PDMS microfluidic capillary systems for patterning proteins on surfaces and performing miniaturized immunoassays Mateu Pla-Roca and

David Juncker Abstract In this chapter, we describe the fabrication and use of microfluidic capillary systems (CSs) made in soft, transparent polydimethylsiloxane (PDMS). 16 microfluidic CSs, each containing a loading pad, a microchannel and a capillary pump are engraved in a single chip. The CSs are used for two applications, first to pattern fibronectin on glass surfaces to locally control the adhesion of cultured cells to the substrate and second to carry out multiplexed miniaturized immunoassays. Keywords: microfluidics, miniaturizated immunoassays, micromosaic immunoassays, protein patterning.

Introduction

Microfluidic systems can transport minute amounts on solutions and replace macroscopic tubes, pipettes, vessels and dishes in a variety of applications. They help save reagents by reducing sample size from milliliters to microliters, decrease the time-to-result by enhancing mass transport, and allow many reactions to be carried out simultaneously on a small chip. These characteristics have driven the adoption of microfluidic systems in a variety of fields and notably in chemistry, biology and medicine.

1.1 Microfluidic capillary systems

A microfluidic capillary system (CS) is made of three elements: a loading pad, a microchannel and a capillary pump, each with a precisely designed geometry. The three elements are structured into a flat substrate, and the recessed (inner) surface is made hydrophilic. A liquid delivered to the loading pad of a CS can therefore spontaneously fill the microchannel and capillary pump by capillary forces alone. The direction and the flow rate are defined by the capillary forces and the flow resistance, both of which depend on the geometry of the three elements of the CS, and which can therefore be controlled (and pre-programmed) by modifying the geometry. Capillary systems and microfluidic networks have both been used to describe arrays of microchannels that are filled by capillary force and used for immunoassays and surface patterning. In this chapter, we use the word CS which reflects the fact that the microchannels have been designed to exploit capillary effects so as to fulfill a particular function akin to other systems.

1.2 Patterning proteins and performing immunoassays

Owing to the reversible sealing properties of the PDMS, capillary systems made of PDMS (PDMS-CSs) are used in the present protocol. A section of the open microchannels of the PDMS-CSs can be covered reversibly by placing a flat substrate onto the engraved PDMS surface, and form a temporally closed microchannel for patterning proteins or carrying out immunoassays on the surface of a substrate (Fig. 1). Solutions are loaded into the loading pads with a conventional pipette and automatically flush through the microchannels by capillary forces (Fig. 1.a-b). The reagents in solution adsorb or react with the surface while faithfully replicating the pattern defined by the microchannels. If the solution is a physiological buffer containing proteins (for example capture antibodies or cell adhesion proteins), the proteins will adsorb to the substrate and form a pattern of parallel lines. After
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rinsing the channels with buffer and double-distilled water, the substrate is peeled off from the PDMS-CSs (Fig. 1.c). The non-patterned areas are back-filled by incubating the substrate with a drop of solution containing a blocking agent, such as the bovine serum albumin protein, which adsorbs to the surface and prevents further binding of other proteins (Fig. 1.d). The patterned surface can be used for cell cultures (Fig. 1.e) if cell adhesion proteins were patterned (1,2) or for performing immunoassays if antibodies were patterned. A drop immunoassay (Fig. 1.A) is performed by incubating the patterned substrate surface with a drop of a sample solution (Fig. 1.f). Each line of immobilized capture antibody specifically binds its target analytes in the sample in a concentration dependent manner. Fluorescent detection antibodies are then delivered over the substrate and bind only to the line where the target analytes have been captured and immobilized (Fig. 1.g). The result of the assay is visualized with a fluorescence microarray scanner or a fluorescence microscope (Fig. 1.h). In one experiment, it is thus possible to detect multiple analytes contained in a small sample (3). Alternatively, it is possible to perform all the steps of an immunoassay without removing the substrate from the engraved PDMS (Fig. 1.B) by sequentially delivering and flowing solutions of blocking agent, samples and labeled detection antibodies through the CSs (Fig. 1.i-m). We call this approach protocol a microchannel immunoassay. Each

