Surface Biopassivation of Replicated Poly(dimethylsiloxane

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Anal. Chem. 2001, 73, 4181-4189

Surface Biopassivation of Replicated Poly(dimethylsiloxane) Microfluidic Channels and Application to Heterogeneous Immunoreaction with On-Chip Fluorescence Detection Vincent Linder,†,‡,§ Elisabeth Verpoorte,‡ Wolfgang Thormann,*,§ Nico F. de Rooij,‡ and Hans Sigrist†

Centre Suisse d’Electronique et de Microtechnique (CSEM), CH-2007 Neuchaˆ tel, Switzerland, Samlab, Institute of Microtechnology, University of Neuchaˆ tel, CH-2007 Neuchaˆ tel, Switzerland, and Department of Clinical Pharmacology, University of Bern, CH-3010 Bern, Switzerland

Poly(dimethylsiloxane) (PDMS) appeared recently as a material of choice for rapid and accurate replication of polymer-based microfluidic networks. However, due to its hydrophobicity, the surface strongly interacts with apolar analytes or species containing apolar domains, resulting in significant uncontrolled adsorption on channel walls. This contribution describes the application and characterization of a PDMS surface treatment that considerably decreases adsorption of low and high molecular mass substances to channel walls while maintaining a modest cathodic electroosmotic flow. Channels are modified with a three-layer biotin-neutravidin sandwich coating, made of biotinylated IgG, neutravidin, and biotinylated dextran. By replacing biotinylated dextran with any biotinylated reagent, the modified surface can be readily patterned with biochemical probes, such as antibodies. Combination of probe immobilization chemistry with low nonspecific binding enables affinity binding assays within channel networks. The example of an electrokinetic driven, heterogeneous immunoreaction for human IgG is described. Starting in the early 1990s with the integration of capillary electrophoresis (CE) techniques onto glass chips, an increasing number of applications have been implemented on microfluidic platforms, such as serial/parallel dilutions,1 pneumatic mixing,2 organic synthesis,3 PCR,4 and cell handling.5 Recent developments are well covered in reviews.6-7 In parallel, advances in material sciences made available polymer-based systems that give quick * Corresponding author: (phone) 41 31 632 3288; (fax) 41 31 632 4997; (e-mail) [email protected]. † Centre Suisse d’Electronique et de Microtechnique. ‡ University of Neucha ˆ tel. § University of Bern. (1) Jacobson, S. C.; McKnight, T. E.; Ramsey, J. M. Anal. Chem. 1999, 71, 4455-4459. (2) Hosokawa, K.; Fujii, T.; Endo, I. Anal. Chem. 1999, 71, 4781-4785. (3) Salimi-Moosavi, H.; Tang, T.; Harrison, D. J. J. Am. Chem. Soc. 1997, 119, 8716-8717. (4) Woolley, A. T.; Hadley, D.; Landre, P.; DeMello, A. J.; Mathies, R. A.; Northrup, M. A. Anal. Chem. 1996, 68, 4081-4086. (5) Li, P. C. H.; Harrison, D. J. Anal. Chem. 1997, 69, 1564-1568. (6) Kutter, J. P. Trends Anal. Chem. 2000, 19, 352-363. (7) Bruin, G. J. M. Electrophoresis 2000, 21, 3931-3951. 10.1021/ac010421e CCC: $20.00 Published on Web 07/27/2001

© 2001 American Chemical Society

access to low-cost, replicated microfluidic networks. Among them, poly(dimethylsiloxane) (PDMS) appeared as a material of choice, mainly due to its ease of handling, good sealing properties, and optical transparency.8 Fast prototyping9 enables PDMS channel layouts to be designed and fabricated within 24 h, while further replication requires only a few hours. The use of replicated PDMS microfluidic platforms has been reported for a wide range of applications,10 but many of them suffer from sample adsorption on the elastomer surface and penetration into the PDMS matrix. In fact, due to its hydrophobicity, PDMS strongly interacts with apolar and partly apolar analytes. The PDMS/aqueous interface behaves like the octane/water system: small molecules, such as toluene, will diffuse into PDMS by sorption mechanisms,11 while large molecules containing apolar domains will adsorb onto the surface.12 Several approaches have been proposed to change the channel surface properties from hydrophobic to hydrophilic. Oxygen plasma oxidation is known to introduce silanol groups at the surfaces,9 but due to its instability, the surface slowly recovers its original hydrophobicity. Silanization of the oxidized PDMS surface,13 and the deposition of alternating layers of positively charged Polybrene and negatively charged dextran sulfate,14 have been described. As a result of the latter treatment, capillary zone electrophoresis (CZE) yielded narrow and symmetrical peaks for dopamine and hydroquinone. In a different approach, changes in ζ potential due to adsorption of detergents or quaternary amines on channel walls have been reported,12 but the influence on adsorption was not explored. Furthermore, PDMS channels were modified with protein A to obtain an IgM binding platform, an approach that was found to suppress but not completely abolish (8) Effenhauser, C. S.; Bruin, G. J. M.; Paulus, A.; Ehrat, M. Anal. Chem. 1997, 69, 3451-3457. (9) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974-4984. (10) McDonald, J. C.; Duffy, D. C.; Anderson, J. R.; Chiu, D. T.; Wu, H.; Schueller, O. J.; Whitesides, G. M. Electrophoresis 2000, 21, 27-40. (11) Baltussen, E.; Sandra, P.; David, F.; Janssen, H. G.; Cramers, C. Anal. Chem. 1999, 71, 5213-5216. (12) Ocvirk, G.; Munroe, M.; Tang, T.; Oleschuk, R.; Westra, K.; Harrison, D. J. Electrophoresis 2000, 21, 107-115. (13) Grzybowski, B. A.; Haag, R.; Bowden, N.; Whitesides, G. M. Anal. Chem. 1998, 70, 4645-4652. (14) Liu, Y.; Fanguy, J. C.; Bledsoe, J. M.; Henry, C. S. Anal. Chem. 2000, 72, 5939-5944.

