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Investigation of dynamically modified optical-fiber sensors for pH sensing at the extremes of the pH scale
Microchemical Journal 69 Ž2001. 123᎐131
Investigation of dynamically modified optical-fiber sensors for pH sensing at the extremes of the pH scale Darrell R. Fry, Donald R. BobbittU Department of Chemistry and Biochemistry, Uni¨ ersity of Arkansas, Fayette¨ ille, AR 72701, USA Received 8 August 2000; received in revised form 13 November 2000; accepted 14 November 2000
Abstract There exist a large number of immobilization strategies useful for preparing optical fiber ŽOF. sensors; one unique approach has been termed dynamic modification. Dynamic modification borrows techniques from reverse-phase high performance chromatography ŽRP-HPLC. to render the optical fiber surface sufficiently hydrophobic to reversibly immobilize hydrophobic probe molecules. Although RP-HPLC stationary phases are known to hydrolyze under conditions of high or low pH, the hydrophobic surface on the optical fiber shows immunity to hydrolysis under similar conditions, including increased temperature Ž65⬚C., and in organic-containing solvents at high and low pH values Ž33% acetonitrile at pH 12 and 2.. Since the surface is stable at the extremes of the pH scale, pH sensing in these regions is possible using this strategy. Two hydrophobically derivatized, pH-sensitive fluorescein dyes were immobilized on the surface of a C-18 optical fiber; however, upon immobilization, the dyes were found to convert into a non-pH-sensitive, non-fluorescent lactone form. pH sensing was achieved over the pH range 7.5᎐10 by controlling the hydrophobicity of the optical fiber surface. The limitations and advantages of this approach are evaluated and discussed. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Optical-fiber sensors; Dynamic modification; Fluorescein-lactone form; Hydrophobic; Immobilization; pH sensor
1. Introduction Optical fibers ŽOFs. have been a promising platform for the development of new sensing strategies since the 1980s. In general, the advantages of using OFs as part of a sensing protocol lie in their ability to propagate light over long distances with little loss, their substantial immuU
Corresponding author. Tel.: q1-501-575-7945; fax: q1501-575-4049. E-mail address: [email protected] ŽD.R. Bobbitt..
nity from electrical interference, their physical flexibility, their chemical resilience and their ease of miniaturization. A specific sensing application where OF sensors may contribute is in the measurement of pH. Typically, the glass pH electrode is used to determine pH; however, it does have limitations that OF pH sensors may overcome. For example, Kopelman and co-workers measured the pH of a single living rat embryo using an OF-based sensor. With a glass pH electrode, approximately 1000 rat embryos would have had to be destroyed
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to obtain the same information and individual variations would have been undetectable w1x. Another advantage OF pH sensors have over the glass pH electrode is in situations where additional variables effect the measurement of pH. For example, several OF probes that measure ionic strength and pH simultaneously have been reported w2,3x. OF pH sensors may offer advantages over the pH electrode in the alkaline region Žabove pH 12. w4x. In this region, the glass pH electrode responds to both hydronium and sodium ions, giving rise to an alkaline error. Ensafi and Kazemezadeh developed an OF sensor to measure pH in the range 9.5᎐13 with reportedly no alkaline error w5x. Although one advantage of using OF pH sensors over the pH electrode is at the extremes of the pH scale, few OF sensors have been reported in this region. The harsh chemical environment would require the development of a renewable immobilization strategy based on a hydrolysis-resilient platform. Most commonly, an optical fiber is transformed into an OF sensor by immobilizing a specific probe molecule at, or near, the optical fiber surface. For relatively small and robust probe molecules, covalent linkage is often used because of its simplicity; however, the immobilization event often pacifies complex probe molecules. Entrapment within, or to, a semi-permeable membrane, or in a sol᎐gel matrix has also been used to immobilize complex probe molecules. For either of these approaches, the analyte must diffuse through the membrane or sol᎐gel, giving rise to a slow response time. Without careful storage, the sol᎐gel matrix has been shown to shrink, leading to cracks and eventual leakage of the probe molecule into the measurement solution w6x. Furthermore, sol᎐gel matrices are typically made from a silica polymer. Silica is known to be soluble in solutions above pH 8 and susceptible to hydrolysis at pH values lower than 2. Recently, Bobbitt and co-workers have developed a different approach for the immobilization of probe molecules on optical fibers based upon dynamic modification w7᎐9x. In this approach, the distal end of an optical fiber is rendered hydrophobic through the attachment of octadecylsilane. Then a hydrophobic probe molecule Žeither
inherent or designed. can be immobilized through a hydrophobic association with this surface. This approach has several advantages. The C-18-derivatized fibers have been extensively characterized by both FT-IR and by detailed studies using the immobilization of hydrophobic probe molecules to probe surface characteristics. These studies have shown the hydrophobic layer to be very dense, ) 4 molrm2 w7x. Second, the probeto-probe reproducibility has been shown to be excellent, of the order of 5᎐7% relative standard deviation ŽR.S.D.. w8x. Third, probe molecules that either irreversibly bind, or are degraded by, the sensing environment can be removed simply by rinsing with methanol. The sensor can then be regenerated by incubating the C-18-derivatized fiber in a solution of the probe molecule. In this publication, the utility of dynamic modification is extended by showing that the hydrophobic surface is immune to hydrolysis, and the immobilization of hydrophobic derivatives of fluorescein, a pH-sensitive dye, potentially allows pH sensing at the extremes of the pH scale. The fluorescein derivatives used are shown to form a non-fluorescent and non-pH-sensitive lactone on the hydrophobic surface. By decreasing the density of the hydrophobic layer, pH sensing can be achieved.