microchannel corresponds to a single analyte only, and repeated delivery of the sample to multiple channels is necessary to analyze multiple analytes. This protocol further reduces sample consumption and incubation time because a few hundred nanoliters can suffice for tens of minutes of continuous flow and sample replenishment within the microchannels, and thus enable high sensitivity assays (4,5). It is also possible to measure multiple analytes in multiple samples simultaneously by first patterning of (m) capture antibodies, then removing the substrate, rotating it 90 degrees,

and sealing it against a second microengraved PDMS-CSs (Fig. 1.C). The second PDMSCSs is used to flow blocking agent and (n) different samples and detection antibodies across the capture lines (Fig. 1.o-q). In this scenario, each intersection represents a unique combination and m n different binding reactions can be performed at once on a single chip. After peeling off the substrate from the CSs (Fig. 1.r), the binding is analyzed by fluorescence imaging and each binding event forms a bright square at the intersection (Fig. 1.s); because the overall pattern appears as a mosaic of squares (Fig. 2), these assays have been dubbed micromosaic immunoassays. Such assays have been used to quantify cross-reactivity between antibodies (6), measure DNA hybridization rates (7), detect protein biomarkers (8) (Fig. 2.D), viruses and bacteria analytes (9) and perform competitive immunoassays (10).

Fig. 1. Using PDMS-CSs for patterning surfaces with proteins and for performing miniaturized immunoassays. (a) A substrate is sealed over the open microchannels of the CSs. (b) Antibodies or proteins are patterned on the substrate by flowing solutions through the microchannels. Three different types of miniaturized immunoassays (drop, microchannel and micromosaic) can be performed following the three different protocols (A, B, C). (A) After peeling off the substrate from the PDMS-CSs, the substrate patterned with lines of capture antibodies is incubated with a drop of sample solution and will bind target analytes in the sample. (B) The substrate is kept in place and each channel is sequentially flushed with minute amounts of different sample solutions thus allowing the detection of one analyte per channel. (C) A micromosaic immunoassay is performed by removing the substrate, rotating it by 90 degrees and flowing different samples in each column across all lines of capture antibodies. When a target analyte corresponding to an immobilized antibody is present in the sample, it will be captured at the intersection of the corresponding line and column. Such a micromosaic immunoassay allows detecting multiple analytes in multiple samples simultaneously.

In the following protocol we provide some basic design guidelines on how to make and use PDMS-CSs and a step by step description on how to pattern proteins and to carry out the miniaturized immunoassays presented in Fig. 1.

Fig. 2. Micromosaic immunoassays performed on a PDMS substrate. (A) Fluorescence image of a capture antibody (against goat immunoglobulin) labeled with a green fluorescent dye that was patterned and immobilized as lines (green fluorescence). (B) Fluorescence image showing the bound analyte (goat immunoglobulin labeled with a red fluorescent dye), bound at the intersections and forming squares (red fluorescence). (C) View of the 1616 intersections of the micromosaic assay. The lanes 8 and 9 were flushed with buffer only and serve as a negative control. The full assay protocol is described in this chapter. (D) Simultaneous detection of the cardiac biomarkers myoglobin (Mb), cardiac Troponin I (cTnI), S100 and C-reactive protein (CRP) and B-type natriuretic peptide (BMP) in human plasma. Four capture antibodies (Mb, cTnI, S100, CRP) were patterned as horizontal lines on a PDMS substrate and used to capture biomarkers spiked into plasma samples (from healthy subjects) delivered in the vertical columns. The captured biomarkers were detected by filling the vertical columns with a cocktail of fluorescently labeled detection antibodies (Reproduced from ref. 8 with permission of Elsevier Science).

