A Far-Field-Viewing Sensor for Making Analytical Measurements in

Jun 12, 1999 - We demonstrate a far-field-viewing GRINscope sensor for making analytical measurements in remote locations. The GRINscope was fabricate...
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Anal. Chem. 1999, 71, 2766-2773

A Far-Field-Viewing Sensor for Making Analytical Measurements in Remote Locations Karri L. Michael, Laura C. Taylor, and David R. Walt*

The Max Tishler Laboratory for Organic Chemistry, Department of Chemistry, Tufts University, Medford, Massachusetts 02155

We demonstrate a far-field-viewing GRINscope sensor for making analytical measurements in remote locations. The GRINscope was fabricated by permanently affixing a micro-Gradient index (GRIN) lens on the distal face of a 350-µm-diameter optical imaging fiber. The GRINscope can obtain both chemical and visual information. In one application, a thin, pH-sensitive polymer layer was immobilized on the distal end of the GRINscope. The ability of the GRINscope to visually image its far-field surroundings and concurrently detect pH changes in a flowing stream was demonstrated. In a different application, the GRINscope was used to image pH- and O2-sensitive particles on a remote substrate and simultaneously measure their fluorescence intensity in response to pH or pO2 changes. Optical fibers have the ability to carry light long distances with minimal transmission loss. The inert materials comprising optical fibers can withstand harsh environments such as high temperatures or salt concentrations. The optical signals are not affected by electromagnetic interference. The flexibility and small dimensions of optical fibers, typically only hundreds of micrometers in diameter, allow them to be deployed in remote or inaccessible places. Since optical fibers are amenable to a wide variety of situations, they are often used in combination with analytesensitive dyes to fabricate chemical sensors and biosensors.1-9 Similarly, Gradient index (GRIN) lenses have been used as fiber-fiber couplers, as light source-fiber couplers, and in chemical sensors to enhance light collection and detection. For example, GRIN-rod lenses have been employed as an optical fiber collimator or connector10,11 and GRIN lenses have been used for (1) Milanovich, F. P.; Hirschfeld, T. B.; Wang, F. T.; Klainer, S. M.; Walt, D. R. Proc. SPIE-Int. Soc. Opt. Eng. 1984, 494, 18-24. (2) Seitz, W. C. R. C. Crit. Rev. Anal. Chem. 1988, 19, 135-173. (3) Preininger, C.; Klimant, I.; Wolfbeis, O. S. Anal. Chem. 1994, 66, 18411846. (4) Collinson, M. E.; Meyerhoff, M. E. Anal. Chem. 1990, 62, 425A. (5) Tan, W. H.; Shi, Z. Y.; Smith, S.; Birnbaum, D.; Kopelman, R. Science 1992, 258, 778-781. (6) Pantano, P.; Walt, D. R. Anal. Chem. 1995, 67, 481A-487A. (7) Bronk, K. S.; Michael, K. L.; Pantano, P.; Walt, D. R. Anal. Chem. 1995, 67, 2750-2757. (8) Michael, K. L.; Ferguson, J. A.; Healey, B. G.; Panova, A. A.; Pantano, P.; Walt, D. R. In Polymers in Sensors: Theory and Practice; Akmal, N., Usmani, A. M., Eds.; American Chemical Society: Washington, DC, 1998. (9) Tabacco, M. B.; Uttamlal, M.; McAllister, M.; Walt, D. R. Anal. Chem. 1999, 71, 154-161. (10) Palais, J. C. Appl. Opt. 1980, 19, 2011-2018. (11) Sakamoto, T. Appl. Opt. 1992, 31, 5184-5190.

