RESUMO
We report on single-molecule nanopore sensing combined with position-encoded DNA molecular probes, with chemistry tuned to simultaneously identify various antigen proteins and multiple RNA gene fragments of SARS-CoV-2 with high sensitivity and selectivity. We show that this sensing strategy can directly detect spike (S) and nucleocapsid (N) proteins in unprocessed human saliva. Moreover, our approach enables the identification of RNA fragments from patient samples using nasal/throat swabs, enabling the identification of critical mutations such as D614G, G446S, or Y144del among viral variants. In particular, it can detect and discriminate between SARS-CoV-2 lineages of wild-type B.1.1.7 (Alpha), B.1.617.2 (Delta), and B.1.1.539 (Omicron) within a single measurement without the need for nucleic acid sequencing. The sensing strategy of the molecular probes is easily adaptable to other viral targets and diseases and can be expanded depending on the application required.
Assuntos
Antígenos Virais , Nanoporos , Humanos , Antígenos Virais/genética , Sondas Moleculares , RNA , RNA Viral/genéticaRESUMO
Scanning ion conductance microscopy (SICM) is a promising tool for visualizing the dynamics of nanoscale cell surface topography. However, there are still no guidelines for fabricating nanopipettes with ideal shape consisting of small apertures and thin glass walls. Therefore, most of the SICM imaging has been at a standstill at the submicron scale. In this study, we established a simple and highly reproducible method for the fabrication of nanopipettes with sub-20 nm apertures. To validate the improvement in the spatial resolution, we performed time-lapse imaging of the formation and disappearance of endocytic pits as a model of nanoscale time-lapse topographic imaging. We have also successfully imaged the localization of the hot spot and the released extracellular vesicles. The nanopipette fabrication guidelines for the SICM nanoscale topographic imaging can be an essential tool for understanding cell-cell communication.
Assuntos
Vesículas Extracelulares , Microscopia , Cintilografia , Comunicação Celular , Membrana Celular , ÍonsRESUMO
The reduction in ion current as a fine pipette approaches a cell surface allows the cell surface topography to be imaged, with nanoscale resolution, without contact with the delicate cell surface. A variety of different methods have been developed and refined to scan the topography of the dynamic cell surface at high resolution and speed. Measurement of cell topography can be complemented by performing local probing or mapping of the cell surface using the same pipette. This can be done by performing single-channel recording, applying force, delivering agonists, using pipettes fabricated to contain an electrochemical probe, or combining with fluorescence imaging. These methods in combination have great potential to image and map the surface of live cells at the nanoscale.
Assuntos
Membrana CelularRESUMO
Clinical use of human mesenchymal stem cells (hMSCs) is limited due to their rapid clearance, reducing their therapeutic efficacy. The inflammatory cytokine IL-1ß activates hMSCs and is known to enhance their engraftment. Consequently, understanding the molecular mechanism of this inflammation-triggered adhesion is of great clinical interest to improving hMSC retention at sites of tissue damage. Integrins are cell-matrix adhesion receptors, and clustering of integrins at the nanoscale underlies cell adhesion. Here, we found that IL-1ß enhances adhesion of hMSCs via increased focal adhesion contacts in an α5ß1 integrin-specific manner. Further, through quantitative super-resolution imaging we elucidated that IL-1ß specifically increases nanoscale integrin α5ß1 availability and clustering at the plasma membrane, whilst conserving cluster area. Taken together, these results demonstrate that hMSC adhesion via IL-1ß stimulation is partly regulated through integrin α5ß1 spatial organization at the cell surface. These results provide new insight into integrin clustering in inflammation and provide a rational basis for design of therapies directed at improving hMSC engraftment.
