Anal. Chem. 2000, 72, 3253-3259
Segregation of Micrometer-Dimension Biosensor Elements on a Variety of Substrate Surfaces Sunday A. Brooks,† Narasaiah Dontha, C. Brandon Davis, Joan K. Stuart,‡ Geoff O’Neill, and Werner G. Kuhr*
Department of Chemistry, University of California, Riverside, California 92521
With the rapid development of micro total analysis systems and sensitive biosensing technologies, it is often desirable to immobilize biomolecules to small areas of surfaces other than silicon. To this end, photolithographic techniques were used to derivatize micrometer-sized, spatially segregated biosensing elements on several different substrate surfaces. Both an interference pattern and a dynamic confocal patterning apparatus were used to control the dimensions and positions of immobilized regions. In both of these methods, a UV laser was used to initiate attachment of a photoactive biotin molecule to the substrate surfaces. Once biotin was attached to a substrate, biotin/avidin/biotin chemistry was used to attach fluorescently labeled or nonlabeled avidin and biotinylated sensing elements such as biotinylated antibodies. Dimensions of 2-10 µm were achievable with these methods. A wide variety of materials, including glassy carbon, quartz, acrylic, polystyrene, acetonitrilebutadiene-styrene, polycarbonate, and poly(dimethylsiloxane), were used as substrates. Nitrene- and carbenegenerating photolinkers were investigated to achieve the most homogeneous films. These techniques were applied to create a prototype microfluidic sensor device that was used to separate fluorescently labeled secondary antibodies. Molecular recognition is widely regarded as the fundamental step responsible for the specificity of biological processes. Biomolecules performing specific biological functions are progressively employed in the development of miniaturized bioassays, biosensors, and bioelectronic devices.1 Recently, the ability to individually modify small areas of a surface has revolutionized biosensing technology. Lab-on-a-chip and micro total analysis systems are rapidly being developed, mainly using silicon-based substrates owing to the availability of numerous covalent modifiers. However, it is often desirable to use a substrate other than silicon. It is also necessary to obtain homogeneous surfaces on the submicrometer scale as systems become smaller and more * Corresponding author. Phone: 909-787-3485. Fax: 909-787-4713. E-mail:
[email protected]. † Current address: Tippecanoe Labs, Eli Lilly and Co., P.O. Box 685, Lafayette, IN 47902. ‡ Current address: Caliper Technologies Corp., 605 Fairchild Dr., Mountain View, CA 94043. (1) Sigrist, H.; Collioud, A.; Clemence, J.; Gao, H.; Sanger, M.; Sundarababu, G. Opt. Eng. 1995, 35, 2339-2348. 10.1021/ac991453t CCC: $19.00 Published on Web 05/31/2000
© 2000 American Chemical Society
sensitive. Finally, derivatization strategies for homogeneous immobilization of proteins on a small scale are increasingly required for proteomics and clinical chemistry applications. Covalent binding of a functional protein to a surface can result in a strong, stable linkage and high surface coverage, both of which are important in the development of functional biomaterialbased devices. Originally the cross-linking reagents synthesized for the preparation of multisubunit enzymes and protein conjugates in solution were also used for immobilization of enzymes on solid supports.2 For example, Bhatia et al. reported that high surface coverages were obtained by using thiol-terminated silanes and heterobifunctional cross-linkers for immobilization of IgG on silica surfaces.3 Fodor et al. have synthesized multiple arrays of different peptides or oligonucleotides on glass surfaces using caged biotin.4 Biotin/avidin chemistry has been used extensively for protein immobilization.5 Once biotin is linked to a surface, it is very simple to immobilize any biomolecule with an avidin/streptavidin label. Streptavidin is a tetrameric protein that has four identical binding sites for biotin. The binding of biotin to streptavidin is almost irreversible, with a binding strength comparable to that of a covalent bond (Ka ) 1 × 1015 M-1).5,6 Because of this strong interaction and the robustness of the protein, the complex is virtually unaffected by extreme pH, temperature, organic solvents, and other denaturing agents. The tetravalency of avidin for biotin allows the construction of a “molecular sandwich” in which the surface-bound avidin can be coupled to a biotinylated probe molecule that has the appropriate characteristics (i.e., capabilities for substrate consumption and product formation) needed for the construction of a biosensor. Photobiotin, specifically the nitrene-generating nitroaryl azide derivative of biotin, has been used primarily to label proteins and nucleic acids.7 Upon ultraviolet (UV) photolysis, the aromatic nucleus absorbs light, followed by vibrational transmission to the azide group.8 Elimination of nitrogen occurs, generating a reactive (2) Mclean, M.; Stayton, P.; Sligar, S. Anal. Chem. 1993, 65, 2676-2678. (3) Bhatia, S.; Shriver-Lake, L.; Prior, K.; Georger, J.; Calvert, J.; Bredehorst, R.; Ligler, F. Anal. Biochem. 