Functionalized 3D-Hydrogel Plugs Covalently Patterned Inside

Chem. , 2009, 81 (19), pp 7967–7973. DOI: 10.1021/ac901138w. Publication Date (Web): September 1, 2009. Copyright .... Published in print 1 October ...
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Anal. Chem. 2009, 81, 7967–7973

Functionalized 3D-Hydrogel Plugs Covalently Patterned Inside Hydrophilic Poly(dimethylsiloxane) Microchannels for Flow-Through Immunoassays Wang-Chou Sung, Huang-Han Chen, Honest Makamba, and Shu-Hui Chen* Department of Chemistry, National Cheng Kung University, No. 1, College Road, Tainan 701, Taiwan Integration of a hydrogel and polydimethylsiloxane (PDMS)based microfluidic device can greatly reduce the cost of developing channel-based devices. However, there are technical difficulties including the hydrophobic and inert surface properties associated with PDMS as well as back pressure and fragile material associated with the use of hydrogel in microchannels. In this study, a strategy to covalently photopattern 3-D hydrogel plugs with functionalized protein G inside microfluidic channels on a hydrophilic PDMS substrate coated with polyelectrolyte multilayers (PEMS) is presented. In this process, a UV-light microscope is applied to initiate the protein G-poly(acryl amide) copolymerization from the bulk substrate to solution areas via the deeply implanted photoinitiator (PI), resulting in sturdy 3D plugs covalently bonded to the upper and lower channel wall, while leaving open spaces in the channel width for the fluid to flow through. In addition, the long-term hydrophilicity and low nonspecific binding property associated with PEMS surface can be conserved for the nonpatterned area, leading to hydrogel plugs in extremely hydrophilic and permeable environment in a restricted channel space for bubble-free fluid transport and affinity interaction. By immobilization of well-oriented antibodies via protein G on the hydrogel plugs in the channel, estrogen receptor r (ERr) is demonstrated to be captured quantitatively with high loading capacity and high specificity. Hydrogel has been widely applied in the field of medicine,1,2 separation,3,4 and biology5 for many years because of their wellordered fibrous structure, physical flexibility, and high waterretaining ability.6 In addition, the functional groups on the hydrogel can be easily modified to immobilize various biomolecules for bioassays. With the aid of the native hydrophilic * To whom correspondence should be addressed. E-mail: [email protected]. (1) Rokhade, A. P.; Kulkarni, P. V.; Mallikarjuna, N. N.; Aminabhavi, T. M. J. Microencapsul. 2009, 26, 27–36. (2) Jhaveri, S. J.; Hynd, M. R.; Dowell-Mesfin, N.; Turner, J. N.; Shain, W.; Ober, C. K. Biomacromolecules 2009, 10, 174–183. (3) Makamba, H.; Huang, J. W.; Chen, H. H.; Chen, S. H. Electrophoresis 2008, 29, 2458–2465. (4) Liu, J. K.; Sun, X. F.; Lee, M. L. Anal. Chem. 2007, 79, 1926–1931. (5) Obaidat, A. A.; Park, K. Biomaterials 1997, 18, 801–806. (6) Zhang, S. G. Nat. Mater. 2004, 3, 7–8. 10.1021/ac901138w CCC: $40.75  2009 American Chemical Society Published on Web 09/01/2009

