Anal. Chem. 1996, 68, 1414-1418
Fluorescent Fiber-Optic Calcium Sensor for Physiological Measurements Michael Shortreed,† Raoul Kopelman,*,† Michael Kuhn,‡ and Brian Hoyland‡
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, and Molecular Probes, Inc., Eugene, Oregon 97402-0414
A new optical sensor based on covalent immobilization of a newly synthesized calcium-selective, long-wavelength, fluorescent indicator has been constructed, with a response dynamic range optimal for physiological measurements. Immobilization occurs via photoinitiated copolymerization of the indicator with acrylamide on the distal end of a silanized 125 µm diameter multimode optical fiber. The working lifetime of this sensor is limited only by photobleaching of the indicator. Due to the inherent hydrophilic nature of the acrylamide polymer, the response time of this new sensor is governed by simple aqueous diffusion of the ionic calcium. This results in sensor response times fast enough to monitor some concentration fluctuations at physiological rates. The ability to monitor calcium concentration fluctuations in a high background level of magnesium is also demonstrated with a calculated selectivity of 10-4.5. The important role that ionic calcium plays in biological systems can hardly be overemphasized. Many physiological processes are triggered, regulated, or influenced by calcium.1 In line with the significance of ionic calcium, analytical chemists have made great strides in recent years to develop means for its measurement. Methods for measuring the concentration of Ca2+ include fluorescence ratio imaging,2 fluorescence lifetime imaging,3,4 flow cytometry,5 electrochemical sensing,6-8 and fiber-optic chemical sensing.9-19 A variety of optical sensors have recently †
University of Michigan. Molecular Probes, Inc. (1) Stryer, L. Biochemistry, 3rd ed.; W. H. Freeman and Co.: New York, 1988. (2) Tsien, R. Y.; Rink, T. J.; Poenie, M. Cell Calcium 1985, 6, 145-157. (3) Lakowicz, J. R.; Szmacinski, H.; Nowaczyk, K.; Johnson, M. L. Cell Calcium 1992, 13, 131-147. (4) Lakowicz, J. R. Laser Focus World 1992, 60-80. (5) Ormerod, M. G. Flow Cytometry a Practical Approach; Oxford University Press: New York, 1990. (6) Junter, G. A. Electrochemical Detection Techniques in the Applied Biosciences; Analysis and Clinical Application; Ellis Horwood Limited: Chichester, England, 1988. (7) Tsien, R. Y.; Rink, T. J. Biochim. Biophys. Acta 1980, 599, 623-638. (8) Weingart, R.; Hess, P. Pflugers Arch. 1984, 402, 1-9. (9) Blair, T. L.; Yang, S.-T.; Smith-Palmer, T.; Bachas, L. G. Anal. Chem. 1994, 66, 300-302. (10) Rosatzin, T.; Holy, P.; Seiler, K.; Rusterholz, B.; Simon, W. Anal. Chem. 1992, 64, 2029-2035. (11) Eckert-Tilotta, S. E.; Scouten, W. H.; Hines, J. Appl. Spectrosc. 1991, 45, 491-495. (12) Werner, T. C.; Cummings, J. G.; Seitz, W. R. Anal. Chem. 1989, 61, 211215. (13) Suzuki, K.; Tohda, K.; Tanda, Y.; Ohzora, H.; Nishihama, S.; Inoue, H.; Shirai, T. Anal. Chem. 1989, 61, 382-384. (14) Morf, W. E.; Seiler, K.; Rusterholz, B.; Simon, W. Anal. Chem. 1990, 62, 738-742. ‡
