Fabrication of Microfluidic Devices Containing Patterned Microwell

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Fabrication of Microfluidic Devices Containing Patterned Microwell Arrays W. Hampton Henley, Patty J. Dennis, and J. Michael Ramsey* Department of Chemistry, University of North Carolina at Chapel Hill, Chapman Hall CB#3216, Chapel Hill, North Carolina 27599 ABSTRACT: A rapid fabrication and prototyping technique to incorporate microwell arrays with sub-10 μm features within a single layer of microfluidic circuitry is presented. Typically, the construction of devices that incorporate very small architecture within larger components has required the assembly of multiple elements to form a working device. Rapid, facile production of a working device using only a single layer of molded polydimethylsiloxane (PDMS) and a glass support substrate is achieved with the reported fabrication technique. A combination of conventional wet-chemical etching for larger (≥20 μm) microchannel features and focused ion beam (FIB) milling for smaller (≤10 μm) microwell features was used to fabricate a monolithic glass master mold. PDMS/glass hybrid chips were then produced using simple molding and oxygen plasma bonding methods. Microwell structures were loaded with 3 μm antibody-functionalized dye-encoded polystyrene spheres, and a sandwich immunoassay for common cytokines was performed to demonstrate proof-of-principle. Potential applications for this device include highly parallel multiplexed sandwich immunoassays, DNA/RNA hybridization analyses, and enzyme linked immunosorbent assay (ELISA). The fabrication technique described can be used for rapid prototyping of devices wherever submicrometer- to micrometer-sized features are incorporated into a microfluidic device. photomasks, sub-10 μm features require the high fidelity achieved by a laser or electron beam-written mask that is considerably more expensive and not amenable to rapid prototyping. The useful lifetime of an SU-8 master mold depends on many factors, but it is generally considered to be 20−50 castings.29 Master molds fabricated from a monolithic material such as Si or glass are not subject to polymer liftoff or degradation and are therefore expected to have an indefinite lifetime. Here, we present a rapid fabrication technique for incorporating microwell arrays within a single layer of microfluidic circuitry. Master molds were fabricated on a monolithic substrate using a combination of conventional photolithography and focused ion beam (FIB) milling. Wet chemical etching is used to etch the microfluidic channels on the device, but as its maximum attainable aspect ratio is 1 μm features, and small beam currents are used to mill high-resolution submicrometer features. Micrometer to submicrometer features

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here is a growing trend toward the use of microfluidicbased architectures for rapid and sensitive analysis of analytes within complex matrixes such as biological samples. Microarrays have proven to be particularly useful as a means to rapidly test multiple analytes within a single assay step, using only a minute amount of sample.1−8 Individual spots of antibodies or oligomers in printed arrays range in size from tens to hundreds of micrometers in diameter, limiting the array size that can be contained within the dimensions of a microfluidic device.9−13 Arrays fabricated from fiber optic bundles containing many thousands of individual sensors within a very small area (∼1 mm2) have been reported, and their use with microtiter plate-scaled sample and reagent volumes has shown low detection limits.14−19 Attempts to incorporate sensor bead arrays within a microfluidic device have also been reported. The methods utilized, however, have relied on manually loading individual sensor beads20−24 into a patterned array and then sealing the array within a polycarbonate holder containing the microfluidic circuitry. Other reports of arrayed assay beads in microfluidic devices have relied on complicated, multistep fabrication using relatively costly components25−27 or multicomponent assembly of the chip after loading with assay beads.28 Photolithography using negative photoresists (such as SU-8) has long been a widely used method for polydimethylsiloxane (PDMS) mold fabrication. Aspect ratios of 20:1 and even 50:1 have been reported for these structures. Molds can be made with features ranging from micrometers to millimeters using a simple photoexposure system consisting of a light source and a photomask. While features with critical dimensions of ≥10−20 μm can be patterned using relatively inexpensive polyester film © 2011 American Chemical Society

Received: September 15, 2011 Accepted: December 22, 2011 Published: December 22, 2011 1776

