Periodic Reporting for period 4 - NVS (Nano Voltage Sensors)
Período documentado: 2020-07-01 hasta 2021-06-30
One of the major goals of neuroscience is to unravel how the brain functions in its entirety and how it generates behavior. The biggest challenge in solving this puzzle is represented by the sheer complexity of nervous systems and the interactions between individual neurons.
This revolutionizing research is expected to lead to new medical treatments and many new answers on how the brain functions. The flexibility in designing semiconductor NPs with exquisite control of sizes, compositions, bandgaps, excited state (exciton) wavefunction and lifetime, makes their development into biomedical reagents highly attractive.
We propose to develop targetable voltage sensing nanoparticles (vsNPs) in the shape of nanorods (vsNRs) that self-insert into the cell membrane and optically record, non-invasively, action potentials (APs) at the single-particle and nanoscale level, at multiple sites, in a large field-of-view.
Once the recognition molecule binds to a target (a specific membrane protein), the vsNR self- inserts into the cell membrane in close proximity to that target. With single-molecule sensitivity, vsNRs could allow nanoscale recording of pre- and post-synaptic membrane potentials during release of- and signaling by- neurotransmitters, record sub-threshold events or monitor individual ion channel activity, simultaneously at many sites, over a large field-of-view, and potentially at a resolution exceeding the diffraction limit.
We and others have recently demonstrated both theoretically and experimentally that engineered NRs could display large QCSE even at room temperature and on the single particle level, and therefore could act as efficient nanoscale vsNRs by displaying changes in fluorescence quantum yield, fluorescence lifetime and/or peak emission wavelength in response to local field perturbation. Moreover, we have tested the feasibility of a functionalization approach for stable membrane insertion of vsNRs into HEK293T cell membranes and demonstrated feasibility of voltage sensing by vsNRs. Our experimental and theoretical studies suggest that vsNRs could afford simultaneous monitoring of the spiking output of a large number of cells.
Here we seek to optimize all aspects of vsNPs synthesis, functionalization, delivery, targeting and detection in order to provide neuroscientists and physiologists a viable and user-friendly technology for the study of AP signals in the brain and in healthy or diseased heart and muscle tissues. Our aims are: (i) development of synthetic routes for heterostructure nanoparticles to produce ultrasensitive vsNPs and characterization of their performance by advanced spectroscopy; (ii) development of functionalization methods based on ligand exchange with transmembrane (amphiphilic) peptides by self-assembly and optimization of vsNPs insertion into the membrane; (iii) development of optical vsNPS recording methodologies and benchmarking of vsNPs performance; (iv) demonstration of vsNPs utility in model neuronal systems and exploration of their use in studying synapses and the molecular dynamics involved in neuronal integration of excitatory and inhibitory inputs.
This revolutionizing research is expected to lead to new medical treatments and many new answers on how the brain functions. The flexibility in designing semiconductor NPs with exquisite control of sizes, compositions, bandgaps, excited state (exciton) wavefunction and lifetime, makes their development into biomedical reagents highly attractive.
We propose to develop targetable voltage sensing nanoparticles (vsNPs) in the shape of nanorods (vsNRs) that self-insert into the cell membrane and optically record, non-invasively, action potentials (APs) at the single-particle and nanoscale level, at multiple sites, in a large field-of-view.
Once the recognition molecule binds to a target (a specific membrane protein), the vsNR self- inserts into the cell membrane in close proximity to that target. With single-molecule sensitivity, vsNRs could allow nanoscale recording of pre- and post-synaptic membrane potentials during release of- and signaling by- neurotransmitters, record sub-threshold events or monitor individual ion channel activity, simultaneously at many sites, over a large field-of-view, and potentially at a resolution exceeding the diffraction limit.
We and others have recently demonstrated both theoretically and experimentally that engineered NRs could display large QCSE even at room temperature and on the single particle level, and therefore could act as efficient nanoscale vsNRs by displaying changes in fluorescence quantum yield, fluorescence lifetime and/or peak emission wavelength in response to local field perturbation. Moreover, we have tested the feasibility of a functionalization approach for stable membrane insertion of vsNRs into HEK293T cell membranes and demonstrated feasibility of voltage sensing by vsNRs. Our experimental and theoretical studies suggest that vsNRs could afford simultaneous monitoring of the spiking output of a large number of cells.