2. Experimental 2.1. Materials The optical fiber used for these studies had a 1000-m core diameter and a protective jacket composed of nylon ŽEnsign-Bickford Optics Co., Avon, CT, type HCNM1000T.. Chlorodimethyloctadecylsilane, chlorotrimethylsilane, toluene, methylene chloride, methanol, sodium chloride, dibasic anhydrous potassium phosphate and sodium tetraborate decahydrate were purchased from Aldrich ŽMilwaukee, WI.. The nitrogen used as a blanket for the synthetic procedures was oxygen-free and was dried by passing it first through a 30 = 5 cm column containing anhydrous calcium chloride and then through a 30 = 5 cm silica gel column. The two probes, N-Žfluo-
D.R. Fry, D.R. Bobbitt r Microchemical Journal 69 (2001) 123᎐131
rescein-5-thiocarbamoyl .-1,2-dihexadecanoyl-snglycero-3-phosphoethanolamine, triethylam monium salt ŽC-15 = 2F. and 5-octadecanoylaminofluorescein ŽC-18F. were obtained from Molecular Probes ŽEugene, OR.. 2.2. Optical set-up The optical configuration for fluorescence measurements using dynamically modified optical fibers has been previously reported in detail w7᎐9x. Briefly, light from an argon ion laser ŽIon Laser Technology, Salt Lake City, UT, Model 490 ASL. operating at 488 nm was modulated using a chopper operating at 550 Hz ŽStanford Research Systems, Inc., Palo Alto, CA, Model SR540.. The resulting laser light was then sent through a dichroic beam splitter ŽCVI Laser Corp., Albuquerque, NM. and into a microscope objective ŽNewport, Model-10X, FL s 14.8 mm, NAs 0.25. which focused the excitation light onto the optical fiber and collimated the emission light. The fluorescence was reflected at the dichroic mirror and collected by a 75-mm focal length, 50-mm diameter plano-convex lens. The collimated light was passed through a holographic filter ŽPhysical Optics Corp., Torrance, CA, Model RHE 488. and into a scanning monochromator ŽCVI, Model DK 240-2.. Light exiting the monochromator was detected with a photomultiplier tube ŽThorn EMI Gencom, Inc., Fairfield, NJ, Model 9816B. which was powered by a constant-voltage power supply ŽBertran Associates, Model 215.. The signal from the PMT was then sent to a lock-in amplifier ŽStanford Research Systems, Inc., Palo Alto, CA, Model SR510.. The demodulated signal was sent to a personal computer for storage. 2.3. Preparation of C-18-modified optical fibers The distal end of the optical fiber surface was prepared by successive polishing with 60-, 9-, 1and 0.3-m lapping film. The polished surface was then activated by refluxing in 2M HCl for 6 h. The fibers were then washed thoroughly with distilled water and dried at 120⬚C overnight. The activated surface was then refluxed for 4 h in a mixture of dry toluene and 0.5g of chlorodimeth-
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yloctadecylsilane and then placed in an ultrasonic bath for an additional 24 h. The solution mixture was removed and 20 ml of toluene and 5ml of chlorotrimethylsilane were added for end capping. Ultrasonic agitation was continued for 12 h. All synthetic steps proceeded in an enclosed anhydrous system under the protection of nitrogen. After modification, the fibers were placed in a soxhlet extractor and washed in turn by methanol, methylene chloride, and hexane. 2.4. Immobilization procedure Immobilization of the probes was accomplished by incubating the C-18-derivatized fiber in a 5 mM solution of the probe molecule for 5 min. The fiber was then removed, air dried to remove the solvent, and rinsed with pH 11 buffer solution. The solvent for C-18F was methanol, while that for C-15 = 2F was propanol.
