak at 985 cm-1. Our results showed excellent performance in detecting glucose within the concentration range from 0.1 µM to 10 mM, with a low detection limitation of 14.72 nM. In addition, the glucose distribution in single HeLa cells was evaluated by real time SERS mapping. By combining noble metal particles and natural oxidases, the nanoreactor possesses both Raman activity and enzymatic functionality, thus enables sensitive glucose detection and facilitates imaging at a single cell level, which offers an insightful monitoring of cellular processes.
Subject(s)
Glucose , Gold , Metal Nanoparticles , Spectrum Analysis, Raman , Spectrum Analysis, Raman/methods , Humans , HeLa Cells , Gold/chemistry , Metal Nanoparticles/chemistry , Glucose/analysis , Glucose/metabolism , Hydrogen Peroxide/analysis , Hydrogen Peroxide/chemistry , Glucose Oxidase/chemistry , Glucose Oxidase/metabolismABSTRACT
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.
ABSTRACT
Hydrogen sulfide (H2S), an important gas signal molecule, participates in intercellular signal transmission and plays a considerable role in physiology and pathology. However, in-situ monitoring of H2S level during the processes of material transport between cells remains considerably challenging. Herein, a cell membrane-targeted surface-enhanced Raman scattering (SERS) nanoprobe was designed to quantitatively detect H2S secreted from living cells. The nanoprobes were fabricated by assembling cholesterol-functionalized DNA strands and dithiobis(phenylazide) (DTBPA) molecules on core-shell gold nanostars embedded with 4-mercaptoacetonitrile (4-MBN) (AuNPs@4-MBN@Au). Thus, three functions including cell-membrane targeted via cholesterol, internal standard calibration, and responsiveness to H2S through reduction of azide group in DTBPA molecules were integrated into the nanoprobes. In addition, the nanoprobes can quickly respond to H2S within 90 s and sensitively, selectively, and reliably detect H2S with a limit of detection as low as 37 nM due to internal standard-assisted calibration and reaction specificity. Moreover, the nanoprobes can effectively target on cell membrane and realize SERS visualization of dynamic H2S released from HeLa cells. By employing the proposed approach, an intriguing phenomenon was observed: the other two major endogenous gas transmitters, carbon monoxide (CO) and nitric oxide (NO), exhibited opposite effect on H2S production in living cells stimulated by related gas release molecules. In particular, the introduction of CO inhibited the generation of H2S in HeLa cells, while NO promoted its output. Thus, the nanoprobes can provide a robust method for investigating H2S-related extracellular metabolism and intercellular signaling transmission.
Subject(s)
Biosensing Techniques , Hydrogen Sulfide , Metal Nanoparticles , Humans , Hydrogen Sulfide/metabolism , HeLa Cells , Spectrum Analysis, Raman/methods , Gold , Nitric Oxide , Cell Membrane/metabolism , CholesterolABSTRACT
Cellular compartments provide confined environments for spatiotemporal control of biological processes and enzymatic reactions. To mimic such compartmentalization of eukaryotic cells, we report an efficient and general platform to precisely control the formation of artificial nanoreactors in single living cells. We introduce an electroosmotic controlled strategy for the synthesis of ZIF-8 at the nanoscale liquid-liquid interface around the tip of a nanopipet, whereby the formed ZIF-8 nanoparticles are driven into a single living cell by the electroosmotic flow. The porous ZIF-8 nanoparticles, as synthetic nanoreactors, are not only able to harvest fluorescent molecules from peripheral cytoplasm but also perform the subsequent photocatalytic degradation, mimicking compartmentalized chemical reactions in eukaryotic cells. Our strategy provides a useful tool for spatiotemporal controlled synthesis of artificial nanoreactors with on-demand functions in single living cells with versatile applications in chemical biology.
ABSTRACT
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.
Subject(s)
Polymers , Schiff Bases , Schiff Bases/chemistryABSTRACT
Oxidative stress is involved in various signaling pathways and serves a key role in inducing cell apoptosis. Therefore, it is significant to monitor oxidative stress upon drug release for the assessment of therapeutic effects in cancer cells. Herein, a glutathione (GSH)-responsive surface-enhanced Raman scattering (SERS) nanoplatform is proposed for ultra-sensitively monitoring the substance related with oxidative stress (hydrogen sulfide, H2S), depleting reactive sulfur species and releasing anticancer drugs to amplify oxidative stress for tumor apoptosis. The Au@Raman reporter@Ag (Au@M@Ag) nanoparticles, where a 4-mercaptobenzonitrile molecule as a Raman reporter was embedded between layers of gold and silver to obtain sensitive SERS response, were coated with a covalent organic framework (COF) shell to form a core-shell structure (Au@M@Ag@COFs) as the SERS nanoplatform. The COF shell loading doxorubicin (DOX) of Au@M@Ag@COFs exhibited the GSH-responsive degradation capacity to release DOX, and its Ag layer as the sensing agent was oxidized to Ag2S by H2S to result in its prominent changes in SERS signals with a low detection limit of 0.33 nM. Moreover, the releasing DOX can inhibit the generation of H2S to promote the production of reactive oxygen species, and the depletion of reactive sulfur species (GSH and H2S) in cancer cells can further enhance the oxidative stress to induce tumor apoptosis. Overall, the SERS strategy could provide a powerful tool to monitor the dynamic changes of oxidative stress during therapeutic processes in a tumor microenvironment.