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been developed, all of them having in common a sensing element whose optical properties change in response to the analyte of interest. Fluorescence, chemiluminescence, absorbance, and reflectance have all been exploited for use in optical sensors. Many schemes exist for positioning the chemically sensitive species at the end of an optical fiber. Entrapment in organic (PVC type) membranes tends to be among the most popular for several reasons. Ionophores developed originally for use in ion-selective electrodes (ISEs) can be used without modification in optode membranes. In the latter, the dynamic range and the pH of optimum response can be adjusted by simple exchange of the chromoionophore within the membrane. On the other hand, the major disadvantage of these systems is that the sensitive components are often leached from the organic membrane phase into the analyte solution. This becomes an even greater problem in biological systems, where the lipophilicity requirements are more stringent. Any leaching of components produces a dramatic effect due to the limited membrane volume, in contrast to ISEs with thick membranes. Covalent immobilization of the reactive components will lengthen the lifetime of the optode. However, one also expects an increase in the response time, owing to much slower diffusion within the membrane phase.10 Other attempts at making optodes include the entrapment of sensitive components behind dialysis membranes. This also results in slow response times and short working lifetimes.9 The sensor described herein, based on a new synthesis, has the advantage of covalent immobilization, i.e., a long working lifetime, yet retains a rapid response time. The rapid response time is a result of immobilization of the sensitive component in a hydrophilic polymer, acrylamide. Finally, the photo-polymerization procedure can be extended to nanooptodes. EXPERIMENTAL SECTION Reagents. Aqueous solutions were prepared with 18 MΩ water from a Barnstead I Thermolyne Nanopure II water purification system (Dubuque, IA). All solvents were purchased from VWR Scientific (Brisbane, CA) and were used without further purification. Pure 5-carboxy-2′,7′-dichlorofluorescein diacetate was recrystallized from ethanol, starting from mixed 5,6-carboxy-2′,7′dichlorofluorescein diacetate (Molecular Probes, Inc., Eugene OR). Aqueous calcium calibration buffer kit II and calcium calibration buffer kit with magnesium II (Molecular Probes, Inc.) (15) Schaffar, B. P. H.; Wolfbeis, O. S. Anal. Chim. Acta 1989, 217, 1-9. (16) Ashworth, D. C.; Huang, H. P.; Narayanaswamy, R. Anal. Chim. Acta 1988, 213, 251-257. (17) Kawabata, Y.; Tahara, R.; Imasaka, T.; Ishibashi, N. Anal. Chim. Acta 1988, 212, 267-271. (18) Saari, L. A.; Seitz, W. R. Anal. Chem. 1984, 56, 810-813. (19) Chau, L.-K.; Porter, M. D. Anal. Chem. 1990, 62, 1964-1971. 0003-2700/96/0368-1414$12.00/0
© 1996 American Chemical Society
were used to measure the response of the sensor. All buffer kit solutions contained 100 mM KCl and 10 mM MOPS, pH 7.2, in deionized water in addition to the ethylene glycol derivative, 1,2bis(β-aminoethoxy)ethane-N,N,N′,N′-tetraacetic acid (EGTA/CaEGTA) buffer. The total EGTA concentration in all buffers was 10 mM. All other reagents were purchased from Aldrich (St. Louis, MO) in the highest available purity and were used as received. Synthesis. Synthesis of 5,5′-Diamino-1,2-bis(4-aminophenoxyethane-N,N,N′,N′-tetraacetic