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Temporally Resolved Fluorescence Spectroscopy of a Microarray-Based Vapor Sensing System Matthew J. Aernecke and David R. Walt* Department of Chemistry, Tufts University, 62 Talbot Avenue, Medford, Massachusetts 02155 This paper describes a method to measure the complete fluorescence spectrum from numerous fluorescent microspheres in a microarray simultaneously during exposure to a vapor. The technique, called spectrally resolved sensor imaging (SRSI), positions a transmission grating directly in front of the microscope objective on a standard epi-fluorescence microscope. This modification produces a hybrid image on the CCD camera that contains a conventional fluorescence image in the zero-order diffracted light and a fluorescence spectral image in the firstorder diffracted light. Three types of surface-functionalized silica microspheres were coated with a solvatochromic dye. The surface functionality on the microspheres influences the maximum emission wavelength of the dye and generates a fluorescence spectral signature that is used to identify each sensor type. These sensors were randomly distributed into a photolithographically fabricated microarray platform, and the spectral signature of each individual sensor was measured. The time resolution of spectral acquisition is short enough to capture dynamic changes in the fluorescence emission as a vapor is presented to the array. The ability to measure the entire fluorescence spectrum from each sensor simultaneously during a vapor exposure increases the dimensionality of the response data and significantly improves the classification accuracy of the system. Artificial nose systems are analytical devices that are designed to detect a myriad of vapors using a collection of semiselective and cross-reactive sensors.1,2 These devices are loosely modeled after mammalian olfactory systems in that vapors produce response patterns that encode a particular vapor and these response patterns can be processed and identified using pattern recognition software. Several platforms have been developed that use a variety of chemical and physical transduction mechanisms such as surface acoustic wave resonators,3,4 polymer5-7 or lowvolatility small-molecule chemiresistors,8 micro-hot-plates,9 calorimeters,10 cantilevers,11,12 and colorimetric sensors.13,14 * To whom correspondence should be addressed. E-mail:
[email protected]. Phone: 617-627-3470. Fax: 617-627-3443. (1) Albert, K. J.; Lewis, N. S.; Schauer, C. L.; Sotzing, G. A.; Stitzel, S. E.; Vaid, T. P.; Walt, D. R. Chem. Rev. 2000, 100, 2595–2626. (2) Rock, F.; Barsan, N.; Weimar, U. Chem. Rev. 2008, 108, 705–725. (3) Hsieh, M. D.; Zellers, E. T. Anal. Chem. 2004, 76, 1885–1895. (4) Rose-Pehrsson, S. L.; Grate, J. W.; Ballantine, D. S.; Jurs, P. C. Anal. Chem. 1988, 60, 2801–2811. (5) Sisk, B. C.; Lewis, N. S. Langmuir 2006, 22, 7928–7935.