Assuntos
Células da Medula Óssea/fisiologia , Adesão Celular , Matriz Extracelular/metabolismo , Integrina alfa5beta1/metabolismo , Interleucina-1beta/farmacologia , Células-Tronco Mesenquimais/fisiologia , Células da Medula Óssea/citologia , Membrana Celular/metabolismo , Movimento Celular , Fibronectinas/metabolismo , Humanos , Integrina alfa5beta1/genética , Células-Tronco Mesenquimais/citologiaRESUMO
Podocyte damage is a hallmark of glomerular diseases, such as focal segmental glomerulosclerosis, typically associated with marked albuminuria and progression of renal pathology. Podocyte structural abnormalities and loss are also linked to minimal change disease and more common diabetic kidney disease. Here we applied the first-time scanning ion conductance microscopy (SICM) technique to assess the freshly isolated human glomerulus's topology. SICM provides a unique opportunity to evaluate glomerulus podocytes as well as other nephron structural segments with electron microscopy resolution but in live samples. Shown here is the application of the SICM method in the live human glomerulus, which provides proof of principle for future dynamic analysis of membrane morphology and various functional parameters in living cells.
Assuntos
Glomérulos Renais/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Podócitos/ultraestrutura , HumanosRESUMO
Exocytosis of peptides and steroids stored in a dense core vesicular (DCV) form is the final step of every secretory pathway, indispensable for the function of nervous, endocrine and immune systems. The lack of live imaging techniques capable of direct, label-free visualisation of DCV release makes many aspects of the exocytotic process inaccessible to investigation. We describe the application of correlative scanning ion conductance and fluorescence confocal microscopy (SICM-FCM) to study the exocytosis of individual granules of insulin from the top, nonadherent, surface of pancreatic ß-cells. Using SICM-FCM, we were first to directly follow the topographical changes associated with physiologically induced release of insulin DCVs. This allowed us to report the kinetics of the full fusion of the insulin vesicle as well as the subsequent solubilisation of the released insulin crystal.
Assuntos
Células Secretoras de Insulina , Insulina , Exocitose , Microscopia Confocal , Microscopia de Fluorescência , Vesículas SecretóriasRESUMO
Understanding the molecular mechanisms involved in the assembly of viruses is essential for discerning how viruses transmit from cell to cell and host to host. Although molecular aspects of assembly have been studied for many viruses, we still have little information about these events in real time. Enveloped viruses such as HIV that assemble at, and bud from, the plasma membrane have been studied in some detail using live cell fluorescence imaging techniques; however, these approaches provide little information about the real-time morphological changes that take place as viral components come together to form individual virus particles. Here we used correlative scanning ion conductance microscopy and fluorescence confocal microscopy to measure the topological changes, together with the recruitment of fluorescently labeled viral proteins such as Gag and Vpr, during the assembly and release of individual HIV virus-like particles (VLPs) from the top, nonadherent surfaces of living cells. We show that 1) labeling of viral proteins with green fluorescent protein affects particle formation, 2) the kinetics of particle assembly on different plasma membrane domains can vary, possibly as a consequence of differences in membrane biophysical properties, and 3) VLPs budding from the top, unimpeded surface of cells can reach full size in 20 s and disappear from the budding site in 0.5 to 3 min from the moment curvature is initially detected, significantly faster than has been previously reported.
Assuntos
HIV-1/metabolismo , Vírion/metabolismo , Montagem de Vírus/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Liberação de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Dynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H+ in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity > 0.01 units, 2 ms response time, and 50 nm spatial resolution. The platform was integrated into a double-barrel nanoprobe combining pH sensing with feedback-controlled distance dependance via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells.