1989, 178, 408-413. (4) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767-773. (5) Wilchek, M.; Bayer, E. A. Anal. Biochem. 1988, 171, 1-32. (6) Fuccillo, D. Biotechniques 1985, 3, 494-501. (7) Forster, A. C.; McInnes, J. L.; Skingle, D. C.; Symons, R. H. Nucleic Acids Res. 1985, 13, 745-761. (8) Iddon, B.; Meth-Cohn, O.; Scriven, E. F. V.; Suschitzky, H.; Gallagher, P. T. Angew. Chem., Int. Ed. Engl. 1979, 18, 900-917.
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uncharged singlet or triplet nitrene.9 Singlet nitrenes react preferentially by insertion into O-H or N-H bonds, but if intersystem crossing occurs to form a triplet, insertion into a C-H bond to form a secondary arylamine is favored.10 Pritchard et al. immobilized avidin on the surface of thiol-covered gold substrates and then added photobiotin, which bound strongly to the immobilized avidin. Illumination of this surface-bound photobiotin resulted in the attachment of antibodies (either rat or rabbit IgG) at specific sites on this substrate.11 Photolithographic masks used in this case created patterns with a spacing of 1.5 µm, near the diffraction limit of the light used. Hengsakul and Cass demonstrated that photobiotin will bind covalently to an organic surface when it is exposed to intense UV light (350-370 nm).12 A polystyrene microtiter plate and a nitrocellulose membrane were illuminated through a mask (50 mesh grid) which formed a pattern corresponding to structures roughly 0.6 mm on a side. Biotin attached to the substrate in this manner was bound with avidin and then with a biotinylated alkaline phosphatase or horseradish peroxidase. Pritchard et al. used photobiotin with masks of 10, 4, and 1.5 µm features to pattern antibodies on silicon and gold surfaces.13 More recently, Flounders et al. described the patterning of antibodies using masks with 15 and 0.4 µm features.14 Mouse monoclonal antibodies were covalently linked through primary amine groups to aminosilanized silicon dioxide films using glutaraldehyde. Photolithographic patterning was performed with a positive photoresist via selective UV exposure through a contact mask. Exposed regions of immobilized antibody were then removed by exposure to a low-power radio frequency oxygen discharge. Successful patterning was demonstrated by challenging surfaces with fluorescently labeled anti-IgG. Previously in this laboratory, a nitrene-generating photobiotin molecule was used to derivatize spatially segregated micrometerdimensional regions for biomolecule attachment.15,16 Two types of maskless patterning techniques were developed. The first technique featured the generation of an interference pattern to create segregated biotinylated lines on glassy carbon.16 Attachment to the carbon surface was achieved by activation of photobiotin in the areas exposed to the UV laser. These regions were then derivatized with Texas Red-labeled avidin (TX Redavidin) and imaged with fluorescence microscopy. Features produced with this technique were approximately 5 µm wide. When the immobilized biotin was derivatized with avidinconjugated alkaline phosphatase and a Vector Red substrate was added, an insoluble fluorescent product was formed via hydrolysis. The detection of this product via fluorescence microscopy with CCD (charge-coupled device) imaging confirms the feasibility of (9) Tsuchiya, T. In CRC Handbook of Organic Photochemistry and Photobiology; Horspool, W. M., Ed.; CRC Press: Boca Raton, FL, 1995; pp 984-990. (10) Bayley, H.; Knowles, J. R. In Methods in Enzymology; Jakoby, W. B., Wilchek, M., Eds.; Academic Press: New York, 1977; Vol. 46, pp 69-114. (11) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Angew. Chem., Int. Ed. Engl. 1995, 34, 91-93. (12) Hengsakul, M.; Cass, A. E. G. Bioconjugate Chem. 1996, 7, 249-254. (13) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Anal. Chem. 1995, 67, 36053607. (14) Flounders, A.; Brandon, D.; Bates, A. Biosens. Bioelectron. 1997, 12, 447456. (15) Brooks, S. A.; Ambrose, W. P.; Kuhr, W. G. Anal. Chem. 1999, 71, 25582563. (16) Dontha, N.; Nowall, W. B.; Kuhr, W. G. Anal. Chem. 1997, 69, 26192625.