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nonspecific binding (NSB) of enzyme-labeled secondary antibodies.15 However, with phospholipid bilayers deposited on PDMS channel walls, no uncontrolled adsorption was observed.16 Immunoassays (IA) are routinely carried out in reaction vials either manually or using robotic instrumentation. Numerous samples are analyzed in batches, the total analysis time being in the range of 30 min to several hours. Other analytical concepts may perform point-of-care analysis and deliver results within minutes without the need for expensive instrumentation. Most homogeneous and heterogeneous IA rely on the competitive binding of labeled antigens (or free tracers, FT) and antigens to a limited number of antibodies. A homogeneous IA refers to a system where all components are in solution, while in a heterogeneous IA, one of the reactants is immobilized on a solid support. The latter approach enables preconcentration of antigens or antibodies from dilute solutions onto a solid support, yielding lower detection limits and higher sensitivity. Moreover, physical isolation of bound immunocomplexes from unreacted FT can be achieved by simple rinsing of the solid support. Both of these assay formats have been implemented in CE and in chip-based instrumentation.17 The performance of on-chip homogeneous IA for serum cortisol has been described by Koutny et al.18 and for serum theophylline by von Heeren et al.19 Furthermore, a complete theophylline IA has been implemented in the chip format, an approach that included all analytical steps from sample preparation to detection.20 Heterogeneous IA have been carried out in fused-silica capillaries, with antibodies immobilized in a segment of the capillary.21-22 More recently, several applications of heterogeneous on-chip IA have been described. In ref 15, PDMS channels were modified with protein A to obtain an IgM binding platform, while Pyrex channels were coated with protein A for binding IgG in ref 23. In another approach, phospholipid bilayers containing antigen were deposited on PDMS channel walls.16 Development of heterogeneous IA in PDMS microchannels requires the deposition of a coating that prevents material adsorption onto the elastomer. Moreover, the coating should impart specificity for the analyte of interest to the channel surface, with little NSB. Such a coating for PDMS channel surfaces has been realized in this study, based on the consecutive deposition of three layers of biopolymers. The coating passivation performance was investigated for adsorption of various fluorescently labeled molecules. Long-term stability of the surface modification was characterized by electroosmotic mobility changes in the channels, using a neutral fluorescent marker. Furthermore, biotinylated bioprobes were incorporated at specific locations of a microchannel network using a flow patterning technique. The combination of controlled passivation of channel walls and bioprobe immobilization enabled the performance of heterogeneous (15) Eteshola, E.; Leckband, D. Sens. Actuators, B 2001, 72, 129-133. (16) Yang, T.; Jung, S.; Mao, H.; Cremer, P. S. Anal. Chem. 2001, 73, 165-169. (17) Schmalzing, D.; Buonocore, S.; Piggee, C. Electrophoresis 2000, 21, 39193930. (18) Koutny, L. B.; Schmalzing, D.; Taylor, T. A.; Fuchs, M. Anal. Chem. 1996, 68, 18-22. (19) Von Heeren, F.; Verpoorte, E.; Manz, A.; Thormann, W. Anal. Chem. 1996, 68, 2044-2053. (20) Chiem, N. H.; Harrison, D. J. Clin. Chem. 1998, 44, 591-598. (21) Phillips, T. M.; Chmielinska, J. J. Biomed. Chromatogr. 1994, 8, 242-246. (22) Ensing, K.; Paulus, A. J. Pharm. Biomed. Anal. 1996, 14, 305-315. (23) Dodge, A.; Fluri, K.; Verpoorte, E.; de Rooij, N. F. Anal. Chem. 2001, 73, 3400-3409.