Assuntos
Imageamento Tridimensional/métodos , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Análise de Célula Única/métodos , Biofísica , Linhagem Celular Tumoral , Diatomáceas/citologia , Humanos , Concentração de Íons de Hidrogênio , Melanoma , Microscopia Eletrônica de VarreduraRESUMO
Dynamin 2 (DNM2) is a GTP-binding protein that controls endocytic vesicle scission and defines a whole class of dynamin-dependent endocytosis, including clathrin-mediated endocytosis by caveoli. It has been suggested that mutations in the DNM2 gene, associated with 3 inherited diseases, disrupt endocytosis. However, how exactly mutations affect the nanoscale morphology of endocytic machinery has never been studied. In this paper, we used live correlative scanning ion conductance microscopy (SICM) and fluorescence confocal microscopy (FCM) to study how disease-associated mutations affect the morphology and kinetics of clathrin-coated pits (CCPs) by directly following their dynamics of formation, maturation, and internalization in skin fibroblasts from patients with centronuclear myopathy (CNM) and in Cos-7 cells expressing corresponding dynamin mutants. Using SICM-FCM, which we have developed, we show how p.R465W mutation disrupts pit structure, preventing its maturation and internalization, and significantly increases the lifetime of CCPs. Differently, p.R522H slows down the formation of CCPs without affecting their internalization. We also found that CNM mutations in DNM2 affect the distribution of caveoli and reduce dorsal ruffling in human skin fibroblasts. Collectively, our SICM-FCM findings at single CCP level, backed up by electron microscopy data, argue for the impairment of several forms of endocytosis in DNM2-linked CNM.-Ali, T., Bednarska, J., Vassilopoulos, S., Tran, M., Diakonov, I. A., Ziyadeh-Isleem, A., Guicheney, P., Gorelik, J., Korchev, Y. E., Reilly, M. M., Bitoun, M., Shevchuk, A. Correlative SICM-FCM reveals changes in morphology and kinetics of endocytic pits induced by disease-associated mutations in dynamin.
Assuntos
Dinamina II/genética , Endocitose/genética , Mutação/genética , Miopatias Congênitas Estruturais/genética , Adulto , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Clatrina/genética , Feminino , Fibroblastos/patologia , Humanos , Cinética , Masculino , Microscopia Confocal/métodos , Microscopia Eletrônica de Varredura/métodos , Microscopia de Fluorescência/métodosRESUMO
Owing to their ability to efficiently deliver biological cargo and sense the intracellular milieu, vertical arrays of high aspect ratio nanostructures, known as nanoneedles, are being developed as minimally invasive tools for cell manipulation. However, little is known of the mechanisms of cargo transfer across the cell membrane-nanoneedle interface. In particular, the contributions of membrane piercing, modulation of membrane permeability and endocytosis to cargo transfer remain largely unexplored. Here, combining state-of-the-art electron and scanning ion conductance microscopy with molecular biology techniques, it is shown that porous silicon nanoneedle arrays concurrently stimulate independent endocytic pathways which contribute to enhanced biomolecule delivery into human mesenchymal stem cells. Electron microscopy of the cell membrane at nanoneedle sites shows an intact lipid bilayer, accompanied by an accumulation of clathrin-coated pits and caveolae. Nanoneedles enhance the internalization of biomolecular markers of endocytosis, highlighting the concurrent activation of caveolae- and clathrin-mediated endocytosis, alongside macropinocytosis. These events contribute to the nanoneedle-mediated delivery (nanoinjection) of nucleic acids into human stem cells, which distribute across the cytosol and the endolysosomal system. This data extends the understanding of how nanoneedles modulate biological processes to mediate interaction with the intracellular space, providing indications for the rational design of improved cell-manipulation technologies.
Assuntos
Sistemas de Liberação de Medicamentos/instrumentação , Endocitose/fisiologia , Nanopartículas/química , Agulhas , Silício/química , Cavéolas/metabolismo , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Clatrina/administração & dosagem , Clatrina/metabolismo , Citosol/metabolismo , Endossomos/metabolismo , Humanos , Espaço Intracelular/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Microscopia Eletrônica/instrumentação , Pinocitose/efeitos dos fármacos , Porosidade , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/metabolismo , Propriedades de SuperfícieRESUMO
Primary cilia are hair-like sensory organelles whose dimensions and location vary with cell type and culture condition. Herein, we employed scanning ion conductance microscopy (SICM) to visualize the topography of primary cilia from different cell types. By combining SICM with fluorescence imaging, we successfully distinguished between surface cilia that project outward from the cell surface and subsurface cilia that are trapped below it. The nanoscale structure of the ciliary pocket, which cannot be easily identified using a confocal fluorescence microscope, was observed in SICM images. Furthermore, we developed a topographic reconstruction method using current-distance profiles to evaluate the relationship between set point and topographic image and found that a low set point is important for detecting the true topography of a primary cilium using hopping mode SICM.
