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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - NVS (Nano Voltage Sensors)

Teaser

Summary

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. To this end, tools need to be developed to allow the investigation of interactions between individual neurons. Multi-electrode recordings have provided important insights but have limited performance when dense local circuits need to be analyzed or when signals from specific types of near-by neurons need to be distinguished. For this reason, considerable efforts have been invested in developing optical recording methods, including the utilization of voltage-sensitive dyes (VSDs) and voltage sensitive proteins (VSPs).
VSDs and VSPs allow for simultaneous direct visualization of neuronal activity over a large number of neurons in a large field-of-view. However, they suffer from several shortcomings: they could alter membrane capacitance, be phototoxic, suffer from photobleaching, have a short retention time in the membrane, and miss-target the membrane (resulting in nonspecific background labeling). We developed voltage sensing nanoparticles (vsNPs) in the shape of nanorods (vsNRs) that self-insert into the cell membrane and could optically record, non-invasively, action potentials (APs) at the single-particle and nanoscale level, at multiple sites, in a large field-of-view.

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.
Significant growth potential for voltage imaging of neurons is expected from the highly demanding and rapidly growing fields of Optogenetics and bioelectronic medicine, where voltage sensing and action potential actuation (stimulation) are desired in specific neuronal circuits.
From a pharmaceutical perspective, neural circuits mapping would allow to design better drugs for neuronal disorders ranging from neurodegenerative disorders such as Alzheimer and Parkinsonâ€™s to mental disorders, the most prevalent of which is depression. In addition, recording aberrant neuronal signals from peripheral neurons would present the opportunity to correct them, through an external feedback circuit (bioelectronics). This in turn will lead to economic benefits in the form of reduced economic burden and social benefits in the form of a healthier, more productive society.
In a broader sense, the implication of high resolution spatiotemporal recordings of neuron circuits will fuel emerging fields like neuroinformatics, medical informatics, Neuromorphic computing and Neurorobotics. As an example, building computational models from analysis of neuron circuits will allow construction of neural networks. Neural networks can provide robust solutions to problems in a wide range of disciplines, particularly areas involving classification, prediction, filtering, optimization, pattern recognition, and function approximation, all are relevant to Artificial Intelligence and big data applications.

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.
In addition, vsNRs could be targeted to specific neurons (or muscle cells), and specific sites within neurons (or muscle cells) by conjugation with specific recognition molecules. 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 pr

Work performed

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 demonstrated that the 12 nm long type-II NRs (sample(v)) exhibit much larger voltage sensitivities (Î”F/F and Î”Î») compared to the other NPs studied, including spherical QDs and 40 nm long quasi-type-I NRs. These NRs, with both positive and negative Î”F/F and Î”Î» due to random orientations in the electric field, are capable of reporting not only the field strength but also the field direction. we were able to provide an estimate for the tradeoff between detection probability and false positive rate of an â€œaction-potential-likeâ€ voltage transient. realistic parameters currently enable a 50% detection probability with a false positive rate well below 1%, figures which are already close to the scale of what is necessary for electrophysiology applications.
We used the 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%.

Functionalization, membrane insertion, & targeting
We are exploring two leading strategies for particle functionalization:
(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 lipid type.
peptide coating: by adsorption of amphipatic and/or hydrophobic trans membrane peptide sequences we prepared water soluble particles which show single particle membrane staining which is peptide dependent.
(2) functionalization by ligand exchange of TOPO coated NRs.
Curvature dependent facet selectivity: We explore facet selective chemistry that is dictated by surface geometry alone and was demonstrated for GNPs. This approach relies on the observation that the pKa of acidic surface ligands depends on ligand density which depends on the curvature (defined as 1/r). For non spherical particles as the NRs we would expect a different pKa for the long axis and for the tips. This should lead to pH dependent chemical reactivity.
DNA Origami: We explored an alternative functionalization method that is based on a library of chemically modified dsDNA molecules, where hydrophobic tags (primarily porphyrins) will act as membrane anchors. Following the work on DNA nanopores, and together with our collaborator, Dr. Stulz (U. Southampton), we have designed and prepared a small library of DNA duplexes for functionalizing the surface of vsNRs for cell membrane insertion.

vsNP-based optical recording methodologies
We have developed several high throughput imaging assays for selecting best performing membrane potential nanosensors. 1. Membrane staining assay: A rapid scan of wells with a high-NA objective is performed in order to test for membrane staining, indicating for some association of the nanoparticles to the membrane. This serves as a fast low content screen, which is used as a preliminary step for subsequent assays. 2. 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.
3. Induced transmembrane voltage: In order to polarize the cell membrane and test for correlation between the fluorescence of membrane-associated nanoparticles and membrane potential, we apply high and short pulses of voltage with a set of external electrodes on a large number of stained cells.
Spherical CHO cells are used, as their induced-polarization is analytically modeled. Nanoparticles that display volt