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sputter (IBS) system (Model IBSe, South Bay Technology, San Clemente, CA) at 8 kV and 6 mA total current for approximately 40 min, depositing a 50 nm layer of chromium measured by a quartz crystal microbalance. FIB milling was performed using a dual focused ion beam system (Helios NanoLab, FEI, Inc., Seattle, WA). A pattern of circular microwells spaced 8.00 μm center-to-center was generated within the FEI software. Ga ions at an ion beam energy of 30 keV and ion current of 21 nA were used to mill an array pattern of between 100 (10 × 10) and ∼900 (28 × 32) wells. Upon completion of FIB milling, the chromium layer was stripped using chrome etch, with subsequent cleaning in 2% sulfuric acid and DI water. The substrate was dried with nitrogen and baked at 95 °C for 20 min. After cleaning the substrate in atmospheric plasma (Model PDC-32G, Harrick Plasma, Ithaca, NY) for 1 min, the substrate was placed in a vacuum desiccator with approximately 40 μL of trichloro(1H,1H,2H,2H-perfluorooctyl)silane. Vacuum was applied for 5 min to allow the silane vapor to saturate the desiccator, and then, the desiccator was sealed for 20−30 min. This silanization process resulted in a low energy surface coating that reduced PDMS adhesion to the substrate. The substrate was then removed from the desiccator and placed in a 95 °C oven for 20 min or allowed to sit at ambient conditions for 1 h to allow the silanization reaction to go to completion. The PDMS layer of the microfluidic device was an exact copy of the original master mold. In order to cast these copies in PDMS, complement or secondary molds were made from the master mold. The silanized substrate was placed etched-side-up into a larger mold form. PDMS was prepared according to the manufacture’s recommendation (10:1 polymer/cross-linker) and poured into the mold form to a depth of approximately 7.5 mm. The mold form was placed in a vacuum desiccator, and vacuum was applied to speed degassing of the PDMS and to ensure filling of the FIB milled microwells. After 15 min, the vacuum pump was turned off and the desiccator was allowed to return to atmospheric pressure. The mold form was then placed in a 60 °C oven for 30 min to begin the PDMS curing reaction and then transferred to a 95 °C oven for 30 min to finish PDMS curing. The mold form was allowed to cool, and the complement mold was then separated from the master mold. After trimming the edges of the complement mold with a razor blade, it was treated with atmospheric plasma for 5 s and then silanized using the same procedure as described for the master mold. The PDMS layer for the microfluidic device was made by pouring approximately 4 mL of PDMS into the complement mold. After degassing in the vacuum desiccator for 10−15 min, a 63.5 mm by 63.5 mm glass slide was placed over the complement mold such that excess PDMS was pushed out and air inclusions were not formed. The PDMS was then cured in a 95 °C oven for 15 min. The low surface energy resulting from the silanization process allowed the PDMS layer to be easily removed from the complement mold. A razor blade was used to trim the PDMS layer, and access holes were punched with a 3 mm biopsy punch (Fisher Scientific) where fluid reservoirs were desired. The PDMS layer was then placed channel-side-up on a glass slide and activated for bonding with a plasma cleaner for 12 s at 30 W. The activated PDMS surface was then quickly pressed against a clean glass slide that was plasma treated alongside the PDMS layer. Irreversible bonding took place between the glass slide and the PDMS within less than a second, but to ensure

can be milled at high aspect ratios if multiple beam passes are used.30,31 The low dead volume achieved by seamless array integration within the microchannel network should significantly increase the performance metrics of assays with small samples. Unlike preprinted antibody arrays sealed within a larger structure or arrays made from rigid materials that must be preloaded with beads before assembly,28 the ability to rapidly load differently functionalized beads into an array after chip manufacture provides much greater flexibility in regards to testing, analysis, and shelf life.15,32−37 For proof-of-concept, an assay using antibody-labeled beads similar to the one described by Blicharz et al.33 was performed using the device.