Acid Tetramethyl Ester (5,5′-DiaminoBAPTA Tetramethyl Ester).20 A solution of 5,5′-dinitro-BAPTA tetramethyl ester (11.0 g, 17.7 mmol), is dissolved in 200 mL of dimethylformamide and shaken under hydrogen at 40 psi for 3 h in the presence of 0.8 g of 10% palladium on charcoal. When the reaction is complete, the mixture is filtered through diatomaceous earth. The clear filtrate is diluted to 500 mL with ethyl acetate, and the solution is washed three times with saturated NaCl and once with water. The organic layer is dried over Na2SO4 and evaporated under reduced pressure to a gray oil. Trituration with methanol yields 7.0 g (12.4 mmol; 70.3% yield) of 5,5′-diamino BAPTA, tetramethyl ester. Synthesis of 5-Carboxy-2′,7′-dichlorofluorescein Diacetate, Isobutyl Anhydride. 5-Carboxy-2′,7′-dichlorofluorescein diacetate (Molecular Probes, Inc.) is dissolved in 20 mL of CH2Cl2 to give a clear solution. The stirred solution is cooled to 0 °C, and 0.43 g (4.36 mmol) of triethylamine is added. After 5 min, 0.64 g (4.67 mmol) of isobutyl chloroformate is added, and the reaction is stirred at 20 °C for 3 h. The reaction is evaporated and redissolved in ethyl acetate. The triethylamine hydrochloride is filtered, and the colorless filtrate is evaporated at 15 °C to give a colorless oil, which dries to 2.2 g (3.5 mmol; 90% yield). 1H NMR (360 MHz, CDCl3): 8.35 (m) 1H; 8.15 (d), 1H; 7.85 (s), 1H; 6.85 2H (s); 7.2 (s), 2H; 4.4 (q), 2H; (q); 3.95 (s), 1H; 2.35 (s), 6H; and 1.55 ppm (s), 6H. Synthesis of 5′-Amino-Calcium Green Tetramethyl Ester/Diacetate (Calcium Green Amine). 5,5′-Diamino-BAPTA tetramethyl ester (0.35 g, 0.64 mmol) is dissolved in 5 mL of dichloromethane, 1 equiv of the mixed anhydride of 5-carboxy-2′,7′-dichlorofluorescein diacetate (0.43 g, 0.64 mmol) is added, and the reaction is stirred at room temperature for 3 h. Thin-layer chromatography using 5% MeOH/CHCl3 shows a new, ninhydrin positive quenching product formed with an Rf of -0.3, which becomes red and weakly fluorescent on exposure to ammonia vapors. The reaction is loaded directly onto a column packed with 150 mL of silica (4070 µm) and eluted with 3% MeOH/CHCl3. Pure fractions are evaporated to a light yellow foam (0.31 g; 45% yield). 1H NMR (360 MHz, CDCl3): 8.51-8.50 (d), 2H; 8.35 (d), 1H; 7.4-7.35 (m), 3H; 7.15 (s), 2H; 6.9 (m), 3H; 6.8 (m), 2H; 6.3 (s), 1H; 6.2 (d), 1H; 4.4 pp m (s), 2H; 4.25 (s), 2H; 4.15 (s), 4H; 4.05 (s), 4H; 3.15 (s), 6H; and 2.4 ppm (s), 6H. Anal. Calcd for C51H46O18N4Cl2‚1H2O: C, 56.57; H, 4.43; N, 5.13. Found: C, 56.23; H, 4.87; N, 5.47. Calcium Green Monomer Preparation. The 5′-amino-Calcium Green tetramethyl ester/diacetate molecule prepared above was further derivatized using a procedure analogous to that of Munkholm and Walt21 for derivatizing fluorescein. Ten milligrams of the Calcium Green amine derivative (Figure 2) was dissolved in 50 µL of acetone. Acryloyl chloride (0.65 µL) was added to (20) Pethig, R.; Kuhn, M.; Payne, R.; Adler, E.; Chen, T. H.; Jaffe, L. F. Cell Calcium 1989, 10, 491-498. (21) Munkholm, C.; Walt, D. R.; Milanovich, F. P.; Klainer, S. M. Anal. Chem. 1986, 58, 1427-1430.
Figure 1. Schematic representation of the apparatus used to make measurements with the optical fiber calcium sensor.
