Promoteur de thèse: Monsieur Ruddy WATTIEZ et co-promoteur Monsieur Christophe Caucheteur
Résumé de la dissertation : Biosensing is a multidisciplinary field of research combining biotechnology with infinite sensing modalities. It has been fruitful and essential in many applications such as medical diagnosis, healthcare, environmental measurements, glucose level monitoring and food safety.
The biosensors market is flourishing for years and will continue to be a growing center of interest. In fact, there is an urgent need for novel techniques that allow fast, low cost, and sensitive biomarkers detection. This is all the more true considering the possibility to directly measure the environment of interest without the need for biopsy or specific sampling. Real-time monitoring and miniaturization can therefore lead to in situ biosensors and increase their interest and performances.
In this way, surface plasmon resonance (SPR) biosensors play a tremendous role. They exploit surface plasmon waves to characterize ultra-low refractive index changes that happen at the sensor surface. Those changes are provoked by interactions between bioreceptors immobilized on the sensor surface and targets of interests, such as diagnostic biomarkers, pathogens, proteins or peptides, cells, etc. The classical configurations of SPR sensors are based on the Kretschmann prism. However, they are expensive and cumbersome tools, limited to laboratory use. A promising alternative is to transpose plasmonics to optical fibers and exploit their numerous assets at a higher degree, such as their small diameter, flexibilitiy, cost-efficiency and reliability. They can be easily inserted into packagings and endoscopes and are sound to screen suspicious tissues directly into the patient’s body in a minimally-invasive way. This work is therefore focusing on in situ biomarker sensing using optical fibers.
To reach biosensing features, photo-imprinted patterns were implemented inside the 8 μm core diameter of standard telecommunication optical fibers. These structures are called tilted fiber Bragg gratings (TFBGs) and are able to excite SPR in the nearinfrared range when a thin gold film of few tens of nanometers is added on its surface. As SPR-TFBGs are highly flexible platforms, four different probes configurations were experimented during this thesis. First, bare-TFBGs (without gold coating) were assayed with different antibodies immobilization strategies to study the inherent properties of the probes. As there is no SPR enhancement in this configuration, the sensor response was lowered but efficient enough to detect the lowest concentrations of analytes. Then, silanization and gold electroless plating (ELP) techniques were studied to better control the gold film deposition and to refine the properties of the sensors by tuning the SPR features. The ELP leads to the formation of gold clusters that excite localized surface plasmon resonance. By modifying the plating time and gold salt concentrations, it was possible to precisely monitor the gold thickness and reach (L)SPR generations. A fourth configuration named “hybrid” was coupling a thin sputtered gold film prior to the electroless deposition, yielding a live monitoring of the metal film growth with sensitivities comparable to those obtained with fully sputtered layers. This can be of interest for many applications where thin films thicknesses have to be monitored in conditions where the use of sputter-coaters or evaporators (under vacuum) is not compatible.
In this context, cytokeratins, mammaglobins, and epidermal growth factor receptors are relevant target biomarkers for lung or breast cancer diagnosis. To catch these molecules, antibodies and aptamers were anchored as bioreceptors on top of SPRTFBGs. The sensors were manufactured from the fiber optic coil to their operational form and were extensively tested in buffered solutions spiked with growing concentrations of the aforementioned targets, but also in serum and porous gels used to mimick physical constraints of tissues. The probes present experimental detection thresholds ranging between 1 pM and 9 pM for the Cytokeratin 17 protein (CK17) and the epidermal growth factor receptor 2 (HER2), respectively. These SPR-TFBGs probes were further used to detect circulating breast cancer cells spiked in PBS buffer. The use of gold nanoparticles as labels was also implemented to amplify the detection,significantly beginning at 10 cancer cells/mL which is roughly the sensing level reported by the best-in-class biosensors.
Finally, this thesis pioneers the use of optical fiber gratings for in vivo plasmonics. Our optical fiber probes were successfully inserted into a bronchoscope and tested in the lungs of anesthetized pig at the Erasmus hospital (Brussels) to reach the first SPRTFBG signature inside an organ. The detection of CK17 biomarkers inside freshly resected human lung tissues was also performed in different lung cancer subtypes and the CK17 expression was verified by immunohistochesmitry on the same patient samples. It allowed the discrimination between healthy and tumoral parts of the analyzed tissues.
This work demonstrates the high potential of SPR-TFBGs biosensors to be further used in biomedical applications that require direct measurements in confined and poorly accessible environments, with a less invasive way than the current available techniques.