Anal. Chem. 1999, 71, 2558-2563
Micrometer Dimension Derivatization of Biosensor Surfaces Using Confocal Dynamic Patterning Sunday A. Brooks, W. Patrick Ambrose,† and Werner G. Kuhr*
Department of Chemistry, University of California, Riverside, California 92521, and Los Alamos National Laboratory, Chemical Science and Technology Division, Los Alamos, New Mexico 87545
Using laser scanning confocal optics in conjunction with avidin/biotin technology, micrometer-sized patterns of biomolecules were fabricated on glassy-carbon and fusedsilica surfaces. Photoactive biotin was immobilized using the 325-nm line of a Helium-Cadmium laser, which was focused through a 25× or 100× quartz microscope objective. A three-dimensional piezoelectric micromanipulator was used to position the sample surface in the focal plane of the microscope objective and to create patterns on the focused surface. Biotin patterns with line widths of 5-20 µm were produced by varying the scan speed of the micromanipulator while exposing the surface to the laser. The integrity of the immobilized biotin was confirmed by subsequent derivatization with fluorescently labeled avidin. Fluorescence microscopy with a cooled charge coupled device (CCD) imaging system was used to visualize the distribution of biotin and fluorescent avidin within the patterns created by the laser. The analysis of very small and complex biological samples with biosensors has been the focus of much analytical research in recent years.1-3 The construction of arrays of micrometer dimension sensor elements each with different selectivity would allow the measurement of many different biological analytes, such as those contained in a pediatric blood sample that is only picoliters in volume. Spatial segregation of biosensor elements can be achieved with photocleavable reagents that are used to protect or deprotect derivatization sites on carbon or other substrate surfaces. Photodeprotection has been widely used to control spatial segregation of biomolecules, chiefly by activation of photosensitive reagents to immobilize proteins.4 Subsequent attachment of proteins has been performed with well-characterized biotin/avidin chemistry.4 The binding of streptavidin, a tetramer protein which binds four biotin molecules is comparable in strength to a covalent bond (Ka ) 1 × 1015 M-1).4,5 Once formed, the complex is stable under conditions of extreme pH and temperature and organic solvents or denaturing reagents. The multiple binding sites of avidin for biotin allow for construction † Los Alamos National Laboratory. (1) Zhai, J. H.; Cui, H.; Yang, R. Biotechnol. Adv. 1997, 15, 43-58. (2) Turner, A. P. F.; Karube, I.; Wilson, G. S. BiosensorssFundamentals and Applications; Oxford University Press: Oxford, 1987. (3) Weetall, H. H. Biosens. Bioelectron. 1996, 11, i-iv. (4) Wilchek, M.; Bayer, E. A. Anal. Biochem. 1988, 171, 1-32. (5) Fuccillo, D. Bio Techniques 1985, 3, 494-501.