1.3 Designing PDMS-CSs

CSs can be made in silicon (Si), glass or PDMS. The advantage of PDMS-CSs is that owing to PDMS spontaneous and reversible sealing properties they can be sealed to any type of smooth surface. This property allows using glass and gold substrates, coated with silanes and thiols respectively, or polymer substrates made of polystyrene, polycarbonate or poly(methylmethacrylate). The glass surfaces used in this protocol are coated with a self-assembled monolayer of a silane with epoxy groups prior to use (11). The epoxy groups on the surface ensure a good attachment of the proteins via a covalent reaction between the epoxy group and their amino groups. PDMS-CSs are made by replica molding (12) of a master which is typically made of a Si wafer structured with an inverse pattern of the microchannels. The PDMS-CSs presented here comprise an array of 16 independent CSs, each containing a loading pad, an open microchannel, and a capillary pump (Fig. 3.A, B, C). Once the loading pad is filled with a solution, the negative capillary pressure at the liquid-air interface at the filling front produces a continuous flow without requiring neither peripheral pumps, nor controllers, nor connections (Fig. 3.D). The key to spontaneous filing is to activate the inside surface of the PDMS microchannels and render it hydrophilic which can be done by exposing the chip to an oxygen plasma or to ozone. The flow rate (Q) of a CS can be calculated and is proportional to the capillary pressure (Pc) formed at the liquid-air interface of the filling front, divided by overall flow rate resistance (R) of the CS, Q ~ Pc/R. Both Pc and R are defined by the cross-section of the conduit as follows: For conduits with a square cross-section, the Pc scales with the width w of the conduit as w-1 and the resistance R scales as w4 (5). Thus, by reducing the crosssection w by a factor of 2 for example, Pc doubles, R increases by a factor 16 and Q decreases by a factor 8. One important parameter that needs to be considered when
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designing a CS is that as the channel is being filled over an increasing length l, the flow resistance R also continuously increases proportionally to the filled length, R ~ l for a channel with a constant cross-section. In addition, the resistance of all upstream sections needs to be added as well. The characteristic dimension of the microstructures in the capillary pumps is made smaller than the one in the loading pad to create a stronger capillary pressure and generate unidirectional flow towards the pump. The arborescent architecture of the capillary pumps provides a high capillary pressure and a large volume while not significantly adding to the overall flow resistance when being filled because all channels run parallel to one another. The capillary pump also serves as a waste, or as a sample concentrator. The total volume of each capillary pump is only a few hundred nanoliters, but larger volumes can be loaded in each CS and flowed through each assay area if a tissue (or any kind of wicking material) or evaporation is used to drain liquid in one or several capillary pumps (Fig. 3.E). A capillary retention valve (CRV) can be used as a control element for entirely self-regulated and autonomous CSs. CRVs prevent undesired drainage of the assay area and trapping of bubbles, and are particularly useful when multiple solutions are added sequentially to many channels, but are beyond the scope of this chapter. Interested readers can find additional information on the design and use of CRVs in refs. 4 and 5.

Capillary pumps

500 m

C
Assay area

16 CSs
microchannel

2 mm
Loading pads

500 m

D
Micropipette (solution 1) Reaction area Substrate Loading pad Filling front Capillary pump

E
Micropipette (solution 2) Substrate Loading pad Flow promoter (tissue) Capillary pump

Fig. 3. (A) PDMS-CSs chip with loading pads, microchannels and capillary pumps. Each channel has been loaded with a different colored solution. (B, C) Detailed view of a loading pad and capillary pumps. (D) Schematic showing how reagent solutions are delivered to the loading pads by means of a micropipette. Capillary pumps continuously flush sample through the reaction area, where the adsorption or binding events are occurring. (E) Multiple solutions and large volumes can be flushed and drained sequentially using a flow promoter (i.e. a clean room paper or a tissue) in contact with the capillary pumps. 10

To design a CS the following parameters need be taken in account: 1) CS chip: The larger the number of CSs, the more difficult it becomes to load them rapidly without error. The chip shown (Fig. 3.A) include 16 CSs each 30 mm long. 2) Loading pads: The size of the loading pads and the spacing between has to be sufficiently large in order to allow manual delivery of solutions with a micropipette. In the presented design the loading pad area is approximately 0.7 mm2. 3) Microchannels: The length (l), width (w), depth (d), and spacing (s) are critical parameters to be considered. The assay area is 8 mm long, and each microchannel is 150 m wide, 30 m deep and the channels are arrayed with a pitch of 300 m. CSs have a high flow rate. For high sensitivity immunoassays we recommend 10-30 m deep channels and aspect ratios between 1:2 and 2:1(5). The length of the microchannels on the assay area of the CS array should be sufficiently long to allow an easy rotation of the substrate when a micromosaic immunoassay is performed. 4) Capillary pumps: A trade-off has to be made between a small pump with a limited capacity and a large pump with a big footprint which will limit the number of CSs that can be arrayed on a chip.

