71 results found
Ren R, Cai S, Fang X, et al., 2023, Multiplexed detection of viral antigen and RNA using nanopore sensing and encoded molecular probes, Nature Communications, Vol: 14, ISSN: 2041-1723
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.
Takahashi Y, Sasaki Y, Yoshida T, et al., 2023, Nanopipette fabrication guidelines for SICM nanoscale imaging., Analytical Chemistry, Vol: 95, Pages: 12664-12672, ISSN: 0003-2700
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.
Novak P, Shevchuk A, Korchev Y, 2022, Nanoscale Electrophysiology Using Scanning Ion Conductance Microscopy, Bioanalytical Reviews, Pages: 123-138
Scanning ion conductance microscopy shares many aspects of its operation with an electrophysiological technique known as the “patch-clamp”, yet the marriage of the two techniques, referred to as “smart patch-clamp”, remains perceived as challenging both in the electrophysiological community and in the SICM community. Here we set out a number of ways how an existing setup, whether it is an SICM or a patch-clamp setup, can be turned into a “smart patch-clamp” setup, and outline strategies for achieving required levels of noise and interference. We describe typical scenarios where we believe the high-resolution spatial awareness of the “smart patch-clamp” is needed as opposed to the low-resolution of conventional patch-clamp. This is followed by a detailed description of the smart patch-clamp procedure and latest addition of controlled nanopipette breaking, offering improvement of the throughput, and partially counteracting the drop in probability of seeing channel activity with increasing spatial resolution. Finally, we discuss the issue of access resistance and conclude that the combination of SICM and patch-clamp technique offers yet unmatched fidelity when it comes to recording true ion currents and membrane voltages with nanoscale resolution.
Bohovyk R, Fedoriuk M, Isaeva E, et al., 2021, Scanning ion conductance microscopy of live human glomerulus, JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Vol: 25, Pages: 4216-4219, ISSN: 1582-1838
Maynard SA, Pchelintseva E, Zwi-Dantsis L, et al., 2021, IL-1β mediated nanoscale surface clustering of integrin α5β1 regulates the adhesion of mesenchymal stem cells, Scientific Reports, Vol: 11, Pages: 1-14, ISSN: 2045-2322
Clinical use of human mesenchymal stem cells (hMSCs) is limited due to their rapid clearance, reducing their therapeutic efficacy. The inflammatory cytokine IL-1β 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-1β enhances adhesion of hMSCs via increased focal adhesion contacts in an α5β1 integrin-specific manner. Further, through quantitative super-resolution imaging we elucidated that IL-1β specifically increases nanoscale integrin α5β1 availability and clustering at the plasma membrane, whilst conserving cluster area. Taken together, these results demonstrate that hMSC adhesion via IL-1β stimulation is partly regulated through integrin α5β1 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.
Bednarska J, Novak P, Korchev Y, et al., 2020, Release of insulin granules by simultaneous, high-speed correlative SICM-FCM, Journal of Microscopy, Vol: 282, Pages: 21-29, ISSN: 0022-2720
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 β‐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.
Bednarska J, Novak P, Korchev Y, et al., 2020, Release of insulin granules by simultaneous, high-speed correlative SICM-FCM
<jats:title>Summary</jats:title><jats:p>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, non-adherent, surface of pancreatic β-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.</jats:p>
Bednarska J, Pelchen-Matthews A, Novak P, et al., 2020, Rapid formation of human immunodeficiency virus-like particles., Proceedings of the National Academy of Sciences of USA, Vol: 117, Pages: 21637-21646, ISSN: 0027-8424
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.
Yang H-Q, Perez-Hernandez M, Sanchez-Alonso JL, et al., 2020, Ankyrin-G Mediates Targeting of both Na<SUP>+</SUP> and K<sub>ATP</sub> Channels to the Cardiac Intercalated Disc, 64th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 270A-270A, ISSN: 0006-3495
Yang H-Q, Perez-Hernandez M, Sanchez-Alonso J, et al., 2020, Ankyrin-G mediates targeting of both Na+ and K-ATP channels to the rat cardiac intercalated disc, eLife, Vol: 9, ISSN: 2050-084X
We investigated targeting mechanisms of Na+ and KATP channels to the intercalated disk (ICD) of cardiomyocytes. Patch clamp and surface biotinylation data show reciprocal downregulation of each other’s surface density. Mutagenesis of the Kir6.2 ankyrin binding site disrupts this functional coupling. Duplex patch clamping and Angle SICM recordings show that INa and IKATP functionally co-localize at the rat ICD, but not at the lateral membrane. Quantitative STORM imaging show that Na+ and KATP channels are localized close to each other and to AnkG, but not to AnkB, at the ICD. Peptides corresponding to Nav1.5 and Kir6.2 ankyrin binding sites dysregulate targeting of both Na+ and KATP channels to the ICD, but not to lateral membranes. Finally, a clinically relevant gene variant that disrupts KATP channel trafficking also regulates Na+ channel surface expression. The functional coupling between these two channels need to be considered when assessing clinical variants and therapeutics.
