Browse
Submit Data
Databases
API
Help

Dataset Information

9 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Real-time sensing of neurotransmitters by functionalized nanopores embedded in a single live cell.

ABSTRACT: Interface between neuron cells and biomaterials is the key to real-time sensing, transmitting and manipulating of neuron activities, which are the long-term pursue of scientists and gain intense research focus recently. It is of great interest to develop a sensor with exquisite sensitivity and excellent selectivity for real-time monitoring neurotransmitters transport through single live cell. Sensing techniques including electrode-based methods, optogenetics, and nanowire cell penetration systems have been developed to monitor the neuron activities. However, their biocompatibilities remain a challenge. Protein nanopores with membrane compatibility and lumen tunability provide real-time, single-molecule sensitivities for biosensing of DNA, RNA, peptides and small molecules. In this study, an engineered protein nanopore MspA (Mycobacterium smegmatis porin A) through site-directed mutation with histidine selectively bind with Cu²⁺ in its internal lumen. Chelation of neurotransmitters such as L-glutamate (L-Glu), dopamine (DA) and norepinephrine (NE) with the Cu²⁺ creates specific current signals, showing different transient current blockade and dwell time in single channel electrophysiological recording. Furthermore, the functionalized M2MspA-N91H nanopores have been embedded in live HEK293T cell membrane for real-time, in situ monitoring of extracellular L-glutamate translocating through the nanopore. This biomimetic neurotransmitter nanopore has provided a new platform for future development of neuron sensors, drug carrier and artificial synapse.

SUBMITTER: Zhang X

PROVIDER: S-EPMC8607392 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

pH tuning of DNA translocation time through organically functionalized nanopores.

Project description:Controlling DNA translocation speed is critically important for nanopore sequencing as free electrophoretic threading is far too rapid to resolve individual bases. A number of promising strategies have been explored in recent years, largely driven by the demands of next-generation sequencing. Engineering DNA-nanopore interactions (known to dominate translocation dynamics) with organic coatings is an attractive method as it does not require sample modification, processive enzymes, or complicated and expensive fabrication steps. In this work, we show for the first time 4-fold tuning of unfolded, single-file translocation time through small, amine-functionalized solid-state nanopores by varying the solution pH in situ. Additionally, we develop a simple analytical model based on electrostatic interactions to explain this effect which will be a useful tool in designing future devices and experiments.

| S-EPMC3584232 | biostudies-literature

Nanoscale metal-organic frameworks for real-time intracellular pH sensing in live cells.

Project description:Real-time measurement of intracellular pH in live cells is of great importance for understanding physiological/pathological processes and developing intracellular drug delivery systems. We report here the first use of nanoscale metal-organic frameworks (NMOFs) for intracellular pH sensing in live cells. Fluorescein isothiocyanate (FITC) was covalently conjugated to a UiO NMOF to afford F-UiO NMOFs with exceptionally high FITC loadings, efficient fluorescence, and excellent ratiometric pH-sensing properties. Upon rapid and efficient endocytosis, F-UiO remained structurally intact inside endosomes. Live cell imaging studies revealed endo- and exocytosis of F-UiO and endosome acidification in real time. Fluorescently labeled NMOFs thus represent a new class of nanosensors for intracellular pH sensing and provide an excellent tool for studying NMOF-cell interactions.

| S-EPMC4156865 | biostudies-literature

Step detection in single-molecule real time trajectories embedded in correlated noise.

Project description:Single-molecule real time trajectories are embedded in high noise. To extract kinetic or dynamic information of the molecules from these trajectories often requires idealization of the data in steps and dwells. One major premise behind the existing single-molecule data analysis algorithms is the gaussian 'white' noise, which displays no correlation in time and whose amplitude is independent on data sampling frequency. This so-called 'white' noise is widely assumed but its validity has not been critically evaluated. We show that correlated noise exists in single-molecule real time trajectories collected from optical tweezers. The assumption of white noise during analysis of these data can lead to serious over- or underestimation of the number of steps depending on the algorithms employed. We present a statistical method that quantitatively evaluates the structure of the underlying noise, takes the noise structure into account, and identifies steps and dwells in a single-molecule trajectory. Unlike existing data analysis algorithms, this method uses Generalized Least Squares (GLS) to detect steps and dwells. Under the GLS framework, the optimal number of steps is chosen using model selection criteria such as Bayesian Information Criterion (BIC). Comparison with existing step detection algorithms showed that this GLS method can detect step locations with highest accuracy in the presence of correlated noise. Because this method is automated, and directly works with high bandwidth data without pre-filtering or assumption of gaussian noise, it may be broadly useful for analysis of single-molecule real time trajectories.

| S-EPMC3606409 | biostudies-literature

Real-Time Imaging of Translation on Single mRNA Transcripts in Live Cells.

Project description:Translation is under tight spatial and temporal controls to ensure protein production in the right time and place in cells. Methods that allow real-time, high-resolution visualization of translation in live cells are essential for understanding the spatiotemporal dynamics of translation regulation. Based on multivalent fluorescence amplification of the nascent polypeptide signal, we develop a method to image translation on individual mRNA molecules in real time in live cells, allowing direct visualization of translation events at the translation sites. Using this approach, we monitor transient changes of translation dynamics in responses to environmental stresses, capture distinct mobilities of individual polysomes in different subcellular compartments, and detect 3' UTR-dependent local translation and active transport of polysomes in dendrites of primary neurons.

| S-EPMC4905760 | biostudies-literature

A Fluorescent Real-Time Plaque Assay Enables Single-Cell Analysis of Virus-Induced Cytopathic Effect by Live-Cell Imaging.

