A Micropillar Array Based Microfluidic Device for Rare Cell Detection and Single-Cell Proteomics
Abstract
:1. Introduction
2. Experimental Design
2.1. Materials
- AZ 400k developer (MicroChemals GmbH, Ulm, Germany)
- Chromium etchant (TechniEtch Cr01, MicroChemals GmbH, Ulm, Germany)
- 5-inch chromium coated glass plate covered with positive photoresist film (Photomask Portal, Richardson, TX, USA)
- 4-inch silicon wafers (University Wafer, South Boston, MA, USA)
- Tweezers for 4-inch silicon wafer (Catalog No. 18-100-946, Fisher Scientific, Newington, NH, USA)
- Acetone (Sigma Aldrich, St Louis, MO, USA)
- Isopropanol (IPA) (Sigma Aldrich, MO, USA)
- 6-inch wide-open round beakers (Fisher Scientific, NH, USA)
- N2 dryer
- Hexamethyldisiloxane (HMDS) (SKU 440191, Sigma Aldrich, MO, USA)
- SU8 3035 photoresist (Kayaku Advanced Materials, Westborough, MA, USA)
- SU8 developer (Kayaku Advanced Materials, MA, USA)
- Polydimethylsiloxane (PDMS) Sylgard 184 kit: silicone elastomer base and curing agent (Dow Corning, Midland, MI, USA)
- Trichloro(1H,1H,2H,2H-perfluorooctyl)silane (TPOS) (SKU 448931-10G, Sigma Aldrich, MO, USA)
- Weighing dish (Catalog No. 01-549-750, Fisher Scientific, NH, USA)
- Petri dish (15 cm) (Catalog No. 09-720-500, Fisher Scientific, NH, USA)
- Weighing scale (Catalog No. 01-922-341, Fisher Scientific, NH, USA)
- Razor blade or scalpel (Catalog No. NC0134996, Fisher Scientific, NH, USA)
- PDMS punches (Catalog No. 12-460-402, 12-460-410, Fisher Scientific, NH, USA)
- Microscope slides (Catalog No. 12-550-102, Fisher Scientific, NH, USA)
- 99% ethanol (SKU 459836, Sigma Aldrich, MO, USA)
- Deionized (DI) water
- 1 mL pipet tips (Catalog No. 05412307, Fisher Scientific, NH, USA)
- Stainless steel dispensing needle with luer lock connection (Catalog No. 11-101-1671, Fisher Scientific, NH, USA)
- Cleaned and bagged high-purity white silicone rubber tubing (Catalog No. 51845K51, McMaster Carr, Elmhurst, IL, USA)
- PTFE light wall tubing (Part No. STT-21, Component Supply Co., Sparta, TN, USA)
- 3D printed supporting structure
- Sterile Luer-Lock syringes (3 mL and 5 mL) (Fisher Scientific, NH, USA)
- Phosphate-buffered saline (PBS) without Ca2+/Mg2+ ions (311-010-CL, Wisent Bioproducts, Saint-Jean-Baptiste, QC, Canada)
- Avidin (Catalog No. MP215004705, Fisher Scientific, NH, USA)
- Bovine serum albumin (BSA) (SKU A7030, Sigma Aldrich, MO, USA)
- Biotinylated anti-EpCAM (eBioscience, San Diego, CA, USA)
- Cell line (e.g., PC9 cells, ATCC)
- Cell culture medium (e.g., RPMI1640 medium,)
- Fetal bovine serum (FBS) (080-150, Wisent Bioproducts, QC, Canada)
- Trypsin-EDTA (0.25% Trypsin with EDTA 4Na) (325-042-CL, Wisent Bioproducts, QC, Canada)
- Vybrant dye DiD (Catalog No. V22887, Thermofisher, Waltham, MA, USA)
- Sterile 1.7-mL centrifuge tubes (Catalog No. 02-682-000, Fisher Scientific, NH, USA)
2.2. Equipment
- Maskless Aligner (Heidelberg uPG 501, Heidelberg Instruments Mikrotechnik GmbH, Heidelberg, Germany)
- Spin coater (H6-23, Laurell Technologies, Lansdale, PA, USA)
- Digital Programmable Stirring Hot Plates (HS40/HS40A, Torrey Pine Scientific, Carlsbad, CA, USA)
- Contact Mask Aligner (Suss MA6 Mask Aligner)
- Desiccator with a vacuum connection (Catalog No. 08-594-15A, Fisher Scientific, NH, USA)
- Programmable Oven (SKU 2958M5, Medicus Health, Kentwood, MI, USA)
- Plasma treater (SKU 12051A, Electro-Technic Products, Chicago, IL, USA)
- Programmable, dual-channel infusion and withdrawal modular syringe pump (Fusion 200-X, Chemyx, Stafford, TX, USA).
