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Page de résumé pour ULgetd-09112020-133351

Auteur : Dumont, Elodie
URN : ULgetd-09112020-133351
Langue : Anglais/English
Titre : Bioanalytical applications of surface-enhanced Raman scattering: The protein corona challenge/Applications de la diffusion Raman exaltée de surface en bioanalyse : Le problème de la couronne protéique
Intitulé du diplôme : Doctorat en sciences biomédicales et pharmaceutiques
Département : Médecine - Département de pharmacie
Jury :
Nom : Titre :
BYRNE, Hugh Membre du jury/Committee Member
DE BLEYE, Charlotte Membre du jury/Committee Member
FLEURY-FRENETTE, Karl Membre du jury/Committee Member
LEBRUN, Pierre Membre du jury/Committee Member
LIEGEOIS, Jean-Fran Membre du jury/Committee Member
OTTEVAERE, Heidi Membre du jury/Committee Member
ROGISTER, Bernard Membre du jury/Committee Member
FILLET, Marianne Président du jury/Committee Chair
HUBERT, Philippe Promoteur/Director
ZIEMONS, Eric Promoteur/Director
Mots-clés :
  • protein corona/couronne protéique
  • bioanalysis/bioanalyse
  • solid SERS substrates/substrats SERS solides
  • gold and silver nanoparticles/nanoparticules d’or et d’argent
  • dopamine/dopamine
  • Surface-enhanced Raman scattering (SERS)/Diffusion Raman exaltée de surface
  • real-time monitoring/suivi en temps réel
  • surface functionalisation/fonctionnalisation de surface
  • cell culture medium/milieu de culture cellulaire
Date de soutenance : 2020-09-11
Type d'accès : Restreint/Intranet
Résumé :

Concurrently with the recent expansion and diversification of bioanalytical applications, a wealth of analytical tools has developed to meet the expectations of bioanalysis. Among these tools, surface-enhanced Raman scattering (SERS) stands out as an attractive alternative to separative techniques. SERS is indeed non-destructive, fast and can be used for identification and quantification purposes. In addition, SERS benefits from an increased sensitivity compared to conventional Raman spectroscopy, making it more suited to the detection of low concentrations of analytes that are often encountered in bioanalysis.

However, some roadblocks on the way towards the use of SERS in routine bioanalyses have been identified. Complex biological matrices often call for either sample preparation or smart SERS substrate surface functionalisation. The former denatures the sample and requires additional steps of sample handling with frequent use of organic solvents, which are harmful for the environment. The latter can be laborious to carry out, needs experienced staff and is limited to some specific analytes. Moreover, in view of routine analyses, repeatability and reproducibility of the SERS substrate preparation must be achieved since they are prerequisites to successfully validate the analysis method. This unfortunately remains an exception in the field.

Consequently, this thesis was dedicated to the investigation of easily implementable strategies to conduct SERS analyses in biological matrices. To guarantee simple method transposal, these strategies essentially made use of suspensions of chemically reduced nanoparticles, which represent the most straightforward and economical SERS substrates. Dopamine was selected as analyte, given its great SERS activity and its involvement in several widespread diseases (e.g. Parkinson’s disease, schizophrenia, addictions). The resulting matrix was the complete culture medium of a model cell line used to study dopamine secretion, namely rat phaeochromocytoma PC-12 cells. A major issue was experienced all thesis long, the protein corona. This corona consists of a layer of proteins that coats the surface of the SERS substrates, preventing nanoparticles aggregation, thus hot-spot formation, and that competes with analytes for the surface of the SERS substrate. As a result, this work focussed on solving protein corona-related problems.

First and foremost, after having identified gold nanoparticles (AuNPs) as the most suitable SERS substrate for dopamine detection, the transfer of their synthesis protocol was assessed between two laboratories. Despite the use of different laboratory hardware, an exhaustive characterisation of the synthesised AuNPs demonstrated that the synthesis protocol could be successfully transferred, providing that critical parameters are under control.

Then, a first strategy was established for the quantification of dopamine in cell culture media through colloidal AuNPs. It was based on the pre-aggregation of the colloid before adding the matrix, in order to circumvent the stabilisation issue brought about by the protein corona. The optimal proportions of reagents, determined by experimental design, enabled dopamine quantification from 0.5 to 50 ppm (2.64 – 264 µM) in the culture medium of PC-12 cells. The specificity of the method towards dopamine was also demonstrated and the exocytosis process of dopamine in PC-12 cells could be studied with the developed methodology.

A preliminary study was afterwards carried out to investigate whether it was possible to increase the sensitivity and the specificity of dopamine detection with smartly functionalised AuNPs. A dual-mode SERS and fluorescence aptasensor was ergo imagined to benefit from both techniques advantages and its synthesis and sample preparation were optimised. The binding of dopamine to its specific aptamer was confirmed by surface plasmon resonance spectroscopy. Nonetheless, the determination of dopamine in a phosphate buffer by fluorescence lacked sensitivity, with an approximate limit of detection of 12.5 ppm (65.9 µM). Dopamine determination by SERS was also tricky because of the complex and noisy spectra obtained.

Another solution to the protein corona stabilisation problem lay in the use of solid SERS substrates, since hot-spots naturally occur in this kind of substrates. The performances of three classes of solid SERS substrates were compared, in terms of dopamine detection in culture medium, of immersion resistance and of biocompatibility. Unlike substrates prepared by advanced physical technologies, polymeric substrates entrapping NPs were able to quantify dopamine in complex matrices. However, cells could not be cultured on these substrates due to a lack of cellular adhesion. The last kind of substrate investigated, glass coated with poly-L-lysine and AuNPs, was biocompatible. Still, dopamine determination in biological matrices was hampered due to protein surface fouling.

This is why an original device capable of removing proteins from the analysed medium was developed. It derived from the use of ultracentrifugation membranes as effective protein retaining element between 2 compartments: the upper one containing the biological sample and the bottom one holding the SERS substrate and a clean collection medium like phosphate buffer. After the investigation of the acquisition and configuration parameters, the device was successfully applied to real-time monitoring. Gradually adding sample complexity, the diffusion process of a SERS label, 2-mercaptopyridine, followed by that of a neurotransmitter, serotonin, were first tracked. The distinction between highly and weakly metastatic pancreatic ductal adenocarcinoma cells was afterwards examined with the device. Based on differential metabolic metabolites production rates, discrimination between both cell types could be established.

Finally, some clues were given about surface functionalisation important features. It was indeed noticed during this work that parameters such as the label nature and concentration, the solvent, the pH or the preservation conditions of the label solutions could impact the intensity and band position in SERS spectra.

In conclusion, the results expounded in this thesis clearly confirm the potential of SERS for bioanalyses. Even so, remaining challenges will need to be taken up to see clinical applications of SERS methods flourishing. Achieving reproducible and efficient SERS substrates preparation and specifically attracting the analytes onto their surface are examples of such challenges to be risen.

Autre version : http://hdl.handle.net/2268/250791
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