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Project

FO-SPR bioassay development for specific and sensitive analysis of extracellular vesicles

Extracellular vesicles (EVs), nanosized cargo carriers of the body, have attracted growing attention of researchers due to their critical role in intercellular communication and signal transduction. Despite their outstanding potential of EVs, this field requires additional improvements in several aspects before reaching applications in clinical settings. Currently, one of the main challenges is lack of standardization, often resulting in interlaboratory variations and biased analysis. Besides, the existing EV detection methods either lack the required sensitivities and specificity or cannot operate directly with complex matrices. Therefore, the objective of this dissertation was to develop standardized fiber optic surface plasmon resonance (FO-SPR) bioassays for specific and sensitive analysis of EVs in complex matrices, thus opening up possibilities for employment of the FO-SPR biosensors as a clinical diagnostic tool in the future. In this context, FO-SPR biosensor has been selected because this type of biosensors emerged over the years as highly-competitive alternative to conventional SPR technologies due to their low-cost probes, low demand in sample volume, fast time-to-result, and possibility for direct detection in complex matrices.

First and foremost, the FO-SPR biosensor was standardized for specific and sensitive EV detection by calibrating the device with recombinant EVs (rEVs) directly in cell culture medium (CCM) and blood plasma. An FO-SPR sandwich bioassay was built for the first time with a 2-step signal amplification strategy by utilizing a biotinylated detection antibody and gold nanoparticles functionalized with an anti-biotin antibody. Remarkable limit of detection values were obtained that were 103 times lower than the expected physiological concentration of EVs in human plasma and 104 times lower than the EV concentration in the plasma of cancer patients. Finally, the developed sandwich bioassay was investigated for its specificity by detecting rEVs, HEK293T endogenous EVs, and MCF7 cell line-isolated EVs in CCM and blood plasma.

The established FO-SPR EV detection bioassay was further expanded towards detecting breast cancer-specific EVs directly in blood plasma. Breast cancer cell lines SK-BR-3 and MCF7 were taken as model systems for breast cancer biomarkers HER2 and EpCAM, respectively. The obtained LODs in blood plasma demonstrated remarkable sensitivity of the established bioassays. Importantly, the specificity of the bioassays was shown using plasma samples from healthy volunteers unknown to be diagnosed with breast cancer. This work demonstrated that, compared to other SPR-based platforms, the FO-SPR platform could offer excellent prospects to bridge the gap between laboratory and clinical settings in utilizing EVs as biomarkers for early (cancer) diagnostics.

In the final part, the FO-SPR biosensor was used to validate the C-X-C chemokine receptor type 4 (CXCR4) as a general EV biomarker for the first time, also exploiting other technologies, including the golden standard SPR platform (i.e. Biacore) and the ExoView. The expression of CXCR4 was demonstrated on EVs isolated from 7 different cell lines with Biacore SPR technology in a label-free manner. Using the established FO-SPR sandwich bioassay, CXCR4-expressing EVs were directly detected in CCM, while also revealing the colocalization of CXCR4 with general EV surface biomarkers. Finally, the results with the ExoView biosensor supported the outcome of obtained colocalization at the single EV level.

This dissertation emphasized the importance of using pre-evaluated biological reference materials to develop robust and specific EV detection bioassays and as such eliminate interlaboratory variations and biased analysis. The obtained results showed (1) the remarkable sensitivity of the FO-SPR device, (2) its capability to measure EVs directly in complex matrices, (3) its capacity to differentiate cancer-specific EVs among others, and (4) perform colocalization analysis to reveal the surface molecular composition of EVs. All these, combined with other intrinsic features of the FO-SPR biosensor, demonstrated its vast potential for the EV research field and use as a clinical tool in the future.

Date:18 Oct 2018 →  19 Oct 2022
Keywords:Biosensors, Extracellular vesicles, Fiber optic surface plasmon resonance
Disciplines:Other chemical sciences, Nutrition and dietetics, Agricultural animal production, Food sciences and (bio)technology, Agriculture, land and farm management, Biotechnology for agriculture, forestry, fisheries and allied sciences, Fisheries sciences, Analytical chemistry, Macromolecular and materials chemistry, Biological system engineering, Biomaterials engineering, Biomechanical engineering, Medical biotechnology, Other (bio)medical engineering
Project type:PhD project