Anal. Chem. 1995, 67, 2750-2757
Combined Imaging and Chemical Sensing Using a Single Optical Imaging Fiber Karen S. Bronk, Karri L. Michael, Paul Pantano, and David R. Walt* The Max Tishler Laboratory for Organic Chemistry, Department of Chemistty, Tufts University, Medford, Massachusetts 02 155
Despite many innovations and developments in the field of fiber-optic chemical sensors, optical fibers have not been employed to both view a sample and concurrently detect an analyte of interest. While chemical sensors employing a single optical fiber or a noncoherent fiberoptic bundle have been applied to a wide variety of analytical determinations, they cannot be used for imaging. Similarly, coherent imaging fibers have been employed only for their originally intended purpose, image transmission. We herein report a new technique for viewing a sample and measuring surface chemical concentrations that employs a coherent imaging fiber. The method is based on the deposition of a thin, analytesensitive polymer layer on the distal surface of a 350-pmdiameter imaging fiber. We present results from a pH sensor array and an acetylcholine biosensor array, each of which contains -6000 optical sensors. The acetylcholine biosensor has a detection limit of 35 pM and a fast (< 1 s) response time. In association with an epifluorescence microscope and a charge-coupled device, these modified imaging fibers can display visual information of a remote sample with 4-pm spatial resolution, allowing for alternating acquisition of both chemical analysis and visual histology. In the last decade, the study of biological processes at the cellular and subcellular levels has prompted the development of a wide range of micrometer-scale measurement For example, voltammetric microelectrodes have monitored the exocytotic secretion of neurotransmitters from individual adrenal chromaffin cells,3and (sub)micrometer fiber-optic chemical sensors have measured intracellular and intraembryonic P H . ~Alternatively, intracellular studies can be conducted by loading living cells with a fluorescent dye sensitive to the chemical variant of interest and monitoring the resulting fluorescence using optical In this manner, a cell's physiological processes may be examined by measuring chemical concentrations or partitioning using fluorescent reporters. However, while these microscopy techniques offer excellent spatial resolution, they are (1) Ewing, A. G.: Strein, T. G.; Lau,Y. Y. Acc. Chem. Res. 1992,25, 440-447. (2) Tan, W.; Shi, Z. Y.; Kopelman, R Anal. Chem. 1992,64, 2985-2990. (3) Kawagoe, K. T.: Jankowski, J. A.: Wightman, R. M. Anal. Chem. 1991,63, 1589-1594. (4) Tan, W.: Shi, Z. Y.; Smith, S.; Bimbaum. D.; Kopelman, R. Science 1992, 258, 778-781. (5) Slavik, J. Fluorescent Probes in Cellular and Molecular Biology; CRC Press: Boca Raton, FL, 1992. (6) Tsien, R. Y. Methods Cell Biol. 1989,30, 127-156. (7) Tsien, R. Y. Annu. Rev. Neurosci. 1989,12, 227-253.
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Analytical Chemistry, Vol. 67, No. 17, September 1, 7995
limited to samples that can be brought to the microscope stage. Conversely, while a (sub)micrometer-sized fiber-optic chemical sensor or a microelectrode is not confined to the stage of a microscope, both require extremely precise positioning and are restricted to monitoring one cell a time. We herein report a new technique for viewing a sample and measuring surface chemical concentrations that employs an optical imaging fiber. An imaging fiber is comprised of thousands of individual 3-4pm-diameter optical fibers melted and drawn together in a coherent manner such that an image can be carried and maintained from one end to the other."1° In the present ,innovation,a 350-pm-diameter distal fiber surface, which contains -6000 optical sensors, is coated with a uniform, planar sensing layer that can measure chemical concentrations with spatial accuracy yet is thin enough that it does not compromise the fiber's imaging capabilities. By combining the distinct optical pathways of the imaging fiber with the spatial discrimination of a chargecoupled device," visual and fluorescence measurements can be obtained with 4pm spatial resolution over tens of thousands of square micrometers. Results are presented from two sensor arrays fabricated via two different covalent polymer/dye immobilization chemistries. A pH sensor was fabricated by spin-coating an N-fluoresceinylacrylamide-derivatized hydroxyethyl methacrylate polymer onto the distal face of an imaging fiber. The fluorescence intensity of the pH indicator is enhanced upon deprotonation. Alternatively, an acetylcholine-sensitive layer was created by first cc-immobilizing acetylcholinesterase in a water-soluble, functionalized prepolymer known as poly(acry1amide-co-N-acryloxysuccinimide).12 The enzyme-derivatized polymer was spin-coated onto an imaging fiber and subsequently reacted with fluorescein isothiocyanate. In this arrangement, the dissociated protons from the enzymegenerated acetic acid quench the fluorescence of the immobilized dye in proportion to the concentration of acetylcholine. The acetylcholine biosensor has a detection limit of 35 pM and a fast (
