Biotinylated bsa

Biotinylated-BSA (1 μg/μl, Thermo) is flowed for 25 min through the device, allowing its binding to the epoxy surface. On top of the biotinylated-BSA, 0.5 μg/μl of NutraAvidin (Pierce, Rockford, IL) is added (flow for 20 min). The ‘button’ valve is then closed, and biotinylated-PEG (1 μg/μl, (PG2-AMBN-5k, Nanocs Inc.) is flowed over for 20 min, passivating the flow layer, except for the buttons area. Following passivation, the ‘button’ valve is released and a flow of 0.2 μg/μl biotinylated anti-GFP antibodies (Abcam; #ab6658, Cambridge, United Kingdom) or 0.01 µg/µl biotinylated anti-Flag antibodies (Cell Signaling; #2908 S Danvers, MA, USA) were applied. The antibodies bound to the exposed NutraAvidin, specifically to the area under the ‘button’, creating an array of anti-GFP - or anti-Flag tag. PBS buffer was used for washing in between steps. In the case of p27 immobilization, surface chemistry was performed with 0.2 μg/ml donkey anti-mouse whole biotinylated anti-IgG antibodies (#715-065-150, Jackson Immuno research laboratories, Maryland, USA) followed by 20 min flow of 6.5 μg/ml anti p27 antibodies (Santa Cruz biotechnology, Heidelberg Germany; #1641 mouse).

Capture domain of the device containing an array of gold microelectrode pairs were modified with anti-MCSP using avidin-biotin chemistry (Supplementary Fig. S2). Prior to functionalization, the electrodes were cleaned by sonication in acetone for 5 min, rinsed with isopropyl alcohol and water for another 2 min, and dried with the flow of nitrogen. They were then incubated in biotinylated BSA (200 μgmL⁻¹ in PBS, Thermo-Fisher scientific, USA) solution for 2 h followed by coupling with streptavidin (100 μgmL⁻¹ in PBS, Invitrogen) for 1 h at room temperature. Subsequently, the electrodes were coated with biotinylated anti-MCSP (5 μgmL⁻¹ in PBS, Invitrogen) for another 2 h. These modification steps were performed manually by filling the microchannel with corresponding solution and after each modification step, channel was flushed with PBS (10 mM, pH 7.0) to remove any unbonded molecules.

For surface immobilization
of protein flat sheets, 10 μg/mL of biotinylated-BSA (29130,
ThermoFisher Scientific) was coated in a 96-well plate for 30 min
at 37 °C. The wells were washed five times with 1 × PBS,
pH 7.4 and then coated with 10 μg/mL streptavidin (21122, ThermoFisher
Scientific) for 30 min at 37 °C. The wells were first coated
with either FS-empty (10.5 nM), FS-α-CD3 (0.5 nM), or FS- FS-α-CD3-CD28
(0.5 nM) in 1× PBS, pH 7.4 for 30 min at 37 °C. After washing
five times with PBS, FS-α-CD3 or FS-α-CD3-CD28-coated
wells were either coated with FS-empty (10 nM), FS-PD-L1 (10 nM),
FS-PD-L1-13 (5 nM), FS-PD-L1-40 (5 nM), or FS-PD-L1-200 (5 nM) for
30 min at 37 °C. After incubation, the wells were subjected to
five washes of PBS. Wells precoated with 5 nM of FS-PD-L1-13, FS-PD-L1-40,
and FS-PD-L1-200 were treated with another coating of 5 nM FS-empty,
to ensure all wells had a similar level of flat sheet coating. After
washing with PBS, PD-1-NFAT cells were seeded at 3 × 10⁴ cells per well and incubated for 3 h at 37 °C, 5% CO₂. Cells were lysed with 1 × Glo Lysis buffer (Promega), and
luciferase reporter activity was measured using Bright-Glo Luciferase
Assay System (Promega) in a Varioskan LUX multimode microplate reader.

To derivatize the slide surface, biotinylated-BSA (1 μg/μl, Thermo) was flown through the device for 30 min allowing the binding of the BSA to the epoxy surface. On top of the biotinylated-BSA, 0.5μg/μl of Neutravidin (Pierce) was added for 30 min. The ‘button’ valve, a micromechanical valve used for both the surface chemistry and MITOMI (55 (link)), was then closed and biotinylated-BSA was flown over for 30 min passivating the rest of the device. Following passivation, the ‘button’ valve was released and a flow of 0.2 μg/μl biotinylated anti-HIS antibody (Qiagen) was applied. The antibody bound specifically to the exposed Avidin surface under the ‘button’ creating an anti-HIS tag array. Hepes (50 mM, Biological Industries) was used for washing unreacted substrates after each of the different surface chemistry steps.

DMEM/F12 medium (HEPES, no phenol red), FBS,
penicillin/streptomycin, biotinylated BSA, and streptavidin were purchased
from Thermo Fisher Scientific (Massachusetts, US). Geneticin sulfate
was purchased from Capricorn Scientific (Ebsdorfergrund, Germany).
Culture 6 channel μ-Slide #1.5 glass bottom was purchased from
Ibidi (Gräfelfing, Germany). Polystyrene nanoparticles of 340
nm were purchased from Spherotech (Illinois, US). Gold nanoparticles
of 80 nm were purchased from Sigma-Aldrich (Missouri, US).

First, 200 μL of 2 μm diameter polystyrene microspheres (#4202 A, Thermo Fisher Scientific Inc.) was centrifuged down at 5.5 × 1000 rpm for 1 min to replace the stock liquid with biotinylated BSA (1 mg/mL; Thermo Fisher Scientific Inc.). After 90 min of incubation on a rotator the biotin-BSA functionalized polystyrene beads were washed with filtered PBS buffer (x0.1, pH 8) for 3 times to remove un-bound biotinylated BSA. The microspheres were then incubated with streptavidin conjugated quantum dots (Q10103MP, Thermo Fisher Scientific Inc.) at a concentration of 1 × 10⁻² μM for 90 min on a rotator. We chose the quantum dot that has a narrow emission maximum at 605 nm and streptavidin modification to form a strong binding through biotin-streptavidin interaction. Finally, quantum dot functionalized microspheres were washed with 0.1× PBS buffer 2–3 times to remove excess quantum dots from the solution. The QD-coated microspheres were used as prepared.