2558 Analytical Chemistry, Vol. 71, No. 13, July 1, 1999
of biotin-avidin-biotin “sandwiches” that can be derivatized with biotinylated enzymes or other biosensor elements. To combine photolithography and biotin/avidin technology one can use a photoactive form of biotin. Photobiotin, a nitro(aryl) azide derivative of biotin, has been used for protein and nucleic acid labeling.6,7 During ultraviolet (UV) photolysis, the aromatic nucleus absorbs light, and this is followed by a vibrational transmission to the azide group.8 Elimination of nitrogen occurs, generating a reactive, 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 Since the functionality of glassy carbon exhibits both alcohol and methyl functional groups, either of these mechanisms should result in a bond to the substrate surface. Photolithographic masks have been used to create surface-bound antibody sites with spacing of 1.5 µm.11 It has also been demonstrated that photobiotin will bind covalently to an organic surface when exposed to UV light.12 Polystyrene or nitrocellulose substrates were exposed through a mask to create patterns that were 600 µm on a side. Previously, in this laboratory, a laser interference pattern was used to immobilize 5-µm-wide lines of photobiotin on glassy-carbon surfaces.13 Biotinylated alkaline phosphatase (ALP) was bound to the exposed lines via a biotin-avidin-biotin linkage. The lines were imaged by addition of a substrate that was converted to a fluorescent product by the ALP. Confocal microscopy has become widely used in threedimensional imaging in recent years because of its excellent lateral resolution and sectioning capabilities.14 In contrast to conventional wide field microscopy, the field of illumination in confocal microscopy is confined to a diffraction-limited spot with a diameter as small as approximately half the wavelength. The objective (6) Forster, A. C.; McInnes, J. L.; Skingle, D. C.; Symons, R. H. Nucleic Acids Res. 1985, 13, 745-761. (7) Lacey, E.; Grant, W. N. Anal. Biochem. 1987, 163, 151-158. (8) Iddon, B.; Meth-Cohn, O.; Scriven, E. F. V.; Suschitzky, H.; Gallagher, P. T. Angew. Chem., Int. Ed. Engl. 1979, 18, 900-917. (9) Tsuchiya, T. In CRC Handbook of Organic Photochemistry and Photobiology; Horspool, W. M., Ed.; CRC Press: Boca Raton, FL, 1995; pp 980-984. (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) Dontha, N.; Nowall, W. B.; Kuhr, W. G. Anal. Chem. 1997, 69, 2619-2625. (14) Pawley, J. B. E. Handbook of Biological Confocal Microscopy; Plenum Press: New York, 1990. 10.1021/ac9814546 CCC: $18.00
© 1999 American Chemical Society Published on Web 05/13/1999
projects an image of the illuminating spot into the sample, thus illuminating only a small region. By moving the light source or the sample, specific regions can be illuminated. Typically, a pinhole is placed in front of a photodetector to detect only the reflected light or fluorescence which is in the focal plane of the objective, thus eliminating out-of-focus light. Confocal imaging has been used in the medical field to image a wide variety of samples such as kidney sections.15 Confocal microscopy has also been utilized for its spatial and depth resolution to image surfaces and surface phenomena. For example, localized corrosion, copper electrodeposition, adsorption of organics,16 and electroetching17 have been investigated using confocal laser scanning microscopy (CLSM). In addition, the small probe volume produced by spatial filtering can be used to lower background scatter enough to detect single fluorescent molecules.18 In this work a laser source focused through a quartz microscope objective was used to initiate the reaction and immobilization of photobiotin to glassy-carbon and fused-silica surfaces. The small area of illumination from the objective lens at the focal point allows spatial control of derivatization when sample movement is controlled in three directions by a piezoelectric positioner. Line patterns of immobilized photobiotin with widths of 5-20 µm were produced by positioning the sample surface in the focal plane of the microscope objective and subsequently exposing the surface at varying scan speeds. Fluorescently tagged avidin was then attached to the patterned biotin surface and visualized using fluorescence microscopy and a cooled CCD system. The variability of biotin deposition and the subsequent binding of fluorescently labeled avidin were characterized. EXPERIMENTAL SECTION Chemicals. Photoactive biotin was obtained from Pierce, Rockford, IL, (EZ-Link Biotin-LC-ASA, 1-(4-azidosalicylamido)-6(biotinamido)hexane) and from Fluka Biochemika ((()biotin{3{3-(4-azido-2-nitroanilino)-N-methylpropylamino}-propylamide}). Texas-Red (TX-Red) avidin was used as received from Molecular Probes, Eugene, OR. (3-Aminopropyl)triethoxysilane (APTES) was obtained from Sigma (St. Louis, MO). Glassy-carbon plates, 1-mmthick (Alfa Aesar, Ward Hill, MA), and fused-silica cover slips (Escoproducts, Oak Ridge, NJ) were used as substrates. Atomic Force Microscopy. Glassy-carbon surfaces were imaged with contact-mode atomic force microscopy (AFM). The surfaces were scanned using a Burleigh Personal AFM, model Aris 3500 (Fishers, NY). Pyramidal-shaped silicon tips from Burleigh were used as the cantilevers. Postprocessing of the images was done with True Image SPM software from Burleigh. Patterning Microscope Apparatus. A diagram of the laser scanning patterning system is shown in Figure 1. For carbon substrates, a 10 mW 325 nm HeCd laser (Omnichrome, Chino, CA) was used to excite the photobiotin. The laser beam was passed through a Leitz-Wetzlar microscope using a dichroic mirror through a 25×, 0.5 N.A. quartz objective. Neutral density filters (15) Pawley, J. B. E. Handbook of Biological Confocal Microscopy; Plenum Press: New York, 1995. (16) Chung, D. S.; Alkire, R. C. J. Electrochem. Soc. 1997, 144, 1529-1536. (17) Gu, Z. H.; Fahidy, T. Z.; Damaskinos, S.; Dixon, A. E. J. Electrochem. Soc. 1994, 141, L153-L155. (18) Ambrose, W. P.; Goodwin, P. M.; Enderlein, J.; Semin, D. J.; Martin, J. C.; Keller, R. A. Chem. Phys. Lett. 1997, 269, 365-370.