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EXPERIMENTAL SECTION Materials and Reagents. B270 glass substrates (101.6 mm by 101.6 mm by 0.9 mm thick) precoated with 120 nm of chromium and 500 nm of AZ 1518 photoresist (Telic Co., Valencia, CA) were used to make the master mold. A film photoplot mask was purchased from Infinite Graphics, Inc. (Minneapolis, MN) with the desired microchannel pattern. AZ 400K developer was obtained from Mays Chemical Corporation (Indianapolis, IN). Chromium mask etchant and 10:1 buffered oxide etch (BOE) were purchased from Transene Company, Inc. (Danvers, MA). Caution: Both solutions require use of protective clothing and a fume hood. Chromium etchant is corrosive and can cause severe burns, and its vapor (nitrogen oxides) can damage eyes and lungs. Do not heat the solution or allow contact with organic materials and be sure to dispose of solutions properly, especially any bearing chromium. Buffered oxide etch contains hydrofluoric acid, and exposure, even latent, can cause severe burns. Sylgard 184 (PDMS) was from Ellsworth Adhesives (Germantown, WI). Trichloro(1H,1H,2H,2H-perfluorooctyl)silane 97% and 10× phosphate buffered saline (PBS) were purchased from Aldrich (St. Louis, MO). Isopropyl alcohol (IPA), ethanol, acetone, and sulfuric acid were all from Fisher Scientific (Fairlawn, NJ). Deionized (DI) water was produced by a Nanopure Diamond system, Barnstead International (Dubuque, IA) and measured 18.0 MΩ/cm2 or greater. Microposit S1813 photoresist was obtained from MicroChem Corp., (Newton, MA). Antibody labeled beads used in the cytokine assay33 were provided by the Walt Laboratory (Tufts University). Assay reagents, including cytokine standards and antibodies, were purchased from R&D Systems (Minneapolis, MN). Alexa Fluor dye was from Invitrogen (Carlsbad, CA), and 90% 2-[methoxy(polyethylenxy)propyl]trimethoxysilane was obtained from Gelest (Morrisville, PA). Fabrication. Master molds were fabricated by first patterning the glass substrates with microchannels using traditional photolithography and wet chemical etching.38,39 An Optical Associates, Inc. J200 mask aligner (San Jose, CA) was used for photoexposures. Channel dimensions were measured using a KLA-Tencor Corp. P-15 profilometer (San Jose, CA) and were approximately 100 μm wide and 30 μm deep. The etched substrates were diced (Basic Dicer II, Dicing Technologies, San Jose, CA) and then stripped of photoresist and washed with IPA. To prepare for focused ion beam (FIB) milling, the nonreflective oxide layer was removed from the chrome layer of the substrate with a 15 s treatment with chrome etch solution. The substrates were then washed in DI water, 2% sulfuric acid, DI water again, and dried with nitrogen. Chromium was sputtered onto the substrate using an ion beam 1777

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RESULTS AND DISCUSSION Figure 1a shows the chip design with four reagent/sample reservoirs and a single waste reservoir where vacuum is applied to move fluid through the chip and over the array. In a point-ofcare application, pinch-type valves may be used at the indicated locations to control flow from each reservoir. For proof of concept, no valving was used, and reagents/samples were simply added sequentially. Figure 1b shows an SEM of an FIB milled array. Arrays of this size (∼900 wells) typically require ∼1 h of milling time with a 21 nA aperture setting. A cross section of two typical FIB milled array features in the glass master mold is shown in Figure 2. A slightly tapered profile

complete bonding, chips were allowed to sit for at least 1 h before use. Assay Bead Loading. The assembled chip was prepared by silanizing the channels with 2-[methoxy(polyethylenxy)propyl]trimethoxysilane40 and then flushing the channels with PBS. This served to form a hydrophilic surface coating on the PDMS and glass channel walls to prevent undesirable protein and bead adsorption. Alternative strategies using blocking agents added to the buffer were equally successful. Equal quantities of beads (nominally 3 μm diameter) labeled with either anti-vascular endothelial growth factor (VEGF) antibodies or anti-interleukin-8 (IL-8) antibodies were mixed together in buffer to form a slurry. Approximately 500 nL of bead slurry was loaded into the reservoir closest to the microwell array, and vacuum was applied at the waste reservoir (Figure 1a). As the

Figure 2. SEM image showing FIB milled cross section of two array wells, each approximately 3 μm deep, 3 μm diameter at the surface, and 2 μm diameter at the base.

Figure 1. (a) Optical image of the microfluidic device. (b) SEM image of the FIB milled array [labeled “Array” in (a)].

bead slurry flowed across the microwell array, the PDMS layer was pressed against the glass layer several times, forcing beads into the microwells. Visual inspection of the array using an optical fluorescence microscope was used to confirm bead loading into the microwells. The channels were then flushed thoroughly with buffer to remove beads that were not held within the array. No surface treatment was needed to remove loose beads in this buffer; however, other groups have reported methods to reduce adsorption of microbeads to PDMS surfaces.41 The chip was then stored in buffer at 4 °C until needed. Assay Protocol. Protein detection was based on a sandwich immunoassay in which monoclonal antibodies were attached to europium-dye-encoded polystyrene beads.33 The anti-IL8 labeled beads were encoded with only a quarter of the concentration of europium dye used to encode the anti-VEGF labeled beads so that the types of beads could be easily distinguished by measuring their fluorescent intensity at 365 nm excitation.33 A standard solution of 50 nM VEGF in PBS w/0.1% BSA was pulled into the chip using vacuum applied at the waste reservoir. After a 30 min incubation time, secondary (biotin-labeled, polyclonal) antibody solution was pulled through the chip and left to incubate for 15 min. Alexa Fluor 488-labeled streptavidin was then pulled over the array and allowed to react with any biotin-labeled antibodies bound to analyte molecules. After a 10 min incubation time, the channels were flushed with tris buffered saline (TBS) w/0.05% Tween 20 for 10 min. Encoding and assay images were taken immediately after the final buffer wash. Optical microscopy images were obtained using a Nikon (Melville, NY) Eclipse Ti−U microscope equipped with a Cascade II electron multiplying charge coupled device (EMCCD; Photometrics, Tucsan, AZ) camera.

