Micro cover glass

In this study, Y+128° X-propagation lithium niobate (LiNbO₃) was used as piezoelectric substrate. The IDT design was patterned by photolithography using a MA/BA6 mask aligner (SUSS MicroTec., Germany). After that, 50 Å of Cr was deposited as an adhesive layer, followed by a 500 Å gold layer for electrode fabrication. The deposition was conducted with an e-beam evaporator (Semicore Corp, USA). Finally, the metal layer was removed with photoresist and IDTs were formed by a lift-off process.
The PDMS/glass hybrid channel was fabricated by a standard soft lithography process, as shown in Figure S1 (Supporting Information). A thin layer of SU8 100 photoresist (MicroChem, USA) was spin-coated and patterned by ultraviolet (UV) exposure on a silicon wafer. A glass slide was placed on the SU8 mold at the designed position where standing acoustic filed was formed. The glass slide was made from micro cover glass (VWR, USA), and was cut to 800 μm × 5 mm by laser cutting. Sylgard 184 Silicone Elastomer Curing Agent and Base (Dow Corning, USA) were mixed at 1:10 and poured on the mold. After setting at room temperature overnight, the PDMS channel was peeled from the mold and bonded to the LiNbO₃ substrate. Before bonding, the surface of the LiNbO₃ substrate and the PDMS channel were treated with oxygen plasma.

The fabrication process for this 3D-AFT device was similar to our previous work.^{24 (link),26} A photo of the device is shown as Figure S1. Briefly, a pair of metal IDTs were patterned on a LiNbO₃ substrate via photolithography, e-beam evaporation and lift-off processes. A microchannel mold was fabricated using SU8 photoresist (MicroChem, USA) and patterned via photolithography. An 800 µm × 5 mm slide glass slide, laser-cut from a micro cover glass (VWR, USA), was placed on the mold structure at a designated position. Then PDMS base and curing agent (Dow Corning, USA) were mixed at a ratio at 10:1 and poured onto the mold. After baking at 65°C for an hour, the PDMS channel with the embedded glass slide was peeled off from the mold, and then bonded to a LiNbO₃ substrate using a plasma treatment. Following bonding, the device was baked at 110 °C overnight.

HT-29 cells were seeded onto micro cover glass (VWR) in 24-well plates (Greiner Bio-One) at 180,000 cells per well. Twelve hours after seeding, TSZ was added to appropriate wells, and samples were fixed 6 hours post-treatment with 4% paraformaldehyde (Sigma) for 30 min at RT. Anti-MLKL phospho S358 antibody (1:200) was added to each sample in immunofluorescence blocking buffer (PBS with 3% BSA, 1% saponin, and 1% Triton X-100) and incubated at 4°C overnight. After washing 5× with 1× phosphate buffered saline (PBS), Alexa488 anti-rabbit antibody (1:500) (Life Technologies), DAPI stain (1:300), and Alexa660 phalloidin (1:50) (Life Technologies) in immunofluorescence blocking buffer were added to each sample and incubated for 1 hour at RT. Samples were washed 5× with 1× PBS. micro cover glasses were then mounted onto microscope slides (Fisher Scientific) with Vectashield (Vector Laboratories Inc). Images were taken with Zeiss LSM 700 confocal microscope and processed with Velocity software.

Acrylamide (AAm) and N,N′-bis(acryloyl)cystamine (BAC) were purchased from Alfa Aesar. Tetramethylethylenediamine (TEMED) and Amplex UltraRed reagent (AUR) were purchased from ThermoFisher Scientific. Peroxidase from horseradish (HRP), 3-(trimethoxysilyl) propyl methacrylate, dextran from Leuconostoc spp (M_r ~ 100000), and anhydrous (≥99.9%) dimethyl sulfoxide were purchased from Sigma-Aldrich. Ammonium persulfate (APS) was purchased from CalabrioChem Inc. Polyethylene(glycol) diacrylate (PEGDA; M_w = 400) was purchased from PolySciences Inc. Ethanol (EtOH) was purchased from Decon Laboratories Inc. Glacial acetic acid was purchased from Macron Fine Chemicals. Water (dd-H₂O) was ionized using Milli-Q Advantage A-10 water purification system (Millipore, U.S.A.). Microscopic glass slides (3” × 1” × 1.2 mm) and micro cover glass (18 mm × 18 mm) were purchased from VWR. Rain-X water repellant manufactured by ITW (Global Bands, TX) was purchased from Home Depot. All UV−vis absorbance spectra were acquired on a Safire2 UV−vis microplate reader (TECAN, Switzerland). The absorbance for the samples were measured at 568 nm in a Nunclon 96-clear Microwell plate. Mechanical characterization tests were performed on a TA Instruments RSA-G2 Solids Analyzer.

Biotin PEG thiol (MW 1000 Da) was purchased from Nanocs. PEG thiol (MW 550 Da) was purchased from Creative PEGWorks. Neutravidin (NA) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Thermo Fisher Scientific. Polystyrene beads (0.6 µm mean particle size), sulfuric acid (95.0–98.0%), hydrogen peroxide (30% w/w in H₂O), and ethyl alcohol (200 proof) were purchased from Sigma-Aldrich. Paraformaldehyde (16% w/v aq soln, methanol free), Dulbecco’s Phosphate Buffered Saline (1×, without calcium and magnesium), and micro cover glass (No. 1, 18 mm × 18 mm) were purchased from VWR. Lysogeny broth and granulated agar were purchased from BD Difco. Biotinylated aptamer (Bt-aptamer, 5′[Bt]-ATACCA-GCTTATTCAATTCCCCCGTTGCTTTCGCTTTTCCTT-TCGCTTTTGTTCGTTTCGTCCCTGCTTCCTTTCTTG-AGATAGTAAGTGCAATCT3′) and fluorescently labeled biotinylated aptamer (6FAM-aptamer-Bt, 5′[6-carboxyfluorescein-ATACCAGCTTATTCAATTCCCCCGTTGCTT-TCGCTTTTCCTTTCGCTTTTGTTCGTTTCGTCCCTG-CTTCCTTTCTTGAGATAGTAAGTGCAATCT-[Bt]3′) were synthesized by Sigma-Aldrich. Deionized (DI) water used in all experiments was prepared using a Milli-Q Gradient water purification system with a resistivity of 18.2 MΩ·cm at 25 °C (Millipore).