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this technique for attaching active biomolecules to surfaces. However, the uniformity of these surfaces was found to vary significantly.17 Our second technique to derivatize segregated micrometerdimension regions of biomolecules utilized a confocal dynamic patterning apparatus.15 UV laser light (325 nm) was focused through a quartz microscope objective and used to initiate attachment of photobiotin to glassy-carbon surfaces. The focused laser spot was used to define areas of derivatization by moving the substrate surface in the x-y directions to “draw” desired patterns. The biotinylated surfaces were then incubated in TX Redavidin and imaged with fluorescent microscopy and CCD imaging. Line widths of 5-10 µm were achieved with this technique. Although these methods have proven successful for the derivatization of sensor surfaces on a micrometer scale, our initial goal was to reduce the dimensions of the feature size. Also, a disturbing trend was observed in the results obtained with the nitrene-generating chemistry. Both techniques described above resulted in films that were extremely heterogeneous along the length of each feature. The uniformity of coverage varied from dense aggregates to bare surfaces within the derivatizated regions.15 Thus, a carbene-generating photolinker is also used here in an effort to obtain homogeneous patterns of biomolecules on glassy-carbon and other substrate surfaces. This molecule uses a (trifluoromethyl)diazarine group which generates a reactive carbene when exposed to UV light.18 With two unbound electrons, the carbene can insert into the C-H and CdC bonds of a substrate surface. The photolinker moiety is attached to a hydrophilic spacer and biotin molecule. Additionally, the substrate was limited to glassy carbon in previous studies, but many other substrate surfaces have been used for biomolecule immobilization and sensor fabrication. For example, the miniaturization of biomolecule analysis to the microchip level makes the use of rigid or flexible polymer substrates desirable. In this work, micrometer-sized domains of glassy-carbon, quartz, and polymer surfaces were chemically modified to attach biosensing elements using a biotin/avidin chemistry similar to that described previously. Both interference patterns and confocal dynamic patterning were used to create derivatized features as small as 2.5 µm on these substrates. A carbene-generating photobiotin was used with a modified protocol to improve the homogeneity of the surface derivatization reaction, leading to better feature reproducibility. Once the surface chemistry was optimized, a prototype flow sensor to detect fluorescently labeled antibodies was fabricated using a polystyrene substrate and PDMS microfluidic channel. EXPERIMENTAL SECTION Chemicals and Materials. Photoactive nitrene-generating biotin was obtained from two sources: EZ-Link Biotin-LC-ASA, 1-(4-azidosalicylamido)-6-(biotinamido)hexane (Pierce, Rockford, IL), and N-(4-azido-2-nitrophenyl)-N′-[3-(biotinylamino)propyl]-N′methyl-1,3-propanediamine (Sigma-Aldrich, St. Louis, MO). The carbene-generating photobiotin N-{4-[3-(trifluoromethyl)diazarin(17) Nowall, W. B.; Dontha, N.; Kuhr, W. G. Biosens. Bioelectron. 1998, 13, 1237-1244. (18) Brunner, J.; Senn, H.; Richards, F. M. J. Biol. Chem. 1980, 255, 33133318.