Assuntos
Cílios/química , Microscopia Eletroquímica de Varredura , Nanopartículas/química , Imagem Óptica , Animais , Células Cultivadas , Cães , Humanos , Células Madin Darby de Rim Canino , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Células NIH 3T3 , Tamanho da PartículaRESUMO
There is a growing realization, especially within the diagnostic and therapeutic community, that the amount of information enclosed in a single molecule can not only enable a better understanding of biophysical pathways, but also offer exceptional value for early stage biomarker detection of disease onset. To this end, numerous single molecule strategies have been proposed, and in terms of label-free routes, nanopore sensing has emerged as one of the most promising methods. However, being able to finely control molecular transport in terms of transport rate, resolution, and signal-to-noise ratio (SNR) is essential to take full advantage of the technology benefits. Here we propose a novel solution to these challenges based on a method that allows biomolecules to be individually confined into a zeptoliter nanoscale droplet bridging two adjacent nanopores (nanobridge) with a 20 nm separation. Molecules that undergo confinement in the nanobridge are slowed down by up to 3 orders of magnitude compared to conventional nanopores. This leads to a dramatic improvement in the SNR, resolution, sensitivity, and limit of detection. The strategy implemented is universal and as highlighted in this manuscript can be used for the detection of dsDNA, RNA, ssDNA, and proteins.
RESUMO
Scanning ion conductance microscopy (SICM) is a super-resolution live imaging technique that uses a glass nanopipette as an imaging probe to produce three-dimensional (3D) images of cell surface. SICM can be used to analyze cell morphology at nanoscale, follow membrane dynamics, precisely position an imaging nanopipette close to a structure of interest, and use it to obtain ion channel recordings or locally apply stimuli or drugs. Practical implementations of these SICM advantages, however, are often complicated due to the limitations of currently available SICM systems that inherited their design from other scanning probe microscopes in which the scan assembly is placed right above the specimen. Such arrangement makes the setting of optimal illumination necessary for phase contrast or the use of high magnification upright optics difficult. Here, we describe the designs that allow mounting SICM scan head on a standard patch-clamp micromanipulator and imaging the sample at an adjustable approach angle. This angle could be as shallow as the approach angle of a patch-clamp pipette between a water immersion objective and the specimen. Using this angular approach SICM, we obtained topographical images of cells grown on nontransparent nanoneedle arrays, of islets of Langerhans, and of hippocampal neurons under upright optical microscope. We also imaged previously inaccessible areas of cells such as the side surfaces of the hair cell stereocilia and the intercalated disks of isolated cardiac myocytes, and performed targeted patch-clamp recordings from the latter. Thus, our new, to our knowledge, angular approach SICM allows imaging of living cells on nontransparent substrates and a seamless integration with most patch-clamp setups on either inverted or upright microscopes, which would facilitate research in cell biophysics and physiology.
Assuntos
Imageamento Tridimensional/métodos , Microscopia de Varredura por Sonda/métodos , Adulto , Animais , Células Cultivadas , Meios de Cultura , Desenho de Equipamento , Feminino , Células HeLa , Humanos , Imageamento Tridimensional/instrumentação , Masculino , Camundongos , Micromanipulação/instrumentação , Micromanipulação/métodos , Microscopia Eletrônica de Varredura , Microscopia de Varredura por Sonda/instrumentação , Nanotecnologia , Técnicas de Patch-Clamp/instrumentação , Técnicas de Patch-Clamp/métodos , Ratos Sprague-DawleyRESUMO
Nanometric field-effect-transistor (FET) sensors are made on the tip of spear-shaped dual carbon nanoelectrodes derived from carbon deposition inside double-barrel nanopipettes. The easy fabrication route allows deposition of semiconductors or conducting polymers to comprise the transistor channel. A channel from electrodeposited poly pyrrole (PPy) exhibits high sensitivity toward pH changes. This property is exploited by immobilizing hexokinase on PPy nano-FETs to give rise to a selective ATP biosensor. Extracellular pH and ATP gradients are key biochemical constituents in the microenvironment of living cells; we monitor their real-time changes in relation to cancer cells and cardiomyocytes. The highly localized detection is possible because of the high aspect ratio and the spear-like design of the nano-FET probes. The accurately positioned nano-FET sensors can detect concentration gradients in three-dimensional space, identify biochemical properties of a single living cell, and after cell membrane penetration perform intracellular measurements.
