Device functionalization

Device functionalization was performed within 10 min after device fabrication, when inner surfaces of the elasticity microcytometer remained hydrophilic. To render confining channels of the elasticity microcytometer inert, non-adhesive, block copolymer pluronics F-127 (Sigma-Aldrich, St. Louis, MO) was injected into the device for 30 min to allow coating of pluronics F-127 on PDMS walls. To quantify EpCAM expression on cancer cells, the elasticity microcytometer was functionalized with monoclonal antibodies against EpCAM using the avidin-biotin chemistry [68]. Briefly, 4% (v/v) of 3-mercaptopropyl trimethoxysilane (Gelest, Morrisville, PA) in ethanol was first flushed into the elasticity microcytometer for 30 min at room temperature. The elasticity microcytometer was then rinsed with pure ethanol, before 1 mM or 0.28% (w/v) N-γ-maleimidobutyryloxy succinimide ester (GMBS) in ethanol was flushed into the device for another 15 min. After rinsing with PBS, 10 µg/mL avidin (Life Technologies Scientific, Grand Island, NY) in PBS was flushed into the elasticity microcytometer at room temperature for 30 min. The elasticity microcytometer was rinsed again with PBS, before 10 µg/mL biotinylated goat anti-human EpCAM (R&D Systems, Minneapolis, MN) in PBS with 1 (w/v) bovine serum albumin (BSA) and 0.09 % (w/v) sodium azide was flushed into the elasticity microcytometer for 30 min. Coating the elasticity microcytometer with GMBS, a heterobifunctional cross-linker, could facilitate covalent conjugation of biotinylated EpCAM antibodies and avidin. To ensure anti-EpCAM activity and avoid antibody hydrolysis processes, cell deformability assays were conducted within 3 hr after device functionalization.