Abstract :
[en] Recently, a huge effort has been put on the measurement of overexpressed membrane receptors at the surface of cancer cells. Indeed, monitoring these markers has huge implications for diagnosis, prognosis, treatment selection and monitoring. SERS nanotags have emerged has an ideal alternative to the traditionally used fluorescent labels for measuring biological markers owing to their unique spectral properties. SERS nanotags are composed of four essential building blocks: (i) a plasmonic nanosubstrate for signal enhancement, (ii) a layer of Raman-reporter molecules for spectroscopic tagging of the nanotag, benefiting from the enhancement effect provided by the nanosubstrate, (iii) a protective coating for physico-chemical stabilisation in complex biological media and (iv) a targeting moiety used to direct the nanotag at a specific target and allow its indirect detection through the SERS signal of the Raman-reporter. They are highly promising tools for measuring biological markers from large tissue sections down to the single cell level when combined with a Raman microscope. However, several limitations still hinder the use of these nanotags in clinically-relevant samples.
In the frame of this thesis, we developed several structure of SERS nanotags with the aim of measuring overexpressed markers on cancer cells. In the first chapter, we used Au@Ag core@shell nanoparticles coated with poly(allylamine). Then, we functionalised these tags to target them either at folate receptor α (FRα) or sialic acids. We could successfully monitor the expression of these markers both independently and simultaneously (multiplexing) on cells and also on tissues samples. However, the performances obtained were not satisfactory (low signal contrast between overexpressing and non-overexpressing samples) due to non-specific interactions between the nanotags and the cells. Therefore, in the second chapter we developed a ratiometric approach by introducing a non-specific nanotag along the nanotags targeted at folate receptor α with the aim of reducing the influence of non-specific binding encountered in the first chapter. We could increase the signal contrast; however, it was at the cost of a more laborious method. In the third chapter, we drastically modified the structure of the nanotags targeted at folate receptor α by switching from poly(allylamine) to polyethylene glycol (PEG). PEG reduced the non-specific binding, allowing us to increase the signal contrast both on cells and on tissue without requiring a second nanotag. Nanotags coated with PEG were also biocompatible, allowing us to perform experiment on live cells with the same performances than on fixed cells, demonstrating the versatility of our nanotags. In the last chapter, we used nanotags coated with PEG and coupled with anti-HER2 antibodies to evaluate the potential of SERS for quantitative measurements directly on tissue samples, which constitutes a serious bottleneck in the current state of the art. We demonstrated that the SERS signal was correlated to the amount of HER2 present in the tumor by comparing SERS and immunohistochemical measurements directly on adjacent sections.
