Anal. Chem. 1993, 65, 2329-2334
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Fiber-optic Sensors for pH and Carbon Dioxide Using a Self-Referencing Dye Jennifer W. Parker, Olga Laksin, Clement Yu, Miu-Ling Lau, Suzanne Klima, Russell Fisher, Ian Scott, and Beauford W. Atwater' Technical Center, The BOC Group, 100 Mountain Avenue, Murray Hill,New Jersey 07974
pH and COz fiber-optic sensors were prepared
using self-referencing dyes from the seminaphthorhodamine family (SNARF).The ratio of the emission from the acidand base tautomers of these dyes is insensitive to photobleaching and variations in the excitation intensity. The photophysics of the dyes has been determined. At high concentrations formation of nonfluorescent aggregates of the dyes results in reducedemission yields. The fluorescent lifetimes of the dyes are unaffected by concentration. The pK. of the dyepolymer composite is sensitive to the structure of the polymer. This sensitivity is attributed to a difference in energy of the conjugate base of the dye as a function of the amount of cross-linking of the polymer. The pH sensors show mpH response in the physiological pH range (6.8-7.8) and fast hydration and response times. The COz sensors show 1-2 Torr response with partial pressures of COZin the range of 10-100 Torr and have response and hydration times on the order of several minutes.
INTRODUCTION Novel optical pH and carbon dioxide sensors are an active area of research.'a Recently, several pH-sensitive dyes have been reported which can be excited in the green part of the spectrum and have emission from both the acid and base tautomers.7-10 Sensors prepared with these dyes have a significant advantage over current sensors. Green excitation allows the use of inexpensive green light emitting diodes (LEDs). The ratio between the emission from the base peak and the isosbestic point, in contrast to the emission from the base peak alone, is impervious to many effects associated with luminescence-based optic fiber sensors: bending loss, connector loss, photobleaching, etc. Consequently, sensors fabricated with these dyes have inherently much lower drift
* Author to whom correspondence should be addressed. (1) Baldini,F. SPIE 1990,1368,184-190 (Chemical,Biochemical,and Environmental Fiber Sensors In. (2) Leiner, M. J. P.; Wolfbeis, 0. S. In Fiber Optic Chemical Sensors; Wolfbeis, 0. S., Eds.; CRC Press: Boca Raton, FL, 1991; Chapter 8. (3) Wolfbeis, 0. S.; Weis, L. J.; Leiner, M. J. P.; Ziegler, W. E. Anal. Chem. 1988,60, 2028-2030. (4) Zen, J.-M.; Patonay, G. Anal. Chem. 1991,63, 2934-2938. (5) Gabor, G.; Walt, D.R. Anal. Chem. 1991,63,793-796. (6) Jordan, D. M.; Walt, D.R.; Milanovich, F. P. Anal. Chem. 1987, 59,437-439. (7) Whitaker,J. E.; Haugland,R. P.;Prendergast,F. G. AnaLBiochem. 1991,194, 330-344. (8) Buckler, K. J.; Vaughan-Jones, R. D.Pfluegers Arch. 1990,417, 234-239. (9) Seksek, 0.;Henry-Toulme, N.;Sureau,F.; Bolard,J.Anal.Biochem. 1991,193,4944, (10) Wolfbeis, 0. S.; Rodriguez, N.V.; Werner, T. Mikrochim. Acta 1992,108, 133. 0003-2700/93/0365-2329$04.00/0
than fluorescent sensors that employ a single excitation band and emit from only one tautomer. Several criteria dictated our choices for both the dye and the polymer support. pH sensors making use of dualexcitation wavelengths and a single-emissionwavelength have been prepared and offer reduced drift through the ratio of the two excitation wavelengths.ll Still, due to the relatively greater stability of detectors over excitation sources, we preferred a dye in which both tautomers fluoresced upon excitation by a single wavelength source. The seminaphthorhodafluor dye SNARF-1 carboxylate7 was chosen due to a favorable pK, in solution (vide infra) and sufficient absorptivity in the green to allow use of a green LED. Our selection of a polymer support for the dye was driven by our desire to have a facile process for preparing the sensors themselves. For both the pH and COZsensors, application of the polymer-dye composite directly to the tip of the fiber via micropipet was favored. Luminescence-based fiber-optic sensors have the advantage over absorption-based or colorimetric fiber sensors in that the polymer composite can be applied directly to the distal end of the fiber; no reflective surfaces need to be applied.12J3 A hydrogel containing 2-hydroxyethyl methacrylate was chosen as the support for the pH sensor since many of its physical properties are similar to those of living tissue.14 The Severinghaus technique of separating a bicarbonate solution from the liquid to be analyzed by a gas-permeable membrane was chosen for the C02 sensor. It was desirable that the sensors be kept dry until just before use. Short hydration times were thus needed. Water transport across gas-permeable membranes can be slow.16 To decrease the hydration time of the COZsensor, the polymer-dye composite contained a fine dispersion of bicarbonate micellesin siloxane. Poly(viny1pprolidone)was added to the bicarbonate solution to stabilize the dispersion.