3. Results and discussion Manufacturers of silica-based RP-HPLC columns recommend that mobile phases do not exceed pH 8, or be less than pH 2. This range is decreased at elevated temperatures. At pH values G 12, not only is the octadecylsilane hydrolyzed, but the silica itself is also soluble in the mobile phase w10,11x. For RP-HPLC columns, stationaryphase hydrolysis is known to increase with temperature w12x. Furthermore, purely aqueous mobile phases are known to collapse the octadecylsilane chains onto the surface and retard hydrolysis; however, organo-aqueous mobile phases do not cause this collapse to occur. It has been shown qualitatively, that water in water᎐ acetonitrile mobile phases is associated with the octadecylsilane layer equally from 0᎐80% acetonitrile w13x. Since the C-18-derivatized optical fibers also use silica, the same factors would be expected to contribute to hydrolysis of the octadecylsilane layer on the fibers. The effect of pH on the stability of the C-18 layer was evaluated under a variety of conditions to better define the range of pH sensing appropriate for this protocol.
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Fig. 1. Surface stability at the extremes of the pH scale. A᎐D represent replicate immobilizations of 3-N-octylriboflavin, labeled i, and the corresponding blank signal, labeled b, on the same C-18-derivatized optical fiber. After each panel, the probe is incubated for 20 min under conditions designed to test the integrity of its surface. The conditions for each are: ŽA. methanol at room temperature; ŽB. pH 12 solution containing 33% acetonitrile at room temperature; ŽC. pH 12 solution containing 33% acetonitrile at 65⬚C; and ŽD. pH 2 solution containing 33% acetonitrile at 65⬚C.
Fig. 1 shows a series of immobilization events performed after incubating a single C-18-derivatized fiber under conditions designed to test the resilience of the hydrophobic surface towards hydrolysis. The conditions depicted in Fig. 1 correspond to situations where RP-HPLC octadecylsilane layers are known to degrade. The conditions are low pH ŽpH 2., high pH ŽpH 12., the presence of organic solvent Ž33% acetonitrile ., and stability at elevated temperature Ž65⬚C.. Previous studies in our laboratory have noted a correlation between the amount of octadecylsilane on the surface and the amount of hydrophobic probe molecule associated with the surface. If the octadecylsilane surface of the C-18-derivatized fiber was being hydrolyzed, then the amount of the fluorescent hydrophobic probe, octylriboflavin, should decrease from immobilization to immobilization. However, the amount of octylriboflavin does not show this behavior, thereby demonstrating that the C-18-derivatized fiber ex-
hibits substantial immunity from hydrolysis under a variety of extreme conditions. The protocol for each panel depicted in Fig. 1 was as follows: incubation of the fiber for 20 min under the test conditions; immobilization of octylriboflavin; measurement of fluorescence; and finally, removal of the octylriboflavin by a methanol rinse prior to the start of the next measurement sequence. Each process was repeated a total of three times. By the end of the sequence depicted in each panel of Fig. 1, the fiber had been exposed to the incubation solution for a total of 60 min. The control experiment, depicted in panel A, was performed by incubating the fiber in roomtemperature pH 7 buffer for 20 min. Panel B represents incubation of the OF in a pH 12, 33% acetonitrile Žvrv. solution for 20 min; and panel C represents incubation in a pH 12, 33% acetonitrile Žvrv. solution at 65⬚C for 20 min. Finally, panel D represents incubation in a pH 2, 33% acetonitrile Žvrv. solution at 65⬚C for 20 min.
