Quantification of Protein Secretion from Circulating Tumor Cells in Microfluidic Chambers

Abstract Cancer cells can be released from a cancerous lesion and migrate into the circulatory system, from whereon they may form metastases at distant sites. Today, it is possible to infer cancer progression and treatment efficacy by determining the number of circulating tumor cells (CTCs) in the patient's blood at multiple time points; further valuable information about CTC phenotypes remains inaccessible. In this article, a microfluidic method for integrated capture, isolation, and analysis of membrane markers as well as quantification of proteins secreted by single CTCs and CTC clusters is introduced. CTCs are isolated from whole blood with extraordinary efficiencies above 95% using dedicated trapping structures that allow co‐capture of functionalized magnetic beads to assess protein secretion. The patform is tested with multiple breast cancer cell lines spiked into human blood and mouse‐model‐derived CTCs. In addition to immunostaining, the secretion level of granulocyte growth stimulating factor (G‐CSF), which is shown to be involved in neutrophil recruitment, is quantified The bead‐based assay provides a limit of detection of 1.5 ng mL−1 or less than 3700 molecules per cell. Employing barcoded magnetic beads, this platform can be adapted for multiplexed analysis and can enable comprehensive functional CTC profiling in the future.

. Size of the microchambers as determined using a profilometer and fluorescent images. The combined information of both techniques enables a precise calculation of the inner volume of the microchamber when the valves are actuated. We calculated a total chamber volume of 82 pL.  inset, which is covered by a lid to set the relative humidity around the chip close to 100% at 5% CO2 and 37°C. c) Fluid flow is controlled with a syringe pump, the pneumatic valves are actuated through a manual valve manifold (d). e) Schematic drawing of the system. The chip has one inlet/outlet and four control lines for the valves.     3005 are spin-coated on the silicon wafer (a) and subsequently patterned through a foil mask (b). Thereafter, the second SU-8 3025 layer is spin-coated onto the wafer and exposed through a second foil mask (c and d). After a post-exposure bake, the SU-8 structures were developed and fixed with a hard bake (e). AZ 40XT positive resist was then spin-coated on top of the SU-8 structures with a speed that results in a lower resist height than the second SU-8 layer (f).
After exposure through a third foil mask (g), the resist was developed (h) and a thermal reflow process realized the desired channel shape with smooth transitions between structures of different heights (j).    Table S3: Fabrication protocol for the multilayer Master structures used for replica molding of the fluid channel in the presented microfluidic device.
Step Name Details 1 Plasma cleaning Plasma clean the blank SI-wafer for 5 min at 400 W to remove any residual organic substances from the silicon surface. 2 Dehydration bake Bake the wafer for 5 min at 200°C to evaporate the hydration layer on the surface of the wafer and increase the bond between resist and wafer material. 3 Spin-coating Spin-coat a 7.5 µm thick SU-8 3005 layer on the wafer. On our device, we used 1500 rpm spinning speed for 30 sec. 4 Soft-bake 1 min at 65°C and subsequently 3 min at 95°C 5 UV-exposure Exposure (i-line) with a light dose of 160 mJ mm -2 (measured intensity at 395 nm) through a foil mask. 6 Spin-coating Spin-coat a 30 µm SU-8 3025 layer on the wafer. On our device, we used 3000 rpm spinning speed for 30 sec. 7 Soft-bake 2 min at 65°C and subsequently 10 min at 95°C 8 UV-exposure Exposure (i-line) with a light dose of 160 mJ mm -2 (measured intensity at 395 nm) through a foil mask. 9 Post-exposure-bake 1 min at 65°C and subsequently 3 min at 95°C 10 Development 3-4 min in mr-Dev 600 under constant agitation, face-down 11 Hard bake 2 h at 160°C (ramp up in 40 min, ramp down slowly by switching the heater off) 12 HDMS coating 300 sec at 50 mbar 13 Spin-coating Spin-coat a AZ 40XT layer (25 µm final height) on top of the existing structures (2300 rpm for 20 sec). Use excess resist to cover all structures before the spinning process is started. This prevents from inclusion of air and bubble formation. 14 Layer relaxation To create smooth transitions between the AZ layer and the protruding SU-8 pillars (future magnetic traps), place the spin-coated wafer on an even surface for 10min.