Assuntos
Trifosfato de Adenosina/análise , Técnicas Biossensoriais/instrumentação , Análise de Célula Única/instrumentação , Transistores Eletrônicos , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Dissulfetos/química , Eletrodos , Enzimas Imobilizadas/metabolismo , Desenho de Equipamento , Hexoquinase/metabolismo , Humanos , Molibdênio/química , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Polímeros/química , Pirróis/química , Saccharomyces cerevisiae/enzimologiaRESUMO
Experimental data on dynamic interactions between individual nanoparticles and membrane processes at nanoscale, essential for biomedical applications of nanoparticles, remain scarce due to limitations of imaging techniques. We were able to follow single 200 nm carboxyl-modified particles interacting with identified membrane structures at the rate of 15 s/frame using a scanning ion conductance microscope modified for simultaneous high-speed topographical and fluorescence imaging. The imaging approach demonstrated here opens a new window into the complexity of nanoparticle-cell interactions.
Assuntos
Membrana Celular/metabolismo , Nanopartículas/química , Linhagem Celular , Humanos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Microscopia de Vídeo/instrumentação , Microscopia de Vídeo/métodosRESUMO
The purpose of this study was to investigate whether caveolin-3 (Cav3) regulates localization of ß2-adrenergic receptor (ß2AR) and its cAMP signaling in healthy or failing cardiomyocytes. We co-expressed wildtype Cav3 or its dominant-negative mutant (Cav3DN) together with the Förster resonance energy transfer (FRET)-based cAMP sensor Epac2-camps in adult rat ventricular myocytes (ARVMs). FRET and scanning ion conductance microscopy were used to locally stimulate ß2AR and to measure cytosolic cAMP. Cav3 overexpression increased the number of caveolae and decreased the magnitude of ß2AR-cAMP signal. Conversely, Cav3DN expression resulted in an increased ß2AR-cAMP response without altering the whole-cell L-type calcium current. Following local stimulation of Cav3DN-expressing ARVMs, ß2AR response could only be generated in T-tubules. However, the normally compartmentalized ß2AR-cAMP signal became diffuse, similar to the situation observed in heart failure. Finally, overexpression of Cav3 in failing myocytes led to partial ß2AR redistribution back into the T-tubules. In conclusion, Cav3 plays a crucial role for the localization of ß2AR and compartmentation of ß2AR-cAMP signaling to the T-tubules of healthy ARVMs, and overexpression of Cav3 in failing myocytes can partially restore the disrupted localization of these receptors.
Assuntos
Caveolina 3/metabolismo , Simulação por Computador , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , Animais , Western Blotting , Caveolina 3/genética , Síndromes Compartimentais/fisiopatologia , Expressão Gênica , Insuficiência Cardíaca/fisiopatologia , RatosRESUMO
Using nanopipettes to locally deliver molecules to the surface of living cells could potentially open up studies of biological processes down to the level of single molecules. However, in order to achieve precise and quantitative local delivery it is essential to be able to determine the amount and distribution of the molecules being delivered. In this work, we investigate how the size of the nanopipette, the magnitude of the applied pressure or voltage, which drives the delivery, and the distance to the underlying surface influences the number and spatial distribution of the delivered molecules. Analytical expressions describing the delivery are derived and compared with the results from finite element simulations and experiments on delivery from a 100 nm nanopipette in bulk solution and to the surface of sensory neurons. We then developed a setup for rapid and quantitative delivery to multiple subcellular areas, delivering the molecule capsaicin to stimulate opening of Transient Receptor Potential Vanilloid subfamily member 1 (TRPV1) channels, membrane receptors involved in pain sensation. Overall, precise and quantitative delivery of molecules from nanopipettes has been demonstrated, opening up many applications in biology such as locally stimulating and mapping receptors on the surface of live cells.