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affinity reactions at microchannel intersections. The findings are illustrated with an immunoreaction model consisting of immobilized goat anti-human IgG as probe and fluorescently labeled human IgG as target. Fluorescence of immunocomplexes was quantified in situ with no need for prior mobilization or separation of target molecules. EXPERIMENTAL SECTION Materials and Chemicals. Polished AF45-glass was purchased from Schott Schleiffer (Feldbach, Switzerland). Corning Pyrex 7740 wafers 100 mm in diameter were obtained from Guinchard (Yverdon, Switzerland). The Dow Corning PDMS kit Sylgard 184 was obtained from Distrelec (Na¨nikon, Switzerland). The Cy5 antibody labeling kit and desalting columns (PD-10, prepacked with Sephadex G-25M) were purchased from Amersham Pharmacia Biotech (Du¨bendorf, Switzerland). BODIPY 493/ 503, BODIPY-digoxigenin, BODIPY-labeled dextran (10 kDa), tetramethylrhodamine-labeled dextran (70 kDa) (TMR-dextran), biotin-conjugated dextran (10 kDa), and neutravidin were Molecular Probes products purchased from Juro Supply (Luzern, Switzerland). Mouse IgG (whole molecule, Immunopure), biotinconjugated goat anti-mouse IgG, and biotin-conjugated goat antihuman IgG were manufactured by Pierce (Rockford, IL) and obtained from Socochim (Lausanne, Switzerland). Human serum plasma and Protein G-coated Sepharose beads (Fast Flow 4B) were purchased from Sigma (Buchs, Switzerland). NHS-LCfluorescein (fluorescein-5(6)-carboxamidocaproic acid N-succinimidyl ester) was obtained from Fluka (Buchs, Switzerland). The urine used was from a healthy volunteer. Other reagents were of research grade. PBS refers to phosphate-buffered saline containing 50 mM phosphate buffer (pH 7.4) and 150 mM NaCl. Pyrex Chip Design and Microfabrication. The layout used is depicted in Figure 1a. The channel network was originally designed for a different application; hence, in the context of this study, the three channels shown in gray remained unused. An injection element for definition of picoliter sample volumes, termed a double T-injector for the two T junctions comprising it (see exploded view in Figure 1a), was incorporated onto the chip.24 Constrictions located close to the central channel decreased effects arising from hydrostatic pressure. Narrow/wide channels were 70/350 µm wide and 20 µm deep, with the rounded side wall profile typical of HF wet etching. The process used to fabricate the Pyrex microfluidic chip has been described elsewhere.25 Briefly, the channel network layout was transferred from a chromium mask into a 200-nm polysilicon masking layer by photolithography on a Pyrex wafer. The Pyrex was then wetetched through the openings in the polysilicon by immersion in concentrated HF (49%). The etching time was adjusted to obtain 20-µm-deep channels, and the etched wafer was then sealed with a second, drilled cover Pyrex plate by fusion bonding at 650 °C. PDMS Channel Replication and Preparation of PDMS/ AF45-Glass Devices. The electrokinetic measurements in the plasma-oxidized PDMS/AF45-glass system were carried out in devices featuring the design illustrated in Figure 1a. For that design, a master for replication into PDMS was prepared by (24) Effenhauser, C. S.; Manz, A.; Widmer, H. M. Anal. Chem. 1993, 65, 26372642. (25) Lichtenberg, J.; Verpoorte, E.; Rooij, N. F. Electrophoresis 2001, 22, 258271.

Table 1. Composition, Concentration, and pH of Buffers Used To Study EOF at Constant Ionic Strength (I ) 13.96 mM)

Figure 1. Channel network layouts employed for (a) electrokinetic determinations in Pyrex and plasma-oxidized PDMS/AF45-glass channels (channels in gray were not used), (b) the TERACOAT study in PDMS/AF45-glass devices, and (c) the TERASYS experiments in PDMS/AF45-glass channels. S refers to the separation channels. Injectors are presented on an expanded scale, and the reservoirs are labeled with numbers.

growing a 3-mm-thick nickel layer onto a structured Pyrex wafer (CSEM, Zu¨rich, Switzerland; for procedure refer to ref 26). The original Pyrex substrate was prepared as described above. The layouts shown in Figure 1b and c were reduced to minimal complexity, to focus on surface passivation/biochemistry aspects and to avoid problems arising from complex fluid handling. These two designs were replicated into PDMS using masters made from micromachined silicon wafers (see below). Experiments related to the passivation study and other tests were performed in the layout illustrated in Figure 1b. The channels, which had a rectangular cross section, were 30 µm wide and 20 µm deep. To prevent electrical short circuits between electrodes, the distance between reservoirs was optimized by bending the two side branches of the cross. The design shown in Figure 1c was used for the immunoreaction experiments evaluated with the fluorescence scanner (see below). Rectangular profile channels were 90 µm wide and 8 µm deep. In that structure, the side branches had to be bended in order to fulfill the format requirements of the scanner (substrate size limitation of 76 × 26 mm). Micromachining of 100-mm silicon wafers provided masters for polymer replication of both layouts. AZ1518 positive photoresist (Shipley, Marlboro, MA) was spin-coated onto a wafer pretreated with gaseous hexamethyldisilazane (HMDS) to improve resist adhesion. The polymer was soft-baked on a hot plate at 100 °C for 1 min prior to photolithography with a mask printed on a transparency.9 The channel layout (see Figure 1b and c) was developed in dilute AZ351B developer (Shipley), and the remaining photoresist was hard-baked for 30 min in an oven at 125 °C. The silicon (26) Gale, M. T. Microelectron. Eng. 1997, 34, 321-339.

buffer system

concn (mM)

pH

sodium acetate sodium acetate sodium phosphate sodium phosphate sodium phosphate sodium phosphate sodium phosphate sodium phosphate sodium tetraborate sodium tetraborate sodium tetraborate