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Soft-bake 7 min at 85°C and subsequently 5 min at 120°C 16 UV-exposure Exposure (i-line) with a light dose of 450 mJ mm -2 (measured intensity at 395 nm) through a foil mask.

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Post-exposure bake 1 min at 85°C and subsequently 2 min at 105°C 18 Development 3 min in AZ 400K developer under constant agitation, face-down 19 Reflow 1 min at 115°C 20 Silanization Place the final wafer for at least 24 h in a dessicator at 300 mbar together with 200 µL trichloro(1H,1H,2H,2H-perfluorooctyl)silane 21 Silanization Place the wafer for another 2h in a desiccator at 300 mbar together with 200 µL chlorotrimethylsilane 22 PTFE-coating Spin-coat the wafer at 500 rpm for 30 sec with a 0.1% Teflon AF solution in fluorinated FC-40 oil.

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Oil evaporation Evaporate residual oil by heating the wafer to 100°C for 5 min. Table S4: Fabrication protocol for the double-layer PDMS chips. As a prerequisite, the two silicon master molds have to be fabricated beforehand (multilayer fluid master fabrication is described in Table S1, the control layer master consists of a single 20 µm SU-8 3025 structure).
Step Name Details 1 Mix PDMS Mix 60 g PDMS monomer and curing agent at a ratio of 10:1.

2
Degas PDMS To remove gas from the PDMS mixture, place the mixture in a desiccator for 15 min under vacuum.

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Cast PDMS onto fluid master Pour 40 g PDMS onto the fluid layer master that has been placed in a plastic petri dish to a final thickness of approximately 4 mm. 4 Store PDMS To avoid PDMS hardening of the remaining PDMS, place it in the fridge at 4°C. 5 Bake fluid layer Bake for 120 min at 80°C.

6
Cut fluid layer Peel cured PDMS from the master and cut the chips to size using a razor blade. 7 Punch inlet and outlet ports In-and outlet ports are punched with 1.5 mm biopsy punchers.

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Spin-coat PDMS onto control layer Use 5 g of the PDMS that was stored in the fridge and spin-coat the PDMS onto the control layer master for 60 sec at 2000 rpm to yield a homogeneous layer covering the control structures with a thin membrane. 9 Bake control layer Place the control layer in the oven at 80°C for 60 min.

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Prepare PDMS to PDMS bonding Spin-coat 1 mL PDMS curing agent onto a blank silicon wafer at 6000 rpm for 40 sec.

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Dip-coat curing agent Dip the prepared PDMS slabs (fluid layer cut to size and with punched ports) onto the blank wafer that is covered with a thin curing agent layer.

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Align PDMS parts Detach PDMS slab from the blank wafer and align it to the control structures on the second wafer with the spin-coated PDMS layer. Redo steps 11 and 12 for all individual chips on the control layer. Finally pour remaining PDMS around the chips.

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Diffusion Let the wafer with the aligned chips sit on the bench for 30 min to allow diffusion of the curing agent. 14 Bake chips Bake the PDMS composite for 60 min at 80°C.

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Cut chips Carefully peel the chips from the control master and punch control ports with a 1 mm biopsy puncher.

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Clean glass slides Clean glass cover slips with isopropanol, ethanol, water, and place them on a hotplate at 80°C for 10min.

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Plasma bonding Plasma activate the glass substrate as well as the chip in air plasma at 0.75 mbar for 40 sec and bond the chips to the glass.

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Bake the composite chip To increase the bonding strength, place the chip onto a hot plate at 80°C for 20 min.

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Storage Store the chips until use in the fridge at 4°C. This prevents from continuous hardening of the chips.