Materials

2.1 General material and reagents

1. Polystyrene Petri dishes (150 mm 15 mm and 60 mm 15 mm, untreated,

Corning, MA). 2. Vacuum desiccator and a vacuum line (1 mm Hg).

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3.

Plasma chamber (Plasma Line 415, Tegal Corporation, CA) connected to air or

oxygen. Alternatively an ozone production system can be used. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. Leveled oven with thermometer. Nitrogen blowgun or duster (ChemTronics, Ultra Jet, GA). Tweezers (SPI, 2A). Round flat tip. Microscope slides (Fisher, 25 cm 75 cm, pre-cleaned). Powder-free gloves (Fisher). Tape (Scotch Ruban Magic tape). Clean room paper (Perotex 100, Perotech Sciences inc., Montreal, Canada). Absorbent paper (Kimwipe, SPI supplies, PA). Nitrogen blowgun or duster (ChemTronics, Ultra Jet, GA). Micropipette (0.5-2 L and 100-1000 L). Scalpel or cutter. Double-distilled water. 96% ethanol and 75% ethanol.

2.2 Fabrication of the mold

The microfluidic PDMS-CSs are made by PDMS replica molding (12) of either SU-8 2015 (MicroChem, Newton, MA) photoresist patterned on a silicon wafer (Fig. 4) or a structured SOI wafer. Mold fabrication is a standard procedure that can be performed in most microfabrication facilities. Molds can be obtained from large universities through their respective microfabrication services (13).

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Capillary pumps

microchannels

Loading pads

1 cm Fig. 4. (A) 4 silicon wafer patterned with CSs made of SU-8 that serve as a mold for PDMS replica molding. (B) Detail of an array of CS with the capillary pumps, loading pads and microchannels.

1.

Photomask with the design (Fineline Imaging, Colorado Springs, CO)

(see Note 1). Patterns have to be clear on the photomask because SU-8 is a negative resist. 2. 3. SU-8 2015 (Microchem, Newton, MA). HMDS, Hexamethyldisilazane (reagent grade 99%, Sigma-Aldrich). SU-8 adhesion

promoter. 4. 5. 6. 7. 8. 9. 10. Spin coater. Photolithographic aligner or collimated UV light source. SU-8 developer (Microchem, Newton, MA). Silicon substrates (4) (Monto Silicon technologies), TPFS, Trichloro-1H,1H,2H,2H-perfluorooctylsilane (97%, Sigma-Aldrich). Digitally controlled hot-plate (VWR, 810-HPS). Glass Petri dish (5).

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2.3
1. MI).

Molding of PDMS-CSs and flat PDMS substrate

Polydimethylsiloxane (PDMS) Sylgard 184 prepolymer (Dow Corning, Midland,

2.4 Coating of cover slips with an epoxy-silane

1. 2. 3. Round or rectangular cover slips (Fisher brand). Toluene (97%, Fisher Scientific, ACS reagent). Epoxy-silane, 3-Glycidoxypropyldimethoxymethylsilane (3-GPS, 97%, Sigma-

Aldrich). 4. Cover slip staining racks (2 units) and staining dishes (2 units) (Fisher Scientific).

2.5 Reagents, proteins and antibody solutions

1. Phosphate buffered saline (PBS) (Fisher Bioreagents, 1X, pH 7.4) and double-

distilled water filtered with syringe filters (FisherBrand, 0.22 m, 13 mm syringe PVDF sterile). 2. Carbonate-Bicarbonate buffer, pH 9.6, prepared following manufacturer

instructions (Carbonate-bicarbonate buffer capsule, Sigma-Aldrich). Filter the buffer with a syringe filter after preparation. 3. Capture antibody solution: Chicken anti-goat immunoglobulin labeled with Alexa g/mL with

Fluor 488 dye (AF488, green fluorescence) from Invitrogen diluted at 100

PBS. Store at 2-8C and protect from light. Bring to room temperature before using it. 4. Blocking solution: 1% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich) in

filtered phosphate buffered saline (PBS) (Fisher Bioreagents, 1X, pH 7.4).

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5.