environment, protein activity can be retained for a longer period of time in the hydrogel compared to the surface-bound proteins. Such superior properties of the hydrogel have been used for making biosensors and enzyme reactors for affinity assays to capture proteins7-15 or cells.16,17 Dense hydrogel beads with different affinity functions can be formed in situ on the surface15-18 to capture proteins or cells. Integration of the hydrogel with microfluidic channels has also been attempted for fabricating functional elements such as valves and pH sensors as well as reactors or concentrators for protein analysis.15,19 Under the restricted channel space, the mass transfer can be enhanced compared to the batch system and cross contamination can be avoided by multichannel design. Until now, however, the hydrogel is more commonly fabricated in microfluidic devices made on a glass substrate. For example, the active hydrogel capable of autonomous control of local flow has been fabricated inside microchannels on a glass substrate via direct photopatterning. The protein-containing hydrogel has also been fabricated on a glass plate covered with poly(dimethylsiloxane) (PDMS) channels to allow the reagent to pass through the micropatch for pH sensing7,8 as well as for glucose quantification.10 Fabrication of the hydrogel as a real functional element on PDMSbased microfluidic devices, however, has not been reported. PDMS possesses many advantages, like high optical transparency, (7) Zhan, W.; Seong, G. H.; Crooks, R. M. Anal. Chem. 2002, 74, 4647–4652. (8) Heo, J.; Crooks, R. M. Anal. Chem. 2005, 77, 6843–6851. (9) Zubtsov, D. A.; Ivanov, S. M.; Rubina, A. Y.; Dementieva, E. I.; Chechetkin, V. R.; Zasedatelev, A. S. J. Biotechnol. 2006, 122, 16–27. (10) Koh, W. G.; Pishko, M. Sens. Actuators, B 2005, 106, 335–342. (11) Dominguez, M. M.; Wathier, M.; Grinstaff, M. W.; Schaus, S. E. Anal. Chem. 2007, 79, 1064–1066. (12) Rubina, A. Y.; Dyukova, V. I.; Dementieva, E. I.; Stomakhin, A. A.; Nesmeyanov, V. A.; Grishin, E. V.; Zasedatelev, A. S. Anal. Biochem. 2005, 340, 317–329. (13) Brueggemeier, S. B.; Kron, S. J.; Palecek, S. P. Anal. Biochem. 2004, 329, 180–189. (14) Rubina, A. Y.; Kolchinsky, A.; Makarov, A.; Zasedatelev, A. S. Proteomics 2008, 8, 817–831. (15) Rubina, A. Y.; Dementieva, E. I.; Stomakhin, A. A.; Darii, E. L.; Pan’kov, S. V.; Barsky, V. E.; Ivanov, S. M.; Konovalova, E. V.; Mirzabekov, A. D. Biotechniques 2003, 34, 1008–1022. (16) Khademhosseini, A.; Yeh, J.; Jon, S.; Eng, G.; Suh, K. Y.; Burdick, J. A.; Langer, R. Lab Chip 2004, 4, 425–430. (17) Cheng, S. Y.; Heilman, S.; Wasserman, M.; Archer, S.; Shuler, M. L.; Wu, M. M. Lab Chip 2007, 7, 763–769. (18) Hu, S.; Ren, X.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. L. Anal. Chem. 2004, 76, 1865–1870. (19) Yang, S. H.; Han, J. S.; Baek, S. H.; Kwak, E. Y.; Kim, H. J.; Shin, J. H.; Chung, B. H.; Kim, E. K. Appl. Biochem. Biotechnol. 2008, 151, 273–282.