94.32 30 13.32 12.51 7.92 6.49 5.13 4.82 6.74 6.74 4.17

4.0 4.7 5.6 6.0 7.0 7.5 8.0 8.5 8.95 9.3 10.0

was etched where unprotected by deep reactive ion etching. Residual photoresist was dissolved in acetone and rinsed off with 2-propanol. Before replication, the silicon surface was treated with octadecyldimethylchlorosilane and fixed in a specially designed holder. PDMS was prepared according to the manufacturer’s directions, by mixing 1 part catalyst with 10 parts base resin. The prepolymer was poured onto the wafer relief and cured at 65 °C for 4 h. The finished PDMS slab was peeled off and 4-mm-diameter access holes to the channels were punched out using a cork borer. The replica was cleaned by ultrasonic treatment in 2-propanol followed by water (5 min each). Thermal aging of the PDMS slab was carried out for 12 h at 115 °C in order to homogenize elastomer surface properties. An unstructured AF45-glass substrate was degreased in hexane (5 min), treated in aqueous HCl 5% (5 min) to render the surface hydrophilic, and rinsed in water. The thermally aged PDMS slab was cleaned in an ultrasonic bath with 2-propanol and then water for 5 min each. Both substrates were dried under vacuum (5 × 10-2 mbar) for 1 h. Reversible sealing of PDMS on AF45-glass was realized without further treatment. Channels were filled with PBS until use. Permanent sealing of the devices was achieved with plasma oxidation of both substrates prior to sealing. Radio frequency-induced 100-W oxygen plasma treatment was carried out for 15 s in a Plasmalab 80plus (Oxford Instruments, Bristol, U.K.) in 0.3 mbar O2. Instrumentation for Assessment of Electrokinetic Chip Properties. The instrument consisted of a modified inverted microscope that was described previously.27 Briefly, argon ion laser light was focused on a microchannel and fluorescence was detected with a photomultiplier tube (H5701.51, Hamamatsu Photonics, Schu¨pfen, Switzerland). Instrumental operation and data acquisition were supported by two DAQPad-6020E boards (National Instruments, Austin, TX). The overall system was computer-controlled, using in-house software written in Labview (National Instruments). Electroosmotic mobility in both Pyrex and PDMS/AF45-glass channels was determined using TMR-dextran as marker compound. A solution of TMR-dextran (50 µM TMR) was prepared in several buffers ranging from pH 4 to 10, with the salt concentration modified in order to keep buffer ionic strength constant at I ) 13.96 mM (see buffer compositions presented in Table 1). Running buffer was electroosmotically pumped through the separation channel for 5-40 s (depending (27) Ramseier, A.; von Heeren, F.; Thormann, W. Electrophoresis 1998, 19, 29692975.

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on the buffer being used). Then, a sample volume was formed for 40-400 s at a double-T (Figure 1a) or a cross (Figure 1b) intersection, under pinching conditions.28-29 Final separation was carried out for 30-80 s, using electric fields of 430-650 Vcm-1. Calculations of the electric field in the separation channel were carried out with Microsim Schematics (8.0 evaluation version, Cadences PCB System Division, Portland, OR). Capillary Zone Electrophoresis in Fused-Silica Capillaries. CZE measurements were carried out on a P/ACE 5510 CE system (Beckman Instruments, Fullerton, CA). Fused-silica capillaries (20/27-cm effective/total length, 50-µm i.d., Polymicro Technologies, Phoenix, AZ) were mounted on a UV absorbance detector assembly (Beckman) working at 214 nm. Fused-silica capillaries were conditioned prior to the measurements with 0.1 M NaOH (6 min), water (6 min), and running buffer (12 min). Sample was injected by positive pressure using 0.5 psi for 5 s. Electrophoretic separation was carried out at 6 kV, while the capillary temperature was maintained at 25 °C. Solutions of TMRdextran (5 µM) and caffeine (10 µM) were prepared in running buffer. Fluorescence Microscopy. The inverted microscope (Axiovert S 100; Carl Zeiss, Zu¨rich, Switzerland) was equipped with a mercury lamp (HBO, 50 W from Osram, Winterthur, Switzerland). Fluorescence measurements were carried out through the bottom AF45-glass substrate. The excitation light was filtered, reflected from a dichroic mirror, and focused onto a microchannel. The collected emission light was directed through the dichroic mirror and filtered. Three set of filters and dichroic mirrors were available for dyes related to fluorescein, rhodamine, or Cy5. The emission light beam was focused on a CF 8/4 DXC black-and-white CCD camera (Kappa, Gleichen, Germany). The camera signal was analyzed with software developed by Kappa. Fluorescence Scanner. A commercial fluorescence scanner (428 Array Scanner) developed by Affymetrix (Santa Clara, CA) was used for quantification of fluorescence intensity in the immunoreaction experiments. The surface was scanned by a moving confocal microscope located above the device. Excitation light was generated by an 8-mW, 635-nm laser diode, and 665-nm filtered emission light was detected by a PMT. Spatial resolution of the scanner was achieved with 10-µm pixelation. Scanning of 90-µm-wide channels revealed quantitative results. Fluorescence measurements were obtained by observation through the PDMS replica. Due to the 1.5-mm working distance of the lenses, the PDMS replica sealed on the devices was made to have a thickness of only ∼0.8 mm. These thinner replicas, obtained by pouring a lesser amount of prepolymer on the master, were separated from the master without difficulties. Despite the decrease in rigidity of the PDMS slab, channels did not show signs of distortion. The channel network was located out of the focal range of the instrument optics. Thus, proper alignment in the z direction was achieved by increasing the substrate thickness using strips of tape 200 µm thick applied along the edges of the device on the glass side. Purification of Biotin-Conjugated Goat Anti-Mouse IgG. Removal of BSA. Commercial preparations of the biotin(28) Jacobson, S. C.; Hergenroder, R.; Koutny, L. B.; Warmack, R. J.; Ramsey, J. M. Anal. Chem. 1994, 66, 1107-1113. (29) Shultz-Lockyear, L. L.; Coyler, C. L.; Fan, Z. H.; Roy, K. I.; Harrison, D. J. Electrophoresis 1999, 20, 529-538.