3-yl]benzoyl}-N′-[3-biotinylaminopropyl]-N′-methyl-1,3-propanediamine in acetate form was obtained from BioLynx LLC (Hagerstown, MD). Texas Red (TX Red) labeled avidin was from Molecular Probes (Eugene, OR), anti-goat IgG tetramethylrhodamine isothiocyanate (TRITC) conjugate and (3-aminopropyl)triethoxysilane (APTES) were obtained from Sigma, and biotinylated IgG was obtained from Pierce. All solutions were prepared with deionized (DI) water from a Barnstead E-Pure system. Rigid polymer substrates (acrylic, polystyrene, acetonitrile-butadienestyrene (ABS), and polycarbonate) were obtained from S&W Plastics (Riverside, CA). Glassy-carbon (GC) plates, 1 mm thick (Alfa Aesar, Ward Hill, MA), and fused-silica cover slips (Escoproducts, Oakridge, NJ) were used as received. Poly(dimethylsiloxane) (PDMS) elastomer was purchased as Dow Corning Sylgard 184 (K. R. Anderson Co), which consists of two components: a base and a curing agent. Preparation of Substrates. GC plates were polished using metacloth and 0.3 and 0.05 µm alumina powder in deionized water on a home-built polishing apparatus. After each polishing, the carbon surfaces were cleaned thoroughly with deionized water to remove any residual polish. The plates were then air-dried in a dust-free environment. Quartz cover slips were cleaned in aqua regia, 3:1 HCl/HNO3 (Caution! This solution is highly corrosive and should be used with extreme care), and washed thoroughly with DI water. After drying, the slips were sonicated in 6 M HCl for 15 min, followed by sonication in DI water. It was necessary to prederivatize these substrates with a carbon-containing reagent in order to facilitate stable insertion of the photobiotin. Therefore, the cover slips were dipped for 5 min each in methanol and acetone and then in 2% APTES solution in acetone for 30 min. Finally, they were rinsed with acetone and dried overnight at 110 °C. The polymer substrates were cleaned with 2-propanol to remove any particles or residues and then rinsed with DI water and air-dried. PDMS elastomer was mixed in a ratio of 10:1 base:curing agent. The solution was stirred for 5 min and degassed for 15 min to eliminate air bubbles. The viscous solution was poured into an aluminum mold and placed in an oven at 100 °C for 1-2 h to cure. The molds were micromachined in the UC Riverside machine shop and featured 75-200 µm wide positive relief features to form microchannels, with pins in either end for inlets/ outlets. Upon extraction, the cured PDMS surface was rinsed with acetone, air-dried, and then exposed to a Tesla coil (Fisher Scientific), which oxidized the surface, making it more hydrophilic. For all substrates but PDMS, approximately 10 µL of photobiotin solution (1 mg/mL in DI H2O) was placed on the surface and dried at room temperature in the dark for 2-3 h. For PDMS, the photobiotin solution (25 µL of 0.5 mg/mL) also contained 0.02% Tween-20 and 50% (v/v) acetone, which improved spreadability and rendered a homogeneous layer of biotin, with a drying time of 1 h. For quartz and glassy-carbon plates, nitrene-generating photobiotin was used. For glassy-carbon, polymer, and PDMS surfaces, carbene-generating photobiotin was used. After drying, the films were patterned using UV light with either a 325 nm HeCd laser or a multiband UV-254/366 nm hand-held lamp (UVP Inc.). Generation of TEM Grid Patterns. For initial experiments on polymer substrates with carbene-generating photobiotin,
transmission electron microscopy (TEM) grids were used as masks. A 400 mesh copper TEM grid (Sigma-Aldrich) was placed onto the dried photobiotin film with a cotton-tipped applicator. Each of the substrates was then exposed to the UV-254/366 nm lamp for 1.5 h to activate insertion of the photobiotin into the substrate surface bonds. Generation of the Interference Pattern. The experimental setup used to produce an interference pattern for photopatterning has been described previously.16 Briefly, a 10 mW UV laser beam (HeCd Omnichrome, 325 nm; Melles-Griot) was divided using a 50% beam splitter and reflected off two mirrors to make two parallel beams of equal intensity. The beams were passed through a 10 cm focal length lens, and the substrate was placed at the focal point where the two beams recombined. The power density of the light used in this case was approximately 1 W/cm2. The reconstruction generated an interference pattern which obeyed Bragg’s law, nλ ) 2D sin(θ/2), where D is the