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laser diode-fiber coupling.12 Such coupling approaches have been used with single-core fibers for Raman microscopy,13,14 solid-state photometers for absorbance-based thin-film chemical sensors,15 thermometric probes,16 and absorbance sensors.17,18 In addition to numerous sensor applications, optical fibers and GRIN lenses have been used together for imaging. In this case, an imaging fiber is employed. An optical imaging fiber is a fused, coherent array containing thousands of optical fibers. Each optical fiber in the array transmits a spatially discrete signal that can be individually interrogated. Together, the individual optical fibers form an image at the fiber’s proximal face that can be projected onto a two-dimensional array detector.6,7,19,20 Coherent images can be carried from one face of the fiber to the other, regardless of the fiber’s orientation. Thus, imaging fibers are attractive for inspecting remote and inaccessible places. However, to image an object, the object must be in the fiber’s near field (e10 nm) or in contact with the fiber’s distal face. By attachment of a GRIN lens to the distal face of the fiber, far-field objects (i.e., objects tens of millimeters from the fiber’s surface) can be imaged. The GRIN lens acts as an objective by forming a real image of the object at the back face of the lens. The back face of the lens is interfaced with the imaging fiber, and the image is transmitted through the fiber to an array detector (Figure 1A). Such imaging fiber-GRIN lens couples are commonly used for endoscopic and holographic imaging.21 Single-core optical fiber-GRIN lens couples have been used to enhance light collection for sensor applications, and fiber arrayGRIN lens couples have been used for endoscopic imaging. To our knowledge, optical fiber-GRIN lens couples have not been used for both imaging and chemical sensing simultaneously. In this paper, we demonstrate a far-field-viewing GRINscope used in two different configurations for remote sensing applications. In one capacity, the GRINscope is modified with an analyte(12) Nishizawa, K.; Nishi, H. Appl. Opt. 1984, 23, 1711-1714. (13) Jiaying, M.; Zhong, L. Appl. Spectrosc. 1991, 45, 1302-1304. (14) Ma, J.; Li, Y.-S. Appl. Spectrosc. 1994, 48, 1529-1531. (15) Jones, T. P.; Coldiron, S. J.; Deninger, W. J.; Porter, M. D. Appl. Spectrosc. 1991, 45, 1271-1277. (16) Conforti, G.; Brenci, M.; Mencaglia, A.; Mignani, A. G. Appl. Opt. 1989, 28, 577-580. (17) Landis, D. A.; Seliskar, C. J.; Heineman, W. R. Appl. Opt. 1994, 33, 34323439. (18) Landis, D. A.; Seliskar, C. J. Appl. Spectrosc. 1995, 49, 547-555. (19) Sweedler, J. V. CRC Crit. Rev. Anal. Chem. 1993, 24, 59-98. (20) Walt, D. R. Acc. Chem. Res. 1998, 31, 267-278. (21) White, R. A.; Klein, S. R. Endoscopic Surgery; Mosby Year Book: St. Louis, MO, 1991. 10.1021/ac990085q CCC: $18.00

© 1999 American Chemical Society Published on Web 06/12/1999

metals;38 and process and waste monitoring.27 In addition, the GRINscope could be used in bioanalytical applications such as multianalyte serum analysis39 and in detection for microchip technologies.40-46

Figure 1. (A) Schematic showing how the GRIN lens is used to project an image of a far-field object onto the distal face of an imaging fiber. (B) Photograph of a GRINscope taken with a video CCD camera as viewed through an optical microscope. The GRINscope diameter is 350 µm. (C) Far-field visual image of the word “TUFTS” viewed through the GRINscope using white light. For this image, boldformatted, 6-point Arial font was printed with black ink on white paper. (D) Far-field fluorescence image of the word “TUFTS” viewed at 530 nm through the GRINscope using 485-nm excitation light. For this image, outline-formatted, 6-point Arial font contained in a black box was printed on fluorescent paper.