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conjugated goat anti-mouse IgG contain bovine serum albumin (BSA) for stabilization purposes (typically IgG/BSA 1:15). Removal of the BSA was achieved by immunoprecipitation of the IgG with protein G-coated Sepharose beads. Beads were sedimented by centrifugation at 5000 rpm for 2 min. A 350-µL aliquot of commercial bead suspension was washed three times with PBS and incubated 90 min with 400 µL of 1 mg/mL biotin-conjugated goat anti-mouse IgG in PBS. Beads were washed three times with PBS and once with 100-fold diluted PBS in water (decrease of buffer capacity prior to addition of acidic chaotropic buffer). A 200-µL aliquot of 100 mM glycine-HCl buffer (pH 2.7) was then added. After a 15-min incubation, the supernatant was collected and the extraction was repeated twice, without incubation. A PD10 column was conditioned with PBS, and glycine extracts were chromatographed in PBS. The IgG solution was diluted, to reach a UV absorption A280 ) 0.134. The final IgG concentration was estimated to be 200 µg/mL. Fluorescent Labeling of IgG with NHS-Activated Dyes. Human IgG was modified with NHS-Cy5 and mouse IgG with NHS-LC-fluorescein. The fluorescent labeling procedure is described for mouse IgG. Mouse IgG (75 µL of an 11.3 mg/mL solution in PBS) was diluted with 75 µL of 50 mM carbonate buffer (pH 9.3) and mixed with NHS-LC-fluorescein (0.5 mg in 10 µL of DMF). The mixture was allowed to react for 2 h at 0 °C. Buffer exchange and excess reagent removal was achieved by PD-10 chromatography in PBS. Absorption measurements of the fractions at 280 and 494 nm yielded both IgG and fluorescein concentrations. Dilution to 200 µg/mL yielded a 0.7 µM bound fluorescein concentration. Three-Layer Coating of PDMS/AF45-Glass Devices (Referred to as TERACOAT (Ternary Affinity Coating)). Each layer was sequentially deposited under the same conditions. The procedure is exemplified here for the first layer. Reservoirs 1-3 (Figure 1b) were filled with a 200 µg/mL solution of BSA-free biotin-conjugated goat anti-mouse IgG in PBS. Vacuum (50 mbar) was applied at reservoir 4 for 2 min. The system was left at rest for 3 min and then all reservoirs were rinsed with PBS. Vacuum was applied once more at reservoir 4 for 2 min. The second layer was formed by treatment with neutravidin (200 µg/mL in PBS) and the third layer by treatment with biotin-conjugated dextran (10 mg/mL in PBS). RESULTS AND DISCUSSION Solute Adsorption to PDMS. Electroosmotic flow (EOF) originates as a result of the presence of surface charges on channel walls. Monitoring EOF thus is a useful tool to investigate surface modification and stability. Electroosmotic mobility in Pyrex and PDMS/AF45-glass channels was measured by recording the detection time of an injected neutral marker in devices having the layout shown in Figure 1a. Two commercially available fluorescent and nominally neutral compounds, TMR-dextran and BODIPY 493/503, both of which could be excited by an argon ion laser, were employed. CZE-UV analysis of TMR-dextran and caffeine (a neutral marker absorbing in the UV) in a fused-silica capillary using three buffers, namely, a 30 mM sodium acetate buffer, pH 4.7, a 7.92 mM sodium phosphate buffer, pH 7.0, and a 6.74 mM sodium tetraborate buffer, pH 9.3 (Table 1), revealed that the two compounds had identical detection times in all three cases. Thus, TMR-dextran could be used as a fluorescent marker

Figure 3. Adsorption/sorption of fluorescent molecules at PDMS/ water interface: (a) 50 µM TMR-dextran (hydrophilic sugar backbone with apolar side groups); (b) 20 µM BODIPY 493/503 (neutral and apolar). Dye solutions in 6.74 mM sodium tetraborate buffer (pH 9.3) were drawn into the channel by vacuum pumping and left 2 min for incubation. The channel was then rinsed with vacuum-pumped PBS.

Figure 2. Use of TMR-dextran for electrokinetic measurements in microchannels with the layout shown in Figure 1a. (a) pH dependence of electroosmotic mobility in Pyrex channels. (b) Electrophoregrams obtained in a plasma-oxidized PDMS/AF45-glass channel 3, 6, and 9 h after oxidation. Channels were filled with PBS directly after oxidation and were kept filled between experiments. Experimental conditions: (a) 20-µm-deep and 70-µm-wide Pyrex channel, total length 6 cm, all buffers ionic strength I ) 13.96 mM (Table 1), electrokinetic transport of marker under an electric field E ) 650 V cm-1 (buffers pH 4.0-8.5) and E ) 430 V cm-1 (buffers pH 8.9510), LIF detection (488-nm excitation), pinched sample injection; (b) 20-µm-deep and 70-µm-wide oxidized PDMS/AF45-glass channel, 5 cm to LIF detector (488-nm excitation); 50 µM TMR-dextran in 7.92 mM sodium phosphate buffer, pH 7.0 (running buffer), pinched sample injection, and electrokinetic transport under an electric field of 500 V cm-1.

to determine the pH dependence of electroosmosis in Pyrex channels (Figure 2a). Although the maximum λex of the tetramethylrhodamine group of TMR-dextran lies at 555 nm, excitation was found to be possible using the Ar ion laser at 488 nm and a TMR-dextran concentration of 50 µM. However, the hydrophobic domains of TMR-dextran showed a great affinity for the PDMS surface, resulting in strong adsorption (see Figure 3a). Interaction of BODIPY 493/503 with PDMS was even larger, as BODIPY appeared to penetrate into the PDMS bulk by sorption mechanisms (see Figure 3b), an effect that was previously also mentioned by Ocvirk et al.12 This effect was not observed for TMRdextran, probably because sorption was prevented by its hydrophilic carbohydrate backbone. In another investigation, the PDMS surface was plasma-oxidized in order to generate a hydrophilic surface and to see whether the fluorescent markers could be used