Here we seek to optimize all aspects of vsNPs synthesis, functionalization, delivery, targeting and detection in order to provide neuroscientists and physiologists a viable and user-friendly technology for the study of AP signals in the brain and in healthy or diseased heart and muscle tissues. Our aims are: (i) development of synthetic routes for heterostructure nanoparticles to produce ultrasensitive vsNPs and characterization of their performance by advanced spectroscopy; (ii) development of functionalization methods based on ligand exchange with transmembrane (amphiphilic) peptides by self-assembly and optimization of vsNPs insertion into the membrane; (iii) development of optical vsNPS recording methodologies and benchmarking of vsNPs performance; (iv) demonstration of vsNPs utility in model neuronal systems and exploration of their use in studying synapses and the molecular dynamics involved in neuronal integration of excitatory and inhibitory inputs.
1. Optimization of size, composition, and shape of vsNRs
The optimal voltage sensors for single particle electrophysiology need to have large voltage sensitivity while maintaining small sizes for membrane insertion. Using a high-throughput QCSE screening assay, we successfully large voltage for spherical QDs and 40 nm long type-I, quasi-type-I and type-II NRs. We used nanocavity-based method for measuring single nanorod quantum yield, and observed that the quantum yield that corresponds to bright states of the same nanorod can be as high as 90%.
2. Functionalization, membrane insertion, & targeting
We explored two leading strategies for particle functionalization:
(2.1) functionalization by ligand adsorption to as-synthesized hydrophobic NRs and QDs
Lipid coating: We have developed a novel method for membrane insertion of large QDs (>5nm diameter) by delivery via a polar solvent. This is achieved by lipid functionalization of the NP surface at specific lipid:QD ratios and controlling lipid composition.
Peptide coating: by adsorption of amphipatic and/or hydrophobic trans membrane peptide sequences we prepared water soluble particles which show single particle membrane staining.
(2.2.) Functionalization by ligand exchange of TOPO coated NRs.
Hetero-functional surface coating by ligand exchange of amphiphilic quantum dots. TGA-GC-MS TGA, GC-MS and ICP analyses were used to analyze the resulting ligand exchange. Facet selective chemistry was explored by coating with ligands of different pKa and by curvature-dependent ligand density.
DNA Origami: Alternative functionalization method based on modified dsDNA molecules was explored, usingprimarily porphyrins as membrane anchors. Due to high cost of synthesizing modified DNA molecules, this approach, although promising, was abandoned.
(2.3.) Direct delivery & Cell Delivery by SUV fusion:
(i) As yet another alternative for membrane insertion, as-synthesized hydrophobic inorganic NPs were inserted into the lipid bilayer of small unilamellar vesicles (SUVs). (ii) We examined the dependence of particle size on membrane incorporation efficiency of QDs and NR by CryoEM. SUVs were fused into large GUVs and analyzed for mode of insertion by CryoEM.
3. vsNP-based optical recording methodologies
(3.1.) Several high throughput imaging assays were developed for selecting best performing membrane potential nanosensors. (i) Membrane staining assay: A rapid scan of wells with a high-NA objective is performed in order to test for membrane staining. This serves as a fast low content screen, which is used as a preliminary step for subsequent assays. (ii) Fluorescence anisotropy: Fluorescence anisotropy microscopy is used for measuring the directionality of anisotropic nanoparticles relative to the membrane. Particles are analyzed and scored according to their directionality relative to the membrane normal. (iii) Induced transmembrane voltage with external electrodes allowed us to correlate and screen fluorescence signal with electrical signals. Spherical CHO cells and primary cultured neuorns were stained with NPs and analyzed for correklated signals. Nanoparticles that display voltage-sensitivity of fluorescence upon membrane polarization are selected for further characterization and full voltage calibration by patch clamping.