spacing between adjacent maxima of the interference pattern, λ is the wavelength of light, θ is the angle between the laser beams, and n is order. Therefore, the spacing could be modified by changing the angle between the beams. The laser interference illuminated the photobiotin-covered carbon surface for 20-300 s. The Confocal Patterning Microscope Apparatus. The dynamic confocal patterning system was described previously.15 The same HeCd laser was used here to activate the photobiotin. The laser beam was passed through a Leitz-Wetzlar microscope equipped with a 25×, 0.5 N.A. quartz objective. A neutral density filter (5% transmission) was used to attenuate the power of the light reaching the substrate. Power densities for this method were approximately 5 kW/cm2. Surface features were brought into focus by varying the z position while the carbon plate was observed through an eyepiece with illumination from an optical fiber fitted with a 590 nm long pass filter. Sample movement in three directions was controlled using Inchworm piezoelectric motors (Burleigh Instruments) and automated using custom software (CE 6000, D. Wipf) to create raster patterns or lines of specified lengths at specified rates. Spatially Localized Immobilization of TX Red-Avidin. Photobiotin-coated substrates were exposed and patterned using one of the above methods. After exposure, each sample was rinsed thoroughly with phosphate buffer and/or a 1% (v/v) solution of Tween-20. Additionally, 1% Tween-20 was placed on the patterned carbon surface and incubated for 1 h. A sessile drop (50-100 µL) of TX Red-avidin (0.25-0.5 mg/mL in PBS or PBS + 0.5% Tween20) was applied to each of the biotin-patterned surfaces for 1 h. They were then normally sonicated for 5-15 min with 1% Tween20 to remove any nonspecifically adsorbed molecules. All samples were rinsed and imaged (excitation ) 595 nm and emission ) 615 nm) for TX Red-avidin fluorescence. Spatially Localized Immobilization of IgG Antibodies. For detection of secondary antibodies in PDMS flow channels, polystyrene substrates were first patterned with carbene-photobiotin, using the confocal apparatus to create a series of lines spaced 25 µm apart. After washing, a PDMS microfluidic chip was placed on the polystyrene such that fluid flow through the 75100 µm wide sealed channels was perpendicularly exposed to the biotin lines. A solution of ExtrAvidin (0.5 mg/mL) was injected into the channels and incubated for 1 h. After the channels were Analytical Chemistry, Vol. 72, No. 14, July 15, 2000
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rinsed with phosphate buffer, a solution of biotinylated IgG (0.5 mg/mL) was injected and incubated for 1 h. The sample was rinsed again, and a final solution of TRITC-labeled anti-IgG (0.25 mg/mL), which specifically recognizes the primary IgG, was injected. The channels were thoroughly rinsed with buffer, and the immobilized secondary antibody was detected via fluorescence microscopy. Visible and Fluorescence Imaging. Substrate surfaces were imaged using an epifluorescence microscope equipped with a 100 mW Hg arc lamp for illumination. Glassy-carbon and quartz images were collected under darkened conditions with a cooled Thompson 7895B charge-coupled device (CCD) operating at -45 °C. Images were collected through a Photometrics NU-200 controller (16-bit, 40 kHz A/D, Macintosh IIci configuration) and saved on a Macintosh IIci. For polymer data, images were collected under darkened conditions with a cooled SpectraSource MCD-600 CCD. Images were collected with SpectraSource software and saved on a personal computer (PC). Image data processing was done with IP-LAB 2.1.1c (Signal Analytics, Vienna, VA) and Spyglass Transform (Spyglass Software). Reflected visible images of patterned surfaces were focused with a 20× or 40× objective after 100-fold attenuation with neutral density filters. Images were collected with 1-5 ms CCD acquisition times and a camera gain of 1. Fluorescent images of derivatized substrate surfaces were collected by passing light from the Hg lamp through an excitation filter specific for TX Red’s absorption band (595 nm), and fluorescence was collected at wavelengths greater than 615 nm with camera collection times of 0.5-3.0 s. For detection of TRITC-tagged antibodies, excitation was filtered for 540 nm and emission was collected above 580 nm. RESULTS AND DISCUSSION The objective of this work was to create micrometer-scale