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biocompatibility, chemical stability, and a simplified bonding process for microfluidic devices via plasma oxidation,20,21 which make it an attractive material for microfluidics. Nevertheless, because of the inherent detrimental effects such as swelling and deliamination caused by nonpolar organic solvents, PDMS is not compatible with many synthesis processes that use organic solvents. PDMS also lacks functional groups for covalent derivatization, and the silanol groups exposed by plasma oxidization do not have long-term stability due to the migration of PDMS chains, leading to recovery of the native hydrophobic nature. Moreover, the hydrophobic surface leads to nonspecific binding that has caused serious problems for bioapplications of PDMS devices and many surface modification methods using either adsorption22-24 or covalent bonding25-27 have been developed. In our previous studies, we successfully developed a surface modification technique to create long-term hydrophilic layers on the PDMS surface by coating polyethyleneimine (PEI) and poly(acrylic acid) (PAA) in sequence and cross-linking to form polyelectrolyte multilayers (PEMS).28,29 PEMS can reduce nonspecific binding by a half and by a dozen times when topping PEMS with poly(ethylene glycol) (PEG) molecules,28 and, thus, improve the affinity binding interaction. We would like to take an advantage of such a long-term hydrophilic coating in making hydrogel plugs within PDMS channels for bioassay development. In a previous report,18 a simple benchtop method was developed for covalently patterning bare PDMS surfaces with poly(acrylic acid) via the use of a photomask and the patterned surface was applied for cell attachment and for antibody immobilization. In microfluidics, the hydrophilic channel wall is essential to reduce surface tension for the fluid transport and for efficient molecular interactions. Besides, the patterned hydrogels must be strong to avoid damages under high-speed flushing. Thus, we would like to construct the hydrogel element covalently bonded to the substrate on hydrophilic surfaces with high permeability and strength to sustain the channel flow. We chose to use poly(acrylamide)-based hydrogels which have been shown to have high porosity for biomolecules and, thus, hold a great potential for affinity analysis on a plate-based protein chip.12,13 One of the most significant properties of the hydrogel is their large pore structure to allow macromolecules such as proteins to pass through. We would also want to functionalize the hydrogel with protein G for antibody immobilization and the device will be validated by the detection and quantification of estrogen receptor. Integration of hydrogel plugs and PDMS-based microfluidic device can greatly reduce the cost in developing channel-based protein chips. (20) Ramachandran, N.; Larson, D. N.; Stark, P. R. H.; Hainsworth, E.; LaBaer, J. FEBS J. 2005, 272, 5412–5425. (21) Mukhopadhyay, R. Anal. Chem. 2006, 78, 5969–5972. (22) Mao, H.; Yang, T.; Cremer, P. S. Anal. Chem. 2002, 74, 379–385. (23) Philips, K. S.; Cheng, Q. Anal. Chem. 2005, 77, 327–334. (24) Qin, M.; Wang, L. K.; Feng, X. Z. Langmuir 2007, 23, 4465–4471. (25) Wu, Y.; Huang, Y.; Ma, H. J. Am. Chem. Soc. 2007, 129, 7226–7227. (26) Roman, G. T.; Culbertson, C. T. Langmuir 2006, 22, 4445–4451. (27) Sui, G.; Wang, J.; Lee, C. C.; Lu, W.; Lee, S. P.; Leyton, J. V.; Wu, A. M.; Tseng, H. R. Anal. Chem. 2006, 78, 5543–5551. (28) Makamba, H.; Hsieh, Y. Y.; Sung, W. C.; Chen, S. H. Anal. Chem. 2005, 77, 3971–3978. (29) Sung, W. C.; Chang, C. C.; Makamba, H.; Chen, S. H. Anal. Chem. 2008, 80, 1529–1535.