EXPERIMENTAL SECTION Materials. SNARF-1 carboxylate (SNARF-lC), SNARF-1 isothiocyanate (SNARF-ITC),and SNARF-1dextran conjugate (SNARF-Des) were obtained from Molecular Probes, Inc. SNARF-ITCand SNARF-Dex were used as received. SNARF1C was treated with KOH in deionized water to pH 10 and then lyophilized to remove the water. The potassium salt of the acid was much easier to dissolve. Poly(hydroxyethy1 methacrylate) (PHEMA) was obtained from Aldrich and purified as follows. A 105% solution of PHEMA in dimethylacetamidewas prepared. The polymer was precipitated by adding a 10-foldgreater volume of pH 7 aqueous buffer. (11) Gehrich,J.L.;L~bbers,D.W.;Opitz,N.;Hansmann,D.;R.;Miller, W. W.; Tusa, J. K.; Yafuso, M. IEEE Trans.Biomed. Eng. 1986,BME-33, 117. (12) Bacci, M.; Baldini, F.; Cosi, F.; Conforti, G.; Scheggi, A. M. Proc. OFS'89,6th Int. Conf. 1989,425-430 (Optical fiber sensors). (13) Vurek, G. G.; Feustel, P. J.; Severinghaus, J. W. Ann. Biomed. Eng. 1983,11,499-510. (14) Brannon-Peppas, L.; Peppas, N.A. Biomateriab 1990,11,636 644. (15) Robb, W. L. Ann. N.Y. Acad. Sci. 1968, 146, 119. 0 1993 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, 1993
The aqueous fraction was exchanged with fresh buffer and stirred overnight at 70 "C. The polymer was then dried at 55 "C to a constant weight. Gel permeation chromatography indicated a molecular weight distribution, M,IM,, of 2.6. Poly(vinylpyrro1idone)(PVP) Kollidon 90 was obtained from BASF and used as received. Siliconeelastomer (McGhan NuSil MED-6210 silicon intraocular lens elastomers A and B), and catalyst (Huls America, Inc. PC072, platinum divinyltetramethylsiloxane complex) were used as received. Optical fiber with a methacrylate core was obtained from Mitsubishi and had a 125-pm outer diameter. Preparation of the Dye-Polymer Composite. The pH sensors were prepared by one of the following two procedures: (a) A 5-g sample purified PHEMA was dissolved in dimethylacetamide to make a 10% solution. To this was added 3 mg of SNARF-ITC with stirring. The solution was heated at 75 "C for 2 h. The polymer was precipitated by adding a 10-fold excess of pH 6 buffer. (b) To 75 mL of a 10% solution of purified PHEMA in dimethylacetamide was slowly added 2.5 g of powdered KOH. The solution was heated for 2 h at 80-90 "C. The solution was allowed to cool and filtered to remove undissolved KOH. To the solution was added 1mg of SNARFITC. The solution was stirred for 4 hat 65 "C. The polymer was precipitated by the addition of a 10-fold excess of pH 6 buffer. The polymer prepared above was soluble in acetone, dioxane, THF, ethanol, and dimethylacetamide. A concentratedsolution of the polymer in one of these solvents was prepared and applied to the distal tip of methacrylate optical fiber, the tip of which had been prepared by cleaving normal to its longitudinal axis with a fresh scalpel blade. The sensors were allowed to dry overnight before use. The COz sensors were prepared using the followingprocedure. A solution of 10mg of the SNARF-1Cin 1mL of 100mM solution of sodium bicarbonate was prepared. To this was added 100 mg of PVP. The vial was heated to 60 "C and shaken until the PVP had dissolved. Equal amounts of the A and B parts of the MED6210 silicone elastomer were put into the microcontainer of an Eberbach mixer. The PVP solution was added, and the contents were mixed for 1min. The uniform, lathery mixture was whitishblue and was stable for 10 h without a catalyst. The composite material was applied to the distal end of methacrylate optical fiber prepared according to the procedure above. The polymer was allowed to cross-link overnight before use. Apparatus. Emission spectra were recorded on either a ISS Greg-200spectrofluorometer or a single-fiberspectrofluorometer discussed below. Absorption spectra were recorded on a PerkinElmer Lambda 5 spectrophotometer. Lifetime measurements were recorded with the ISS Greg-200 spectrofluorometer using excitation from a Coherent 700 dye laser with rhodamine 6G synchronously pumped with a frequency-doubled Coherent Antares mode-locked Nd:YAG laser. The laser pulse width was