modifications of a wide variety of surfaces. Most experiments have been directed toward constructing microscopic regions with a specific type and density of immobilized molecules to provide a surface that is optimized for use as a sensor device. The spatially localized modification of carbon ultramicroelectrodes (electrodes of micrometer dimensions) has been accomplished to allow spatial segregation of enzyme-binding sites from electron-transfer sites.16,19 Patterning allows the construction of microscopic arrays of active enzyme sites on a carbon-fiber substrate while leaving other sites underivatized to facilitate electron-transfer reactions of redox mediators and to maximize enzyme activity and detection of the enzyme mediator. In this work, the beam-focusing lens used previously was replaced with one of 10 cm focal length. When the angle between the two beams was adjusted to achieve the smallest features, the recombination point of the two beams resulted in an interference pattern with 2.5 µm spacing. From Bragg’s law, we calculate that the spacing observed results from an angle of 3.6°. Exposure of the photobiotin film to UV light was done over different time intervals ranging from 30 to 200 s. Figures 1 and 2 show the spatial distributions of nitrene-generating photobiotin and TX Red-avidin with 2.5 µm spacings on carbon and quartz surfaces, respectively. Figures 1A and 2A show the (19) Rosenwald, S. E.; Dontha, N.; Kuhr, W. G. Anal. Chem. 1998, 70, 11221140.
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Figure 1. Glassy-carbon substrates derivatized with photobiotin using a laser diffraction pattern: (A) white reflected light image of the pattern formed after exposure (light integrated for 1 ms); (B) fluorescence image of TX Red-avidin attached to the biotin-patterned surface (fluorescence integrated for 1 s).
Figure 2. Quartz substrates derivatized with photobiotin using a laser diffraction pattern: (A) white reflected light image; (B) fluorescence image of TX Red-avidin attached to the biotin-patterned surface. Images were obtained similarly to those in Figure 1.
visible reflected interference patterns of biotin molecules. After the surfaces were patterned with biotin, the substrates were rinsed and TX Red-avidin solutions were placed on the surfaces. Figures 1B and 2B show the TX Red-avidin fluorescence images. The features achieved with these modified optics were twice as small as those previously achieved. However, as before,15,16 some lines were not continuously labeled with TX Red-avidin. It was of interest to investigate the source of the heterogeneity of the resulting fluorescent patterns. One possible source to consider was the functionality of the substrate surfaces. Glassy carbon is well-known for exhibiting a wide range of functional groups, including carboxylic acids, ketones, and alcohols, which could react differently with the nitrene photolinker. However, the heterogeneity was also observed on quartz surfaces, which are covered here with aminopropylsilane. Another possibility was the irregularity of the photobiotin film after evaporation of the solvent. A third possibility was the chemistry of the nitrene photolinker itself. Films of both nitrene- and carbene-based photolinkers were dried on glassy-carbon plates to compare the homogeneities of
Figure 4. White light reflectance (A) and TX Red-avidin fluorescence (B) of carbene-generating photobiotin derivatized on polystyrene. The photobiotin was exposed for 1.5 h through a 400 mesh TEM grid with a UV hand-held lamp. Image A shows little or no observable pattern on the plastic surface. Image B shows very distinct fluorescence in the pattern of the TEM grid. Magnification was 40×.
Figure 3. White light reflectance (A, C, E) and TX Red-avidin fluorescence (B, D, F) images of photobiotin immobilized on various substrates using the laser confocal dynamic patterning apparatus. Patterns A and B were produced using nitrene-generating photobiotin on glassy carbon and imaged at 5× magnification. Patterns C and D were produced using carbene-generating photobiotin on glassy carbon and imaged at 20× magnification. Patterns E and F were produced using carbene-generating photobiotin on acrylic, again with 20× magnification. The samples were moved in a raster pattern at scan rates of 50-100 µm/s and slightly different travel distances. CCD images were obtained with the same conditions as indicated in Figure 1.