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EXPERIMENT SECTION Materials. The PDMS, sylgard 184 kit, was obtained from Dow Corning. Polyethyleneimine (PEI, MW 75 000 Da), poly(acrylic acid) (PAA, MW 1 000 000 Da), 1-[3-(dimethylamino)propyl]-3ethylcarbodiimide hydrochloride (EDC), benzophenone, N-hydroxysuccinimide (NHS), and toluidine blue were acquired from Sigma-Aldrich (St. Louis, MO), and all these chemicals were reagent grade. FITC-labeled IgG, protein G, HRP-conjugated IgG, and phosphate buffer saline (PBS) were obtained from Pierce. Bovine serum albumin (BSA) was purchased from Sigma (St. Louis, MO). Water was purified with the Barnstead E-pure system (Waltham, MA). Tween 20 and acrylamide/bisacrylamide was from J.T. Baker (Phillipsburg, NJ). NHS-PEG-acrylate (MW 5000 Da) was from Nektar (San Carlos, CA). The chemiluminescence reagents (NEL104 and NEL105) were bought from PerkinElmer Life Science (Waltham, MA). 17R-Cy3-estradiol (Cy3-E2) was synthesized in-house following the procedure reported previously.30 Surface Modification in the PDMS Channel. The prepared PDMS reagent (oligomer/curing reagent ) 10:1) was poured onto the chromium master to form the channel structure and cured at 90 °C for 60 min. Each channel has a dimension of 1000 µm in width and 20 µm in depth. Eight channels can be formed in a 1 × 2 cm2 dimension PDMS plate. The PDMS plate with channels was oxidized by oxygen plasma and sealed with another plasma-treated flat PDMS film to form an enclosed channel. PEI and PAA solution (0.05%, v/v) were added into the channel in sequence to form the multiple coating layer on the PDMS surface. After coating steps, the mixture containing EDC (0.04 g/mL in PBS buffer) and NHS reagents (0.02 g/mL in PBS buffer) were applied to form amide bonds between the PEI/ PAA layers. Next, ethylene amine (0.1% in PBS buffer, v/v) was incubated in the channel to block the NHS residue in the modified layer. Finally, the channel was dried with the nitrogen gas and stored at 4 °C for further applications. Conjugation of Acrylate-PEG Group with Protein G. NHS-PEG-acrylate (NPA) solution (20 µL, 1000 µg /mL in PBS, pH 7.4) was mixed with the protein G solution (20 µL, 1000 µg /mL in PBS, pH 7.4) at room temperature for 2 h. Subsequently, excess NPA was removed from the acrylate-PEG-protein G conjugate using a centrifuge column (microcosm, 30 kDa cutoff). The purified acrylate-PEG-protein G was resuspended in PBS buffer and stored at 4 °C for further copolymerization. Fabrication of Protein G Functionalized Hydrogel in PDMS-Based Microfluidics. At first, the solution contained 0.1 mg/mL benzophenone, was dissolved in acetone, and was loaded into the polyelectrolyte multilayer modified PDMS channel. After 5 min incubation, unabsorbed benzophenone was washed away by ethanol and water in sequence. The gel precursor aqueous solution containing 5% acrylamide (w/v), 0.12% bis-acrylamide (w/ v), 50% glycerol (v/v), and acrylate-PEG-protein G (500 µg/ mL in PBS buffer) was transferred to the channel and irradiated with UV light (365 nm) for 4 min. Without application of any photomask, the UV light was directly irradiated on the channel area through the objective (40×, Olympus). After polymerization, the unreacted monomer was washed away with the solution (30) Chen, C. C.; Yen, S. F.; Makamba, H.; Tsai, M. L.; Chen, S. H. Anal. Chem. 2007, 79, 195–201.

Scheme 1. Fabrication Scheme for the Protein G-Immobilized Hydrogel Microfluidic Protein Chipa

a (a) The PDMS channel surface was coated with PEMS by layer by layer modification; (b) absorption of PI into the PEMS-modified PDMS channel wall; and (c) protein G was covalently bonded to NPA molecules for copolymerization with the acrylamide/bisacrylamide. (d) The UV light emitted from the objective irradiates on the incubated precursor solution for in situ synthesis of hydrogel beads in the PEMS-modified PDMS channel. (e) Hydrogel plugs were formed within three PDMS channels.

containing the 20% ethanol (v/v) in PBS buffer. The device fabrication is shown in Scheme 1. Three hydrogel plugs were formed in each channel, and the distance between each plug was about 600 µm. Hydrogel plugs can also be patterned in the microchannels without protein G by following the same procedures using underivatized acrylate-PEG in the precursor solution. Finally, the channel was left filled with PBST and stored in the refrigerator for further applications. A sandwich assay using chemiluminescence detection was carried out to validate whether the protein G is incorporated into the hydrogel plugs and capable of immobilizing antibodies. A solution containing 20 µg/mL mouse IgG (monoclonal, prepared in PBST buffer) was incubated in the channel for 2 h. After the channel was washed with PBST buffer, it was incubated in the blocking solution (1 mg/mL BSA, in PBS buffer) for 1 h at room temperature. Subsequently, 5 µg/mL HRP-conjugated antimouse IgG in PBST buffer was added into the channel. After 1 h incubation, the color reagent was loaded into the channel and the emitted luminance from the hydrogel was captured immediately via an objective (40×) and collected by a cooling CCD camera (F-view, Olympus). In addition, fluorescence detection was also carried out to investigate the channel cross section of the patterned hydrogel using toluidine blue dye which will stain the carboxylic acid group of immobilized protein G. PI-implanted channels with and without polymerized hydrogel were loaded with 0.1% toluidine blue solution (% wt in PBS buffer, pH 7.4) for 10 min, washed with the PBS buffer, and dried by vacuum. Then, the channel end was cut and fixed on a glass slide with a side on for fluorescence imaging (excitation, 550 nm; emission, 570 nm) under the microscope incorporated with a CCD (Olympus).