(3.2.) Conditions for loading vsNPs into primary dissociated cortical neurons were optimized, and simultaneous fluorescence and electro physiological recordings were performed. A high NA fluorescence microscope equipped with patch-clamp recording hardware was built and a dedicated home-written software was developed for validation and calibration of vsNR signals. Single vsNRs recordings were demonstrated. An automated software for analysis of NR fluorescence in neurons, including algorithms that identify and remove blinking (intermittency) periods was developed and used in this study.
The optimal voltage sensors for single particle electrophysiology need to have large voltage sensitivity while maintaining small sizes for membrane insertion. Using a high-throughput QCSE screening assay, we successfully large voltage for spherical QDs and 40 nm long type-I, quasi-type-I and type-II NRs. We used nanocavity-based method for measuring single nanorod quantum yield, and observed that the quantum yield that corresponds to bright states of the same nanorod can be as high as 90%.
2. Functionalization, membrane insertion, & targeting
We explored two leading strategies for particle functionalization:
(2.1) functionalization by ligand adsorption to as-synthesized hydrophobic NRs and QDs
Lipid coating: We have developed a novel method for membrane insertion of large QDs (>5nm diameter) by delivery via a polar solvent. This is achieved by lipid functionalization of the NP surface at specific lipid:QD ratios and controlling lipid composition.
Peptide coating: by adsorption of amphipatic and/or hydrophobic trans membrane peptide sequences we prepared water soluble particles which show single particle membrane staining.
(2.2.) Functionalization by ligand exchange of TOPO coated NRs.
Hetero-functional surface coating by ligand exchange of amphiphilic quantum dots. TGA-GC-MS TGA, GC-MS and ICP analyses were used to analyze the resulting ligand exchange. Facet selective chemistry was explored by coating with ligands of different pKa and by curvature-dependent ligand density.
DNA Origami: Alternative functionalization method based on modified dsDNA molecules was explored, usingprimarily porphyrins as membrane anchors. Due to high cost of synthesizing modified DNA molecules, this approach, although promising, was abandoned.
(2.3.) Direct delivery & Cell Delivery by SUV fusion:
(i) As yet another alternative for membrane insertion, as-synthesized hydrophobic inorganic NPs were inserted into the lipid bilayer of small unilamellar vesicles (SUVs). (ii) We examined the dependence of particle size on membrane incorporation efficiency of QDs and NR by CryoEM. SUVs were fused into large GUVs and analyzed for mode of insertion by CryoEM.
3. vsNP-based optical recording methodologies
(3.1.) Several high throughput imaging assays were developed for selecting best performing membrane potential nanosensors. (i) Membrane staining assay: A rapid scan of wells with a high-NA objective is performed in order to test for membrane staining. This serves as a fast low content screen, which is used as a preliminary step for subsequent assays. (ii) Fluorescence anisotropy: Fluorescence anisotropy microscopy is used for measuring the directionality of anisotropic nanoparticles relative to the membrane. Particles are analyzed and scored according to their directionality relative to the membrane normal. (iii) Induced transmembrane voltage with external electrodes allowed us to correlate and screen fluorescence signal with electrical signals. Spherical CHO cells and primary cultured neuorns were stained with NPs and analyzed for correklated signals. Nanoparticles that display voltage-sensitivity of fluorescence upon membrane polarization are selected for further characterization and full voltage calibration by patch clamping.
(3.2.) Conditions for loading vsNPs into primary dissociated cortical neurons were optimized, and simultaneous fluorescence and electro physiological recordings were performed. A high NA fluorescence microscope equipped with patch-clamp recording hardware was built and a dedicated home-written software was developed for validation and calibration of vsNR signals. Single vsNRs recordings were demonstrated. An automated software for analysis of NR fluorescence in neurons, including algorithms that identify and remove blinking (intermittency) periods was developed and used in this study.
voltage recordings from individual NPs; insertion of large QDs to membranes; expected – AP recording from single NP
Voltage recordings from individual NPs
Alternative approaches
We examined the potential of polystyrene and Nano-disc particles as voltage-sensors in a FRET-based system We proved Site directed membrane potential voltage sensing in neurons.
Voltage recordings from individual NPs
Alternative approaches
We examined the potential of polystyrene and Nano-disc particles as voltage-sensors in a FRET-based system We proved Site directed membrane potential voltage sensing in neurons.