biotin films. The films were exposed to UV light using the confocal dynamic patterning apparatus at scan rates of 75-100 µm/s. The software was programmed to draw stair-step raster patterns. Figure 3 shows the reflected light and TX Red-avidin fluorescence images for both nitrene-based (Figure 3A,B) and carbene-based photolinkers (Figure 3C-F). The line widths produced were 5-9 µm. As shown, the films formed by the carbene-generating photobiotin were observably more homogeneous than those produced by the nitrene-generating molecule. It is evident from these data that the photolinker was the determining factor in
producing homogeneous films of biomolecules, owing either to differences in reactivity with the surfaces or to differences in solution distribution/solubility. An additional goal of this work was to investigate, in addition to quartz and glassy carbon, other materials as substrates for the production of sensors on microfluidic chips. The ability to derivatize polymer surfaces using this chemistry was first evaluated using an acrylic substrate. The carbene-generating photolinker was immobilized on the surface using the confocal dynamic patterning apparatus in the same manner as described above for glassy carbon. The reflected light and TX Red fluorescence results for the acrylic substrate, presented in Figure 3E,F, show the homogeneity of derivatization achieved with the combination of carbene-generating photolinker and polymer substrate. Line widths of this pattern were approximately 5 µm. The carbene-generating photolinker was also tested on other polymers and flat PDMS microchips. To assess whether derivatization was possible on these surfaces, photobiotin solution was distributed over a wide area. This was done on multiple samples to determine the initial exposure time necessary to immobilize biotin on the surface. Transmission electron microscopy (TEM) grids (400 mesh) were placed on the substrates as masks to aid in distinguishing the success of immobilization. The films were exposed with a hand-held UV lamp at 254 nm. Average exposure time was 1.5 h; the power density of the lamp was much lower than that of the laser. After being rinsed with Tween-20, the patterned substrates were incubated with 1 mg/mL TX Red-avidin solution and then rinsed and sonicated in Tween-20 to reduce nonspecific adsorption of the avidin to the substrates. Figure 4 shows reflected light and TX Red-avidin fluorescence images on polystyrene after exposure of the photolinker film using a 400 mesh grid as a mask. Unlike the case of glassy carbon, there is no easily observable pattern when reflected light is used to image the polystyrene surface. However, the fluorescence image confirms the presence of immobilized biotin/TX Red-avidin regions in the TEM grid pattern. The squares alternate with bare lines, where the grid was masking the photolinker film; therefore, no derivatization is seen in these areas. There is some fluorescence observed between squares, indicating that nonspecific Analytical Chemistry, Vol. 72, No. 14, July 15, 2000
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Figure 5. White light reflectance (A) and TX Red-avidin fluorescence (B) of carbene-generating photobiotin derivatized on PDMS, observed with a magnification of 20×. Exposure was done in the same manner as indicated in Figure 4.
adsorption was not eliminated completely. Even so, the squares are easily distinguished from areas that were not exposed. The films produced were stable, even after 20 min of sonication in surfactant. Fluorescence was still observed after 1 month of dry storage (data not shown), indicating that, once formed, the biotin/ avidin surface is stable for long time periods. The carbenegenerating photolinker on polymer surfaces proved to be versatile, with similar results obtained for polycarbonate and ABS surfaces (data not shown). Also of interest was derivatization of PDMS surfaces. The molding of PDMS elastomer is a simple and inexpensive method of fabricating microfluidic channels for microchip-based analysis.20 PDMS is well-known for its uses in microcontact printing,21 and is known to be chemically inert. The above procedure using a TEM grid as a mask was repeated on PDMS substrates. Reflected light and TX Red fluorescence images of the grid pattern are shown in Figure 5. A 20× objective was used to observe the uniformity over a larger area. Once again, the TX Red-avidinlabeled biotin regions are easily distinguished from areas where no exposure occurred. The resulting films are homogeneous, similar to those obtained on glassy carbon and rigid polymers. The use of Tween-20 surfactant and acetone in the photobiotin solution lowered the surface energy and resulted in even spreading and distribution of the linker molecules on the hydrophobic surface. Some additional reactivity was undoubtedly encouraged by preactivation of the surface with the Tesla coil. The uniform distribution of avidin can be inferred from the fluorescence images. It is difficult to estimate surface density from fluorescence because of uncertainties in the degree of labeling, fluorophore environment, etc. However, we have acquired atomic force microscopy images of these surfaces (not shown) which show a uniform height distribution over derivatized squares, with heights of approximately 6 nm, corresponding to a monolayer of avidin. This chemistry proved successful at derivatizing a surface that was traditionally considered nonreactive, making the fabrication of microfluidic sensors with PDMS more feasible. Once derivatization was performed on polymer and PDMS surfaces, this technique was further applied for detection of secondary antibodies in a microchannel flow system. The dynamic (20) Effenhauser, C. S.; Bruin, G. J. M.; Paulus, A.; Ehrat, M. Anal. Chem. 1997, 69, 3451-3457. (21) Xia, Y.; Whitesides, G. M. Langmuir 1997, 13, 2059-2067.