Gorelkin P, Erofeev A, Kolmogorov V, et al., 2020, Scanning Ion Conductance Microscopy (SICM) for Low-stress Directly Examining of Cellular Mechanics, ISSN: 1431-9276
Zhang Y, Takahashi Y, Hong SP, et al., 2019, High-resolution label-free 3D mapping of extracellular pH of single living cells, Nature Communications, Vol: 10, Pages: 1-9, ISSN: 2041-1723
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 technology was incorporated into a double-barrel nanoprobe integrating pH sensing with feedback-controlled distance sensing 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.
Gorelkin P, Erofeev A, Kolmogorov V, et al., 2019, Nanoscale Bioimaging with Scanning Ion Conductance Microscopy, Joint 12th EBSA European Biophysics Congress / 10th IUPAP International Conference on Biological Physics (ICBP), Publisher: SPRINGER, Pages: S157-S157, ISSN: 0175-7571
Ali T, Bednarska J, Vassilopoulos S, et al., 2019, Correlative SICM-FCM reveals changes in morphology and kinetics of endocytic pits induced by disease-associated mutations in dynamin, The FASEB Journal, Vol: 33, Pages: 8504-8518, ISSN: 0892-6638
Dynamin 2 (DNM2) is a GTP-binding protein that controls endocytic vesicle scission and defines a wholeclass of dynamin-dependent endocytosis, including clathrin-mediated endocytosis bycaveoli. It has been suggestedthat mutations in theDNM2gene, associated with 3 inherited diseases, disrupt endocytosis. However, how exactlymutations affect the nanoscale morphology of endocytic machinery has never been studied. In this paper, we used livecorrelative scanning ion conductance microscopy (SICM) and fluorescence confocal microscopy (FCM) to study howdisease-associated mutations affect the morphology and kinetics of clathrin-coated pits (CCPs) by directly followingtheir dynamics of formation, maturation, and internalizationinskinfibroblastsfrompatients with centronuclearmyopathy (CNM) and in Cos-7 cells expressing corresponding dynamin mutants. Using SICM-FCM, which we havedeveloped, we show how p.R465W mutation disrupts pit structure, preventing its maturation and internalization, andsignificantly increases the lifetime of CCPs. Differently,p.R522H slows down the formation of CCPs without affectingtheir internalization. We also found that CNM mutations inDNM2affect the distribution of caveoli and reduce dorsalruffling in human skin fibroblasts. Collectively, our SICM-FCM findings at single CCP level, backed up by electronmicroscopy data,argue for the impairment of several forms of endocytosis inDNM2-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.CorrelativeSICM-FCMreveals changes in morphology and kinetics of endocytic pitsinduced by disease-associated mutations in dynamin.
Gorelkin P, Erofeev A, Shevchuk A, et al., 2019, Scanning Ion Conductance Microscope as a new tool for bionanotechnology, Publisher: WILEY, Pages: 169-169, ISSN: 2211-5463
Gopal S, Chiappini C, Penders J, et al., 2019, Porous silicon nanoneedles modulate endocytosis to deliver biological payloads, Advanced Materials, Vol: 31, ISSN: 0935-9648
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.
Gorelkin P, Erofeev A, Alova A, et al., 2018, Nanopipette navigation system as a new tool for nanoscale investigation of living cells, FEBS OPEN BIOMED, Publisher: WILEY, Pages: 480-480, ISSN: 2211-5463
Zhou Y, Saito M, Miyamoto T, et al., 2018, Nanoscale imaging of primary cilia with scanning ion conductance microscopy, Analytical Chemistry, Vol: 90, Pages: 2891-2895, ISSN: 0003-2700
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.
Gorelkin P, Erofeev A, Komarova A, et al., 2017, Nanopipette biosensors as a new tool for metabolism and signaling investigation, 42nd Congress of the Federation-of-European-Biochemical-Societies (FEBS) on From Molecules to Cells and Back, Publisher: Wiley, Pages: 372-372, ISSN: 0014-2956
Cadinu P, Paulose Nadappuram B, Lee DJ, et al., 2017, Single molecule trapping and sensing using dual nanopores separated by a zeptoliter nanobridge, Nano Letters, Vol: 17, Pages: 6376-6384, ISSN: 1530-6984
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.