D.R. Fry, D.R. Bobbitt r Microchemical Journal 69 (2001) 123᎐131
Other incubation times were investigated, for example up to 1-h incubations were evaluated, and 2-propanol was also used as the organic modifier. Although there is a slight difference, approximately 10% of the signal from panel to panel, there is no gross decrease in signal, and therefore the amount of octylriboflavin immobilized on the fiber is, within experimental error, invariant. Again, these results suggest that the C-18-derivatized fiber is immune to hydrolysis in the pH range 2᎐12, at elevated temperatures, and in the presence of organic modifiers. Other groups have found similar results; long-chain stationary phases, such as octadecylsilane, slow the rate of hydrolysis w11,14x. In our case, no hydrolysis was observed. In RP-HPLC separations, care is taken to limit the pH range of the mobile phase to 2᎐8. Under conditions outside of this range, the stationary phase, octadecylsilane, is found to be ‘stripped’ from the column. The retention time of a hydrophobic species thereby decreases dramatically due to the interaction with the reduced amount of octadecylsilane on the stationary phase. In contrast, the C-18-deri¨ atized fiber, also modified with octadecylsilane, retains the same amount of octylriboflavin, even after an extensive series of incubations. One possible explanation for the observed ruggedness concerns the amount of excess derivatizing reagent used in the synthetic procedure. The OF surface being modified is extremely small, 1 mm in diameter, as compared to high surface-area silica stationary phases, which have surface areas of the order of 400᎐600 m2rg w15x. Even under conditions where 10᎐20 fibers are simultaneously derivatized, the amount of octadecylsilane reagent exceeds the available surface SiOH groups by approximately nine orders of magnitude. Thus, a very large excess of derivatizing reagent is possible for chemical modification of the optical fiber. Theoretically, the maximum coverage of an octadecylsilane monolayer is limited by the coverage of silanol groups on the surface, which has been determined to be of the order of 8 molrm2 w15x. In comparison, most RP-HPLC stationary phases based on octadecylsilane have a surface coverage of between 1 and 3 molrm2 of octadecylsilane w15x. These results suggest that the dynamic modification protocol
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can be applied to measurement problems involving harsh experimental conditions that were not previously amenable to optical fiber-based sensors. Hydrophobic derivatives of fluorescein were chosen as probe molecules for a number of reasons. Fluorescein is a well-characterized, high quantum yield, pH-sensitive dye w16᎐18x. In addition, hydrophobic derivatives of fluorescein are commercially available. A Hitachi F-2500 fluorescence spectrometer was used to generate a pH vs. florescence intensity plot for each derivative. Each data point is an average of three measurements. A fluorescence intensity vs. pH plot for C-18F is given in Fig. 2a; from this figure, the p K a is estimated to be 9. A spline line was added to emphasize the shape of the curve. The range of each replicate measurement is smaller than the size of the solid circles. The fluorescence intensity does reach a maximum at a pH of approximately 10, and the fluorescence decreases at higher pH values. For the other fluorescein derivative, C-15 = 2F, its p K a is reported to be approximately 6.2 w19x. Reversible association Žimmobilization. of a hydrophobic probe molecule with the C-18-derivatized fiber is key to the dynamic modification sensing scheme. Furthermore, measurement of pH in mixed solvent systems is of general interest. In the case of dynamic modification, the minimum hydrophobicity required of a probe molecule in order to assure efficient association with the fiber surface and allow sensing in a mixed solvent system is of interest. For the experiments summarized in Table 1, a 5 mM solution of each derivative was incubated with a hydrophobic fiber for 5 min. The fiber was removed, air dried and rinsed with pH 12 buffer. The fiber was then immersed in the test solvents listed in Table 1 at pH 9 for 10 min. Finally, the fiber was immersed in methanol. Each entry was repeated at least five times. Immobilization of the probe molecule can be judged in a number of ways; however, the simplest is a two-part question: does the test solvent contain fluorophore after the fiber has been removed, and does the methanol rinse contain any fluorophore? As Table 1 shows, the C-15 = 2F derivative does not leak from the fiber, even in a 15% methanol
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Fig. 2. Ža. Fluorescence vs. pH for C-18F in solution. Žb. Fluorescence vs. pH for C-18F immobilized on a C-18-derivatized optical fiber.
solution. In contrast, the C-18F derivative partially dissociates from the fiber, even in a 5% methanol solution. In both cases, it was found
that the hydrophobic derivatives of fluorescein can be reversibly associated with a single fiber in an aqueous buffer. Fig. 3 shows four ‘sensing’ events in pH 9 buffer for C-18F. The most obvious result is the lack of difference between the blocked signal and the signal from immobilized C-18F. After the ‘sensing’ event in pH 9 buffered solution, the C-18-derivatized fiber was rinsed with methanol. As the non-fluorescent fiber was rinsed with methanol, the fluorescent C-18F was clearly observed diffusing away from the C-18-derivatized fiber. In addition, no C-18F was found in the aqueous solution. C-15= 2F gave similar results Žnot shown.. Fluorescein is known to form a nonfluorescent inner lactone, as depicted in Fig. 4, in hydrophobic solvent systems w17,18x. Furthermore, Dutta and Salesse incorporated C-18F into a palmitic acid Langmuir᎐Blodget film w20x. They found that the fluorescence of C-18F was quenched as the surface pressure, and thus the hydrophobicity of the film, was increased. They also performed spectroscopic measurements on the film and in a variety of different solvent systems. These results implied that the lactone form was found in the film and in non-polar hydrophobic solvents, while the zwitterionic form was found in polar solvents. The dense hydrophobic portion of a C-18-derivatized fiber closely resembles the hydrophobic Langmuir᎐ Blodget film of palmitic acid. The literature cited, along with the visual observation of the C-18F and C-15 = 2F being removed by methanol from the C-18-derivatized fiber, strongly suggests that C-18F is associated with the fiber in the lactone non-fluorescent form.