Characterization of PDMS Surfaces. The properties of the PDMS surface with different treatment were analyzed by attenuated total reflection Fourier transform infrared spectroscopy (ATRFT-IR) and contact angle measurements. The ATR-FT-IR instrument (Waltham, MA) was applied to confirm the functional groups on the surface, 36 scans were collected for each plug at a resolution of 4 cm-1, and the background signal detected from bare PDMS was subtracted to eliminate interferences. The contact angle measurements were carried out with 5 µL of DI water droplets at room temperature with an optical meter (CADT, Kyowa, Japan). All the PDMS plates were dried by nitrogen gas before contact angle measurements. On each piece of plate, the contact angle was an average value from five randomly selected locations. The hydrophilic stability of PDMS surface under different treatments were also monitored after 4 weeks of storage in the refrigerator (temperature at 4 °C). Molecular Diffusion in the Hydrogel. Both Cy3-labeled E2 (MW 972 Da) and FITC-labeled IgG (150 kDa) were used to estimate the diffusion coefficient in the hydrogel. For this investigation, Cy3-labeled E2 (30 µg/mL) or FITC-labeled immunoglobulin (30 µg/mL monoclonal) was incubated in the microchannel patterned with the hydrogel plugs and the fluorescence emitted from the hydrogel was recorded every minute by using a photomultiplier-tube (PMT). The diffusion coefficient (DGEL) of the molecule in the gel was calculated by dividing the square area of hydrogel plug R by the time (TD) required to reach a homogeneous fluorescence color on the whole hydrogel plug:9

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Sandwich Type Immunoassay Using Fluorescence Detection for ERr. Sandwich type immunoassay using fluorescence detection was adopted for detecting ERR in the hydrogel channel chips. All the sample solutions were loaded by capillary force and pumped out by vacuum. The protein G-functionalized hydrogel channel array was first activated with the monoclonal anti-ERR (mouse IgG, 20 µg/mL in PBST buffer) for 2 h. After washing with PBST buffer, blocking solution (1% BSA in PBS buffer in weight ratio) was applied and incubated for 1 h at room temperature. Then, the prepared ERR solutions were loaded into each channel, incubated in the channels for 2 h, washed with PBST buffer, and then treated with 30 µM Cy3-labeled E2 for fluorescence detection. After a PBST wash, the fluorescence intensity was captured by a 40× objective and detected by PMT with an enhanced voltage of 750 V and the fluorescence images of gel plugs were captured by a cooling CCD camera with an exposure time of 20 s. For quantitative analysis, standard solutions at a concentration of 500, 250, 125, 62.5, 31.25, and 0 ng/mL in PBST buffer were prepared from recombinant ERR and the intensity detected by PMT was used for quantification. Complex sample was prepared by spiking ERR into the bovine serum at a final concentration of 50 ng/mL. Competitive Assay on Protein G-Immobilized Hydrogel. For competitive assays, the anti-ERR (20 µg/mL in PBST buffer) and ERR (500 ng/mL in PBST buffer) were loaded into the channel with protein G-immobilized hydrogel in sequence. Then, a serial of mixtures containing 30 µM Cy3-labeled E2 and nonlabeled E2 at a concentration of 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, and 0 M, respectively, were coincubated in the channel for 2 h at room temperature. The unbound elements were washed away by PBST buffer, and the emitted fluorescence from the bound Cy3-E2 was recorded by a cooling CCD camera. RESULTS AND DISCUSSION Characterization of the PI-Implanted PEMS PDMS Surface. In this experiment, the photoinitiator (PI), benzophenone, was preincubated in the PEMS modified PDMS channel before photopolymerization as shown in Scheme 1. Once penetrated into the bulk substrate, benzophenone can be retained in the hydrophobic PDMS inner layer.18 Therefore, photopolymerization can be initiated from the bulk substrate upon UV irradiation. This is a good strategy for in situ fabrication of the hydrogel element in the PDMS microchannel since the polymerization is initiated from the irradiated upper and lower channel wall to form photodefined 3D hydrogel constructs within a microchannel with covalent bonding. Contact angle measurements were first carried out to find out whether the absorbed benzophenone affects the hydrophilic property of the modified PEMS coating. As shown in parts b and c of Figure 1, the contact angle for the freshly prepared PEMS and PI-implanted PEMS surface was 5.87° and 7.81°, respectively, and increased to 13.09° and 21.61°, respectively, after a 4-week exposure to the air. Compared to the native PDMS surface that was measured to have a contact angle of 83.01°,29 the PI-implanted PEMS surface conserves durable hydrophilicity and allows the use of capillary forces for filling the solution into the microchannel. With the use of the native hydrophobic PDMS as the substrate for microfluidics, solutions are hardly loaded by capillary forces 7970