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Figure 6. Fluorescence image of secondary antibody detection in a PDMS flow channel. The photobiotin was immobilized on the polystyrene face with the laser confocal apparatus in lines spaced 25 µm apart. The PDMS channel, placed perpendicular to the lines, allowed isolation of avidin, biotinylated IgG, and TRITC-labeled secondary IgG. After incubation and rinsing, the TRITC fluorescence was imaged.
patterning apparatus was used to derivatize a series of biotin lines spaced 25 µm apart on a polystyrene substrate. After sequential immobilization of avidin, biotinylated IgG, and fluorescently labeled secondary IgG onto the patterned area inside the channel, the fluorescence of the TRITC was then imaged with the CCD through the polystyrene and buffer solution (Figure 6). Flow of solution was perpendicular to the fluorescent lines. As the figure indicates, fluorescence was observed only along the lines inside the flow channel, where derivatization was initiated with the dynamic patterning apparatus. The cloudiness of the image is due to the buffer solution and polystyrene that are in the focal path of the microscope. A slight gradient in fluorescence is observed at and beyond the edges of the channel, presumably due to incomplete sealing of the PDMS against the polystyrene surface. The presence and segregation of the target antibody of interest are easily detected using this patterning and immobilization scheme. CONCLUSIONS With this work, we have extended our derivatization methods for the patterning of biomolecules. A carbene-generating photolinker has allowed fabrication of homogeneous regions of immobilized biotin for probe biomolecule attachment to multiple substrates. With the development of sensitive detection methods and rapid immobilization of multiple biological probes, the limitation of miniaturized bioassays can often be economic. The use of polymeric substrates can potentially facilitate the development of disposable test chips by eliminating the need for multiple micromachining steps. Maskless photopatterning using either an interference pattern or a dynamic confocal patterning apparatus allows us to create micrometer-sized domains on carbon, quartz, polymer, and elastomer substrate surfaces. Photopatterning also allows control of the spatial distribution of biomolecules such as antibodies and active enzymes. This technique has been applied here to antibody detection in a microchannel flow-based sensor. In future work, these derivatization techniques will be applied to produce multielement microfluidic sensors with different
biorecognition molecules to detect disease-specific markers. The dynamic confocal patterning apparatus makes possible the attachment of numerous different ligands by sequential spatial addressing of individual zones along a microfluidic channel. In addition, by the incorporation of multiple channels, different avidin-labeled probes can be injected into each channel for capture. Biotin/avidin chemistry provides a robust foundation for further derivatization with biotinylated oligonucleotide sequences and hybridization of complementary DNA targets from complex solutions. In addition, any protein, peptide, or ligand that can be biotinylated or conjugated with avidin can be immobilized using this technique. Thus, these types of devices can see widespread use as bioanalytical and clinical diagnostic tools. Immobilized probes can extract complementary targets from complex solutions. After washing, the targets can be eluted under appropriate conditions and
detected downstream using one of many methods (i.e., fluorescence, electrochemistry, or mass spectrometry). The use of photoactivatable biotin has facilitated the spatial patterning of surfaces that have traditionally been difficult to modify, thus opening new pathways for biosensor fabrication. ACKNOWLEDGMENT This work was supported by the UC BioSTAR Project and by a grant from PE Biosystems. S.A.B. was the recipient of a National Institutes of Health National Research Service Award (5F32DK09770-02). Received for review December 20, 1999. Accepted April 8, 2000. AC991453T
Analytical Chemistry, Vol. 72, No. 14, July 15, 2000
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