Identificación de las áreas del conocimiento en los procedimientos, formularios e

2.2.1 Chip fabrication

The microfluidic channel network layout is shown in Chapter 1, Figure 1.1. All microchips were fabricated from 50 mm x 50 mm, 0.9 mm thick B270 crown glass using traditional photolithography techniques and wet chemical etching methods.40-42 The etchant used was a 10:1 buffered oxide etch (BOE) (Transene Company, Danvers, MA). Channel access holes were drilled through the substrate at the channel ends using aluminum oxide powder blasting (Microblaster, Comco Inc., Burbank, CA). The etched substrate was then fusion bonded to a 0.9 mm thick B270 cover slip to create a closed channel network.40 Buffer and sample reservoirs were created by attaching cloning cylinders (Fisher Scientific, 4 mm i.d.) around the access holes using Norland 63 optical adhesive (Norland Products, Inc., Cranbury, NJ). Each reservoir holds a volume of approximately 100 L. To connect a syringe pump to the chip, a 1/16˝ barbed female Luer lock (Upchurch Scientific, Oak Harbor, WA) was altered by removing the barb and drilling an ~ 3 mm hole into the bottom using a

Dremel tool. The altered Luer lock was then epoxied onto the top of the cell waste reservoir and 1/16˝ i.d. Tygon tubing was attached to the female Luer lock via a 1/16˝ barbed male Luer lock (Upchurch Scientific, Oak Harbor, WA).

Several different microchips were used for this study. Channel depths and widths were determined using a stylus-based surface profiler (P-15; KLA-Tencor, Mountain View, CA). Widths are reported as the full channel width measured at the top of the channel. All channels were etched to a depth between 20 - 25 m. The cell flow (CF) channel and the cell waste (CW) channel were 65-80 m wide. The analysis (A) channel from the lysis

intersection to the 90º turn and the narrow section of the separation buffer (SB) channel were 45-60 m wide. The broad sections of all the channels were 255-270 m wide. The analysis channel from the lysis intersection to the 90º turn was 20.0 mm long, and the distance

between the tee intersection of the focusing (F) channel with the sample cell flow channel and the lysis intersection was approximately 105 m.

For some coating studies, 25.4 mm x 50.8 mm chips were fabricated that had only three straight channels. For each chip, the channels were 19.5 m deep and 110 m wide. A maskless system (SF-100, Intelligent Micro Patterning, LLC, St. Petersburg, FL) was used to expose the photoresist on the substrates. All other photolithography, etching and bonding steps were the same as described above.

2.2.2 Cell culture and preparation

Jurkat Cells (ATCC TIB-152, American Type Culture Collection, Rockville, MD; obtained from the University of North Carolina Tissue Culture Facility) were used for all experiments. The cell cultures were maintained at 37ºC and 5% CO2 in RPMI 1640 1X medium (Gibco BRL, Gaithersburg, MD) supplemented with 10% (v/v) FBS, 100 g/mL

penicillin and 100 g/mL streptomycin. The cells were grown in 25-mL polystyrene culture flasks (Nalge Nunc International, Rochester, NY) to densities of approximately 1x106/mL before passage.

To load the cells with dye, ~5 x 105 cells were pelleted (1000g for 3 minutes) and the supernatant discarded. The cells were then re-suspended in a solution of 10 to 50 M

Oregon Green 488 carboxylic acid diacetate 6-isomer in extracellular buffer (ECB: 135 mM NaCl, 15 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, pH 7.4). Oregon Green in diacetate form is membrane permeable and cell loading was achieved through simple incubation (~20 minutes) in a centrifuge vial while maintaining the cells at 37ºC. The cells were then washed three to four times by pelleting with ECB and re-suspended in ECB containing 10 mM Glucose.

2.2.3 Channel coating procedures

2.2.3.1 PDMS/Pluronic coating

To coat the glass channels with PDMS, the channels were rinsed for approximately five minutes with 1 N NaOH followed by deionized (DI) water for five minutes. The channels were then thoroughly dried by pulling vacuum on one reservoir of the chip. A mixture of 1:10 PDMS (w/w, 1 part curing agent to 10 parts monomer, Sylgard, Dow

Corning, Midland, MI) was prepared. A 20% mixture (v/v) of the PDMS in hexane was then prepared and pulled through the channels for approximately five minutes. Next, 100% hexane was pulled through the channels for 30 to 45 minutes to remove excess PDMS. The channels were dried by pulling air through the channels and the PDMS was cured in a vacuum oven at 100-110C for 8 to 10 hours.