Subject(s)
Hydrogen Sulfide , Nanoparticles , Neoplasms , Humans , Nanoparticles/chemistry , Doxorubicin/pharmacology , Doxorubicin/chemistry , Neoplasms/drug therapy , Oxidative Stress , Tumor MicroenvironmentABSTRACT
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.
ABSTRACT
Nanopipette, which is fabricated by glasses and possesses a nanoscale pore in the tip, has been proven to be immensely useful in electrochemical analysis. Numerous nanopipette-based sensors have emerged with improved sensitivity, selectivity, ease of use, and miniaturization. In this minireview, we provide an overview of the recent developments of nanopipette-based electrochemical sensors based on different types of nanopipettes, including single-nanopipettes, self-referenced nanopipettes, dual-nanopipettes, and double-barrel nanopipettes. Several important modification materials for nanopipette functionalization are highlighted, such as conductive materials, macromolecular materials, and functional molecules. These materials can improve the sensing performance and targeting specificities of nanopipettes. We also discuss examples of related applications and the future development of nanopipette-based strategies.
ABSTRACT
The abnormal change in the expression profile of multiple cancer biomarkers is closely related to tumor progression and therapeutic effect. Due to their low abundance in living cells and the limitations of existing imaging techniques, simultaneous imaging of multiple cancer biomarkers has remained a significant challenge. Here, we proposed a multi-modal imaging strategy to detect the correlated expression of multiple cancer biomarkers, MUC1, microRNA-21 (miRNA-21) and reactive oxygen (ROS) in living cells, based on a porous covalent organic framework (COF) wrapped gold nanoparticles (AuNPs) core-shell nanoprobe. The nanoprobe is functionalized with Cy5-labeled MUC1 aptamer, a ROS-responsive molecule (2-MHQ), and a miRNA-21-response hairpin DNA tagged by FITC as the reporters for different biomarkers. The target-specific recognition can induce the orthogonal molecular change of these reporters, producing fluorescence and Raman signals for imaging the expression profiles of membrane MUC1 (red fluorescence channel), intracellular miRNA-21 (green fluorescence channel), and intracellular ROS (SERS channel). We further demonstrate the capability of the cooperative expression of these biomarkers, along with the activation of NF-κB pathway. Our research provides a robust platform for imaging multiple cancer biomarkers, with broad potential applications in cancer clinical diagnosis and drug discovery.
Subject(s)
Biosensing Techniques , Metal Nanoparticles , Metal-Organic Frameworks , MicroRNAs , Neoplasms , Humans , Biomarkers, Tumor , Gold , Reactive Oxygen Species , Biosensing Techniques/methods , Neoplasms/diagnosis , MicroRNAs/genetics , Spectrum Analysis, RamanABSTRACT
Nanomaterials have presented great potential for cancer therapy. However, their therapeutic efficacy is not always satisfied because of inefficient biocompatibility and targeting efficacy. Here, we report engineered extracellular vesicle (EV)-encapsuled nanoreactors for the targeting and killing of cancer cells. EVs are extracted from engineered cancer cells with surface N-glycans cut and intracellular microRNA-21 (miR-21) silenced to generate cancer-targeting membranes for the following coating of gold-polydopamine (PDA) core-shell nanoparticles. The encapsuled nanoparticles are decorated with doxorubicin (Dox), glucose oxidase (GOx), and miR-21-indicative DNA tags. Once endocytosed, the acidic pH, together with the photothermal effect of the PDA shell, can promote the release of Dox and GOx-catalyzed H2O2 generation/glucose consumption, while the DNA tags allow enhanced fluorescence imaging of miR-21 to indicate the targeting effect. The coadministration of EV-assisted delivery and cascade treatment represents a promising strategy for combination therapy.
Subject(s)
Extracellular Vesicles , MicroRNAs , Neoplasms , Hydrogen Peroxide , Doxorubicin/pharmacology , Glucose Oxidase , MicroRNAs/genetics , Nanotechnology , Neoplasms/drug therapyABSTRACT
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.
Subject(s)
Apoptosis , Gold , Reactive Oxygen Species/metabolism , Gold/chemistry , Adenosine TriphosphateABSTRACT
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.
