Monocouches peptidiques auto-assemblées et applications dans le domaine des biocapteurs de résonance de plasmon de surfaces

The work presented in this thesis aims to extend the use of surface plasmon resonance (SPR) biosensors to generate more rapid, cost efficient and simple to use diagnostic tools to diagnose or follow serious medical conditions. This task required the development of a new SPR instrument that relies on an inversion prism (dove) and is able to reach a limit of detection (LOD) in the 10-6 refractive index unit (RIU) range, a value comparable to more complex commercial instruments.  The developed SPR instrumentation is inexpensive, robust and very simple to manipulate. The other work presented in this thesis is based on reducing nonspecific interactions between the surface of SPR sensors and components in biological matrices such as urine, cell lysate, serum and whole blood. These nonspecific interactions induce SPR responses that have typically prohibited the use of SPR in these complex matrices. Amino acidshavebeen investigated for reduction of nonspecific binding (NSB) because they offer a wide variety of physico-chemical properties capable of tuning the physical properties of surfaces in a self-assembled monolayer (SAM) format. Initially, the attachment of one of 19 physiological 20 amino acids to 3-mercaptopropionic acid (3-MPA) allowed the formation of amino acid SAMs. Exposure of these surfaces to bovine serum revealed nonspecific interactions ranging from 400 ng/cm² to 800 ng/cm². Detection assays for β-lactamase (an enzyme produced by drug resistant bacteria at a micromolar level) demonstrated that the amino acid SAM is suitable for SPR biosensing. By using a solid phase approach, peptides were of 2 to 5 residues were synthesized to investigate NSB properties. The result of this study showed that adding amino acids decreased nonspecific interactions up to a peptide length of 5 amino acids. The best performing peptide, 3-MPA-(Serine)5-OH, resulted in low nonspecific adsorption of bovine serum proteins to a level of 180 ng/cm².  This value is similar to nonspecific adsorption obtained under identical conditions for one of the best reported surfaces: polyethylene glycol-based SAMs at 100 ng/cm².  The 3-MPA-(Serine)5-OH based SAM was used to calibrate β-lactamase, leading to its direct quantification in crude cell lysate. The detection limit for this analyte was 10 nM.  A third generation of peptide, which is binary patterned, decreased significantly nonspecific adsorption to a level as low as 23 ± 10 ng/cm², a value comparable to the best surfaces known.  This surface SAM allowed the calibration of matrix metalloproteinase-3 (MMP-3), a potential indicator of cancer. Direct quantification assays of MMP-3 in whole blood serum were achieved with the binary patterned peptides developed. The LOD for MMP-3 was 0.2nM over a 50 nM linear domain. A fourth generation of peptide based surfaces was developed, reducing the level of nonspecific adsorption of blood serum proteins to 12 ± 11 ng/cm2. These new surfaces were modified to attach His-tagged biomolecules enabling rapid screening of small ligands targeting the Cluster of differentiation-36 (CD36).  Finally, the electroformation of peptide monolayers was studied to determine the optimal conditions needed to form an ultralow biofouling surface.  It was demonstrated that the difference in potential applied during the formation of a peptide based layer influences the kinetics of formation and the arrangement of this layer.  An optimal layer of 3-MPA-HHHDD-OH could be obtained in less than 6 min by applying a potential of 200mV vs Ag/AgCl to the SPR sensor.

Keyphrases: Nonspecific interactions, Surface plasmon resonance, Biosensors, Chemical sensors, Self-assembled monolayers, Peptides, Pharmaceutical, Electroformation, Diagnostic tools, Chemical layers, Adsorption