Trichloro

3″ silicon test wafers (University Wafer, Catalog #447, Boston, MA) were rinsed extensively with acetone and isopropyl alcohol, then placed on a hotplate at 300 °C for a minimum of two hours to drive off water. Wafers were then coated with SU8 2025 photoresist (MicroChem Corp., Westborough, MA) and spun on a WS-400-6NPP spin coater (Laurell Technologies Corporation, North Wales, PA) using empirical protocols. After soft bake, photoresist was exposed on a Karl Suss MJB3 mask aligner (Suss MicroTec, Garching bei München, Deutschland) using transparency masks (printed by CAD/Art Services, Bandon, OR) mounted on a quartz slide (Chemglass Life Sciences, Vineland, NJ). Wafers were then post-exposure baked according to MicroChem protocol and developed using SU8 Developer (MicroChem). The thickness of resultant channel masks was confirmed at multiple locations using a NewView 7300 optical profilometer (Zygo Corp., Middlefield, CT), and re-verified after device assembly with fluorescence imaging. To facilitate later release of molded devices, wafers were treated with 1% v/v Trichloro(1 H,1 H,2 H,2H-perfluorooctyl)silane (Sigma-Aldrich Corp., St. Louis, MO) in hexanes for 5–30 minutes, dependent on ambient humidity, at room temperature and washed extensively with isopropyl alcohol before overnight hard bake at 80 °C in a hybridization oven.

Polycaprolactone (PCL) (M_w: 45,000), dichloromethane and trichloro(1H,1H,2H,2H-perfluorooctyl) silane were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyurethane acrylate (PUA) and polyethylene terephthalate (PET) were purchased from Minuta Tech (311 RM; Minuta Tech, Osan, Republic of Korea).

The PDMS microchannels were fabricated using a standard PDMS soft lithography method where PDMS was casted onto a master containing the negatives of the channels. The master was created using standard photolithography techniques where two layers of 100 µm-thick SU-2075 (MicroChem, Westborough, MA, USA) photoresist were initially spin-coated onto a clean silicon wafer. After the spin-coating first and second photoresist layer, the layers were baked for 40 and 90 min, respectively, at 95 °C. The patterns of the channels were exposed onto the photoresist using 400 mJ/cm² of ultraviolet light. After the exposure, the photoresist was further baked for 90 min at 95 °C followed by development in SU-8 developer (MicroChem) to remove unexposed photoresist. The silicon wafer was then silanized (20 µL of Trichloro (1H,1H,2H,2H-perfluorooctyl) silane (Sigma-Aldrich, Castle Hill, NSW, Australia)) before casting with Sylgard-184 PDMS (Dow Corning, Midland, MI, USA) with a curing agent-to-base weight ratio of 1:10. The cast was cured at 65 °C for 5 h, and inlets and outlets of the channels were cut using a 0.75 mm hole puncher. The channels were sealed using #1.5H coverslips (Menzel-Gläser, Braunscheig, Germany) bonded using air plasma at 300 mTorr for 45 s.

Goat anti-mouse IgG (H + L) labeled with Alexa Fluor 532 and chicken anti-goat IgG (H + L) labeled with Alexa Fluor 647 were purchased from Life Technologies. Cognate antibody pairs and proteins used are listed in SI Table 1. Streptavidin-conjugated Cy5 was obtained from Rockland. Phosphate buffered saline (PBS) tablets and glass slides were purchased from Fisher Scientific. Glycerol, 1,3-butanediol, betaine, trichloro(1H,1H,2H,2H-perfluorooctyl)silane, acetone, hydrochloric acid, Tween-20, argatroban monohydrate, phenylmethanesulfonyl fluoride (PMSF) and leupeptin hydrochloride were purchased from Sigma-Aldrich. Bovine serum albumin (BSA) was purchased from Jackson ImmunoResearch Laboratories, Inc. BSA-free StabilGuard Choice Microarray Stabilizer was purchased from SurModics, Inc. Human alpha-thrombin (HCT-0020), fluorogenic substrate (SN20) and the inhibitor D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) were purchased from Haematologic Technologies Inc. Nitrocellulose coated slides were purchased from Grace Bio-Laboratories, and aminosilane coated slides were purchased from Schott North America.

Positive masters and pillar array casting were described previously (Poole et al., 2014 (link)). Briefly, positive silicon masters were silanised using vapour phase Trichloro(1H,1H,2H,2H-perfluorooctyl) silane (Sigma-Alrich) for 16 hr. Negative masters were cast from this substrate in polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning), mixed at a ratio of 1:10 and cured at 110°C for 15 min. Negative masters were silanised as above and used to cast pillar arrays. Arrays were coated with degassed PDMS (1:10) and left for 30 min. A thickness two coverslip activated with oxygen plasma generated using a low pressure Zepto plasma system (Diener, Germany) was placed over the still liquid PDMS. Pillar arrays were cured for 1 hr at 110°C. Pillar arrays were activated using the oxygen plasma system and either cells were directly seeded onto this activated surface or arrays were first functionalised by coating with 10 μg/ml LM-511 (BioLamina, Sweden) for one hour at 37°C. Cells were seeded at a concentration of 2 × 10⁴ cells/mL in complete media and incubated overnight.

Silicon wafers were O2-plasma activated (300 W, 2 min) and vapor phase silanized in vacuum (Trichloro(1H,1H,2H,2H-perfluorooctyl)silane, 1 h, Sigma Aldrich). The silanized wafers were spin coated with a release agent (Ease Release 205, 3000 rpm, 20 s, Mann Release Technologies). PDMS (Sylgard 184, Dow Corning) was spin coated (500 rpm, 30 s) and semi-cured on a hotplate (65 °C, 10 min). Membranes with deposited AgNWs were baked (100 °C, 10 min) and put in contact with the PDMS layer under pressure and heat (100 °C, 5 min). The membranes were peeled of leaving the AgNWs on the PDMS surface. SEM images were taken with a Zeiss SUPRA 50 VP (acceleration voltage 5–10 kV).

The designs for the poly(dimethyl)siloxane (PDMS) chip devices were prepared using Autocad CAD software and designs are shown in Supplementary Fig. 4. The corresponding CAD files can be downloaded from http://openwetware.org/wiki/DropBase (a database of microfluidic device designs). The devices were fabricated with standard soft lithographic procedures59 (link). The photoresist material SU-8 2015 was used to obtain a 15 μm channel height. PDMS monomer and curing agent were mixed at a ratio 10:1 and then poured onto the lithographic plate before degassing. After PDMS solidification (65 °C, 4 h), PDMS was activated by exposure to an oxygen plasma and devices were sealed onto a microscope glass slide (or cover slip (thickness: 0.13 mm) for the sorting chip). Hydrophobic modification of the channels surface was achieved by injecting a solution of 1% (v/v) trichloro(1H,1H,2H,2H-perfluorooctyl)silane (Sigma) in HFE-7500 oil into the channels. Electrodes for the sorting devices were prepared using low melting point indium composite solder (51 In/32.5 Bi/16.5 Sn, Indium Corporation).