Table 1 Summary of association measurements for C-18F and C-15 = 2F a Probe
Buffer
MeOH rinse after buffer
5% MeOH buffer
MeOH rinse after 5% MeOH buffer
10% MeOH buffer
MeOH rinse after 10% MeOH buffer
15% MeOH buffer
MeOH rinse after 15% MeOH buffer
C-18F C-15 = 2F
No No
Yes Yes
Yes No
Yes Yes
Yes No
No Yes
Yes No
No Yes
a Immobilization is judges by a two-part question: does the solvent contain fluorophore after the fiber has been removed; and does the methanol ŽMeOH. rinse contain fluorophore?
D.R. Fry, D.R. Bobbitt r Microchemical Journal 69 (2001) 123᎐131
Fig. 3. Immobilization of C-18 on C-18-derivatized optical fiber. Immobilized C-18F, a, and pH 9 blank, b, are indistinguishable; g represents residual light with the laser blocked. The C-18F immobilization was confirmed by immersing the probe in methanol, where the fluorophore could be seen diffusing away from the probe tip Žnot shown..
From these observations, a model of how C-18F and C-15= 2F interact with a C-18-derivatized fiber can be proposed. A model, describing the decay of the signal in Fig. 3, can be proposed based on the following premises: Ž1. the solvent environment of the octadecylsilane chains affects the degree of extension of the chains, and there-
Fig. 4. Solvent-dependent equilibrium of fluorescein-free acid form and non-fluorescent inner lactone form.
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fore the local environment of the probe molecule; and Ž2. the C-18-derivatized fiber is a very densely packed layer of octadecylsilane. First, in an organic solvent the octadecylsilane chains are extended, and the probe molecule selectively partitions from the organic solvent into the chains. In addition, a portion of the organic solvent also partitions into the chains. The volatile organic solvent is easily removed by application of a stream of warm air to the fiber, leaving the probe molecule behind. As the C-18-derivatized fiber is rinsed with aqueous buffer, the outside chains, which are exposed to the aqueous environment, collapse onto the inside chains. Finally, as the C-18-derivatized fiber is placed in an aqueous solution, the probe molecules are in contact with the aqueous environment. As time progresses, the chains collapse onto themselves, thereby changing the local environment experienced by the probe. In this extremely hydrophobic environment, it is reasonable to expect the probe molecule to form the lactone, which is non-fluorescent. In an effort to open the chains so that probe molecules can be more effectively exposed to the surrounding solution, a small % of methanol was added to the buffer. These results are summarized in Table 1. At 5% methanol, the C-18F did fluoresce; however, it was also not immobilized onto the fiber. In contrast, the C-15= 2F was immobilized in the inner lactone form with up to 15% methanol; however, above 15%, C-15 = 2F was not immobilized on the fiber. Returning to the model explained above, it appears that if the hydrophobicity of the OF surface could be reduced by decreasing the amount of octadecylsilane on the surface, the fluorescein derivatives would be less likely to form the non-fluorescent lactone form, since this process is so clearly tied to the hydrophobicity of the environment surrounding the probe. The hydrophobic derivatization of the OF does provide several mechanisms for producing reduced-coverage fibers. One possibility is to stop the nitrogen purge gas 3 h into the reaction, so that the moisture-sensitive octadecylsilane reagent will react with the moisture in the atmosphere, thereby decreasing the amount of octadecylsilane and therefore the surface coverage of octadecylsilane
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D.R. Fry, D.R. Bobbitt r Microchemical Journal 69 (2001) 123᎐131
Fig. 5. The two figures, A and B, represent immobilization events of C-8R on two different C-18-derivatized optical fibers. Panel A depicts the amount of C-8R immobilized on a low-coverage C-18-derivatized optical fiber, while B depicts an increased amount of C-8R immobilized on a normal-coverage C-18-derivatized optical fiber. The blocked signal is represented by a, the blank signal by b, and immobilization of C-8R by i.