- Pipet (P10, P200, P1000) (Eppendorf) (Fisher Scientific, NH, USA)
- Serological pipet (Catalog No. 9541, Thermofisher, USA)
- CO2 incubator for cell culture (Catalog No. 11-676-604, Fisher Scientific, NH, USA)
- Biosafety hood (1300 Series, Fisher Scientific, NH, USA)
- Centrifuge (Eppendorf) (Catalog No. 05-413-112, Fisher Scientific, NH, USA)
- Mini centrifuge (Catalog No. 75002541, Fisher Scientific, NH, USA)
3. Procedure
3.1. Chrome Mask Writing
- Draw the layout of the micropillar-based microfluidic device using CAD software and convert the file into DXF form.
- Load the chromium and positive photoresist coated glass plate to the maskless aligner.
- Pattern writing on the photoresist layer of the glass plate using the maskless aligner.
- Dilute the AZ 400K developer 5 times (e.g., 40 mL of AZ 400K developer and 160 mL of DI water).
- Develop the glass plate with the diluted AZ 400K developer for 20 s.
- Rinse the glass plate with running DI water for 10 s.
- Soak the glass plate in the chrome etchant for 1 min.
- Rinse the glass plate with running DI water for 10 s.
- Strip off the undeveloped photoresist in undiluted AZ 400K developer for 30 s.
- Rinse the chrome mask with running DI water for 10 s.
- Dry the chrome mask with the N2 gun.
3.2. Silicon Mold Fabrication
- 12.
- Bath a 4-inch silicon wafer in acetone for 10 min.
- 13.
- Spray wash the silicon wafer with IPA to remove acetone residues.
- 14.
- Bake the wafer at 120 °C for 10 min until fully dry and cool it down to room temperature.
- 15.
- Vaporize HMDS onto the silicon wafer to create hydrophobic surfaces.
- 16.
- Spin-coat a layer of 50 µm-thick SU-8 3035 photoresist (2000 rpm for 30 s).
- 17.
- Soft bake the wafer on a hotplate at 60 °C for 1 min.
- 18.
- Ramp up the hotplate temperature to 95° C at 450 °C/h.
- 19.
- Incubate the wafer at 95 °C for 10 min.
- 20.
- Expose the wafer using the contact mask aligner (200 mJ/cm2).
- 21.
- Post exposure bake the silicon wafer at 95 °C for 5 min.
- 22.
- Develop the silicon wafer in SU-8 developer for 7 min.
- 23.
- Hard bake the wafer at 125 °C in the oven for 15 min.
3.3. Micropillar-Based Microfluidic Device Fabrication
- 24.
- Place the silicon mold with a small weighing bowl in the vacuum desiccator.
- 25.
- Dispense 20 µL of TPOS in the weighing bowl.
- 26.
- Turn on the vacuum for 1 min to create a low-pressure environment for TPOS evaporation.
- 27.
- Incubate the silicon mold for 30 min.
- 28.
- Place the silicon mold in a 150 cm petri dish.
- 29.
- Mix 30 g of PDMS prepolymers (base: curing agent = 10:1) in a weighing boat.
- 30.
- Place the PDMS mixture in a vacuum for 15–30 min to remove bubbles.
- 31.
- Dispense PDMS mixture on the silicon mold.
- 32.
- Incubate PDMS in the oven at 75 °C for 2–4 h.
- 33.
- Cut off the polymerized PDMS substrate with fluidic feature from the silicon mold using a scalpel or razor blade.
- 34.
- Punch holes at the inlet and outlet of the PDMS substrate using a punch with inner diameter of 1.2 mm.
- 35.
- Treat an unused silicon wafer with TPOS as described from Step 24–27.
- 36.
- Apply 10 g of degassed liquid PDMS mixture on the silicon wafer.
- 37.
- Spin the wafer at 1000 rpm for 1 min.
- 38.
- Incubate the PDMS coated silicon wafer at 100 °C for 5 min.
- 39.
- Treat the PDMS substrate with an oxygen plasma treater for 1 min.
- 40.
- Treat the PDMS film on the silicon wafer with the oxygen plasma treater for 1 min.
- 41.
- Bind the PDMS substrate with the PDMS film.
- 42.
- Incubate the sandwiched layers at 100 °C for 1 min in the oven.
- 43.
- Cool the bonded device down to room temperature.
- 44.
- Gently cut off the excessive PDMS thin film using the scalpel.
- 45.
- Peel the microfluidic device off the silicon wafer.
- 46.
- Physically attach the microfluidic device on a microscope slide for sample processing.
3.4. Antibody Immobilization in the Microfludic Device
- 47.
- Prepare 2 sections of silicone rubber tubes using a scalpel or razor blade. One with a length of 5 cm. The other with a length of 10 cm.
- 48.
- Prepare 2 sections of PTFE light wall tubes with a length of 8 mm using a scalpel or razor blade.
- 49.
- Couple the silicone rubber tubes with the PTFE light wall tubes by inserting the PTFE light wall tube to one end of the silicone rubber with a depth of 4 mm.
- 50.
- Connect the short-length silicone rubber tube to the inlet of the chip by inserting the coupled PTFE light wall tube.
- 51.
- Connect the long-length silicone rubber to the outlet of the chip by inserting the coupled PTFE light wall tube.
- 52.