sensitive polymer layer to create a sensor that can visually image its far-field surroundings and concurrently detect a single analyte. This single-analyte far-field viewing and sensing configuration has potential applications for corrosion assessment and monitoring in aircraft,22 ship hulls and offshore pipelines,23 gas distribution systems,24 hostile environments,25 and possibly metal prosthetic implants.26 The GRINscope sensor could also be used for industrial and environmental process and waste monitoring,27,28 for measuring blood chemistry, such as blood gases, while viewing angioplasty29 and endovascular procedures,30 and for monitoring pH or glucose levels during photodynamic therapy cancer treatments.31-33 In a different capacity, the GRINscope is used to image and monitor multiple sensing particles on a remote substrate. This far-field, multianalyte sensing configuration has potential applications for remote analysis of phytoplankton;34 monitoring water pollutants such as nitrates,35 pesticides,36 herbicides,37 and heavy (22) Li, D.; Ma, Y.; Flanagan, W. F.; Lichter, B. D.; Wikswo, J. P. Corrosion 1997, 53, 93-98. (23) Engel, D.; Richter, B. Mater. Corros. 1998, 49, 790-795. (24) Croall, S. Mater. Perform. 1997, 36, 14-17. (25) Dickie, N. Anti-Corros. Methods Mater. 1997, 44, 41. (26) Jacobs, J. J.; Skipor, A. K.; Patteson, L. M.; Hallab, N. J.; Paprosky, W. G.; Black, J.; Galante, J. O. J. Bone Jt. Surg. 1998, 80A, 1447-1458. (27) Dickens, J. E.; Sepaniak, M. J. J. Microcolumn Sep. 1999, 11, 45-51. (28) Lucovsky, G.; Kim, S. S.; Fitch, J. T.; Wang, C.; Rudder, R. A.; Fountain, G. G.; Hattangady, S. V.; Markunas, R. J. J. Vac. Sci. Technol., A 1991, 9, 10661072. (29) Voigtlander, T.; Rupprecht, H. J.; Britten, M.; Stahr, P.; Nowak, B.; Otto, M.; Kirkpatrick, C. J.; Brennecke, R.; Meyer, J. I. J. Cardiac Imaging 1998, 14, 65-70. (30) Calligaro, K. D.; Dougherty, M. J.; Patterson, D. E.; Raviola, C. A.; DeLaurentis, D. A. Ann. Vasc. Surg. 1998, 12, 296-298. (31) Moore, J. V.; Waller, M. L.; Zhao, S.; Dodd, N. J. F.; Acton, P. D.; Jeavons, A. P.; Hastings, D. L. Eur. J. Nucl. Med. 1998, 25, 1248-1254. (32) Miccoli, L.; Beurdeley-Thomas, A.; DePinieux, G.; Sureau, F.; Oudard, S.; Dutrillaux, B.; Poupon, M. F. Cancer Res. 1998, 58, 5777-5786. (33) Musser, D. A. Res. Commun. Chem. Pathol. Pharmacol. 1988, 60, 361369.

EXPERIMENTAL SECTION Chemicals. EPO-TEK 301 epoxy was obtained from Epoxy Technology, Inc. (Billerica, MA); (3-aminopropyl)triethoxysilane, poly(methyl methacrylate) (PMMA), and triethylenetetramine (TET) were purchased from Aldrich Chemical Co. (Milwaukee, WI); acrylamide, N-(acryloxy)succinimide, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), phosphate-buffered saline (PBS), fluorescein isothiocyanate (FITC), and tris(2,2′-bipyridine)ruthenium(II) chloride hexahydrate (RuBipy) were obtained from Sigma Chemical Co. (St. Louis, MO); 2,2′-azobis-2-methylpropionitrile (AIBN) was obtained from Pfaltz and Bauer, Inc. (Waterbury, CT). All chemicals were used as received. GRINscope Fabrication. The GRINscope was fabricated by permanently affixing the back face of a Gradient index (GRIN) lens on the distal face of an optical imaging fiber. First, the proximal and distal faces of a 350-µm-diameter optical imaging fiber (Sumitomo Electric Industries, Torrance, CA) were polished on 30-, 12-, 9-, 3-, 1-, and 0.3-µm lapping films (Mark V Laboratory, East Granby, CT) using the standard figure-eight technique. Residual polishing material was removed from the fiber faces using an acetone-soaked swab, followed by brief sonication. A 350-µmdiameter Selfoc GRIN lens (NSG America, Inc., Somerset, NJ) was optically aligned with the imaging fiber using one of two methods. In one method, the imaging fiber and the GRIN lens were held horizontally in separate fiber chucks and aligned with x-y positioners using a dissecting microscope. In the second method, the imaging fiber and the GRIN lens were aligned using a 350-µm-i.d. piece of Teflon tubing. In either method, optically transparent EPO-TEK 301 epoxy was used to adhere the GRIN lens to the imaging fiber. The epoxy was placed in a 65 °C oven for 10-15 min to begin polymerization. The thickened epoxy was applied to the fiber’s distal face, and the GRIN lens was brought into contact with the fiber. Note that care was taken not to foul the distal face of the GRIN lens with the epoxy. The GRINscope (34) Barbini, R.; Colao, F.; Fantoni, R.; Micheli, C.; Paulucci, A.; Ribezzo, S. ICES J. Mar. Sci. 1998, 55, 793-802. (35) Belz, M.; Dress, P.; Klein, K. F.; Boyle, W. J. O.; Franke, H.; Grattan, K. T. V. Water Sci. Technol. 1998, 37, 279-284. (36) Rubtsova, M. Y.; Samsonova, J. V.; Egorov, A. M.; Schmid, R. D. Food Agric. Immunol. 1998, 10, 223-235. (37) Rodriguez, M.; Orescan, D. B. Anal. Chem. 1998, 70, 2710-2717. (38) Herdan, J.; Feeney, R.; Kounaves, S. P.; Flannery, A. F.; Storment, C. W.; Kovacs, G. T.; Darling, R. B. Environ. Sci. Technol. 1998, 32, 131-136. (39) Shaw, R. A.; Kotowich, S.; Leroux, M.; Mantsch, H. H. Ann. Clin. Biochem. 1998, 35, 624-632. (40) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767-773. (41) Fodor, S. P. A.; Rava, R. P.; Huang, X. C.; Pease, A. C.; Holmes, C. P.; Adams, C. L. Nature 1993, 364, 555-556. (42) Pease, A. C.; Solas, D.; Sullivan, E. J.; Cronin, M. T.; Homes, C. P.; Fodor, S. P. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5022-5026. (43) Matson, R. S.; Rampal, J.; Pentoney, S. L. J.; Anderson, P. D.; Coassin, P. Anal. Biochem. 1995, 224, 110-116. (44) Chee, M.; Yang, R.; Hubbell, E.; Berno, A.; Huang, X. C.; Stern, D.; Winkler, J.; Lockheart, D. J.; Morris, M. S.; Fodor, S. P. A. Science 1996, 274, 610614. (45) McGall, G.; Labadie, J.; Brock, P.; Wallraff, G.; Nguyen, T.; Hinsberg, W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13555-13560. (46) Eggers, M.; Ehrlich, D. Hematol. Pathol. 1995, 9, 1-15.