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Figure 1. Contact angle measurements on freshly prepared PDMS plates (blue column) and after 4 weeks storage (red column) with different modifications: (a) bare PDMS plate, (b) PEMS-modified PDMS plate, and (c) PEMS-modified PDMS plate with absorbed benzophenone.

and air bubbles are easily formed and trapped inside the microchannel. Whether or not the benzophenone was trapped inside the PIimplanted PEMS surface was investigated by ATR-FT-IR measurement. Figure 2 shows the spectra obtained from the native PDMS surface, PEMS surface, and PI-implanted PEMS surface. Two major peaks at 1550 and 1750 cm-1 belonging to the stretching bands of the N-H amide and N-CdO amide of the PDMS material were found for all surfaces investigated, which match the results found in the previous reports.28,29 The ketone and the aromatic alkenes (CdC) bands were found at 1720 and 1660 cm-1, respectively, after treating with benzophenone. These two IR bands confirm the retention of benzophenone inside the PDMS surface, which can be applied to initiate photopolymerization. Photopatterning and Characterizing the Hydrogel Plugs Inside the Microfluidic Channels. As illustrated in Scheme 1, photopatterning is performed on the PI-implanted PEMS surface using UV irradiation (365 nm) under the fluorescence microscope. In this manner, polymerization can be restricted in the area exposed to UV light that emitted directly from the objective without applying any photomask. Because the PI was not in the free monomer solution but trapped inside the channel substrate, the induced radicals can only support limited polymerization in the free solution and polymerization in the nonradiated solution area caused by the diffusion of free radicals was prevented. Moreover, polymerization was initiated from both the upper and lower irradiated channel walls rather than a planar surface, leading to 3D hydrogel constructs which shape can be defined by the microscope objective. Figure 3b shows the cross section fluorescence image of the hydrogel patterned inside the PDMS microchannel. The fluorescence was emitted from the PDMS in a depth of 20-30 µm after polymerization, supporting the fact that PI could penetrate into the substrate to form covalent bonding. Figure 4 shows the fluorescence image of three hydrogel plugs patterned in situ in a microfluidic channel. The formed hydrogel was around 600 ± 25 µm in the dehydrated form and increased to 810 ± 18 µm in solution-filled form, which difference is expected since hydrogels will swell in hydrophilic buffers. However, the size of the hydrogel is smaller than the channel width which was 1000 µm. Thus, the resulting open channels in the width around the hydrogel plug could allow the sample and the buffer to flow through with no back pressure. Since the shape and size of the

Figure 2. Comparison of the ATR-FT-IR spectrum on PDMS plates with different treatments: (a) bare PDMS, (b) PEMS-modified PDMS, and (c) PEMS-modified PDMS with absorbed benzophenone. The inset is the enlarged IR spectra between the wavelengths of 1200 and 2000 cm-1. The stars indicate the IR stretching bands of the ketone and aromatic alkene groups at the wavelengths 1720 and 1660 cm-1, respectively.