under these conditions. It was observed that TMR-dextran adsorption on the surface was strongly reduced, but BODIPY 493/ 503 sorption into the elastomer bulk was still substantial. Electrokinetic characterization of the oxidized channels was carried out with TMR-dextran. Detection times were observed to be stable, but peak intensity was found to decrease rapidly (see Figure 2b). No peaks could be detected 9 h after plasma treatment, though the oxidized channels had been in constant contact with phosphate buffer at room temperature. This indicated that surface reconstitution of plasma-oxidized PDMS was taking place within hours after treatment. This change was determined to have a minor influence on the ζ potential (detection times were stable; see Figure 2b) but severely affected the adsorption/sorption properties of PDMS. The mechanism responsible for that phenomenon was not investigated. However, electroosmosis in oxidized PDMS/Pyrex channels just after plasma oxidation could be estimated using TMR-dextran as marker. For instance, in 7.92 mM phosphate buffer (pH 7.0), the electroosmotic mobility was calculated to be 4.2 × 10-4 cm2 s-1 V-1 a value that compares well to that of plain PDMS reported elsewhere.12 BODIPY 493/503 could not be used for untreated and plasma-oxidized PDMS/Pyrex channels. These preliminary observations demonstrated the need for a stable hydrophilic surface treatment of PDMS. TERACOAT Biopassivation of the PDMS Surface. Adsorption on the native PDMS surface was controlled by deposition of a coating made of three successive layers of biopolymers (Figure 4). The strong adsorptive properties of PDMS were exploited for the physisorption of biotin-conjugated IgG. Neutravidin was then linked by affinity interaction, producing a second layer. Finally, a layer of biotin-conjugated dextran was bound to not-occupied biotin binding sites of tetravalent neutravidin. The overall surface Analytical Chemistry, Vol. 73, No. 17, September 1, 2001

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Figure 4. Schematic diagram of the coating used for passivation: (1) biotin-conjugated goat anti-mouse IgG; (2) neutravidin; (3) biotinconjugated dextran.

treatment was called TERACOAT. The presence of each layer of TERACOAT was demonstrated by fluorescence microscopy using labeled species. The coating capacity to reduce adsorption was tested for a wide range of fluorescently labeled molecules. IgG interacts strongly with a native PDMS surface, but after TERACOAT deposition, Cy5- and fluorescein-labeled mouse IgG in 200 µg/mL solutions in PBS did not adsorb onto channel walls. Similarly, TMR-dextran (50 µM TMR, prepared in 6.74 mM tetraborate buffer, pH 9.3) and BODIPY-dextran (150 µM BODIPY, prepared in 6.74 mM tetraborate buffer, pH 9.3), both of which contain a large sugar backbone modified with apolar fluorescent side groups, did not interact with the coated surface. Smaller molecules such as BODIPY-digoxigenin (20 µM in PBS) were also prevented from adsorption by TERACOAT channel modification (see Figure 5). However, completely apolar and neutral molecules penetrated into the PDMS bulk despite the presence of the coating. Such a sorption mechanism was observed with BODIPY 493/503 (25 µM in 1:10 (v/v) methanol/7.92 mM phosphate buffer, pH 7.0) (data not shown). Coating stability in various liquids was studied by fluorescence microscopy using a TERACOAT containing fluorescein-labeled neutravidin. Desorption was monitored by recording the loss of fluorescence in the coating. TERACOAT was shown to be stable for 1-h exposure to water, 7.92 mM phosphate buffer (pH 7.0), and 6.74 mM tetraborate buffer (pH 9.3). All solutions were continuously drawn through the channel using vacuum. Moreover, no degradation was observed when biological matrixes such as serum or human urine were applied. However, the coating was slowly damaged by weak detergents. After 1-h exposure to Tween 20 (0.02% (v/v) in 7.92 mM phosphate buffer, pH 7.0) and 1-min rinsing with 7.92 mM phosphate buffer (pH 7.0), fluorescence was reduced by ∼50%. TERACOAT desorption was observed in the presence of sodium dodecyl sulfate (SDS, 10 mM in 6.74 mM tetraborate buffer, pH 9.3), or 0.1 M NaOH. After 1 h of exposure and 1-min rinsing with 7.92 mM phosphate buffer (pH 7.0), the fluorescence due to the TERACOAT could not be distinguished from background. The long-term stability of TERACOAT was investigated by CZE using a 6.74 mM tetraborate buffer (pH 9.3). Over one month, intermittent measurements were made with two microchannel structures having the layout shown in Figure 1b. Data obtained with one structure are presented in Figure 6a. The data of the 4186 Analytical Chemistry, Vol. 73, No. 17, September 1, 2001

Figure 5. Changes in adsorption after TERACOAT deposition in PDMS/AF45-glass channels: (a) no treatment; (b) with TERACOAT modification. Fluorescence microscope pictures were acquired using identical settings. A 20 µM solution of BODIPY-digoxigenin in PBS was drawn into the channel by vacuum pumping and left 2 min for incubation. The channel was then rinsed with vacuum-pumped PBS.

second structure were similar and are thus not shown. Electroosmotic mobility was calculated from the detection time of TMRdextran, with the detector placed 1.35 cm distant to the cross injector. Measurements were obtained employing a two-stage sequence, consisting of (i) the formation of a plug under pinching conditions and (ii) plug transport under an electric field of 825 V cm-1. For every time point, 10 measurements were taken within ∼1 h. Mean values of detection times are shown to decrease ∼2fold, the largest drop being observed during the first 100 h (Figure 6a). Consequently, calculated electroosmotic mobilities were found to increase from about 1.5 × 10-4 to 2.8 × 10-4 cm2 s-1 V-1. These values were found to be considerably smaller compared to those observed in uncoated Pyrex (Figure 2a), PDMS/AF45-glass (see above), and plain PDMS12 channels. RSD values of detection times were found to be less than 2%, indicating that operation of the chip was reproducible on the hour time scale. For all these experiments, the shape of the TMR-dextran peak did not change (Figure 6b). Thus, the favorable passivation properties of TERACOAT appeared to be stable under CZE conditions over longer periods of time and the devices could thus be employed in the presence of an internal standard. No further investigations to elucidate the reasons for the temporal mobility change were undertaken. Antibody Patterning and On-Chip Heterogeneous Immunoreaction. A variation of TERACOAT deposition enabled the immobilization of a biochemical probe in a specific area of the channel network. For that purpose, the structure depicted in Figure 1c was used. This surface preparation was named TERASYS (referring to ternary affinity system). The first layer (biotinconjugated goat anti-mouse IgG) and second layer (neutravidin)

