LABEL FREE SINGLE CELL DETECTION OF IMMUNE CELL CYTOTOXICITY TOWARDS TUMOR CELLS USING DIELECTROPHORESIS AND ELECTRICAL IMPEDANCE SPECTROSCOPY

Abstract

This research aimed to develop an innovative Lab-On-a-Chip (LOC) system for accurately and efficiently assessing the cytotoxicity of immune cells against cancer cells. It was pivotal in addressing significant limitations associated with conventional clinical methods, notably their high cost, intensive labor, and extended processing times. The primary goal was to design a system leveraging Dielectrophoresis (DEP) and electrical impedance spectroscopy to trap and monitor cancer cell viability. First, substantial groundwork was laid through extensive literature review, identifying optimal methods for capturing and evaluating cancer cell interactions with immune cells, particularly Natural Killer (NK) cells. Next, significant progress was made with the design and fabrication of micro-electrode arrays capable of single-cell analysis, aiming to overcome limitations posed by traditional methods that lacked control over target cell populations. These arrays were successfully fabricated, integrating them into a microfluidic system coupled with an electronic readout, effectively creating a controlled environment for cell capture and impedance measurement. However, challenges emerged related to the interface between the electrical chips and the electronic readout system. Initially, a wire bonding method was employed but faced issues with scalability and reliability. Attempts to use anisotropic Pressure-Sensitive Adhesive tapes revealed practical limitations due to the necessity for sustained external pressure. Ultimately, a refined spring-loaded connector approach resolved these challenges, ensuring consistent and reliable chip connections. Next further advancements were done, particularly the integration of microfluidics and improved impedance measurement methods. A new microfluidic channel design drastically reduced the sample volume, improving signal-to-noise ratios and allowing better control over cell positioning and interactions. To address detection challenges, a parallel approach involving label-less image cytometry using artificial intelligence algorithms was successfully tested, providing an efficient method for real-time cytotoxicity assessments alongside impedance spectroscopy. Then, considerable breakthroughs were achieved. The system was optimized for effective and selective trapping of cancer cells, with careful tuning of DEP conditions. Challenges such as electrolysis in the cell medium and premature cell apoptosis due to environmental fluctuations were systematically resolved through design improvements, including an improved sealing mechanism for the assay environment. Ultimately, the system successfully demonstrated statistically significant detection of apoptosis events, clearly distinguishing between NK targeted cells and controls using impedance spectroscopy. This critical milestone marked the achievement of the project's core objective, confirming the viability of this novel LOC platform for reliable cytotoxicity assays. Throughout this research, we encountered and methodically addressed multiple technical obstacles, ranging from microfabrication complexities and interface issues to cell environment management. Each obstacle was strategically navigated, leveraging innovative engineering solutions, rigorous experimentation, and interdisciplinary collaboration, significantly advancing the state-of-the-art in biomedical device technology. At its current form, the project introduced a proof-of-concept device that holds substantial potential for future clinical applications, notably in personalized immunotherapy, CAR-T cell therapy efficacy testing, and deeper explorations into immune-cancer cell interactions.