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was cured for 1 h at 65 °C. When alignment method 1 was employed, the GRINscope was left overnight for further curing before removal from the fiber chucks. When alignment method 2 was used, a second aliquot of epoxy was applied to strengthen the fiber-lens interface and the GRINscope was cured at 65 °C for an additional hour. pH-Sensitive GRINscope Fabrication. A pH-sensitive polymer layer was covalently immobilized on the distal face of the GRINscope using spin-coating techniques described previously.7 The lens surface was immersed in a 10% solution of (3-aminopropyl)triethoxysilane in acetone for 1.5-2 h to functionalize the surface with primary amino groups. This procedure facilitates covalent attachment of the copolymer poly(acrylamide-co-N(acryloxy)succinimide) (PAN)47 to the silica surface of the lens. The presynthesized PAN copolymer was prepared for spin coating using the following protocol. First, 25 mg of PAN and 100 µL of buffer (0.3 M HEPES, pH 7.5) were thoroughly mixed for 30 s. Second, cross-linker (10.6 µL of 0.5 M TET) was added to the dissolved copolymer and the reaction was allowed to proceed for another 30 s. Third, a 25-µL aliquot of buffer was added, followed by mixing for 10 s to ensure the correct polymer viscosity. Fourth, 20 µL of the viscous polymer was placed on the amino-functionalized surface of the GRINscope. Finally, the GRINscope was spun at 2000 rpm for 10 s to achieve a thin, uniform polymer layer. The polymer-coated GRINscope was cured for 1 h at room temperature and then allowed to react with a pH-sensitive dye solution (0.4 mM FITC in 0.3 M HEPES, pH 7.5) for 15 min in the dark. Fluorescein isothiocyanate (FITC) was covalently immobilized in the polymer layer by reaction with the free amine groups on TET. Unreacted FITC was rinsed from the polymer layer by soaking the sensor two or three times in 0.3 M HEPES (pH 7.5) for 30 min each, followed by equilibration in PBS (pH 7.4). pH-Sensitive Particle Fabrication. pH-sensitive particles were fabricated using 37-74-µm (200-400-mesh) aminopropyl porous glass particles (Sigma Chemical Co., St. Louis, MO). Approximately 18 mg of the porous particles (average pore size 170 Å) was allowed to react with a solution of FITC (4.28 × 10-4 M in 10 mM PBS, pH 7.4) at room temperature for 30 min. The particles were rinsed with PBS a minimum of three times to remove unreacted FITC. O2-Sensitive Particle Fabrication. Oxygen-sensitive particles were fabricated from 100-µm Type B porous silica particles (Matech Advanced Materials, Westlake Village, CA). Approximately 18 mg of the silica particles was soaked overnight in a 1.78 × 10-3 M solution of RuBipy in a 4:1 methylene chloride/ ethanol solution containing 10% PMMA. The dyed particles were vacuum-filtered using a 0.5-µm Millipore Durapore filter (Fisher Scientific, Springfield, NJ) and air-dried. Before use, the oxygensensitive particles were rinsed three times with PBS, pH 7.4. Instrumentation. An optical imaging system built in our laboratory7 was used to acquire both visual and fluorescence images. This instrument has the ability to collect concurrent visual images (by shuttering an external white light) and fluorescence ratiometric measurements (by interchanging excitation and emission filters) throughout the experiment using computer-controlled (47) Pollak, A.; Blumenfeld, H.; Wax, M.; Baughn, R. L.; Whitesides, G. M. J. Am. Chem. Soc. 1980, 102, 6324-6336.