Figure 1. Simplified diagram of laser scanning confocal microscope. Experimental details in text.
(5% transmission) were used to attenuate the power of the light reaching the substrate. Surface features were brought into focus by varying the z position while observing the carbon plate through an eyepiece with illumination provided by a tungsten lamp and fitted with a 590-nm long-pass filter. Sample movement in three directions was controlled using Burleigh Inchworm piezoelectric motors (Fishers, NY). For exposure of fused-silica substrates, a 100×, 1.3 N. A. neofluar objective from Zeiss (Thornwood, NY) was used. Glycerol was used as an immersion fluid between the fused-silica disk and the objective. Patterning of Carbon Surfaces. Glassy-carbon plates were polished on a home-built apparatus using Metacloth and 0.5-µm alumina powder. The plates were sonicated in deionized water to remove any residual alumina and then dried at 110 °C. Photoactive biotin (from Pierce, 0.5 mg/mL in 75% ethanol) was applied to the carbon by pipetting 5-10 µL of solution onto the polished surface afterwhich the solvent was dried in a dark room. To expose the photobiotin, samples were placed on the translation stage, brought into focus, illuminated with the focused laser through the objective while moving the sample in programmed raster line patterns. Exposure time was controlled by variation of the scan or raster rate, which ranged from 30 to 100 µm/s. After patterning, the samples were rinsed with deionized water and ethanol to remove unbound biotin. Patterning of Silica Surfaces. Fused-silica disks were cleaned in successive steps with aqua regia, hydrochloric acid, methanol, and acetone. The silica surfaces were modified with 2% APTES in acetone for 30 min and dried at 110 °C for 6 h. After modification, 10 µL of photobiotin solution (Fluka, 1 mg/mL) was dried on the APTES-coated slips while in the dark. All subsequent work was performed under yellow lights to prevent photochemical modification of the photobiotin. Macros were written for the sample positioner to draw alphabetic letters. Scan speeds were approximately 5-10 µm/s. After focusing the laser at the photobiotin/fused-silica surface, the sample was moved 50 µm to a fresh spot, and a macro was executed to write a character. When a character was finished, the sample was immediately moved laterally by 50 µm to prevent further exposure. After exposure, the disks were soaked in deionized water to remove unexposed photobiotin. Localized Derivatization of TX-Red Avidin. The patterned samples were first treated with a dilute solution of Tween-20 surfactant for 30 min to reduce nonspecific adsorption. After the surfaces were rinsed with DI water, they were then covered with a TX-Red avidin solution (0.25 mg/mL in D.I. water) for at least Analytical Chemistry, Vol. 71, No. 13, July 1, 1999
2559
1 h. After rinsing with DI water, the samples were imaged for TX-Red fluorescence. Visible and Fluorescence Imaging. Glassy-carbon surfaces were imaged using an epifluorescence microscope using a 100-W Hg arc lamp for epi-illumination. Images were collected in darkened conditions with a cooled Thompson 7895B charge coupled device (CCD) operated 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. Image data processing was done with IP-LAB 2.1.1c (Signal Analytics, Vienna, VA) and Spyglass Transform (Spyglass Software, Champaign, IL). Reflected visible images of patterned surfaces were focused with the Hg arc lamp after 100-fold attenuation with