Figure 2. Amine-containing Calcium Green derivative.
this solution. The reaction vessel was capped and placed in a refrigerator at 4 °C for 3 days. Unreacted acryloyl chloride and acetone were then removed with a speed-vacuum. The result of this reaction was conversion of the added amine functionality to an amide with an extending vinyl group. The vinyl group is necessary to covalently link the fluorescent indicator to the acrylamide polymer through free radical polymerization. The copolymer solution was then prepared as described by Hicks and Updike,22 slightly modified to accommodate the special solubility requirements of the aminated form of Calcium Green. Five milliliters of a 1:1:1 mixture (by volume) of THF, ethanol, and H2O was added to the vial containing the Calcium Green monomer. To this solution were added 0.376 g of acrylamide and 0.086 g of N,N′-methylenebis[acrylamide]. This solution was used in the sensor fabrication described below. Apparatus. All silica multimode optical fiber with 105 µm core and 125 µm core plus cladding diameters was used to fabricate the optical sensors (General Fiber Optics, Inc., Cedar Grove, NJ). An argon ion laser was used for photoinitiation of the polymerization process as well as excitation of the fluorescent dye during measurements (Ion Laser Technology, Salt Lake City, UT). Fluorescence was collected through the objective (LWD CDPlan 0.4 NA; Olympus, Lake Success, NY) of an inverted optical microscope (Olympus IMT-2). Laser rejection was done with the use of an internal dichroic mirror. The microscope output was coupled into a 0.25 m f/4 spectrograph (Jarrell Ash) with a linear array detector (EG&G PARC) by using a 1.5× projection photo(22) Hicks, G. P.; Updike, S. J. Anal. Chem. 1966, 38, 726-730.
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eyepiece (Olympus) and two matched achromats (Melles Griot, Irvine, CA). Figure 1 shows a schematic of the entire arrangement of equipment. Response time and photobleaching measurements were carried out using the same optical arrangement with the exception of the replacement of the acromats and spectrograph with a photomultiplier tube (Pacific Instruments). The voltage on the photomultiplier tube was set at +500 V with a power supply (Bertan Associates, Inc., Model 205A-05R). An amplifierdiscriminator (Model 1120) and a digital synchronous computer (both SSR Instruments Co., Santa Monica, CA) were connected to the photomultiplier. Signal was recorded on a 486 computer via GPIB interface using software programmed in house. Sensor Fabrication. Multimode optical fibers were cut in 1 m lengths, and approximately 2 cm of the jacket was removed from each end with CH2Cl2. The ends were cleaved flat with a sapphire cleaver. Once cleaved, one end of the fiber was glued in a 10 cm long capillary tube for added stability. The fiber end extending from the capillary tube was then silanized using a procedure adapted from Mosbach.23 The silanization solution (2% v/v) was prepared by dissolving 200 µL of 3-(trimethoxysilyl)propyl methacrylate in 10 mL of H2O that was previously adjusted to pH 3.45 with HCl. The fiber ends were placed in the silanization solution for 1 h and were then rinsed with deionized H2O and placed under nitrogen for 1 h. The silanized ends were then sensitized by placing them in a solution of 2 g of benzophenone in 10 mL of cyclohexane for 15 min. Munkholm and Walt21 and later Tan, Shi, and Kopelman24 described the use of acrylamide with fluoresceinamine in a copolymer to form the sensitive region of an optical pH sensor. These procedures have been modified for use in fabricating the sensitive region of this optical calcium sensor. Into a small vial was placed 1 µL of catalyst, prepared by mixing 0.4 g of potassium persulfate in 10 mL of H2O. Then, 70 µL of the Calcium Green monomer solution was added. The vial was placed in a constant temperature oil bath at 60 °C. The sensitized end of the optical fiber was placed in contact with the monomer solution. This assures size control of the formed polymer. A 30 mW laser beam at 4880 Å was coupled into the opposite end of the optical fiber via a microscope objective with a 0.25 NA, which initiated the polymerization. This polymerization was allowed to continue for 1-2 min. The sensors were placed in an aqueous solution of NaOH at pH 12.5 for 5 h at 4 °C. This final step in the sensor fabrication was necessary to cleave the ester groups from the Calcium Green dye, providing a site for chelation of calcium. The sensors were rinsed in deionized water and then placed in a neutral pH phosphate buffer for 24 h prior to use. Experimental Procedure. Fluorescence Measurements. The fluorescent indicator-labeled polymer material, immobilized on the end of the optical fiber, was excited by an Ar+ laser lasing at 488 nm. The laser light was coupled into the optical fiber such that approximately 1 mW emanated from the end on which the sensitive polymer was bound. During each measurment, the fluorescent material was illuminated for approximately 0.1 s. The sensor was placed in a small beaker with approximately 2 mL total volume with an optically clear bottom directly above the objective of the microscope. The position of the sensor was optimized to achieve the highest possible throughput of fluores(23) Mosbach, K. In Methods in Enzymology; Colowick, S. P., Kaplan, N. O., Eds.; Academic Press, Inc.: New York, 1976; Vol. XLIV. (24) Tan, W.; Shi, Z.-Y.; Kopelman, R. Anal. Chem. 1992, 64, 2985-2990.