on the surface. Fig. 5 shows two immobilizations of octylriboflavin on a normal fiber and two immobilizations on a reduced-coverage fiber. As is evident, the normal fiber holds significantly more octylriboflavin than the reduced-coverage fiber. On the latter, C-18F was immobilized, and its fluorescence proprieties changed with pH. Fig. 2b shows the calibration curve for four immobilizations of C-18F. A spline line was added to emphasize the shape of the curve. Comparison of the fluorescence response versus pH for C-18F in solution, Fig. 2a, and C-18F immobilized on a reduced-coverage fiber, Fig. 2b, confirms that the immobilized probe is responsive to pH. The shapes and slopes of the curves are nearly identical. Furthermore, the probe was immobilized on a renewable surface. Although the reduced coverage fibers can immobilize C-18F and C-15= 2F in a fluorescent and pH-sensitive form, they do not show the same resilience to hydrolysis. For instance, after several w8x immobilizations and measurement in pH 10 or 11 buffered solutions, the reduced-coverage fibers failed to hold the immobilized C-18F. The fluorphore could be seen diffusing away from the OF surface. However, other OF sensors have shown similar inadequacies. For example, sol᎐gel matrices are known to leak probe molecule if not stored and handled carefully. An extensive list of fluorescent pH indicators is available in most
chemistry reference books w21,22x. Obviously, one consideration of any potential probe molecule should be its fluorescent properties in hydrophobic solvents. We are currently pursuing several other probe molecules for pH sensing. Previous studies in our laboratory have shown that placement of the C-18-derivatized fiber in the sensing solution alone leads to approximately 5᎐7% R.S.D. in measurements. For the experiments summarized in Fig. 2b, the R.S.D. was approximately 10%. When the R.S.D. of the each pH measurement was subtracted from the R.S.D. of the blank signal, the result was nearly zero. This implies that fiber placement within the sensing vessel is a limiting factor in the sensing protocol. To overcome this limitation, a pH-insensitive dye with a larger Stokes shift could be immobilized concurrently with the pH-sensitive dye. The ratio of the pH-insensitive dye to the pHsensitive dye would be invariant to placement. We immobilized octylriboflavin four times and monitored its fluorescence intensity at two wavelengths, 525 and 535 nm, without moving the fiber. Although the R.S.D. was 7% from immobilization-to-immobilization, the ratio of the response at the two wavelengths had an R.S.D. which was, within experimental error, f 0%. This shows that the dynamic modification event can be used to prepare a suitable mixed-probe surface, which can respond to a specified analyte while
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providing immunity from measurement-to-measurement variations.
4. Conclusions The dynamic modification protocol is suitable for pH sensing at the extremes of the pH scale, as the high-density octadecylsilane surface provides significant immunity from hydrolysis, and the surface provides a renewable sensing surface. Two pH-sensitive derivatives of fluorescein were immobilized on this platform. Fluorescein is known to form a lactone, which is non-fluorescent and non-pH sensitive in hydrophobic solvents. The derivatives of fluorescein were shown to be nonfluorescent and non-pH sensitive when immobilized on a dense C-18-derivatized fiber. Renewable pH sensing was achieved by reducing the density of the octadecyl chains attached to the fiber; however, the octadecyl chains were more easily hydrolyzed, causing the sensor to degrade. The sensor responds over the pH range 7.5᎐10. These results provide the basis for future applications involving pH sensing. First, the development of specific hydrophobic, pH-sensitive dyes for use in pH sensing protocols is currently being explored. Second, new strategies for controlling the hydrophobicity of the OF surface, while providing immunity to hydrolysis, are under investigation. For example, the use of a mixture of monomeric short- and long-chain hydrocarbon phases and a polymeric surface are both being explored.
Acknowledgements Support from the National Science Foundation for portions of this work through grant no. EPS9977778 is acknowledged.
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Investigation of dynamically modified optical-fiber sensors for pH sensing at the extremes of the pH scale Darrell R. Fry, Donald R. BobbittU Department of Chemistry and Biochemistry, Uni¨ ersity of Arkansas, Fayette¨ ille, AR 72701, USA Received 8 August 2000; received in revised form 13 November 2000; accepted 14 November 2000
Abstract There exist a large number of immobilization strategies useful for preparing optical fiber ŽOF. sensors; one unique approach has been termed dynamic modification. Dynamic modification borrows techniques from reverse-phase high performance chromatography ŽRP-HPLC. to render the optical fiber surface sufficiently hydrophobic to reversibly immobilize hydrophobic probe molecules. Although RP-HPLC stationary phases are known to hydrolyze under conditions of high or low pH, the hydrophobic surface on the optical fiber shows immunity to hydrolysis under similar conditions, including increased temperature Ž65⬚C., and in organic-containing solvents at high and low pH values Ž33% acetonitrile at pH 12 and 2.. Since the surface is stable at the extremes of the pH scale, pH sensing in these regions is possible using this strategy. Two hydrophobically derivatized, pH-sensitive fluorescein dyes were immobilized on the surface of a C-18 optical fiber; however, upon immobilization, the dyes were found to convert into a non-pH-sensitive, non-fluorescent lactone form. pH sensing was achieved over the pH range 7.5᎐10 by controlling the hydrophobicity of the optical fiber surface. The limitations and advantages of this approach are evaluated and discussed. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Optical-fiber sensors; Dynamic modification; Fluorescein-lactone form; Hydrophobic; Immobilization; pH sensor
1. Introduction Optical fibers ŽOFs. have been a promising platform for the development of new sensing strategies since the 1980s. In general, the advantages of using OFs as part of a sensing protocol lie in their ability to propagate light over long distances with little loss, their substantial immuU
Corresponding author. Tel.: q1-501-575-7945; fax: q1501-575-4049. E-mail address: [email protected] ŽD.R. Bobbitt..
