BLUE FINGERS STUDENT AWARD

Title: Diaphragm-actuated microfluidic sorting of cellular aggregates on impedance-based size 

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The ability to enrich and selectively sort cellular aggregates within defined size and viability ranges is central to advancing biosensing, regenerative medicine, and targeted therapeutics. Conventional detection methods often rely on fluorescent staining for viability or destructive secretion assays, which preclude the direct use of the sorted populations for transplantation. As a result, there is a critical need for label-free, physiologically gentle platforms capable of dynamically classifying and isolating aggregates based on both their biophysical and dielectric cues. Our research addresses this challenge by developing diaphragm-actuated microfluidic systems that will eventually integrate inline impedance cytometry with real-time pneumatic sorting. Our prior work established diaphragm-enabled nanoconfinement for enrichment of DNA molecules [1], and diaphragm actuated biophysical cytometry based on nucleus phenotypes [2]. Building upon these innovations, we explore application of diaphragms for label-free sorting of multicellular aggregates. Using low-melting-point Field’s metal to form sidewall electrodes, a microchannel with 3D electrode for multifrequency impedance cytometry is achieved. Model particles and multicellular aggregates, hydrodynamically focused into a dual-electrode impedance sensing region, are characterized for size, membrane integrity, and viability metrics based on low-frequency impedance magnitude (|ZLF|) to estimate size, and high-frequency electrical opacity (|ZHF|/|ZLF|) to infer membrane integrity [3]. Calibration using polystyrene beads (33-150 μm) reveals a strong linear correlation between particle size and |ZLF|, validating the size-detection metrics for cellular aggregates. For active sorting, a pneumatically actuated sidewall diaphragm is integrated adjacent to the flow channel. Upon image and impedance-based detection of a specific cell aggregate, a brief pressure pulse (10-50 psi) deforms the diaphragm wall to deflect the aggregate into the designated outlet. The diaphragm geometry (500 μm length, 200 μm width, 35 μm thickness) is optimized to achieve rapid actuation (~20ms) while minimizing shear stress. This system will be integrated in future work for cell aggregate detection, real-time classification on viability metrics and activated sorting for isolation of specific cell aggregates. This integrated approach, combining label-free impedance phenotyping with diaphragm-based microfluidic actuation, can provide a scalable and modular solution for cell aggregate sorting, which will be essential for multimodal cytometry and transplant sample isolation systems.