neutral density filters. Images were collected with a 1-ms CCD acquisition time and a camera gain setting of 1. Fluorescent images of patterned carbon surfaces were collected by passing light from the Hg arc lamp through an excitation filter specific for TX-Red’s adsorption band (595 nm), and fluorescence was collected at wavelengths greater than 615 nm with a camera collection time of 0.5-1.0 s. Fused-silica samples were imaged using epi-illumination with a cooled CCD camera. For illumination, the source was 50 mW of 514.5-nm Argon-ion laser light focused through a 22×, 0.65 N.A. Leitz oil immersion objective. Zeiss 518C immersion oil was applied to the backside of the sample to couple the sample to the objective. Fluorescence images were obtained with a 1-second exposure time with a Princeton Instruments LN2 512 × 512 Site CCD camera. RESULTS AND DISCUSSION Previously, in this laboratory, it was shown that a laser interference pattern could be used to produce micrometer-sized, spatially segregated lines of immobilized biotin on glassy carbon.13 Spatial control of derivatization sites is significant not only for production of regions of different selectivity, for example in a sensor array on glass, but also for electron transfer to the electrode surface of a carbon electrode. The objective of this work was to determine a procedure for using an objective-focused laser to “write” desired biomolecule patterns on the surface of a carbon or fused-silica substrate. This technique would allow different sized biotinylated areas and patterns to be defined on one sensor surface without the use of multiple steps or masks. Also, the resolution theoretically possible at the focal point of a microscope objective (half of the wavelength) should allow construction of submicrometer-sized derivatization sites. Before photopatterning and immobilization of biotin was investigated, the glassy carbon surface and the initial application of the photoactive molecules were characterized. An atomic force microscope (AFM) image was obtained of a bare glassy carbon surface (Figure 2a). The surface topography of the polished bare carbon was flat, with 45 nm of variation in the z direction. The surface was then coated with 10 µL of photoactive biotin solution and allowed to dry. The topography of the photoactive biotin film is shown in Figure 2b. In this image, a film several hundred nanometers thick was observed, as can be seen by the increased thickness in the z direction. In addition to the film, large aggregates of biotin were also observed, resulting in large peaks in the AFM image. This indicated that the application of the biotin 2560 Analytical Chemistry, Vol. 71, No. 13, July 1, 1999
Figure 2. Atomic force microscope images of bare glassy carbon and of the same surface after application of 10 µL of photobiotin solution. Each image is a 50 µm × 50 µm section of the carbon surface. The image was processed with plane removal, line removal, and a median filter.
Figure 3. White-light reflectance (a) and TX-Red avidin fluorescence (b) images of photobiotin immobilized on glassy carbon with an unfocused 325-nm HeCd laser using a 100-mesh TEM grid as a pattern. The samples were held stationary and exposed to the laser for 1.5 min. The squares represent immobilized photobiotin, while the lines represent bare substrate. Each data point represents the CCD output and includes a system offset of 1100. The image in (a) was integrated for 1 ms on the CCD camera, while the image in (b) was integrated for 1 s.