nity from electrical interference, their physical flexibility, their chemical resilience and their ease of miniaturization. A specific sensing application where OF sensors may contribute is in the measurement of pH. Typically, the glass pH electrode is used to determine pH; however, it does have limitations that OF pH sensors may overcome. For example, Kopelman and co-workers measured the pH of a single living rat embryo using an OF-based sensor. With a glass pH electrode, approximately 1000 rat embryos would have had to be destroyed
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to obtain the same information and individual variations would have been undetectable w1x. Another advantage OF pH sensors have over the glass pH electrode is in situations where additional variables effect the measurement of pH. For example, several OF probes that measure ionic strength and pH simultaneously have been reported w2,3x. OF pH sensors may offer advantages over the pH electrode in the alkaline region Žabove pH 12. w4x. In this region, the glass pH electrode responds to both hydronium and sodium ions, giving rise to an alkaline error. Ensafi and Kazemezadeh developed an OF sensor to measure pH in the range 9.5᎐13 with reportedly no alkaline error w5x. Although one advantage of using OF pH sensors over the pH electrode is at the extremes of the pH scale, few OF sensors have been reported in this region. The harsh chemical environment would require the development of a renewable immobilization strategy based on a hydrolysis-resilient platform. Most commonly, an optical fiber is transformed into an OF sensor by immobilizing a specific probe molecule at, or near, the optical fiber surface. For relatively small and robust probe molecules, covalent linkage is often used because of its simplicity; however, the immobilization event often pacifies complex probe molecules. Entrapment within, or to, a semi-permeable membrane, or in a sol᎐gel matrix has also been used to immobilize complex probe molecules. For either of these approaches, the analyte must diffuse through the membrane or sol᎐gel, giving rise to a slow response time. Without careful storage, the sol᎐gel matrix has been shown to shrink, leading to cracks and eventual leakage of the probe molecule into the measurement solution w6x. Furthermore, sol᎐gel matrices are typically made from a silica polymer. Silica is known to be soluble in solutions above pH 8 and susceptible to hydrolysis at pH values lower than 2. Recently, Bobbitt and co-workers have developed a different approach for the immobilization of probe molecules on optical fibers based upon dynamic modification w7᎐9x. In this approach, the distal end of an optical fiber is rendered hydrophobic through the attachment of octadecylsilane. Then a hydrophobic probe molecule Žeither
inherent or designed. can be immobilized through a hydrophobic association with this surface. This approach has several advantages. The C-18-derivatized fibers have been extensively characterized by both FT-IR and by detailed studies using the immobilization of hydrophobic probe molecules to probe surface characteristics. These studies have shown the hydrophobic layer to be very dense, ) 4 molrm2 w7x. Second, the probeto-probe reproducibility has been shown to be excellent, of the order of 5᎐7% relative standard deviation ŽR.S.D.. w8x. Third, probe molecules that either irreversibly bind, or are degraded by, the sensing environment can be removed simply by rinsing with methanol. The sensor can then be regenerated by incubating the C-18-derivatized fiber in a solution of the probe molecule. In this publication, the utility of dynamic modification is extended by showing that the hydrophobic surface is immune to hydrolysis, and the immobilization of hydrophobic derivatives of fluorescein, a pH-sensitive dye, potentially allows pH sensing at the extremes of the pH scale. The fluorescein derivatives used are shown to form a non-fluorescent and non-pH-sensitive lactone on the hydrophobic surface. By decreasing the density of the hydrophobic layer, pH sensing can be achieved.