Assay buffer: 0.1% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich) and

0.1% (w/v) Tween-20 (Sigma-Aldrich) in PBS. 6. Sample solution: Goat immunoglobulin labeled with Alexa Fluor 647 dye

(AF647, red fluorescence) from Invitrogen is diluted at 0.1 g/mL with assay buffer. Store at 2-8C and protect from light. Bring to room temperature before using it. 7. Fibronectin solution: sterile cell culture tested fibronectin (Sigma-Aldrich) is

diluted at 100 g/mL with carbonate-bicarbonate buffer. No BSA should be added. Store at 2-8C and bring to room temperature before using it.

2.6 Patterning of fibronectin on epoxy-coated cover slips for cell culture applications
1. 2. 3. PDMS microfluidic capillary systems (PDMS-CSs). Epoxy-silane coated cover slips. PBS, fibronectin solution and blocking solution. The solutions should be used at

room temperature. 4. Dulbecco's Modified Eagle Media (DMEM) and fetal bovine serum (sterile-filtered

and cell culture tested) both from Sigma-Aldrich. 5. 6. 7. 4% (w/v) paraformaldehyde (95% powder, Sigma-Aldrich) solution in PBS. Toluene (97%, Fisher scientific, ACS reagent). Myoblast cells suspension at 1 106 cells/mL (C2C12 cell line, American Type

Culture Collection, VA). 8. Microscope with DIC (Differential interference contrast) or phase contrast.

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2.7 1. 2. 3. 4.

Miniaturized immunoassay PDMS-CSs. Microscope slide (Fisher, 22 cm 75 cm, pre-cleaned). Square PDMS substrate cut to size (~1 cm2). Blocking, capture antibody and sample solutions. The solutions should be used at

room temperature. 5. Fluorescence scanner with 488 nm and 633 nm laser excitations and FITC and Cy5

dyes emission filters or fluorescence microscope with the corresponding filters.

Methods

3.1 Fabrication of PDMS-CSs and substrates 3.1.1 Fabrication of PDMS-CSs

1. Activate the surface of the CSs mold in a plasma chamber for 1 min (see Note 2).

Place the activated mold immediately in a desiccator together with a few drops of TPFS (Trichloro-1H,1H,2H,2H-perfluorooctylsilane) on a cover slip. Connect to vacuum for 1-5 min, close the desiccator valve and keep the mold for 1 h in the silane atmosphere. The TPFS coating will prevent PDMS from sticking to the mold during replica molding. 2. Transfer the coated mold into a glass Petri dish and place it in an oven at 110C for

10 min. This will ensure the complete reaction of the silane with the surface. 3. Place the CSs mold on the bottom of a polystyrene Petri dish (150 mm 15 mm)

and use tape to fix it to the Petri dish and cover the edges (Fig. 5). Pour PDMS prepolymer (Sylgard 184) until it forms a 3 mm thick layer.

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Fig. 5. Tape the edges of the 4 silicon CSs mold on a plastic Petri dish before pouring PDMS prepolymer.

4.

Degas by placing the dish in a vacuum chamber for 5-30 min depending on the

vacuum level. Check with a microscope to ensure that the structures are bubble-free. If not, place under vacuum for an additional 5 -10 min. 5. 6. Fully cure the PDMS in the oven at 60C for 12 h (see Note 3). Use a scalpel to cut the cured PDMS along the edge of the silicon wafer. Use flat

tweezers to peel off the replica from the mold. If handled carefully, the mold can be reused many times. The TPFS needs to be applied every few months only. Inspect the mold under a microscope after each replication. If PDMS residues are observed, especially in the channels area, discard the mold. 7. Cut the PDMS-CSs from the PDMS replica and place tape on top of the engraved

CSs to prevent dust contamination of the patterned surface. Keep the rest of the replicas face down in a Petri dish or in a dust free environment for future use.

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3.1.2 Fabrication of flat PDMS substrate

1. A flat PDMS substrate is made by curing PDMS on a polystyrene Petri dish (150

mm 15 mm). Pour the PDMS mixture to reach a level of at least 1 mm. Degas for 5 min. Fully cure the PDMS in the oven at 60C for 3-12 h and cut out a PDMS block with the desired size. The surface of PDMS cured in contact with the Petri dish is used as assay surface.

