Real-time study of CTC-cluster growth and secretions on dedicated microfluidic platform coupled with multiplexed biosensor

Cancer is the proliferation of purposeless cells taking place in any part of the body while the spread of cancer to different part via the circulation is defined as metastasis. In recent years, circulating tumour cells (CTCs) have come to the forefront for their role in metastatic progression. Their detection and analysis can provide valuable information for early cancer diagnosis, screening, and monitoring. The objective of this project is to develop an integrated biosensing-microfluidic setup that can trap and cultivate such rare cells for their biomarker analysis. For biosensing, label-free optical modality has been selected through plasmonic materials (gold, Au) and nano-structuration (nanohole array, NHA). The gold surfaces were functionalized with mixed alkylated/PEGylated monolayer allowing the covalent immobilization of anti-CTC biomarker antibodies. Two covalent immobilization strategies were studied: (i) novel site-specific via enzymatic activation, copper free strain promoted azide-alkyne click chemistry (SPAAC) using dibenzocyclooctyne and (ii) conventional carbodiimide/NHS chemistry via amine coupling for comparative analysis. Surface characterization was performed through XPS analysis, AFM, and Infra-Red spectroscopy. SPRi analysis was used to evaluate biosensing efficiency. Comparative analysis between conventional and copper free click chemistry exhibited SPAAC immobilized target recognition to be ~3 times higher. Thus, optimal coupling chemistry was demonstrated leading to site-specific and oriented antibody immobilization on functionalized gold surfaces. Then, to trap cancer cells, PDMS microfluidic device was fabricated via moulding protocol. Characterization for valve actuation and trap unit functionality was performed first using microbeads and then metastatic breast cancer MCF7 cell line. For analysis of biomarkers secreted directly by cancer cells, ELISA was performed for metastatic ovarian cancer SK-OV-3 cells for Interleukin-6 (IL-6) and fibronectin, where around 5-50 pg/nL for IL-6 and 600-800 pg/nL for fibronectin concentrations were analysed per cell. An integrated setup was developed with a label-free optical NHA biosensor coupled within a pneumatically actuated microfluidic device for directed real-time and multiplexed analysis of such biomarker concentrations. Cancer associated biomarkers i.e., IL-6 and soluble Tumour Necrosis Factor Receptor 2 (TNFR2) were injected into the integrated microsystem for characterization of biosensing efficiency using the optimal surface immobilization approach. We were able to highlight significant difference between specific IL-6 signal interaction vs non-specific TNFR2 signal interaction, however for subsequent lowered concentration (sub ng/mL to pg/mL), this setup is not sensitive enough to detect such small biomarkers. This could be due to inconsistencies showcased for the gold film deposition protocol employed for NHA fabrication, where significant difference between hole size and deposition depth was showcased. The system developed in this work provide an avenue for studying the effects of different chemotherapeutic drugs on cultured CTCs from cancer patient blood samples. The results obtained are expected to further contribute towards the development of precise cancer diagnosis and personalized treatments.<p></p>