Malavika Kayyil will defend her PhD thesis entitled “Exploring and improving fluorescence correlation spectroscopy (FCS) sensitivity from nanoprobe to label-free proteins for biosensing and nanopore applications.” on Thursday, 29th January 2026 at 01:30 p.m. in room Pierre Cotton, Institut Fresnel, campus St Jerome in Marseille.
The defence will be given in English.
The jury is composed of :
– Dr. Thomas PONS, LPEM, INSERM, Paris, Reviewer
– Dr. Karen PERRONET, LuMIn, CNRS, Gif sur Yvette, Reviewer
– Dr. Serge MONNERET, Institut Fresnel, CNRS, Marseille, Examiner
– Prof. Antoine DELON, LIPhy, University of Grenoble Alpes, President
– Dr. Jerome WENGER, Institut Fresnel, CNRS, Marseille, PhD supervisor
Abstract : Fluorescence correlation spectroscopy (FCS) is a powerful and versatile technique that analyzes fluorescence intensity fluctuations within a small volume to reveal dynamic molecular processes such as diffusion, chemical reactions, and conformational changes, and has become an invaluable tool across physics, chemistry, and biology. But some factors act as limitations to FCS, like background noise, spectral overlap, photobleaching, heterogeneous labelling, etc. This thesis explores strategies to reduce these limitations and enhance the sensitivity and detection limit of FCS. The thesis starts with an introduction to the technique and gives a broad overview of its applications, advantages, and limitations. Further, we explore the capability of FCS to study the photophysics of nanoprobes like gold nanoclusters and lanthanide nanoparticles at a single particle level. This could be challenging due to their long lifetime, leading to low brightness and early saturation. Later, we explore Fluorescence lifetime correlation spectroscopy (FLCS), a variant of FCS that uses fluorescence lifetime information to distinguish the contribution of emitters and background. FLCS can be used to overcome spectral overlap and it also substantially reduces background, improving the signal-to-background ratio (SBR). We investigated the multiplexing capability of FLCS in a ternary mixture of three components with overlapping emission spectra. Then we explored the detection limit of FLCS in the low concentration range and determined the parameters responsible for this limit. We also introduced an FLCS correction factor to further improve the detection limit. We used our understanding to estimate the association rate constant of the streptavidin-biotin binding interaction. Labelling of proteins is known to create structural and functional damage to the protein. Hence, in the last part, we moved to deep UV, and we focused on label-free protein detection utilising the intrinsic autofluorescence of aromatic amino acid residues. We discussed the limitations of UV microscopy and the significance of buffer stabilisation and antioxidants to reduce those limitations. We also showed how optimising the solvent condition and microscope configuration of the confocal setup can improve the detection limit of label-free streptavidin. We also figured out that stirring the sample can substantially reduce photobleaching and improve the brightness of the streptavidin protein. This improved brightness was used to understand streptavidin-biotin cooperativity. Since all the techniques discussed lack specificity to identify the protein, we also put forwarded a future perspective on using plasmonic nanopores based on an optical readout strategy. Our approach to limit background and improving protein photostability can be helpful for nanopore applications. Conclusively, this thesis presents different strategies to overcome the FCS limitations and improve the sensitivity of the technique for biosensing applications. This approach can be helpful when dealing with weak emitters and ultra-low concentrations with low SBR.
Keywords : Fluorescence correlation spectroscopy (FCS), Fluorescence lifetime correlation spectroscopy (FLCS), Single molecule sensing, UV autofluorescence, Confocal microscopy, Nanopores