3.1.3 Coating of cover slips with epoxy-silanes

1. 2. Fill a staining dish with 99 mL of toluene and cover it with its lid. Place cover slips on the staining rack and plasma treat for 1 min in order to activate

the glass surface. 3. Add 1 mL of 3-GPS (3-Glycidoxypropyldimethoxymethylsilane) to the toluene and

mix it well. Immediately immerse the activated cover slips for 20 min. 4. Rinse the cover slips with fresh toluene and blow dry with nitrogen (30 s). Place the

cover slips on a new staining rack and cure in the oven for 30 minutes at 110C.
5.

Rinse with ethanol, blow dry and keep in a dust-free environment for future use.

3.2 Patterning fibronectin on cover slips for cell culture applications

Fibronectin is a well-characterized multifunctional extracellular matrix protein that mediates cell adhesion, migration and plays an important role in many cellsurface interactions and wound healing. Fibronectin-coated surfaces promote cell attachment. To maintain sterility, the following steps should be performed in a cell culture environment or in a biosafety cabinet. Tweezers should be sterilized under a flame or by immersion in a 75% ethanol solution before using them.

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1. 2.

Use a scalpel to cut out several 2 cm 5 cm pieces of clean room paper. Use gloves in order to manipulate the PDMS-CSs. Rinse the gloves with ethanol

before using them. Peel off the protecting tape from the PDMS-CSs. Rinse the microstructures with ethanol and dry it with nitrogen (1 min). 3. Place the cleaned PDMS-CSs on a glass slide, microstructures facing up. This will

allow better handling of the PDMS-CSs during the following steps. Place the PDMS-CSs in the plasma chamber for 1 min in order to render the PDMS-CSs hydrophilic (see Note 4). Stamp the activated PDMS-CSs several times against a flat PDMS surface. This treatment will neutralize the reactive groups on the surface of the CS but will keep the inside of microchannels hydrophilic. This step is important to prevent solution delivered to the loading pads from spreading over the surface, and generally helps minimize leakage and ensure proper function of the CS (e.g. spontaneous filling). 4. Place an epoxy coated cover slip over the hydrophilized PDMS-CSs. Make sure not

to block the loading pads with the cover slip. With a glass scribe mark a small R (for Rear) near the edge of the cover slip to mark the back side. Slightly press with the tweezers in order to ensure conformal contact with the PDMS-CSs. Sterilize the setup for a few minutes under UV light and place it in a polystyrene Petri dish (150 mm 15 mm) along with a wet tissue or paper). This will prevent evaporation during incubation. 5. Load 2 L of fibronectin solution in the channels, cover the Petri dish with its lid,

and incubate for 30 min (see Note 5). Shorter incubation times will result in lower levels of protein attached on the surface. 6. Place the short side of the clean room paper piece (2 cm 5 cm) over the capillary

pumps until all channels are drained. Slightly humidify the paper edge prior to use in order to ensure a better contact with the capillary pumps.

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7.

Rinse with PBS (3 2 L) and peel off the cover slip. Replace the clean room

paper whenever it is saturated. 8. Peel off the PDMS-CSs and place the patterned cover slip with the fibronectin lines

facing up in a small Petri dish. Use the previously inscribed small R as an orientation mark. Incubate for 2 h at 4C in order to ensure fibronectin adhesion to the surface. Rinse the cover slip whit PBS (2 2 mL) and add 2 mL of blocking solution. Ensure that the cover slip is perfectly immersed. Incubate for 1 h at room temperature. Remove the blocking solution and rinse several times with filtered PBS (see Note 6). 9. Transfer the cover slip in a new small Petri dish and add 2 mL of a myoblast cells

suspension at a density of 1 106 cells/mL in DMEM media with 20% (v/v) fetal bovine serum media. After 24 h incubation (37C, and 5% CO2 atmosphere) the attachment of the cells to the areas where the fibronectin was patterned can be visualized using a microscope (Fig. 6). For imaging purposes the cells can be fixed by adding 2 mL of 4% (w/v) paraformaldehyde PBS solution to the Petri dish for 10 min at 37C. Rinse the cover slips with PBS (3 2 mL) and (1 2 mL) double-distilled water.