to the surface is not uniform but has areas of high and low density of molecules. Photobiotin was then immobilized over a wide area on glassy carbon using the unfocused HeCd laser beam, which had a diameter of approximately 1 mm and an irradiance of 1 W/cm2. This was done on a stationary sample to determine the exposure time necessary to immobilize biotin to the surface over a large area. Transmission electron microscopy (TEM) grids (100 mesh square copper) were placed on the carbon plates as masks to aid in distinguishing the success of immobilization. Glassy-carbon plates with a dried photobiotin film were exposed directly to the laser beam for times varying from 30 s to 10 min to initiate photolysis and insertion of the molecule into the carbon surface bonds. The optimal exposure time to produce uniform coverage of the surface averaged 1.5 min, which corresponded to a fluence of 90 J/cm2. After rinsing the unexposed (not immobilized) photobiotin, a grid pattern of biotin and bare lines was produced (Figure 3a). The average square side was 240 ( 5 µm, with an average line width of 43 ( 7 µm (n ) 5) when viewed with white light. After photopatterning, TX-Red avidin solution was applied to the sample and allowed to bind. The TX-Red fluorescent tag was used to image avidin bound to biotin using fluorescence microscopy. The distribution of TX-Red avidin fluorescence (Figure 3b)
correlates well with the biotin pattern observed with visible light (Figure 3a). The average fluorescent square size was 239 ( 9 µm, with an average line width of 44 ( 7 µm (n ) 5), confirming that avidin was bound specifically within the immobilized-biotin areas. The immobilized-biotin/avidin films could be reproducibly constructed using the above technique. It was observed that, within the squares defined by the TEM grid, the biotin film was heterogeneous, with some areas bare while others were densely covered (Figure 3). Following the exposure of a large area of photobiotin to UV light as described above a 25× quartz objective was used to focus the excitation source down to micrometer dimensions. The approximate radius of the laser spot in the x-y direction at the focal plane can be calculated assuming a uniform beam: rAiry ) 0.61λo/NAobj. Here, λo is the wavelength of light used and NAobj is the numerical aperture of the microscope objective. For experiments with glassy carbon, a N.A. ) 0.5 objective was used with 325-nm light, resulting in a theoretical Airy radius of 0.4 µm. Focused down to a diameter of 0.8 µm (the smallest possible case), the power density of the 10 mW laser used in this work was approximately 2 MW/cm2, or approximately 6 orders of magnitude larger than the unfocused beam. For writing on carbon surfaces, two neutral density filters (5% transmission each) were used to adjust the irradiance to approximately 5 kW/cm2. The experimental scan rates were varied to obtain fluences near 90 J/cm2. Laser treatments have been used in the past to activate electrode surfaces.19-21 Short, intense pulses using Nd:YAG or N2 lasers were shown to increase electron-transfer rates through a combination of active site generation and surface cleaning.22 The effect of the HeCd laser used here on the substrate surfaces was not investigated directly. However, the power densities used in previous studies were much higher (25-110 MW/cm2) than that used in this work (5kW/cm2-1W/cm2). Below a threshold of approximately 20 MW/cm2, little or no effect was seen.20 Therefore, we believe that the laser is initiating the photochemical immobilization reaction without altering the carbon surface in any other way. Glassy-carbon samples coated with unexposed photobiotin were placed on a stage controlled by a three-dimensional piezoelectric positioner. After focusing, the UV light was allowed to pass through the objective to the sample, which was moved in a raster line pattern at a specified scan rate by the positioner. The rate at which the sample was moved (scan rate) varied from 20 to 150 µm/s. Above approximately 75 µm/s, exposure of the photobiotin was incomplete, resulting in little or no immobilization to the carbon surface. Below 25 µm/s, overexposure occurred, resulting in a sparse film which was unstable during TX-Red derivatization and exhibited very weak, if any, fluorescence. It was found that scan rates in the range of 45-75 µm/s were most successful for producing immobilized-biotin patterns that retained the binding affinity for avidin. Figure 4a shows a reflected visible light image of a stair-step raster pattern of biotin bound to a glassy(19) Jaworski, R. K.; McCreery, R. L. J. Electroanal. Chem. 1994, 369, 175181. (20) Rosenwald, S. E.; Dontha, N.; Kuhr, W. G. Anal. Chem. 1998, 70, 11221140. (21) McDermott, M. T.; McDermott, C. A.; McCreery, R. L. Anal. Chem. 1993, 63, 937-944. (22) Sternitzke, K. D.; McCreeery, R. L. Anal. Chem. 1990, 62, 1339-1344.