Figure 3. The fluorescence images acquired from the cross section of the PDMS channel: (a) the photoinitiator-implanted PDMS microchannel before polymerization and (b) the polymerized hydrogel in the PDMS channel. The dotted line indicates the interface between the channel and the PDMS inner layer.

3D patterning is controlled by the microscope objective without a mask, the method is straightforward, convenient, and applicable for most laboratories. From the images shown in Figure 4, the size of the patterned plug can be controlled by the exposure time and the objective lens. Molecular Diffusion in the Hydrogel. Because the pore size of the hydrogel changes with the percentage of cross-linking reagent added, it is necessary to see whether the sample can easily diffuse within the fabricated gel for affinity interaction. A solution containing FITC-labeled antibody (MW 150 kDa) and Cy3-labeled E2 (MW 952 Da) were applied to study the diffusion kinetics within the hydrogel. Under PMT detection, the fluorescence intensity of the hydrogel plugs gradually increased upon adding the dye-labeled molecules into the channel and reached a plateau

constant which was below the maximum hit value of the PMT. From the intensity-time curve shown in Figure 5, the saturation time for Cy3-E2 and FITC-IgG was estimated to be 24 and 52 min, corresponding to a diffusion coefficient of 1.96 × 10-6 and 9.06 × 10-7cm2/s, respectively. Although the difference between two measured diffusion coefficients is relatively small as one may expect based on the 150× difference between the two solute molecular weights, we think there are a number of factors which may be attributed to such a discrepancy. For example, the radius of the two molecules may not differ as large as their molecular weights and the detection sensitivity may differ slightly by using two different dyes. The order of the magnitudes measured, however, are comparable to those measured from the conventional protein chip.9 Thus, these results suggest Analytical Chemistry, Vol. 81, No. 19, October 1, 2009

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Figure 4. The CCD images of (a) the hydrogel plugs in the PDMS microfluidic channels and (b) the enlarged channel image of the patterned hydrogel. (c) The chemiluminscence of protein G emitted from the hydrogel.

Figure 5. Diffusion curves for (a) Cy3-E2 and (b) FITC-labeled IgG. The signal saturation time for Cy3-E2 and FITC-IgG was estimated to be 24 and 52 min.

the poly(acrylamide)-based hydrogels we patterned inside the microchannels have porous and permeable structure allowing free diffusion of molecules with a wide range of molecular weights. Quantitative and Competitive Immunoassays. Protein G is known to have a high binding affinity ranging from 109 to 1010 M-1 with many kinds of immunoglobulins31 and was being chosen to copolymerize with the hydrogel for immobilizing antibodies. Protein G was first bound to the NHS-PEG-acrylate monomer through the amine group by carbodiimide coupling and then imbedded within the hydrogel via the subsequent photopolymerization. The NPA compound has a long PEG chain which can minimize nonspecific bindings. The successful immobilization of protein G within the hydrogel was examined by chemiluminsence detection using a secondary antibody. As shown in Figure 4c, strong chemiluminescence was clearly detected within the hydrogel with a very weak background from the rest of the channel area. We further compared the background emissions from bare, BSA-coated, and PEMS-coated PDMS (31) Neubert, H.; Jacoby, E. S.; Bansal, S. S.; Iles, R. K.; Cowan, D. A.; Kicman, A. T. Anal. Chem. 2002, 74, 3677–3683.