1416 Analytical Chemistry, Vol. 68, No. 8, April 15, 1996
Figure 3. Fluorescence spectra of the optical sensor in various concentrations of free calcium. From top to bottom: 39.8 µM, 1.35 µM, 602 nM, 225 nM, 100 nM, 37.6 nM, and zero free calcium.
cence to the detector prior to the measurement. The position was then held constant. The solution in the vial was changed after each measurement. The sensor was allowed to equilibrate for 2 min in the new solution prior to the next measurement. Free calcium concentrations were determined by the ratio of EGTA/ CaEGTA in the buffer solutions. All of the measurements were done at pH 7.2. Response Time Measurements. The sensor was positioned over the objective as just described. The tip of the sensor was placed into a single drop of deionized water. The signal of the sensor in the water was allowed to reach steady state, and then about 2 mL of the 39.8 µM free Ca2+ EGTA buffer solution was injected. The fluorescent signal was monitored with a photomultiplier tube instead of the spectrograph and array detector. The laser line was rejected by a dichroic mirror. Photobleaching Measurements. Approximately 2 µL of a dilute aqueous solution of the fluorescent material was placed in a borosilicate glass capilliary tube with 0.86 mm i.d. and a 1.5 mm o.d. The solution was illuminated by an optical fiber which was placed inside the capillary tube with the sample. The 488 nm line of the argon ion laser (20 mW) was coupled into the fiber. The emission was collected by the microscope objective and directed to the photomultiplier tube. The integration time was either 0.01 or 0.1 s. RESULTS AND DISCUSSION The fluorescence spectra of this sensor in various concentrations of free calcium are shown in Figure 3. The increase in fluorescence produced by increasing the concentration from zero free calcium to 38 nM is clearly resolved and has a good signalto-noise ratio. The intensity data were normalized between the minimum intensity (zero free calcium) and the maximum intensity (39.8 µM free calcium) and are shown in Figure 4. This range of calcium concentrations lies conveniently in the physiological range (50 nM-2.5 µM) of many cells.25 A linear regression curve was (25) Ammann, D. Ion-Selective Micro-electrodes; Springer-Verlag: Berlin-Heidelberg, 1986.
Figure 4. Fluorescence intensity of the sensor at each concentration of free calcium, normalized between the minimum fluorecence intensity, found at zero free calcium (shown on the graph as 1 nM), and the maximum fluorescence intensity, found at 39.8 µM free calcium.
Figure 5. Fluorescence spectra of the optical calcium sensor in various concentrations of free calcium containing 1 mM Mg2+. From top to bottom: 39.8 µM, 1.35 µM, 602 nM, 225 nM, 100 nM, 37.6 nM, and zero free calcium.
fitted to the five intermediate values (38 nM-11.35 µM free calcium) of Figure 4. The point at which this line crossed the ordinate axis was taken as the detection limit and equaled approximately 25 nM free calcium. To further show the potential utility of this sensor in biological samples, the sensor’s calcium response was measured with a physiological background level of magnesium (1 mM). In Figures 5 and 6, the spectra and the linearized response of this set of experiments are shown. The ability to distinctly monitor a small change in free calcium concentration, zero free calcium to 38 nM, with such a high background of ionic magnesium indicates that this sensor should have adequate selectivity for most intracellular applications. The detection limit of this sensor for calcium in a 1 mM magnesium background was slightly higher (31 nM free calcium). The fixed interference method, used to measure selectivity values for ion-selective electrodes,25 was adapted to
Figure 6. Normalized response (see Figure 4 caption) of the fluorescence signal to changing calcium concentrations in the presence of 1 mM Mg2+.