- Connect the short-length silicone rubber tube to a 1 mL pipet tip used as a sample reservoir. Keep the pipet tube vertical to the chip using a 3D printed hanger or fixture.
- 53.
- Fix a 5 mL Luer-locked syringe on the syringe pump.
- 54.
- Cap the syringe with a blunt-tip mixer nozzle.
- 55.
- Connect the long-length silicone rubber tube to the blunt-tip mixer nozzle.
- 56.
- Introduce 300 µL of 99% ethanol to the sample reservoir.
- 57.
- Withdraw 250 µL the ethanol at a flow rate of 2 µL/s using the syringe pump.
- 58.
- Introduce 200 µL of DI water to the sample reservoir and withdraw 200 µL at a flow rate of 2 µL/s.
- 59.
- Introduce 200 µL of PBS to the sample reservoir and withdraw 200 µL at a flow rate of 2 µL/s.
- 60.
- Introduce 100 µL of avidin to the sample reservoir and withdraw 100 µL at a flow rate of 2 µL/s.
- 61.
- Incubate for 15 min at room temperature.
- 62.
- Introduce 300 µL of PBS to the sample reservoir and withdraw 300 µL at a flow rate of 2 µL/s.
- 63.
- Dilute anti-EpCAM 25 times (4 µL of antibodies in 100 µL of PBS at a concentration of 20 µg/mL)
- 64.
- Introduce 100 µL of anti-EpCAM to the sample reservoir and withdraw 100 µL at a flow rate of 2 µL/s.
- 65.
- Incubate for 20 min at room temperature.
- 66.
- Introduce 300 µL of 1% BSA to the sample reservoir and withdraw 300 µL at a flow rate of 2 µL/s.
3.5. Capture of Rare Cells from a Spiked Sample
- 67.
- Trypsinize PC9 cancer cells by adding 5 mL of 0.25% trypsin-EDTA followed by incubating for 5 min at 37 °C.
- 68.
- Neutralize the trypsin-EDTA by adding 10 mL of whole culture medium (RPMI 1640 supplemented with 10% FBS and 1% penicillin streptomycin).
- 69.
- Spin down the cells at 1200× g for 4 min.
- 70.
- Discard the supernatant and wash the cells with 10 mL of PBS at 1200× g for 4 min.
- 71.
- Discard the supernatant and resuspend the cells in 1 mL of PBS.
- 72.
- Determine the concentration of cells using a hemocytometer.
- 73.
- Aliquot 1 mL of PC9 cells in PBS at a concentration of 1 × 106 cells/mL.
- 74.
- Add 5 µL of Vybrant DID dye in the cell sample and mix cell by pipetting.
- 75.
- Incubate the mixture at 37 °C for 15 min.
- 76.
- Spin down the cells at 1200× g for 4 min.
- 77.
- Wash the cells 3 times with 1 mL of PBS.
- 78.
- Resuspend the cells in 1 mL of PBS.
- 79.
- Determine the concentration of cells using a hemocytometer.
- 80.
- Aliquot 1 mL of cell sample with a concentration of 103 cells/mL through serial dilution.
- 81.
- Add 50 µL of cells into 1 mL of whole blood sample supplemented with 1.8 mg/mL EDTA and mix well.
- 82.
- Load the spiked sample to the sample reservoir of the microfluidic device.
- 83.
- Withdraw 1.05 mL of the sample at a flow rate of 1 µL/s.
- 84.
- Wash the microfluidic device by withdrawing 300 µL of PBS at a flow rate of 2 µL/s.
- 85.
- Repeat Step 83 two times.
- 86.
- Clamp the silicone tubes of both inlet and outlet.
- 87.
- Dislodge the microfluidic device from the sample reservoir and the syringe.
- 88.
- Inspect the microfluidic device under a fluorescence microscope. Fluorescently labeled PC9 cell can be detected under the APC channel.
- 89.
- Mark the positions with captured PC9 cells using a marker.
- 90.
- Detach the microfluidic device from the microscope slide.
- 91.
- Punch though the microfluidic device of the captured PC9 position using a PDMS punch with an inner diameter of 5 mm.
- 92.
- Collect the dislodged piece of the PDMS with captured PC9 cells in a 1.7 mL centrifuge tube.
- 93.
- Fill the centrifuge tube with 1 mL of cell culture medium to keep the viability of the captured PC9 cells
4. Expected Results
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Chen, K.; Wang, Z. A Micropillar Array Based Microfluidic Device for Rare Cell Detection and Single-Cell Proteomics. Methods Protoc. 2023, 6, 80. https://doi.org/10.3390/mps6050080
Chen K, Wang Z. A Micropillar Array Based Microfluidic Device for Rare Cell Detection and Single-Cell Proteomics. Methods and Protocols. 2023; 6(5):80. https://doi.org/10.3390/mps6050080
Chicago/Turabian StyleChen, Kangfu, and Zongjie Wang. 2023. "A Micropillar Array Based Microfluidic Device for Rare Cell Detection and Single-Cell Proteomics" Methods and Protocols 6, no. 5: 80. https://doi.org/10.3390/mps6050080