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150 m

100 m

Fig. 6. Differential interference contrast (DIC) micrographs showing the preferential attachment of myoblast cells on fibronectin patterns after 24 h incubation (37C and 5% CO2 atmosphere).

3.3 Miniaturized immunoassays

1. 2. Use a scalpel to cut several (2 cm 5 cm) pieces of clean room paper. Use a scalpel to cut a 1 cm2 piece of PDMS substrate. Before peeling it off from the

Petri dish, make a notch in one of the corners. This notch will help proper alignment of the substrate in later procedures (see Note 7). Using the tweezers, peel off the substrate, pulling from the edges in order to avoid tearing of the PDMS. Place the PDMS substrate in a Petri dish in order to protect it from dust. 3. Peel off the tape that protects the PDMS-CSs from dust, rinse the microstructures

with ethanol and dry it under a stream of nitrogen for several seconds. Use powder-free gloves to manipulate the PDMS-CSs. 4. Place the cleaned PDMS-CSs chip in a microscope glass slide, microstructures

facing up. Place the PDMS-CSs in a plasma chamber for 1 min in order to hydrophilize the surface (see Note 4). Bring the hydrophilized PDMS-CSs several times into contact with a

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flat PDMS surface. This treatment will minimize leakages and facilitate the loading of solutions on the microchannels. 5. Place the PDMS substrate on top of the PDMS-CSs channels. Press slightly in order

to ensure conformal contact. Make sure not to block the loading pads with the substrate. (Fig. 7. A). Place the microscope slide with the immunoassay setup surrounded by a wet tissue or paper in a polystyrene Petri dish (150 mm 15 mm). This will prevent evaporation during incubation procedures. 6. Load 2 L of PBS buffer in microchannels 8 and 9 as negative controls. Load all L of capture antibody solutions (chicken anti-goat

other microchannels with 2

immunoglobulin labeled with AF488 has been used in the present protocol for demonstration purposes) (Fig 7. A). Cover the Petri dish with its lid in order to prevent evaporation and incubate antibodies for 1 min. 7. Place the short side of the clean room paper (2 cm 5 cm) over the capillary pumps

until all channels are drained (Fig 7. B). Do not let dry for an extended time (see Note 8).

Any of the three miniaturized immunoassay variants can be performed at this stage (Fig. 1. A, B, C, see Subheading 1). The protocol for the drop immunoassay (Fig. 1, protocol A) follows the same steps as for the microchannel (Fig. 1, protocol B) and the micromosaic immunoassay (Fig. 1, protocol C) which are described below, but, with the exception that whenever filling of the microchannels is described, a drop of solution is to be applied to the patterned PDMS surface instead. This drop further needs to be spread over the entire surface of the substrate using a glass cover slip, and incubated for ten times longer than the time indicated for steps carried out in a microfluidic channel.

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8.

Remove

the

wet

clean

room

paper

and

load

the

channels

with

2 L of BSA blocking solution. Incubate for 1 min. Unload the channels with a new piece of clean room paper and keep it in place. 9. Rinse the channels (3 2 L) with PBS and (1 2 L) with double-distilled water.

Replace the clean room paper if saturated. 10. Remove the clean room paper. Load the channels with 1% BSA and incubate for 1

min. Drain the channels with a tissue or clean room paper. 11. (C only) Peel off the PDMS substrate and blow it dry with the nitrogen blowgun

(10 s). Use the tweezers for this process. Rotate the PDMS substrate 90 degrees and place it over a new PDMS-CSs previously activated in the plasma chamber. Use the notch for orientation (Fig. 7).

Fig. 7. (A) Loaded CSs with capture antibody solution. A PDMS substrate with a notch has been placed on the assay area. (B) Draining capture antibody solution by means of the clean room paper placed over the capillary pumps. (C) Microchannels loaded with analyte solutions. The notch on the PDMS substrate is used as orientation in the course of the 90 substrate rotation. For visualization purposes color dye has been added to the loaded solutions. Scale bar 2 mm. 12. Load 2 L of sample solution into each CS and incubate for 3 min (see Note 9).

Drain the channels with clean room paper. The incubation time will be dependent on the

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binding capacity of the capture antibodies used and the analyte concentration to be detected. The longer the incubation, the higher the sensitivity. In the present protocol a solution containing goat immunoglobulin AF 647 was used as the sample, and the corresponding result is shown in Fig. 2.A. For a full micromosaic immunoassay as the one shown in Fig. 2.B, steps 13 and 14 needs to be carried out to deliver the labeled detection antibodies that will bind to the immobilized analyte.