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We have previously reported a fluorescence-based artificial nose system that uses an etched imaging fiber-optic bundle as a platform to house micrometer-sized porous silica microspheres.15 The silica microspheres are coated with the solvatochromic dye Nile Red. This dye is used as an indicator because its optical properties vary as a function of the microenvironmental polarity. The silica microspheres can be functionalized with different chemical moieties using silane chemistry, and this silane layer acts as a semiselective layer for partitioning various vapors. The silane modifies the intrinsic polarity of the microsphere resulting in a change in the Nile Red microenvironment. When the microspheres are exposed to vapors, the vapors partition to different extents into the different microspheres, depending on their composition. The temporal fluorescent signal measured from the sensors upon exposure to the vapors is indicative of the polarity of the analyte vapor and the degree to which it partitions into the functionalized microsphere. Different types of microsphere sensors can be incorporated into the high-density array format and monitored simultaneously, enabling a high degree of signal averaging resulting in enhancements to the signal-to-noise ratio of the array response.16 We have used this system to detect volatile organic compounds (VOCs),17 explosive vapors,18 and nerve agent simulants.19,20 Recent trends in artificial nose research have focused on extracting multidimensional responses from a collection of sensors (6) Lonergan, M. C.; Severin, E. J.; Doleman, B. J.; Beaber, S. A.; Grubbs, R. H.; Lewis, N. S. Chem. Mater. 1996, 8, 2298–2312. (7) Doleman, B. J.; Lonergan, M. C.; Severin, E. J.; Vaid, T. P.; Lewis, N. S. Anal. Chem. 1998, 70, 4177–4190. (8) Maldonado, S.; Garcı´a-Berrı´os, E.; Woodka, M. D.; Brunschwig, B. S.; Lewis, N. S. Sens. Actuators, B 2008, 134, 521–531. (9) Raman, B.; Hertz, J. L.; Benkstein, K. D.; Semancik, S. Anal. Chem. 2008, 80, 8364–8371. (10) Lerchner, J.; Caspary, D.; Wolf, G. Sens. Actuators, B 2000, 70, 57–66. (11) Vancˇura, C.; Ruegg, M.; Li, Y.; Hagleitner, C.; Hierlemann, A. Anal. Chem. 2005, 77, 2690–2699. (12) Lang, H. P.; Baller, M. K.; Berger, R.; Gerber, C.; Gimzewski, J. K.; Battiston, F. M.; Fornaro, P.; Ramseyer, J. P.; Meyer, E.; Gu ¨ntherodt, H. J. Anal. Chim. Acta 1999, 393, 59–65. (13) Bang, J. H.; Lim, S. H.; Park, E.; Suslick, K. S. Langmuir 2008, 24, 13168– 13172. (14) Rakow, N. A.; Suslick, K. S. Nature 2000, 406, 710. (15) Dickinson, T. A.; Michael, K. L.; Kauer, J. S.; Walt, D. R. Anal. Chem. 1999, 71, 2192–2198. (16) Albert, K. J.; Walt, D. R. Anal. Chem. 2000, 72, 1947–1955. (17) Albert, K. J.; Walt, D. R.; Gill, D. S.; Pearce, T. C. Anal. Chem. 2001, 73, 2501–2508. (18) Albert, K. J.; Myrick, M. L.; Brown, S. B.; James, D. L.; Milanovich, F. P.; Walt, D. R. Environ. Sci. Technol. 2001, 35, 3193–3200. (19) Bencic-Nagale, S.; Sternfeld, T.; Walt, D. R. J. Am. Chem. Soc. 2006, 128, 5041–5048. (20) Bencic-Nagale, S.; Walt, D. R. Anal. Chem. 2005, 77, 6155–6162. 10.1021/ac900589b CCC: $40.75 2009 American Chemical Society Published on Web 06/11/2009
by incorporating partitioning layers into a vapor flow channel.21-23 These devices mimic the functioning of the nasal mucosa in biological olfactory systems and serve to attenuate the flow rates of odorants as they traverse the channel and interact with the sensors. This added dimensionality in individual sensor responses has been shown to provide superior discriminating ability for individual vapors and vapor mixtures. The fluorescence-based artificial nose system described above has traditionally only monitored changes in the intensity at the maximum emission wavelength of a sensor during exposure to an analyte vapor. Herein, we demonstrate that it is possible to increase the dimensionality of a sensor response by monitoring multiple wavelengths simultaneously. Current microanalytical approaches employing fluorescence detection have typically focused on single-wavelength systems where the excitation and emission light are optimized for a single