indicative of high-current FIB milling is easily noted, and this profile may facilitate bead loading. Figure 3a shows an SEM of

Figure 3. (a) SEM of the PDMS layer with polystyrene beads loaded into the microwells fabricated using the described technique. The loading efficiency was 91%. This SEM was obtained after an assay, and residual crystallized buffer components are visible on the surface of the PDMS. (b) SEM of an FIB milled PDMS microwell (cross section) containing an antibody labeled bead. The bead was bisected perpendicular to the plane of the PDMS surface. The SEM image was taken at a 52° angle.

beads loaded into an array fabricated using the described technique. Approximately 80−100% of microwells within an array are routinely filled using this simple technique. Several variations of the microwell dimensions were milled to find the optimal diameter and depth to hold 3.00 μm diameter microspheres. Using the FIB mill’s direct write features, many different geometries could be written onto a single substrate and evaluated in parallel, greatly expediting device development. Microwell dimensions of 2.75 μm in diameter and 3.00 μm in depth were found to best facilitate the retention/loading of the microspheres into the wells. An FIB milled cross section of a loaded microwell (Figure 3b) demonstrates how the PDMS conforms to the bead surface, holding the beads firmly. Bead retention is high, as the beads could not be removed from the array once loaded, even with prolonged sonication. 1778

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fabrication Laboratory (CHANL) for help in developing the FIB milling techniques.

Optical microscopy images were taken of a bead array using an excitation wavelength of 365 nm to excite the europium encoding dye so that the location of each type of bead could be determined (Figure 4a). Assay images (495 nm excitation/519

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Figure 4. False-color optical fluorescent microscopy images of the array. The highest intensities are red, midlevel intensities are blue, and the lowest intensities are black. The array had a 97% bead loading efficiency. Left: Encoding image showing the europium encoding dye (365 nm excitation). VEGF labeled beads (red) were approximately 4× more intense than IL8 labeled beads (light blue). Center: Image obtained after performing the assay using only VEGF in the sample. Only the beads identified as VEGF from the encoding image are visible. Right: Image obtained after IL8 was subsequently added to the chip and the assay was repeated. After addition of all samples and reagents, the signal is strong for all beads as expected.

nm emission) showing the array of beads after sequential VEGF (Figure 4b) and IL8 (Figure 4c) assays are shown. The corresponding signals for each analyte are as predicted. A novel technique for integrating high aspect ratio, sub-10 μm features within a microfluidic device has been demonstrated using microwell arrays and simple microfluidic architecture. The absence of dead volume and reduced sample requirements attributable to the full integration of microchannels and microwells within a single device layer is expected to show significant performance enhancements for immunoassay and DNA hybridization assays currently performed within microtiter plates or microcentrifuge tubes. Future work will include development and device optimization for cytokine assays, DNA hybridization assays, and signal amplification methods that will greatly expand the utility of these devices.42 Access to FIB instrumentation ultimately limits applicability of this technique as a rapid prototyping system; however, similar two-step fabrication protocols for master molds using more commonly available instrumentation such as reactive ion etching can be easily adopted and will be addressed in future work. While the fabrication techniques were initially developed to generate a grid to hold assay beads in a microfluidic structure, they have wide applicability for the rapid prototyping of any device requiring the incorporation of micrometer or submicrometer features within larger microfluidic elements.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: (919) 962-4952.

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REFERENCES

ACKNOWLEDGMENTS

The authors thank Professor David Walt and Timothy Blicharz of Tufts University for providing the antibody-labeled beads and initial starting points for the assay protocol. The authors also thank Carrie Donley of Chapel Hill Analytical Nano1779

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dx.doi.org/10.1021/ac202445g | Anal. Chem. 2012, 84, 1776−1780