13.

Rinse the microchannels (3 2 L) with PBS and (1 2 L) with double-distilled

water. Replace the clean room paper whenever it is saturated. 14. Load the microchannels with the corresponding fluorescently labeled detection

antibodies in assay buffer and incubate them for 20 min. The recommended concentration is 1 g/mL (see Note 10). 15. Rinse the microchannels (3 2 L) with PBS and (1 2 L) double-distilled water.

Use a clean room paper in order to drain each solution. 16. Peel off the PDMS substrate and dry it under a stream of nitrogen (30 s). The

immunoassay result is revealed by imaging with a fluorescence microscope or scanner (see Note 11). The result of the demonstration mosaic immunoassay is shown in Fig. 2.A (see Subheading 1).

Notes 1. The design of the photomask can be made using Inkscape (open source software),

CorelDraw (Corel Corporation) or Adobe Illustrator (Adobe systems), or more specialized software such as Clewin 4.0 (Vieweb, Netherlands) or Autodesk (AutoCAD, Autodesk).

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The photomask can be made of mylar foil or glass depending on the requirements of the photolithographic process (i.e. aligner). 2. Plasma will clean the mold surface and activate it for functionalization. Time and

power vary depending on the plasma chamber used and the processing time should be kept short to prevent heating and damaging the SU-8 features on the mold. Alternatively an ozone chamber (Ozomax Corporation, www.ozomax.com) can be used for activating but the processing time needs to be increased. 3. Curing of PDMS can be accelerated by using a higher temperature up to 90C. At

90C curing takes only 1 h, but shrinkage of PDMS becomes more pronounced and most of the Petri dishes melt at 70C. 4. PDMS surfaces after plasma treatment remain activated (and hydrophilic) only a

few minutes because uncured low-molecular weight PDMS migrates from the bulk to the air-surface interface. 5. Proteins attach covalently to epoxy coated slides due to the reaction between the

protein lysine residues with the 3-GPS epoxy groups at basic pH. 6. In the present protocol, bovine serum albumin (BSA) has been used as the blocking

agent in order to reduce the attachment of the cells on the areas where fibronectin is not present. Alternatively, Pluronic F127 (Ethylene Oxide/Propylene Oxide Block Copolymer, BASF) can also be used to reduce the attachment of the cells (14). 7. Use tape to remove residual PDMS debris. This is a really effective method to clean

the PDMS surface. 8. More advanced CSs with a capillary retention valve can be designed in order to

prevent the drying of the microchannels on the assay area (5). 9. Incubation time of the samples will depend on the concentration of the analytes to

be detected and the affinity of the capture antibodies used. An incubation time of 12 min

25

with continuous flow driven by evaporation was used in order to detect TNF- (Tumor necrosis factor ) at a concentration of 20 pg/mL in cell culture media (15). 10. If the signal is too weak the concentration can be varied, although problems due to

non-specific adsorptions of detection antibodies over the substrate may increase as well. 11. When using a fluorescence microarray scanner, we recommended sticking the

substrate to a microscope slide or a cover slip, with the assay substrate surface stuck to the glass to facilitate focusing and manipulation of the sample. This necessitates a scanner that can scan through the glass (e. g. LS Reload Tecan Laser Scanner or Agilent Technologies DNA microarray Scanner).

Acknowledgments The authors would like to thank Emmanuel Delamarche and Ute Drechsler (IBM Research Center, Zurich) for the fabrication of the molds. We also are very grateful to Saravanan Sundararajan and Haig Djambazian (Genome Quebec, McGill University, Montreal) for providing the myoblast cells and the DIC microscopy imaging of the cells. M. P acknowledges financial support of the Spanish Ministry of Science post-doctoral fellows. References 1. Lang, S., von Philipsborn A. C., Bernard, A., Bonhoeffer, F., Bastmeyer, M. (2008)

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(2005) General Method for Patterning Gradients of Biomolecules on Surfaces Using Microfluidic Networks. Anal. Chem. 77, 2338-47.

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