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channel surfaces and included the observations in the Supporting Information. It was found that both BSA and PEMS coating can reduce nonspecific binding substantially (70-90%), and PEMS can reduce nonspecific binding 2-5 times more than that by BSA. These observations not only indicate that NPA-modified protein G was successfully incorporated into the hydrogel with conserved immuno-activity but also demonstrate that the extremely hydrophilic PEMS channel surface could effectively reduce nonspecific binding on the patterned microfluidic devices. The developed device was investigated for quantitative immunoassays in detecting ERR. ERR is a nuclear receptor protein found in many organs such as liver and kidney and can be activated by binding with 17β-estradiol (E2).32 Since E2 has strong binding affinity with ERR, Cy3-labeled E2 was applied as the fluorescence probe for quantitative immunoassays in detecting ERR. In order to capture ERR protein, the oriented monoclonal anti-ERR (mouse, 20 µg/mL) was bound to protein G immobilized in the hydrogel. ERR solutions with concentrations ranging from 500 to 31.25 ng/mL prepared from the recombinant protein were introduced into the hydrogel channels; ERR was captured by antiERR on the hydrogel plug and then detected by flushing through Cy3-labeled E2. Figure 6 shows the fluorescence images obtained from the measurements, and the calibration curve deduced from the detected signals can be fit into the linear equation: y ) 1.2351x + 27.914 with R2 ) 0.9794. In addition, the relative standard deviations (RSD) were found to be less than 9.43% for repetitive measurements. To examine the accuracy of quantification using the constructed calibration curve, ERR (50 ng/mL) was spiked into bovine serum and detected by the channel-based protein chips containing protein G-immobilized hydrogel plugs. The concentration of the (32) Dahlman-Wright, K.; Cavailles, V.; Fuqua, S. A.; Jordan, V. C.; Katzenellenbogen, J. A.; Korach, K. S.; Maggi, A.; Muramatsu, M.; Parker, G. M.; Gustaffson, J. Pharmacol. Rev. 2006, 58, 773–781.

E2 by orders of magnitudes due to the hindrance effect caused by the short linker length of Cy3-E2. These results confirm that the signals detected are specific for ERR, and the background nonspecific binding is minimal. We have further compared the sensitivity of the immnoassays obtained from hydrogel plugs with that obtained from the direct binding of protein G on the channel wall without hydrogel plugs. Under the same experimental conditions using the same ERR solution, the fluorescence intensity obtained from the hydrogel plug was found to be about 13 times stronger than that obtained from the channel wall, indicating an increased capture capacity by the use of the porous hydrogel microstructure.

Figure 6. Immunoassay of ERR in the hydrogel plugs with immobilized anti-ERR: (a) fluorescence images obtained from ERR solutions with different concentrations and (b) calibration curve constructed from the measurements of part a. The error bars indicate the standard deviation for each measurement with n ) 3. (c) Competitive assays by coincubating various concentrations (as indicated) of nonlabeled E2 with 30 µM Cy3-E2 in each ERR-bound channel.

spiked sample was determined to be around 48.93 ng/mL based on the calibration equation shown in Figure 6, which gives about only 3% deviation from the exact amount (50 ng/mL) spiked, indicating a good accuracy for complicated samples. The specificity of the signal detected for ERR was also investigated by competitive assays performed by coincubating 30 µM Cy3-E2 with various concentrations of nonlabeled E2 solutions ranging from 10-13 to 0 M. Results shown in Figure 6c indicate that the fluorescence intensity decreases with increasing the concentration of nonlabeled E2. There was virtually no signal detected when the E2 concentration was greater than 10-17 M due to a complete displacement of Cy3-E2 by E2. The binding affinity of Cy3-E2 with ERR is weaker than that of nonlabelded

CONCLUSIONS In summary, we have demonstrated a successful integration of a poly(acrylamide)-based hydrogel and PDMS microfluidic devices in fabricating channel-based protein chips by covalently photopatterning affinity plugs in an extremely hydrophilic channel surface. The fabricated device could allow the solution to flow through with almost no resistance and no destruction on the covalently patterned hydrogel microstructure. We also showed that the developed protein G-immobilized hydrogel plugs could be used for immuno-assays in detecting ERR quantitatively with high specificity and high loading capacity. The strategy holds great promises to be used in fabricating other functional microfluidic devices at low cost. ACKNOWLEDGMENT The work was supported by National Science Council in Taiwan. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review May 25, 2009. Accepted August 17, 2009. AC901138W

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