Shevchuk A, Tokar S, Gopal S, et al., 2016, Angular approach Scanning Ion Conductance Microscopy, Biophysical Journal, Vol: 110, Pages: 2252-2265, ISSN: 1542-0086
Scanning ion conductance microscopy (SICM) is a super-resolution live imagingtechnique that uses a glass nanopipette as an imaging probe to produce 3D images of cell surface.SICM can be used to analyze cell morphology at nanoscale, follow membrane dynamics, preciselyposition an imaging nanopipette close to a structure of interest, and use it to obtain ion channelrecordings 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 isplaced right above the specimen. Such arrangement makes the setting of optimal illuminationnecessary for phase contrast or the use of high magnification upright optics difficult. Here wedescribe the designs that allow mounting SICM scanhead on a standard patch-clampmicromanipulator and imaging the sample at an adjustable approach angle. This angle could be asshallow as the approach angle of a patch-clamp pipette between a water immersion objective andthe specimen. Using this angular approach SICM, we obtained topographical images of cells grownon non-transparent nanoneedle arrays, of islets of Langerhans, and of hippocampal neurons under 2upright optical microscope. We also imaged previously inaccessible areas of cells such as the sidesurfaces of the hair cell stereocilia and the intercalated disks of isolated cardiac mocytes, andperformed targeted patch-clamp recordings from the latter. Thus, our new angular approach SICMallows imaging of living cells on non-transparent substrates and a seamless integration with mostpatch-clamp setups on either inverted or upright microscopes, which would facilitate research incell biophysics and physiology.
Ali T, Shevchuk A, 2016, Charcot-Marie-Tooth and Centronuclear myopathy induced mechanistic impairment in endocytosis, Publisher: PERGAMON-ELSEVIER SCIENCE LTD, Pages: S4-S4, ISSN: 0960-8966
Zhang Y, Clausmeyer J, Babakinejad B, et al., 2016, Spearhead Nanometric Field-Effect Transistor Sensors for Single-Cell Analysis., ACS Nano, Vol: 10, Pages: 3214-3221, ISSN: 1936-086X
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.
Takahashi Y, Shevchuk AI, Novak P, et al., 2014, Erratum: Multifunctional nanoprobes for nanoscale chemical imaging and localized chemical delivery at surfaces and interfaces (Angewandte Chemie - International Edition (2011) (50) DOI: 10.1002/anie.201102796), Angewandte Chemie - International Edition, Vol: 53, ISSN: 1433-7851
Novak P, Shevchuk A, Ruenraroengsak P, et al., 2014, Imaging single nanoparticle interactions with human lung cells using fast ion conductance microscopy, Nano Letters: a journal dedicated to nanoscience and nanotechnology, Vol: 14, Pages: 1202-1207, ISSN: 1530-6984
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.
Wright PT, Nikolaev VO, O'Hara T, et al., 2014, Caveolin-3 regulates compartmentation of cardiomyocyte beta2-adrenergic receptor-mediated cAMP signaling, JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, Vol: 67, Pages: 38-48, ISSN: 0022-2828
Actis P, Tokar S, Clausmeyer J, et al., 2014, Electrochemical nanoprobes for single-cell analysis, ACS Nano, Vol: 8, Pages: 875-884, ISSN: 1936-0851
The measurement of key molecules in individual cells with minimal disruption to the biological milieu is the next frontier in single-cell analyses. Nanoscale devices are ideal analytical tools because of their small size and their potential for high spatial and temporal resolution recordings. Here, we report the fabrication of disk-shaped carbon nanoelectrodes whose radius can be precisely tuned within the range 5–200 nm. The functionalization of the nanoelectrode with platinum allowed the monitoring of oxygen consumption outside and inside a brain slice. Furthermore, we show that nanoelectrodes of this type can be used to impale individual cells to perform electrochemical measurements within the cell with minimal disruption to cell function. These nanoelectrodes can be fabricated combined with scanning ion conductance microscopy probes, which should allow high resolution electrochemical mapping of species on or in living cells.
Babakinejad B, Joensson P, Lopez Cordoba A, et al., 2013, Local Delivery of Molecules from a Nanopipette for Quantitative Receptor Mapping on Live Cells, ANALYTICAL CHEMISTRY, Vol: 85, Pages: 9333-9342, ISSN: 0003-2700
Novak P, Gorelik J, Vivekananda U, et al., 2013, Nanoscale-targeted patch-clamp recordings of functional presynaptic ion channels, Neuron, Vol: 79, Pages: 1067-1077, ISSN: 0896-6273
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 Ca2+ channels. This semiautomated approach allows direct investigation of the distribution and properties of presynaptic ion channels at small central synapses.
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