Figure 4. White-light reflectance (a) and TX-Red avidin fluorescence (b) images of photobiotin immobilized on glassy carbon using the laser focused through a 25×, 0.5 N.A. microscope objective. Magnification for these images was 5×. The sample was moved in a raster pattern at a scan rate of 75 µm/s. CCD images were obtained with the same conditions as those indicated in Figure 3.
carbon surface at 5× magnification. The scan rate for this pattern was 75 µm/s. Lines parallel to the y-axis measured 261 ( 12 µm, while lines parallel to the x axis were 113 ( 7 µm in length. Line widths were 14 ( 2 µm (n ) 5). After derivatization with TX-Red avidin, the fluorescence image was obtained, as shown in Figure 4b. The spots that exhibit fluorescence correlate well with the spots observed with visible-light imaging, as was observed with the TEM grid experiment. The lines parallel to the y axis in Figure 4b were 254 ( 14 µm in length; lines parallel to the x axis were 103 ( 10 µm in length, and line widths were 12 ( 3 µm (n ) 5), indicating that the dimensions of the lines did not change appreciably after derivatization. The width of the raster-pattern lines were an order of magnitude larger than predicted using the Airy equation. One possible reason for the increased line width is the position of the sample relative to the focal plane of the objective (z direction). The sample was initially manually focused by sight at two points on the carbon surface. However, during the scan the sample is not refocused; bumps or variations in the sample surface could cause the sample to be out of the focal plane, exposing it to a larger diameter laser spot. During future experiments, a feedback system will be implemented which will automatically adjust the sample such that the surface is continuously in the focal plane. It was also observed that the biotin immobilization (and therefore the fluorescence) does not form a uniform layer but a grainy pattern, with some areas more densely covered, as was observed with the AFM images of the unexposed-biotin film. This pattern heterogeneity is illustrated in Figure 5, which is a 20× magnification of the sample in Figure 4. To investigate the uniformity of biotin coverage, photobiotin was dissolved in several solvents and applied to clean glassy carbon surfaces. Water, methanol, ethanol, 2-propanol, and dimethyl sulfoxide (DMSO) were used as solvents for photobiotin and either drop- or spincoated onto glassy-carbon plates and then patterned using the procedure described above (data not shown). DMSO caused visible contamination of the carbon, resulting in nonuniform surface coverage. The methanol and 2-propanol solvents produced films that were less uniform than those obtained by water and ethanol. The solvent used to produce the data presented here Analytical Chemistry, Vol. 71, No. 13, July 1, 1999
2561
Figure 5. 20× magnification white-light reflectance (a) and TX-Red fluorescence (b) images of the glassy-carbon sample imaged in Figure 4. Note the heterogeneity of the photobiotin spots within the lines.
Figure 6. Lateral profile of immobilized photobiotin produced on a glassy-carbon surface by exposure to 325-nm light in a raster pattern. The intensities of reflected light (upper plot) and TX-Red avidin fluorescence (lower plot) were plotted as a function of lateral distance along one entire line length of immobilized biotin (parallel to the y axis in Figure 4).