fluorophore; however, monitoring a system at one wavelength is often insufficient, particularly when applied to multiplexed analyses where the sensors do not share a common emission wavelength. Many different methods have been used to collect spectral information from microsystems. Multiple images at discrete wavelength bands can be obtained by scanning either a series of band-pass filters or a liquid crystal tunable filter (LCTF).24 The temporal resolution of these serial techniques is limited by the switching speed of either the filter wheel or the LCTF and is generally too long to capture many dynamic events. Faster scan speeds are possible by using an acousto-optical tunable filter (AOTF)25 or prism-based spectrograph.26,27 Although these techniques have demonstrated excellent wavelength and temporal resolution of the analytical signal, they require additional, often costly, instrumental components. Other approaches have used optical fibers to ferry the emission light from multiple points to an external spectrometer, but this approach is limited in the number of points that can be addressed. Simpler alternatives to multiwavelength detection have incorporated a transmission grating into the optical train of a microscope. As the fluorescence emission passes through this dispersive element, the diffracted orders are collected and projected onto the image sensor. An analysis of the first-order diffracted light, which is spatially separated by wavelength, enables one to obtain spectral information about the fluorescence emission in a single image, albeit at a lower resolving power than more complex spectroscopic systems. This modification has proven sufficient for the rapid measurement of the complete fluorescence spectra from single molecules28,29 and cells30 and for collecting absorption (21) Stitzel, S. E.; Stein, D. R.; Walt, D. R. J. Am. Chem. Soc. 2003, 125, 3684– 3685. (22) Woodka, M. D.; Brunschwig, B. S.; Lewis, N. S. Langmuir 2007, 23, 13232– 13241. (23) Gardner, J. W.; Covington, J. A.; Tan, S.-L.; Pearce, T. C. Proc. R. Soc. London, Ser. A 2007, 463, 1713–1728. (24) Albert, K. J.; Gill, D. S.; Pearce, T. C.; Walt, D. R. Anal. Bioanal. Chem. 2002, 373, 792–802. (25) Wachman, E. S.; Niu, W.; Farkas, D. L. Biophys. J. 1997, 73, 1215–1222. (26) Frank, J. H.; Swartling, A. D. E. J.; Venkitaraman, A. R.; Jeyasekharan, A. D.; Kaminski, C. F. J. Microsc. 2007, 227, 203–215. (27) Frederix, P. L. T. M.; Asselbergs, M. A. H.; van Sark, W. G. J. H. M.; van den Heuvel, D. J.; Hamelink, W.; de Beer, E. L.; Gerritsen, H. C. Appl. Spectrosc. 2001, 55, 1005–1012. (28) Han, R.; Zhang, Y.; Dong, X.; Gai, H.; Yeung, E. S. Anal. Chim. Acta 2008, 619, 209–214.
Table 1. Microsphere Sensor Materials sensor name
bead size (µm)
bead surface functionality
manufacturer
Luna C8 Luna CN Luna OH
3 3 3
aliphatic (C8) cyano hydroxy
Phenomenex (Torrance, CA) Phenomenex (Torrance, CA) Phenomenex (Torrance, CA)
spectra from numerous points within a microfluidic channel.31 The transmission grating approach offers a simple and inexpensive instrumental modification and can provide complete spectral measurements on a time scale that is rapid enough to resolve dynamic chemical events. In this paper, we present an approach to increase the dimensionality of a single sensor response by monitoring several wavelengths simultaneously in a fluorescence-based artificial nose system. This approach incorporates a photolithographically fabricated microwell array platform housing 3 µm diameter surfacefunctionalized silica microspheres coated with the solvatochromic dye Nile Red. We adapt a transmission grating approach previously employed to make dynamic visible absorption measurements in microscale systems31 and use it to make temporally resolved fluorescence spectral measurements of a microsensor array containing several different sensing elements each with a distinctive spectral signature. We demonstrate the ability of this technique, which we call spectrally resolved sensor imaging (SRSI), to measure the complete fluorescence emission spectra of many individual 3 µm sensors simultaneously with high spatial resolution. We use the spectral