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filter wheels and shutters. The proximal face of the GRINscope is positioned in the focal point of an objective using an x-y micrometer and a modified microscope stage. For fluorescence imaging, filtered excitation light from a 75-W xenon arc lamp is reflected by a dichroic mirror and focused onto the proximal face of the GRINscope with a 20× microscope objective. The filtered light is transmitted through the GRINscope and excites an analytesensitive fluorescent dye located either on the distal face of the GRINscope or on a remote substrate. The fluorescence signal propagated through the GRINscope is collected by the microscope objective, transmitted through the dichroic mirror, filtered at the appropriate emission wavelength, and imaged onto an intensified charge-coupled device (ICCD) detector (Intensified PentaMAX512EFT, Princeton Instruments Inc., Trenton, NJ). To obtain visual images, the fiber is illuminated externally with white light. The distal end of the GRINscope is held by a micropipet holder (Narishige Co. Ltd.). A micropositioner is used to adjust the GRINscope working distance with respect to the target (e.g., a Photopore mask for visual images during sensing with (1) an immobilized analyte-sensitive polymer layer or (2) multianalytesensitive particles on a remote substrate). pH-Sensitive GRINscope Data Analysis. For continuous fluorescence measurements, a ratiometric sequence was acquired by exciting the indicator at 485 and 440 nm and monitoring the emission at 530 nm using computer-controlled shutters and filter wheels. After a sequence of fluorescence images was acquired, IPLab imaging software (IPLab Spectrum IO version 3.0, Signal Analytics Co., Vienna, VA) was used to obtain the mean fluorescence intensity at 530 nm for each excitation wavelength. These data were then ratioed (485 nm/440 nm), and the mean fluorescence intensity ratio data were plotted vs time. Analyte-Sensitive Particle Image Analysis. Images of the pH- and O2-sensitive particles were obtained by exciting the particles at 485 nm and collecting their fluorescence emission at 530 and 605 nm, respectively. Using the IPLab imaging software, a region of interest (ROI) was drawn around each particle in the GRINscope’s field of view. The mean fluorescence intensity for each particle type was obtained and plotted as a function of pH or oxygen response. The images were normalized and contrastadjusted for visual enhancement. RESULTS AND DISCUSSION GRINscope Characterization. Optical imaging fibers have the ability to carry a coherent image from one face of a fiber to the other; however, the fiber’s face must be in contact with the object it is imaging. GRINscopes were fabricated by permanently adhering a micro-Selfoc GRIN lens to the distal face of an imaging fiber. By affixing a GRIN lens to the distal face of the fiber, one can image far-field objects (i.e., objects beyond the fiber’s surface). The GRIN lens acts as an objective by collecting a far-field image and forming a real image of the object at the back face of the lens. Since the back face of the lens is coupled to the imaging fiber, the image is transmitted through the fiber to the detector (Figure 1A). Figure 1B is a photograph of a GRINscope fabricated using a 350-µm-diameter, 1-mm-long Selfoc GRIN lens and a 350µm-diameter imaging fiber containing 6000 individual fiber elements. These small dimensions, along with the GRINscope’s flexibility, allow environments to be explored that are otherwise inaccessible. Parts C and D of Figure 1 demonstrate the GRIN-

Figure 2. (A) Schematic of GRINscope working distance characterization using an Air Force resolution target. (B) Visual images of an Air Force resolution target (group 0 element 1 and group 1 element 6) viewed through the GRINscope from various working distances. These images were acquired in air for 150 ms using white light from an external source.