Assuntos
Capsaicina/metabolismo , Gânglios Espinais/química , Nanotecnologia/instrumentação , Canais de Cátion TRPV/metabolismo , Animais , Capsaicina/química , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Tamanho da Partícula , Ratos , Ratos Sprague-Dawley , Propriedades de Superfície , Canais de Cátion TRPV/químicaRESUMO
Direct electrical access to presynaptic ion channels has hitherto been limited to large specialized terminals such as the calyx of Held or hippocampal mossy fiber bouton. The electrophysiology and ion-channel complement of far more abundant small synaptic terminals (≤ 1 µm) remain poorly understood. Here we report a method based on superresolution scanning ion conductance imaging of small synapses in culture at approximately 100-150 nm 3D resolution, which allows presynaptic patch-clamp recordings in all four configurations (cell-attached, inside-out, outside-out, and whole-cell). Using this technique, we report presynaptic recordings of K(+), Na(+), Cl(-), and Ca(2+) channels. This semiautomated approach allows direct investigation of the distribution and properties of presynaptic ion channels at small central synapses.
Assuntos
Ativação do Canal Iônico/fisiologia , Canais Iônicos/fisiologia , Neurônios/citologia , Terminações Pré-Sinápticas/fisiologia , Animais , Animais Recém-Nascidos , Fenômenos Biofísicos/fisiologia , Cálcio/metabolismo , Células Cultivadas , Espinhas Dendríticas/fisiologia , Espinhas Dendríticas/ultraestrutura , Estimulação Elétrica , Eletrodos , Corantes Fluorescentes/metabolismo , Hipocampo/citologia , Imageamento Tridimensional , Canais Iônicos/ultraestrutura , Potenciais da Membrana/fisiologia , Microscopia de Tunelamento , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/ultraestrutura , RatosRESUMO
Determining the organization of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signalling, is a major challenge in cell biology and requires methods with nanoscale spatial resolution and high temporal resolution. Here, we review biophysical tools, based on scanning ion conductance microscopy and single-molecule fluorescence and the combination of both of these methods, which have recently been developed to address these issues. We then give examples of how these methods have been be applied to provide new insights into cell membrane organization and function, and discuss some of the issues that will need to be addressed to further exploit these methods in the future.
Assuntos
Membrana Celular/química , Rastreamento de Células/métodos , Receptores de Antígenos de Linfócitos T/química , Biologia Celular , Clatrina/química , Clusterina/química , Endocitose , Fluorescência , Humanos , Substâncias Macromoleculares/química , Microscopia de Fluorescência/métodos , Doenças Neurodegenerativas/patologia , Mapeamento de Interação de Proteínas , Transporte ProteicoRESUMO
AIM: To investigate the effect of surface charge of therapeutic nanoparticles on sarcolemmal ionic homeostasis and the initiation of arrhythmias. MATERIALS & METHODS: Cultured neonatal rat myocytes were exposed to 50 nm-charged polystyrene latex nanoparticles and examined using a combination of hopping probe scanning ion conductance microscopy, optical recording of action potential characteristics and patch clamp. RESULTS: Positively charged, amine-modified polystyrene latex nanoparticles showed cytotoxic effects and induced large-scale damage to cardiomyocyte membranes leading to calcium alternans and cell death. By contrast, negatively charged, carboxyl-modified polystyrene latex nanoparticles (NegNPs) were not overtly cytotoxic but triggered formation of 50-250-nm nanopores in the membrane. Cells exposed to NegNPs revealed pro-arrhythmic events, such as delayed afterdepolarizations, reduction in conduction velocity and pathological increment of action potential duration together with an increase in ionic current throughout the membrane, carried by the nanopores. CONCLUSION: The utilization of charged nanoparticles is a novel concept for targeting cardiac excitability. However, this unique nanoscopic investigation reveals an altered electrophysiological substrate, which sensitized the heart cells towards arrhythmias.