information to assign each sensor to one of three groups and to resolve changes in the fluorescence emission spectrum over time as a vapor is presented. More importantly, we demonstrate that monitoring a single sensor response at several wavelengths significantly improves the discrimination ability of the artificial nose system. MATERIALS AND METHODS Materials. Toluene, acetone, (Fisher Scientific, Fairlawn, NJ) heptane, ethanol, methanol, isopropyl alcohol, acetone, Nile Red (Aldrich, St. Louis, MO), and benzene (Arcos Organics, Morris Plains, NJ) were used as received. Sensor and Array Fabrication. The microsphere sensors used in this study (Table 1) were prepared by placing a 10 mg aliquot of commercially available functionalized silica microspheres into a 2 mg/mL solution of Nile Red in toluene. The mixture was stirred for 1 h after which the sensors were filtered and dried overnight in an oven at 60 °C. Fluorescence spectra from a sample of each sensor type were measured on a benchtop microtiter plate spectrometer (Tecan, San Jose, CA) using an excitation wavelength of 480 nm. Aliquots of each sensor type were combined into a mixture that was used to fabricate sensor arrays. Microwell arrays used to contain the microsphere sensors were fabricated using the procedure outlined in Figure 1a. A 7.5 cm diameter 500 µm thick Pyrex wafer was cleaned by sonication in acetone for 5 min. A 300 nm layer of chromium was deposited (29) Ma, Y.; Shortreed, M. R.; Yeung, E. S. Anal. Chem. 2000, 72, 4640–4645. (30) Isailovic, D.; Li, H. W.; Phillips, G. J.; Yeung, E. S. Appl. Spectrosc. 2005, 59, 221–225. (31) Damean, N.; Sia, S. K.; Linder, V.; Narovlyansky, M.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 10035–10039.
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Figure 1. (a) Schematic of the fabrication procedure used to produce the microwell array used in this study. (b) Scanning electron micrograph of the array loaded with 3 µm microsphere vapor sensors. Each microwell is framed in a 300 nm layer of chromium and is designed to hold a single microsphere vapor sensor.
onto the cleaned wafer by e-beam evaporation after which a thin coat of positive photoresist (Micropoist 1805, Shipley Company, Marlborough, MA) was applied via spin-coating (ReynoldsTECH, East Syracuse, NY) at 4000 rpm for 40 s. The photoresist-coated wafer was prebaked at 115 °C for 2 min followed by exposure to UV light (365-405 nm) for 7 s through a transparency mask patterned with 3 µm diameter circles on a mask aligner (Suss Microtec MA6, Garching, Germany). The microcircle pattern was transferred to the underlying chromium layer by developing the photoresist in developer (Microposit CD-30, Shipley Company, Marlborough, MA) for 90 s followed by a 30 s O2 plasma descum (Technics Microstripper Series 220, Advanced Technologies, Newport News, VA) and a 2 min treatment with chromium etch solution. Microwells were etched into the underlying Pyrex substrate in the exposed areas by reactive ion etching (RIE) (Nexx Systems, Billerica, MA). The fabricated arrays were characterized using scanning electron microscopy (Jeol Ltd., Tokyo, Japan) and profilometry (Dektak 6M, Vecco, Plainview, NY). Microsphere sensors were loaded into the microwell array by placing an aliquot of dry sensor mixture onto the array surface and gently tapping with a glass coverslip. Excess sensors were removed using a stream of air from a compressed gas duster. Vapor Preparation and Delivery. Analyte vapors were prepared using an automated gas delivery system (GDS) (Sensor Research and Development, Orono, ME) described previously.20 Briefly, 20 mL of liquid analyte was placed in a 120 mL bubbler (Ace Glass, Vineland, NJ) and connected to one of the seven vapor delivery lines of the GDS. The bubblers were housed in an oven maintained at 25 °C. Each vapor delivery line is regulated by a mass flow controller and solenoid valves to maintain stable flow rates and enable automated switching between analyte vapors. Saturated vapor streams were prepared by passing a 100 standard cubic centimeters per minute (sccm) flow of ultrazero air (total hydrocarbon content (THC)
