Final Report Summary - VSNS (Voltage-Sensitive Plasmon-Resonant Nanoparticles, Novel Nanotransducers of Neuronal Activity)
The VSNS project has made considerable progress in its various fronts over the past 36 months. A range of functionalised nanoparticles (NPs) have been developed as planned, including core-shell (SiO2/Au) structures realised by attachment of 10 nm Au to SiO2 NPs and subsequent cathalytic enlargement. Further, optimal protocols to improve membrane-binding of the NP/NRs and to preclude internalisation using CTAB were developed. As an alternative to binding the nanosensors directly to the membrane, protocols for electropolimerisation-based substrate attachment have been optimised. To this end thioaniline-functionalised Au NPs were electropolymerised and zwitterionic ligands were used to bind and sense amino-acids (specifically, the neurotransmitter glutamate). A method to imprint specific molecular recognition sites, again with emphasis in neurotransmitter detection as indicator of neuronal activity, was also developed. Electropolymerisation was performed in the presence of the target neurotransimtter resulting in specific sensors of L-glutamic acid or D-glutamic acid.
Further work has been performed on single-nanoparticle spectroscopy to gain insight on the fundamental processes leading to changes in membrane-bound Nanoparticle plasmon resonance (NPPR) of relevance to the measurement of membrane potential. Three distinct voltage / electric field ranges could be identified where the plasmon reacts differently, possibly due to three completely different physico-chemical processes: a 'classical' capacitor charging region in which the changes in spectra are due to changes of electron density, a non-linear spectral shift region and an oxidation region resulting in hystheresis. These results have opened a plethora of new applications for the NP/NRs used in the project, including the use of second harmonic generation in NP-based biosensing but also in optoelectronics.
Work on dual optical / electrical measurements on cultured neurons and slices has made substantial progress. Firstly, the E-squared (E2) technology was developed to achieve dual optical and electrical recordings from identified axon bundles. This technology was patented and a spin-off launched. Toxicity of the nanorods was then evaluated as low and dual electrical / optical measurements with them were attempted. A major effort focussed on avoiding aggregation and attachment to the cellular membrane. Good results were eventually obtained using CTAB capping layers. Initial results showed poor signal-to-noise ratios in scattering and cross-polarised modes. Non-linear properties of NPs, a high-impact result of the project, prompted interest in second-harmonic generation as an approach to achieve higher signal-to-noise ratios. First experiments with fluorophore-stained neurons under extracellular stimulation showed large SHG changes correlated with electrical stimulation. Future work will address the enhancement of this effect by means of the NP developed in the project.
Regarding instrumentation, dark-field microscopy was complemented with Thermal lensing microscopy (TLM) and a phase-sensitive setup has been put in place to test this approach. In principle TLM should be particularly suited for highly scattering preparations, such as slices. Simulations have also been carried out to study the effect of the applied voltage on the resonance spectrum of the NPs under test. Further, work package 6 has produced a prototype amplifier with sync inputs in order to correlate multichannel electrophysiology and optical traces using randomly plated neurons, as opposed to identified axon bundles in the E2 technology.
Further work has been performed on single-nanoparticle spectroscopy to gain insight on the fundamental processes leading to changes in membrane-bound Nanoparticle plasmon resonance (NPPR) of relevance to the measurement of membrane potential. Three distinct voltage / electric field ranges could be identified where the plasmon reacts differently, possibly due to three completely different physico-chemical processes: a 'classical' capacitor charging region in which the changes in spectra are due to changes of electron density, a non-linear spectral shift region and an oxidation region resulting in hystheresis. These results have opened a plethora of new applications for the NP/NRs used in the project, including the use of second harmonic generation in NP-based biosensing but also in optoelectronics.
Work on dual optical / electrical measurements on cultured neurons and slices has made substantial progress. Firstly, the E-squared (E2) technology was developed to achieve dual optical and electrical recordings from identified axon bundles. This technology was patented and a spin-off launched. Toxicity of the nanorods was then evaluated as low and dual electrical / optical measurements with them were attempted. A major effort focussed on avoiding aggregation and attachment to the cellular membrane. Good results were eventually obtained using CTAB capping layers. Initial results showed poor signal-to-noise ratios in scattering and cross-polarised modes. Non-linear properties of NPs, a high-impact result of the project, prompted interest in second-harmonic generation as an approach to achieve higher signal-to-noise ratios. First experiments with fluorophore-stained neurons under extracellular stimulation showed large SHG changes correlated with electrical stimulation. Future work will address the enhancement of this effect by means of the NP developed in the project.
Regarding instrumentation, dark-field microscopy was complemented with Thermal lensing microscopy (TLM) and a phase-sensitive setup has been put in place to test this approach. In principle TLM should be particularly suited for highly scattering preparations, such as slices. Simulations have also been carried out to study the effect of the applied voltage on the resonance spectrum of the NPs under test. Further, work package 6 has produced a prototype amplifier with sync inputs in order to correlate multichannel electrophysiology and optical traces using randomly plated neurons, as opposed to identified axon bundles in the E2 technology.