Figure 6. Long-term stability of TERACOAT treatment in PDMS/ AF45-glass channels. (a) Mean detection time (n ) 10) of TMRdextran (right-hand y-axis) and calculated EO mobility (left-hand y-axis) determined by intermittent measurements over a 1-month period. (b) Recorded peak of TMR-dextran 3 and 700 h after TERACOAT deposition. Experimental conditions: running buffer (RB), 6.74 mM tetraborate buffer (pH 9.3); sample, 50 µM TMR-dextran in RB; E ) 825 V cm-1; LIF detection (488-nm excitation); pinched sample injection.

were deposited as described above for TERACOAT. Afterward, reservoir 4 was filled with the biotin conjugate of a bioprobe (e.g., 200 µg/mL antibody in PBS). Both neighboring reservoirs (1 and 3) were filled with biotin-conjugated dextran (10 µg/mL in PBS), and vacuum was applied at reservoir 2 for 2 min (see Figure 7a). While suction pumping was maintained, all three reservoirs were rinsed with PBS, and channel rinsing was carried out for 2 min. At the channel intersection, three converging laminar flows do not mix, a phenomenon that has been observed previously.30-33 This spatial separation of laminar flows takes place because, at milliliter per minute flow velocities, diffusion is not fast enough to enable solute mixing. Thus, spatially resolved deposition of the molecules on the surface can be achieved and no antibodies could be detected outside the patterned area. Immobilization of the bioprobe took place in the central part of the cross, while biotinconjugated dextran was bound along either side (see Figure 7a). The TERASYS concept was demonstrated with a biotin-conjugated (30) Zhao, B.; Moore, J. S.; Beebe, D. J. Science 2001, 291, 1023-1026. (31) Kenis, P. J.; Ismagilov, R. F.; Whitesides, G. M. Science 1999, 285, 83-85. (32) Kamholz, A. E.; Weigl, B. H.; Finlayson, B. A.; Yager, P. Anal. Chem. 1999, 71, 5340-5347. (33) Ermakov, S. V.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1998, 70, 44944504.

Figure 7. Schematic representation of the flow patterns applied for an on-chip immunoreaction. (a) Bioprobe patterning: the biotinconjugated goat anti-human IgG was immobilized on the neutravidincoated surface. The immobilization pattern followed the laminar flow profiles generated by vacuum pumping at reservoir 2. (b) Immunoreaction is taking place at the intersection where antibodies are immobilized and in contact with sample (hatched area). Cy5-human IgG (antigen) was delivered across the antibody-coated area using a two-step electrokinetic process. Side buffer flow was kept much smaller than sample flow, such that no flow patterning was observed at the cross. Sample was brought to the cross area during the first step under an electric field of 300 V cm-1 (75 s, -600 V at reservoir 1, -420 V at reservoirs 2 and 4, ground at reservoir 3), whereas incubation was carried out during the second step under an electric field of 60 V cm-1 (360 s, -120, -84 and 0 V at reservoirs 1, 2/4, and 3, respectively). (c) Unbound antigen was removed by electrokinetic pumping (60 s, -72 V at reservoirs 1 and 3, -600 V at reservoir 2, ground at reservoir 4) without removing the bound antigen (hatched area). All reservoirs were then rinsed with running buffer.

goat anti-human IgG/Cy5-human IgG (immobilized probe/target) immunosystem. Biotin-conjugated goat anti-human IgG was patterned into the channel network, and channels were stored in running buffer consisting of PBS. As shown in Figure 7a, antibodies were immobilized along a strip running from reservoirs 4 to 2, while the remaining channels were effectively passivated with TERACOAT. A solution of Cy5-human IgG (200 µg/mL (1.33 µM) in PBS) was electrokinetically transported from reservoirs 1 to 3 (see Figure 7b). It is interesting to note that, under the chosen conditions, Cy5-human IgG is negatively charged and migrates against EOF and is thus brought to the sensing area by electrophoresis and not electroosmosis. Incubation was carried out for 6 min, and the sensing area was subsequently rinsed with running buffer for 1 min using electroosmotic fluid pumping (Figure 7c). Three important pieces of information can be extracted from the imaged sensing area determined by the fluorescence scanner (Figure 8). First, the binding of Cy5-human IgG to the immunoprobe can be quantified. Second, light detected in the bottom left (or top right) channel reveals the system background value (such as scattered light at material interfaces, for instance). Third, NSB of Cy5-human IgG to the coated surface can be extracted from Analytical Chemistry, Vol. 73, No. 17, September 1, 2001

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Figure 8. Bound antigen image at the cross area obtained with the fluorescence scanner. Signal is observed at the intersection where the biotin-conjugated goat anti-human IgG pattern is immobilized (Figure 7a) and immunostained with Cy5-human IgG (Figure 7b). Information on background levels and NSB can be extracted from the same experiment. Quantification was performed by averaging the fluorescence intensity from four pixels (total area of 400 µm2) in the regions of interest.