Project description:Conventional plaque assays rely on the use of overlays to restrict viral infection allowing the formation of distinct foci that grow in time as the replication cycle continues leading to countable plaques that are visualized with standard techniques such as crystal violet, neutral red, or immunolabeling. This classical approach takes several days until large enough plaques can be visualized and counted with some variation due to subjectivity in plaque recognition. Since plaques are clonal lesions produced by virus-induced cytopathic effect, we applied DNA fluorescent dyes with differential cell permeability to visualize them by live-cell imaging. We could observe different stages of that cytopathic effect corresponding to an early wave of cells with chromatin-condensation followed by a wave of dead cells with membrane permeabilization within plaques generated by different animal viruses. This approach enables an automated plaque identification using image analysis to increase single plaque resolution compared to crystal violet counterstaining and allows its application to plaque tracking and plaque reduction assays to test compounds for both antiviral and cytotoxic activities. This fluorescent real-time plaque assay sums to those next-generation technologies by combining this robust classical method with modern fluorescence microscopy and image analysis approaches for future applications in virology.

| S-EPMC8310316 | biostudies-literature

Real-Time Sensing of Single-Ligand Delivery with Nanoaperture-Integrated Microfluidic Devices.

Project description:The measurement of biological events on the surface of live cells at the single-molecule level is complicated by several factors including high protein densities that are incompatible with single-molecule imaging, cellular autofluorescence, and protein mobility on the cell surface. Here, we fabricated a device composed of an array of nanoscale apertures coupled with a microfluidic delivery system to quantify single-ligand interactions with proteins on the cell surface. We cultured live cells directly on the device and isolated individual epidermal growth factor receptors (EGFRs) in the apertures while delivering fluorescently labeled epidermal growth factor. We observed single ligands binding to EGFRs, allowing us to quantify the ligand turnover in real time. These results demonstrate that this nanoaperture-coupled microfluidic device allows for the spatial isolation of individual membrane proteins while maintaining them in their cellular environment, providing the capability to monitor single-ligand binding events while maintaining receptors in their physiological environment. These methods should be applicable to a wide range of membrane proteins.

| S-EPMC5537690 | biostudies-literature

Real-time single-cell response to stiffness.

Project description:Living cells adapt to the stiffness of their environment. However, cell response to stiffness is mainly thought to be initiated by the deformation of adhesion complexes under applied force. In order to determine whether cell response was triggered by stiffness or force, we have developed a unique method allowing us to tune, in real time, the effective stiffness experienced by a single living cell in a uniaxial traction geometry. In these conditions, the rate of traction force buildup dF/dt was adapted to stiffness in less than 0.1 s. This integrated fast response was unambiguously triggered by stiffness, and not by force. It suggests that early cell response could be mechanical in nature. In fact, local force-dependent signaling through adhesion complexes could be triggered and coordinated by the instantaneous cell-scale adaptation of dF/dt to stiffness. Remarkably, the effective stiffness method presented here can be implemented on any mechanical setup. Thus, beyond single-cell mechanosensing, this method should be useful to determine the role of rigidity in many fundamental phenomena such as morphogenesis and development.

| S-EPMC2944728 | biostudies-literature

Real-time sensing of cell morphology by infrared waveguide spectroscopy.

Project description:We demonstrate that a live epithelial cell monolayer can act as a planar waveguide. Our infrared reflectivity measurements show that highly differentiated simple epithelial cells, which maintain tight intercellular connectivity, support efficient waveguiding of the infrared light in the spectral region of 1.4-2.5 µm and 3.5-4 µm. The wavelength and the magnitude of the waveguide mode resonances disclose quantitative dynamic information on cell height and cell-cell connectivity. To demonstrate this we show two experiments. In the first one we trace in real-time the kinetics of the disruption of cell-cell contacts induced by calcium depletion. In the second one we show that cell treatment with the PI3-kinase inhibitor LY294002 results in a progressive decrease in cell height without affecting intercellular connectivity. Our data suggest that infrared waveguide spectroscopy can be used as a novel bio-sensing approach for studying the morphology of epithelial cell sheets in real-time, label-free manner and with high spatial-temporal resolution.

| S-EPMC3485211 | biostudies-literature

Single molecule sensing of amyloid-β aggregation by confined glass nanopores.

Project description:We have developed a glass nanopore based single molecule tool to investigate the dynamic oligomerization and aggregation process of Aβ1-42 peptides. The intrinsic differences in the molecular size and surface charge of amyloid aggregated states could be distinguished through single molecule induced characteristic current fluctuation. More importantly, our results reveal that the neurotoxic Aβ1-42 oligomer tends to adsorb onto the solid surface of nanopores, which may explain its instability and highly neurotoxic features.

| S-EPMC7020925 | biostudies-literature

Selectively Sized Graphene-Based Nanopores for in Situ Single Molecule Sensing.

Project description:The use of nanopore biosensors is set to be extremely important in developing precise single molecule detectors and providing highly sensitive advanced analysis of biological molecules. The precise tailoring of nanopore size is a significant step toward achieving this, as it would allow for a nanopore to be tuned to a corresponding analyte. The work presented here details a methodology for selectively opening nanopores in real-time. The tunable nanopores on a quartz nanopipette platform are fabricated using the electroetching of a graphene-based membrane constructed from individual graphene nanoflakes (ø ∼30 nm). The device design allows for in situ opening of the graphene membrane, from fully closed to fully opened (ø ∼25 nm), a feature that has yet to be reported in the literature. The translocation of DNA is studied as the pore size is varied, allowing for subfeatures of DNA to be detected with slower DNA translocations at smaller pore sizes, and the ability to observe trends as the pore is opened. This approach opens the door to creating a device that can be target to detect specific analytes.

| S-EPMC4543996 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data