Figure 7. Response time measurement for a step change in concentration from zero to 39.8 µM free Ca2+.
optical sensors. The values for the activity of calcium, aCa2+, at the detection limit in the presence of a constant background activity of magnesium, aMg2+, are used in calcultating the selectivity. We find that, based on eq 1, the selectivity for calcium over magnesium is 10-4.5.
log KCa2+,Mg2+ ) log
aCa2+ aMg2+zCa2+/zMg2+ log
)
3.1 × 10-8 M ) -4.5 (1) 1 × 10-3 M
The results of the response time measurements, discussed above, are shown in Figure 7. The 10-90% response is 0.6 s. The buffer capacity of the EGTA undoubtedly had a positive effect on the response time of the sensor. While without a buffer the response may be much slower, we note that the activity of calcium in biological cells is highly buffered and regulated, not unlike that Analytical Chemistry, Vol. 68, No. 8, April 15, 1996
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of the EGTA buffers used for these measurements. To our knowledge, this is the fastest response of any optical Ca2+ sensor to date.9-11,13-17,19,26 As a measure of the working lifetime of the sensor, fluorescence photobleaching measurements were carried out. The fluorescence photobleaching rate of fluorescein was used here as a bench-mark for comparison with the photobleaching rate of the Calcium Green amine monomer. The 1/e time constant of the dilute aqueous fluorescein solution was 1.74 s, whereas that of the Calcium Green amine monomer derivative was 5.89 s. Decay curves of both solutions under identical conditions (laser flux, wavelength, volume, and concentration) are shown in Figure 8. We note that our actual sensor measurments require much less excitation power than was used to produce this data set. The data indicate that the working lifetime of the calcium sensor should be more than 3 times that of the similarly constructed pH sensor of Tan et al.24,27 The later sensors were workable even for 0.2 µm sizes. Thus, in principle, this sensor could be miniaturized to the submicrometer scale. The advantages of this device over similar optical calcium sensors include (1) a linear response in the physiological range of calcium found in most biological cells; (2) sensor response at a physiological pH of 7.2; and (3) good selectivity against magnesium, permitting measurements under normal physiological concentrations (1-5 mM Mg2+).25 In the future, we intend to miniaturize this sensor to the 1 µm size or smaller for use in single living cells. This work is analogous to previous work of this (26) Vo-Dinh, T.; Viallet, P.; Ramirez, L.; Pal, A. Anal. Chem. 1994, 66, 813817. (27) Tan, W.; Shi, Z.-Y.; Smith, S.; Birnbaum, D.; Kopelman, R. Science 1992, 258, 778-781. (28) Rosenzweig, Z.; Kopelman, R. Anal. Chem. 1995, 67, 2650-2654.
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Figure 8. Fluorescence photobleaching decay curves of fluorescein and the Calcium Green amine monomer derivative under identical conditions.
group.24,27,28 Also under development is a means for internal calibration of this sensor which will eliminate concerns over changes in the collection geometry during data aquisition. ACKNOWLEDGMENT This work was supported in part by grants from the National Institutes of Health (1-RO-1-GM50300-01) and Proctor and Gamble. Special thanks go to Shawn Stevens, Erland Stevens, and Erik Hembre for many useful discussions and help with the derivatisation. We also thank Dr. Xiaolin Zhao for his help with the photobleaching measurements. Received for review September 21, 1995. January 31, 1996.X AC950944K X
Abstract published in Advance ACS Abstracts, March 1, 1996.
Accepted