Figure 9. Fluorescence quantification determined with the fluorescence scanner of (a) background, (b) nonspecifically bound antigen, and (c) specifically interacting antigen (logarithmic scale). TERASYS consisted of immobilized goat anti-human IgG at the cross section and Cy5-human IgG as antigen (200 µg/mL ) 1.33 µM). Data represent average values from eight disposable TERASYS devices.

the top left (or bottom right) channel. The extent of analyte-specific binding is obtained by subtracting the NSB value from the measured signal, and sample compatibility with TERACOAT is quantified by the difference between the background value and NSB. Eight disposable TERASYS chips were tested, and signal intensity appeared to be reproducible (Figure 9). Quantitative fluorescence measurements were obtained using the fluorescence scanner, because its sensitivity was far beyond any other instrument used in this study. An average value of 18380 RFU was obtained, associated with a chip-to-chip RSD of 18%. Background 4188 Analytical Chemistry, Vol. 73, No. 17, September 1, 2001

Figure 10. Immunoactivity of the IgG located in the first layer of the TERASYS device assessed by fluorescence microscopy. Binding of fluorescein-conjugated mouse IgG to goat anti-mouse IgG present in the third layer of the TERASYS is observed at the center of the cross. Weaker fluorescence is detected in the channel running from top to bottom, where fluorescein-conjugated mouse IgG was electrokinetically introduced. The experiment was carried out in a PDMS/ AF45-glass device, with the design depicted in Figure 1b. The immunoreaction was performed with the same sequence as described in Figure 7. Goat anti-mouse IgG was immobilized between reservoirs 2 and 4. Fluorescein-mouse IgG (antigen) was electrokinetically pumped in a two-step procedure across the antibody-coated area. Sample was supplied to the cross during the first step under an electric field of 1130 V cm-1 (15 s, -1440 V at reservoir 3, -1140 V at reservoirs 2 and 4, ground at reservoir 1). Incubation was carried out during the second step under an electric field of 113 V cm-1 (240 s, -144, -114, and ground at reservoirs 3, 2/4, and 1, respectively). Free antigen was removed by electrokinetic pumping (60 s, -1320 V at reservoir 2 and ground at all other reservoirs). All reservoirs were then rinsed with running buffer.

values reached 40 RFU. Fluorescence related to Cy5-human IgG NSB was 89 RFU, about twice the background value. A signalto-noise ratio of 206 was achieved. Hence, the system was found to be fully adequate for a Cy5-human IgG immunoreaction. The activity of the IgG located in the first (bottom) layer of the system (biotin-conjugated goat anti-mouse IgG) was qualitatively investigated using a different TERASYS. That device was prepared as described above, using the same biotin-conjugated goat anti-mouse IgG for the ground layer and the patterned third layer. Fluorescein-conjugated mouse IgG (antigen) was then electrokinetically pumped across the channel intersection. Antigen was shown to react with the immobilized IgG located in the third layer. However, some binding was also observed with the IgG situated in the ground layer (see Figure 10). This demonstrates that antigen can penetrate into the coating and interact with its corresponding antibody. Care should therefore be taken when designing an assay to avoid cross reactivity between sample and any coating constituent. Advantages of the TERACOAT and TERASYS Approach. The approach developed by Eteshola and Leckband15 relies on physisorption of BSA on the channel wall and glutaraldehyde binding of protein A. When ELISA assays were run, the NSB of the enzyme-labeled secondary antibodies appeared relatively high. That drawback was circumvented by addition of serum to the sample matrix. The TERACOAT approach shows superior passi-

vation efficiency and suppresses the requirement for a blocking agent during an assay. The phospholipid bilayer principle reported by Yang et al.16 appeared very efficient for suppressing NSB. In that work, antigens were introduced into the system in the form of phospholipid-bound antigens. Due to the mobility of individual lipids within the bilayer, phospholipid-bound antigens became homogeneously distributed over the entire microchannel network. The latter aspect may become a major drawback for setting up parallel immunoassays within a unique microchannel network. For such a system, the bioprobes have to be localized into definite zones, where the signal readings can be carried out individually. Preliminary results described in this contribution show that the TERASYS concept enables the immobilization of defined zones of different antibodies on structures containing multiple channel intersections. Individual addressability of different immobilized bioprobes on a device is also a crucial point for building up parallel immunoassays. This is made possible through use of microfluidic networks such as those presented in this study. CONCLUSIONS A simple coating procedure has been established to passivate the hydrophobic PDMS surface while maintaining a residual surface charge capable of producing a modest EOF. The coating is stable in biological fluids and detergent-free CE buffers. Strongly reduced adsorption (low NSB) makes its use possible for heterogeneous affinity assays. The coating concept enables

integration of any biotinylated probe into the third layer. Moreover, the probe-patterning technique used in the channel network permits the quantification of bound target, NSB to the coated surface, and background fluorescence, on a single TERASYS device. Here, a representative example showed antibody immobilization and subsequent immunoreaction performance with a fluorescent antigen. The signal-to-noise ratio was found to be 206, while fluorescent antigen NSB to the treated surface was suppressed to a large degree. Bound antigen quantification showed that fluorescence intensity was reproducible chip to chip and day to day. Data from eight disposable chips showed a relative chip-to-chip standard deviation of 18%. In the future, changes in the assay format will be investigated in order to perform parallel competitive immunoassays for clinically relevant substances. ACKNOWLEDGMENT V.L. is a recipient of a CSEM university collaboration grant. The authors are grateful to Dr. J. So¨chtig for producing the nickel shim. This project was partly sponsored by a grant from the Swiss National Science Foundation (31-58946.99)

Received for review April 11, 2001. Accepted June 18, 2001. AC010421E

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