consisted of 75% ethanol/25% water. A second type of photobiotin, previously used in this laboratory,10 produced the most uniform films when dissolved in water. The spatial distribution of biotin and TX-Red avidin along one line scan of the laser was further investigated by plotting intensity as a function of lateral distance along the line (Figure 6). This plot was generated from a slice through a biotin line parallel to the y axis in Figure 4. Both the visible intensity (top plot) and the fluorescence (bottom) confirm the observation that the biotin is distributed in patches along each line. However, the correlation of the fluorescence peaks with the visible-light valleys indicates that the TX-Red avidin is indeed binding specifically to the immobilized biotin. Additional features present in the visible-light plot indicate that TX-Red avidin does not bind to 100% of the immobilized biotin. This could be an indication that a percentage of the biotin is damaged or decomposed during immobilization, rendering it unable to bind avidin. A second possibility is that contaminants in the photobiotin solution are also immobilized or nonspecifically adsorbed. The uniformity of the immobilized-biotin lines was also investigated across the width of a single line. Figure 7 is a plot of visible and fluorescence intensity as a function of lateral distance. The values were taken from a slice through the middle of the line pattern, parallel to the x axis in Figure 4. The width indicated by fluorescence was narrower (22 µm) in this case than that 2562 Analytical Chemistry, Vol. 71, No. 13, July 1, 1999
Figure 7. Lateral profile of one line width of immobilized photobiotin from Figure 4. The intensities of reflected light (upper plot) and TXRed avidin fluorescence (lower plot) were plotted as a function of distance across one line (parallel to the x axis in Figure 4).
Figure 8. Lateral profile across the center of several lines in Figure 4. The upper plot represents reflected visible light intensity, and the lower plot represents the TX-Red avidin fluorescence as a function of distance.
measured using the visible intensity (25 µm). However, the intensity of both measurements is uniform across the entire line, indicating that the film is uniform over the distance of one line width. The uniformity of biotin across several line features was also characterized. Figure 8 indicates the visible and fluorescence intensities of a slice taken across several line features in Figure 4. Both the line widths (12 ( 2 µm, n ) 5) and line spacing (74 ( 7 µm, n ) 5) were uniform across the length of the scan. This indicates that the piezoelectric positioners used are successfully controlling the area of exposure of the sample. Therefore, it is possible, using objective focused laser excitation in conjunction with precise control of sample motion, to spatially control micrometer-dimension deposition of biosensor elements. UV immobilization of photobiotin on fused-silica surfaces was investigated at Los Alamos. Figure 9 shows several letters that were UV-written into the interface between the photobiotin and the APTES/fused-silica layers and subsequently derivatized with TX-Red avidin. Letters were selected to spell out our institutional acronymssLANL and UCR. The letters were 25 µm high, with line widths of 5-10 µm. As can be seen in the figure, other spots of fluorescence were present in addition to the patterned letters. The spots result from nonspecific binding of TX-Red avidin to the fused-silica surface, which was more prevalent on the APTES/ fused silica compared with the glassy carbon. This figure does, however, serve to illustrate that this form of photolithography can be used to write structures on a glass surface as well as on carbon.
single and multiple analytes. Optimization of the confocal optics will be investigated to decrease the line widths/areas of photobiotin-immobilization sites. Upon further investigation, this technique could be capable of producing microscopic arrays of sensor elements that are limited by diffraction to approximately 160 nm. Also, a feedback system that will keep the sample surface in the focal plane will be investigated. Future work with fusedsilica surfaces will focus on prevention of nonspecific binding that occurs during fluorescence derivatization.
Figure 9. Characters written by exposing photoactive biotin to a 325 nm laser through a 100 ×, 1.3 N. A. objective on a fused-silica surface. The images show fluorescence from derivatized TX-Red avidin. The additional spots of fluorescence are due to nonspecific binding of TX-Red avidin to the silica surface. Each letter is 25 µm high with line widths of 5-10 µm. The scale bar in the figure is 10 µm.
The use of CLSM for spatially controlled derivatization of biomolecules on carbon and glass substrates has been demonstrated. This technique will be further investigated to develop micrometer- and submicrometer-dimensioned biosensors for
ACKNOWLEDGMENT This work was supported, in part, by the Environmental Protection Agency (R821325-01) and conducted under the auspices of the US Department of Energy, supported (in part) by funds provided by the University of California for the conduct of discretionary research by Los Alamos National Laboratory. S.A.B. was the recipient of a National Institutes of Health National Research Service Award (5F32DK09770-02).
Received for review December 31, 1998. Accepted March 25, 1999. AC9814546
Analytical Chemistry, Vol. 71, No. 13, July 1, 1999
2563