scope’s ability to efficiently relay and preserve both a visual and a fluorescence far-field image, respectively. The imaging capabilities of the GRINscope were characterized by viewing a standard Air Force resolution target from various working distances (i.e., different distances from the distal face of the lens to the target). The GRINscope was positioned above the resolution target using a micropositioner and illuminated with white light from an external source (Figure 2A). Visual images containing group 0 element 1 and group 1 element 6 were acquired through the GRINscope from 1- to 10-mm optical working distances. The effects of the various working distances on image quality and magnification are shown by the images in Figure 2B. Line pair calculations using the Air Force resolution target revealed the highest resolution image was obtained at a distance of 5 mm. This 5-mm optimal working distance corresponds to the working distance specified by the GRIN lens manufacturer. As the GRINscope was moved closer to (1-4 mm) or farther from (6-10 mm) the target, only a moderate loss of resolution was observed. Similar working distances have been employed in

surgical applications using endoscopes.48 Using the present GRINscope at a 5 mm working distance, the best resolution obtained was approximately 20 micrometers (calculated using group 2 element 5). Far-Field Viewing and Chemical Sensing Using a Thin, Analyte-Sensitive Polymer Layer. A GRINscope sensor capable of monitoring a single analyte while simultaneously imaging the surrounding environment was fabricated. Spin-coating techniques and amine chemistry were used to covalently coat the GRINscope’s distal surface with a uniform polymer layer. The polymer layer was modified with the indicator FITC to create a pH sensor. The pH-sensitive GRINscope was calibrated in a series of buffers differing by 0.5 pH unit. The fluorescence intensity of the FITC polymer layer was measured at 530 nm using 485- and 440-nm excitation light. Ratiometric measurements were used to reduce the effects of dye photobleaching, leaching, or lamp fluctuations on fluorescence intensity. Figure 3 is a representative calibration (48) Yamakawa, K.; Kondo, T.; Yoshioka, M.; Takakura, K. Acta Neurochir. Suppl. 1992, 54, 47-52.

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Figure 3. Representative titration curve of a pH-sensitive GRINscope fiber. The sensor was submerged in 10 mM PBS of varying pH for 3 min. Fluorescence intensity ratio measurements were collected for 150 ms at 530 nm using 485 and 440-nm excitation light.

Figure 4. Schematic of real-time, far-field viewing and chemical sensing using a thin, analyte-sensitive polymer layer. Using white light, a visual image of the Photopore mask can be acquired through the GRINscope. Using fluorescence, pH measurements can be made as a peristaltic pump delivers buffers with differing pHs past the pHsensitive GRINscope.

curve of the pH-sensitive GRINscope and indicates a precision of ∼0.17 pH unit in the linear region of the curve. To demonstrate the GRINscope sensor’s potential use in practical applications, an environment having both physical and chemical variables was created (Figure 4). A 4-mm-i.d. glass tube was used to fabricate a cavity. The tubing was cut into two pieces, and a Photopore mask (Vacco, South El Monte, CA) was epoxied between them. The mask contained 250 µm × 750 µm pores providing an object that could be imaged but would not hinder analyte transport to the sensor. A flowing stream was created by connecting a peristaltic pump (model 203, Scientific Industries, Inc., Bohemia, NY) to the distal end of the cavity. To vary the stream’s chemical environment, the tubing inlet was submerged in different pH buffer reservoirs. In operation, both a micromanipulator and the GRINscope’s real-time imaging capability were used to guide the sensor through the tube. During the first experiment, the GRINscope sensor was held at a fixed distance from the mask while buffer was delivered through the cavity. The inset in Figure 5 is a visual image of the Photopore mask acquired through the GRINscope sensor using 2770 Analytical Chemistry, Vol. 71, No. 14, July 15, 1999

Figure 5. The inset is a visual image of the Photopore mask viewed through the pH-sensitive GRINscope from a 3-mm fixed distance. The plot shows the pH-sensitive GRINscope’s fluorescence intensity response to a flowing stream of varying pH over time. The fluorescence was monitored at 530 nm using 485- and 440-nm excitation light. The visual image was acquired using an ICCD with a 150-ms acquisition time; the fluorescence images were acquired using an ICCD with a 50-ms acquisition time.