Pluronic F-127, a triblock copolymer of polypropylene oxide and polyethylene oxide, was coated onto the surface of the PDMS-coated channels. The PDMS-coated channels were prepared by first rinsing with methanol followed by DI water to ensure that the channels were free of air bubbles. The chip was cooled on an ice block and a solution of 30% (w/w) Pluronic F-127 in water was pulled through the channels for 20 minutes by applying vacuum to the waste reservoir. After 20 minutes, the reservoirs and channels were rinsed thoroughly with DI water. After the DI water rinse, the chip was ready for use.

2.2.3.2 Pluronic-only coating

To coat the channels with Pluronic F-127, the channels were prepared by rinsing with 1 N NaOH for approximately 1 hour followed by a DI water rinse. With the chip on an ice block to keep it cool, a solution of 30% Pluronic F-127 (w/w) was pulled through the channels for approximately 1 hour.

2.2.3.3 Poly(ethylene glycol) (PEG) coatings

2.2.3.3.1 Aminopropyltriethoxysilane (APTES) coating

Chip channels were rinsed with 1:1 (v/v) MeOH:HCl for approximately five minutes followed by a DI water rinse. The channels were then thoroughly dried under vacuum. To ensure complete dryness, the chip was placed in a 90C oven for 20 minutes. Next, 3% (v/v) APTES in ethanol (EtOH) was pulled through the channels and allowed to react for 15 minutes. The channels were then flushed with EtOH to wash out the excess APTES and the chip was placed in a 120C oven overnight.

2.2.3.3.2 PEG succinimidylpropionate (PEG-SPA)

Chip channels were first coated with APTES according to the above procedure. After APTES coating, a 1 mg/mL PEG-SPA solution in 20 mM sodium phosphate buffer (pH 8)

was pulled through the channels and reacted for 24 hours at 4C. The channels were then flushed with DI water. The procedure was taken directly from Chuang et al.43

2.2.3.3.3 N-(triethoxysilylpropyl)-O-poly(ethylene) oxide urethane (PEG-Urethane)

Channels were rinsed with 1 N NaOH for 15 minutes followed by a 10 minute DI water rinse. Channels were flushed with anhydrous toluene for 10 minutes and dried by applying vacuum to the waste reservoir. A 6 mg/mL solution of N-(triethoxysilylpropyl)-O- poly(ethylene) oxide urethane (MW 400-500) (PEG-Urethane) in anhydrous toluene was pulled through the channels and allowed to react for 4 to 12 hours. After reaction, the channels were flushed with anhydrous toluene and the microchip placed in an 80C oven for at least two hours.

2.2.3.3.4 2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane (PEG-Silane)

Channels were rinsed with 1 N NaOH for approximately one hour followed by a DI water flush. The channels were dried by pulling vacuum on the waste channel. Channels were rinsed with concentrated HCl for 30 minutes and then rinsed again with DI water. A 100% solution of the PEG-silane was pulled through the channels and allowed to react for 30 minutes. After the reaction, the channels were rinsed with DI water and dried in a 95C oven for 30 minutes.

2.2.3.3.5 Carboxytetramethylrhodamine, succinimidyl ester(TAMRA) Coating

Chip channels were first coated with APTES according to the above procedure. The APTES-coated channels were filled with a 1 mg/mL solution of 5(6)-TAMRA in 20 mM sodium phosphate buffer (pH 8). The TAMRA solution was allowed to react for 24 hours at 4C. Channels were then flushed with DI water and filled with the appropriate buffers for analysis.

2.2.3.4 Fetal Bovine Serum (FBS) channel pretreatment

The channels were pre-rinsed with 2X Nanostrip (Cyantek, Fremont, CA) followed by DI water. Channels were then filled with 100% FBS (Invitrogen, Carlsbad, CA) and allowed to incubate for 15 to 30 minutes at room temperature. The reservoirs were refilled with fresh FBS and the fresh FBS was flushed through the channels.