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              <p>The UFO project was initiated with the objective of developing a biologicallyfunctionalized optical nanosensor designed to detect biological electric fields, such as neuronal action potentials and transmembrane potential variation induced by electroporation. Er3+ and Yb3+ doped BTO nanocrystals (NanoBTO) developed with this funding demonstrated a notable up-conversion (process converting two or more low- energy photons into higher-energy photons) signal upon exposure to a laser with a wavelength of typically 977 nm. These nanocrystals re-emitted a spectrum with several peaks, notably one in the green at 545 nm (G2), which increased in intensity in presence of an electric field. Indeed, given that NanoBTO are ferroelectric, a variation in the external electric field decrease the interatomic distance, thereby facilitating re-emission at G2.NanoBTO present multiple advantages. The near IR wavelength of 977 nm allows for easier tissue penetration compared to most fluorescent probes. Additionally, they can be functionalised and their nanometric size of 200 nm makes them less invasive, making them ideal for cellular applications. Functionalisation with PEG enhances their biocompatibility, while cholesterol functionalisation enables them to interact with cell membranes, making them potential effective sensors for transmembrane potentials.Developed by our partners [1], the NanoBTO exhibited a reversible 14% up-conversion variation in G2, under significant DC electric fields (500 kV/cm). Our testing protocol involved exposing the nanocrystals to various environments, including air, deionised water, and phosphate-buffered saline (PBS), under an AC stimulation (5 kV/cm 200 Hz). Up-conversion variation was evaluated using a specific confocal microscope and 977 nm laser excitation. We observed a reversible increase in the G2 peak with a response time of 0.5 s only when the NanoBTO were in the highly ionic environment of PBS.We then tested four different types of NanoBTO on human induced neural stem cells (iNSC): PEG-cholesterol functionalised nanocrystals with or without hydroxylation, zwitterion-functionalised nanocrystals for pH sensitivity, and non-functionalised BTO nanocrystals in PBS. Our investigations were done on iNSCs as we have previous knowledge on their electroporation behaviour and they can be maturated in neurons capable of action potentials firing. The four different NanoBTO cytotoxicity on iNSC was measured using a lactate dehydrogenase (LDH) test over a 3-day period at concentrations ranging from 100 μg/ml to 5 μg/ml.The successful cell membrane attachment of the PEG-cholesterol functionalizedNanoBTO was assessed using two-photon microscopy and the second harmonicgeneration (SHG) signals generated by the NanoBTO.</p>
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