white light. This image shows the imaging capabilities of the device are not hindered by the immobilized sensing layer or the liquid medium. To demonstrate the sensing capabilities of the GRINscope sensor, buffers varying by 1 pH unit were delivered though the cavity at a rate of 3 mL/min. Fluorescence images were acquired every 30 s using computer-controlled shutters and filter wheels. The plot of fluorescence intensity versus time in Figure 5 shows the sensor’s response to pH changes introduced by the peristaltic pump. These data illustrate the sensor’s ability to rapidly detect changes in analyte. The polymer layer withstood the sheer forces of the buffer; i.e., the sensing layer remained intact during the experiment. Overall, this experiment has shown a GRINscope sensor can both image and monitor an analyte from a fixed distance. A second set of experiments was conducted to examine the imaging and sensing capabilities of the GRINscope sensor from various working distances relative to the Photopore mask. In these experiments, both white light and fluorescence images were acquired every 30 s using computer-controlled filter wheels and shutters. The GRINscope’s initial working distance was 3 mm from the mask. After every three fluorescence measurements, the sensor was moved 1 mm. The first five movements increased the working distance (3 mm to 8 mm); the last five movements decreased the working distance (8 mm to 3 mm). Figure 6A displays the visual images of the Photopore mask at each working distance. The GRINscope maintains a quality image at each working distance. The GRINscope sensor was even able to image the dark rings produced by air bubbles that formed on the mask’s surface (second row: 8-, 7-, and 6-mm images). These bubbles resulted from an air plug, introduced to mark the change in buffer pH, traveling through the mask. Figure 6B is a plot showing the sensor’s response to 1 unit pH changes was not altered by its movement. Overall, this experiment has shown a GRINscope sensor can both image and monitor an analyte from a changing distance. Far-Field Viewing and Chemical Sensing of Multiple Analytes on a Remote Substrate. In addition to the application

Figure 6. (A, B) Alternating visual and fluorescence data acquired simultaneously in a flowing stream with various working distances. (A) Visual images (a-k) of the Photopore mask viewed through the GRINscope from varying working distances. (B) Fluorescence intensities measured at the working distances shown in (A) while pH was simultaneously changed via flow delivery of different buffers. Note the stability in the readings as a function of time and working distance. The fluorescence was monitored at 530 nm using 485- and 440-nm excitation light. After every three fluorescence measurements, the sensor was moved 1 mm. Data points with gray circles denote a change in the GRINscope’s working distance with respect to the Photopore mask. The initial working distance was 3 mm. The first five movements (a-f) increased the working distance to 8 mm; the last five movements (f-k) decreased the working distance to 3 mm. The visual images were acquired between each fluorescence image using a 150-ms ICCD (duplicate images are not shown); the fluorescence images were acquired using a 50-ms ICCD.

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Figure 7. Schematic of far-field viewing and sensing of multianalytesensitive particles on a remote substrate.

described above, the GRINscope can be used for multianalyte sensing. Situations often occur where monitoring several analytes simultaneously is desired for a total analysis. Several efforts have been made to fabricate sensors for such circumstances using photodeposition,8,49,50 ink-jet printing,51-53 and photolithography.40-46 These techniques require one to design an array in which each sensor location is predetermined. We recently demonstrated a new technique that allows high-density optical sensor arrays to be fabricated in a random fashion by making each sensor addressable.54,55 These arrays are prepared by randomly distributing a mixture of microsphere sensors on an optical substrate containing thousands of micrometer-scale wells. Although attaching the sensing chemistry directly to the sensor surface is useful for a wide variety of situations, there are some instances where such sensors may not be practical or cost efficient. For example, microchip technologies employed with capillary electrophoresis and DNA sequencing necessitate the automated exchange of multiple reaction surfaces. Such technologies also require an objective to view the substrate, thus limiting the sensor to a benchtop microscope stage. Since the GRINscope consists of an optical imaging fiber and a GRIN lens, it can be used to view and monitor sensing regions or particles in the far field, i.e., from a preset optical distance on a remote platform.56 The GRINscope’s ability to image and monitor multiple species simultaneously was demonstrated using pH- and O2-sensitive particles in a remote microtiter plate (Figure 7). The pH-sensitive particles were fabricated by reacting 37-74-µm amino-functionalized porous glass particles with a solution of FITC. Oxygensensitive particles were fabricated by adsorbing a thin PMMA layer containing RuBipy on 100-µm porous silica particles. The pH- and O2-sensitive particles were mixed in approximately equal amounts. Separate aliquots of the particles were placed in the bottoms of two microtiter plate wells, each containing a different (49) Li, L.; Walt, D. R. Anal. Chem. 1995, 67, 3746-3752. (50) Healey, B. G.; Walt, D. R. Anal. Chem. 1995, 67, 4471-4476. (51) Kimura, J.; Kawana, Y.; Kuriyama, T. Biosensors 1988, 4, 41. (52) Newman, J. D.; Turner, A. P. F.; Marrazza, G. Anal. Chim. Acta 1992, 262, 13-17. (53) Lemmo, A. V.; Fisher, J. T.; Geysen, H. M.; Rose, D. J. Anal. Chem. 1997, 69, 543. (54) Michael, K. L.; Taylor, L. C.; Schultz, S. L.; Szurdoki, F.; Walt, D. R. Proceedings of the International Society of Optical Engineering; 1998; pp 34-41. (55) Michael, K. L.; Taylor, L. C.; Schultz, S. L.; Walt, D. R. Anal. Chem. 1998, 70, 1242-1248. (56) Walt, D. R.; Michael, K. L.; Chadha, S. U.S. Patent 5,814,524, 1998.