Subject(s)
DNA, Catalytic , Neoplasms , Telomerase , Humans , DNA, Catalytic/chemistry , Tumor Suppressor Protein p53/genetics , Telomerase/metabolism , DNA/chemistry , Neoplasms/diagnostic imaging , Neoplasms/genetics , Neoplasms/pathologyABSTRACT
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%.
ABSTRACT
Herein, a fluorescence and surface-enhanced Raman spectroscopy dual-mode system was designed for cholesterol detection based on self-assembled plasmonic nanojunctions mediated by the competition of rhodamine 6G (R6G) and cholesterol with ß-cyclodextrin modified on gold nanoparticles (HS-ß-CD@Au). The fluorescence of R6G was quenched by HS-ß-CD@Au due to the fluorescence resonance energy transfer effect. When cholesterol was introduced as the competitive guest, R6G in the cavities of HS-ß-CD@Au was displaced to recover its fluorescence. Moreover, two of HS-ß-CD@Au can be linked by one cholesterol to form a more stable 2:1 complex, and then, plasmonic nanojunctions were generated, which resulted in the increasing SERS signal of R6G. In addition, fluorescence and SERS intensity of R6G increased linearly with the increase in the cholesterol concentrations with the limits of detection of 95 and 74 nM, respectively. Furthermore, the dual-mode strategy can realize the reliable and sensitive detection of cholesterol in the serum with good accuracy, and two sets of data can mutually validate each other, which demonstrated great application prospects in the surveillance of diseases related with cholesterol.
Subject(s)
Gold , Metal Nanoparticles , Gold/chemistry , Metal Nanoparticles/chemistry , Fluorescence Resonance Energy Transfer , Cholesterol , Spectrum Analysis, Raman/methodsABSTRACT
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.
Subject(s)
Biosensing Techniques , DNA, Catalytic , Neoplasms , Humans , DNA, Catalytic/chemistry , Neoplasms/drug therapy , Tumor MicroenvironmentABSTRACT
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.
Subject(s)
Metal Nanoparticles , Metal-Organic Frameworks , Ascorbic Acid , Gold , Hypochlorous Acid , Oxidation-Reduction , Spectrum Analysis, Raman/methodsABSTRACT
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.
Subject(s)
Fluorescent Dyes , Nanotechnology , DNA , Fluoresceins , Isothiocyanates , Nanotechnology/methods , PerfusionABSTRACT
There is an unmet clinical need to develop noninvasive liquid biopsy tools for systemic lupus erythematosus (SLE) diagnosis and therapeutic effect evaluation. Extracellular vesicles (EVs), which are abundant in body fluids, have emerged as a valuable resource for liquid biopsy. Herein, we describe a simple and robust EV detection platform that is based on a plasmonic nanoparticle-embedded polydopamine substrate that is modified with EV-capture molecules and detection probes. We investigated three EV biomarkers, namely, programmed cell death protein-1 (PD-1), microRNA-146a (miRNA-146a) and sialic acid (SA), in serum and urine from SLE patients and healthy controls. This platform prevents complex pretreatment while enabling highly efficient EV capture to the substrate surface, and the multiple functionalization of the detection interface with specific biomarker probes enables simultaneous detection of PD-1, miRNA-146a and SA that are carried by EVs via fluorescence (FL) imaging at the single-vesicle level. Via comparison of EV biomarker profiles, SLE patients can be distinguished from normal controls and classified into treated and untreated groups. Due to its ease of preparation, simplicity and stability, our approach shows good potential in the design of EV-based biosensors for clinical use.
Subject(s)
Body Fluids , Extracellular Vesicles , Lupus Erythematosus, Systemic , MicroRNAs , Nanoparticles , Biomarkers/metabolism , Body Fluids/metabolism , Humans , Indoles , Lupus Erythematosus, Systemic/diagnosis , Lupus Erythematosus, Systemic/metabolism , MicroRNAs/metabolism , Polymers , Programmed Cell Death 1 Receptor/metabolismABSTRACT
Carbon dots (CDs), as new type of carbon-based nanoparticles, are considered to be an aggregate with irreversible polymerization. Achieving the reversible tunability of CDs luminescence based on their reversible polymerization is a challenging subject. Herein, we, for the first time, design and construct the blue-emitting CDs with reversible polymerization by a room-temperature Schiff base reaction between tannic acid and ethylenediamine. The formation of CDs is proven to be due to the crosslinking polymerization of precursors caused by imine bond. As a dynamic covalent bond, imine bond endows CDs with controllable structural transformation properties, and the prepared CDs can be depolymerized and polymerized reversibly by pH-controlled imine bond cleavage and re-formation. These properties of reversible fluorescence photoswitching make the CDs have a good application prospect in reversible information encryption.