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Figure 8. (A-D) Fluorescence images of the two mixtures, each containing both pH- and O2-sensitive particles. Mixture 1 buffer contained 50% pO2 at pH 6. Mixture 2 buffer contained 100% pO2 at pH 7. (A) Fluorescence image of the O2-sensitive particles in mixture 1. (B) Fluorescence image of the O2-sensitive particles in mixture 2. (C) Fluorescence image of the pH-sensitive particles in mixture 1. (D) Fluorescence image of the pH-sensitive particles in mixture 2. The pH-sensitive particles in each mixture were monitored at 530 nm, and the O2-sensitive particles were monitored at 605 nm. White corresponds to high fluorescence intensity. (E) Bar graph showing the quantitative change in fluorescence intensity of each particle type. The letters on the bottom of each bar correspond to images A-D.

buffer environment. One well contained pH 6 buffer with 50% pO2; the other well contained pH 7 buffer with 100% pO2. The GRINscope was used to image the sensor particles from a 5-mm working distance. The fluorescence responses of the pH- and O2sensitive particles to each environment were measured at 530 and 605 nm, respectively, using 485-nm excitation light. The images in Figure 8 show the particles’ qualitative response to the two buffer environments at each emission wavelength. The bar graph shows the quantitative change in fluorescence intensity for each environment. These data demonstrate remote, multianalyte sensing particles can be imaged and their chemical sensitivity can be

both qualitatively and quantitatively measured using the GRINscope far-field-viewing technique. CONCLUSION We have demonstrated a far-field-viewing GRINscope sensor for making analytical measurements in remote locations. The GRINscope comprises just two components and allows us to obtain both chemical and visual information. In one application, a thin, analyte-sensitive polymer layer can be immobilized on the distal end of the GRINscope to create a sensor that can visually image its far-field surroundings and concurrently detect a single analyte. Since the GRINscope’s working distance can be prechosen by selecting the appropriate GRIN lens, the far-field-viewing technique can be used for a variety of applications. Such applications could include investigating small body cavities, monitoring oxygen levels during angioplasty surgery, determining the effectiveness of broncho- or vasodilator medications, and observing in vitro and/ or in vivo fertilization technologies or cancer diagnosis without biopsy. In addition to monitoring biological analytes, the GRINscope sensor could also be used for industrial, nuclear, and environmental applications such as process monitoring, inspecting pipes, engines, and tankers for corrosion, and monitoring groundwater and waste streams. The resolution of the current GRINscope (i.e., 20 µm) is more than sufficient for most anticipated applications. However, the resolution can be improved by employing

imaging fibers containing smaller diameter fibers, as well as by employing GRIN lenses with different specifications. In a different capacity, the GRINscope can be used to image an array of sensors on a remote substrate and simultaneously measure their response to multiple analytes. This far-field viewing and chemical sensing technique is advantageous for many situations. For example, if sensor lifetime must be extended over many months, fresh particles can be easily added or the substrate can be exchanged without having to remove and reposition the GRINscope. Far-field sensing provides the benefit of larger sensing areas compared to sensors where the sensing site is immobilized directly on the sensor’s surface. Finally, optical encoding and size discrimination techniques allow the sensing particles to be randomly added to any surface, thus eliminating the need for sensor blueprints and complicated fabrication schemes. ACKNOWLEDGMENT We gratefully acknowledge the National Institutes of Health (Grant GM-48142) for financial support and Dr. Paul Pantano for helpful discussions.

Received for review January 28, 1999. Accepted April 19, 1999. AC990085Q

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