2. Experimental 2.1. Materials The optical fiber used for these studies had a 1000-m core diameter and a protective jacket composed of nylon ŽEnsign-Bickford Optics Co., Avon, CT, type HCNM1000T.. Chlorodimethyloctadecylsilane, chlorotrimethylsilane, toluene, methylene chloride, methanol, sodium chloride, dibasic anhydrous potassium phosphate and sodium tetraborate decahydrate were purchased from Aldrich ŽMilwaukee, WI.. The nitrogen used as a blanket for the synthetic procedures was oxygen-free and was dried by passing it first through a 30 = 5 cm column containing anhydrous calcium chloride and then through a 30 = 5 cm silica gel column. The two probes, N-Žfluo-
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rescein-5-thiocarbamoyl .-1,2-dihexadecanoyl-snglycero-3-phosphoethanolamine, triethylam monium salt ŽC-15 = 2F. and 5-octadecanoylaminofluorescein ŽC-18F. were obtained from Molecular Probes ŽEugene, OR.. 2.2. Optical set-up The optical configuration for fluorescence measurements using dynamically modified optical fibers has been previously reported in detail w7᎐9x. Briefly, light from an argon ion laser ŽIon Laser Technology, Salt Lake City, UT, Model 490 ASL. operating at 488 nm was modulated using a chopper operating at 550 Hz ŽStanford Research Systems, Inc., Palo Alto, CA, Model SR540.. The resulting laser light was then sent through a dichroic beam splitter ŽCVI Laser Corp., Albuquerque, NM. and into a microscope objective ŽNewport, Model-10X, FL s 14.8 mm, NAs 0.25. which focused the excitation light onto the optical fiber and collimated the emission light. The fluorescence was reflected at the dichroic mirror and collected by a 75-mm focal length, 50-mm diameter plano-convex lens. The collimated light was passed through a holographic filter ŽPhysical Optics Corp., Torrance, CA, Model RHE 488. and into a scanning monochromator ŽCVI, Model DK 240-2.. Light exiting the monochromator was detected with a photomultiplier tube ŽThorn EMI Gencom, Inc., Fairfield, NJ, Model 9816B. which was powered by a constant-voltage power supply ŽBertran Associates, Model 215.. The signal from the PMT was then sent to a lock-in amplifier ŽStanford Research Systems, Inc., Palo Alto, CA, Model SR510.. The demodulated signal was sent to a personal computer for storage. 2.3. Preparation of C-18-modified optical fibers The distal end of the optical fiber surface was prepared by successive polishing with 60-, 9-, 1and 0.3-m lapping film. The polished surface was then activated by refluxing in 2M HCl for 6 h. The fibers were then washed thoroughly with distilled water and dried at 120⬚C overnight. The activated surface was then refluxed for 4 h in a mixture of dry toluene and 0.5g of chlorodimeth-
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yloctadecylsilane and then placed in an ultrasonic bath for an additional 24 h. The solution mixture was removed and 20 ml of toluene and 5ml of chlorotrimethylsilane were added for end capping. Ultrasonic agitation was continued for 12 h. All synthetic steps proceeded in an enclosed anhydrous system under the protection of nitrogen. After modification, the fibers were placed in a soxhlet extractor and washed in turn by methanol, methylene chloride, and hexane. 2.4. Immobilization procedure Immobilization of the probes was accomplished by incubating the C-18-derivatized fiber in a 5 mM solution of the probe molecule for 5 min. The fiber was then removed, air dried to remove the solvent, and rinsed with pH 11 buffer solution. The solvent for C-18F was methanol, while that for C-15 = 2F was propanol.
3. Results and discussion Manufacturers of silica-based RP-HPLC columns recommend that mobile phases do not exceed pH 8, or be less than pH 2. This range is decreased at elevated temperatures. At pH values G 12, not only is the octadecylsilane hydrolyzed, but the silica itself is also soluble in the mobile phase w10,11x. For RP-HPLC columns, stationaryphase hydrolysis is known to increase with temperature w12x. Furthermore, purely aqueous mobile phases are known to collapse the octadecylsilane chains onto the surface and retard hydrolysis; however, organo-aqueous mobile phases do not cause this collapse to occur. It has been shown qualitatively, that water in water᎐ acetonitrile mobile phases is associated with the octadecylsilane layer equally from 0᎐80% acetonitrile w13x. Since the C-18-derivatized optical fibers also use silica, the same factors would be expected to contribute to hydrolysis of the octadecylsilane layer on the fibers. The effect of pH on the stability of the C-18 layer was evaluated under a variety of conditions to better define the range of pH sensing appropriate for this protocol.
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Fig. 1. Surface stability at the extremes of the pH scale. A᎐D represent replicate immobilizations of 3-N-octylriboflavin, labeled i, and the corresponding blank signal, labeled b, on the same C-18-derivatized optical fiber. After each panel, the probe is incubated for 20 min under conditions designed to test the integrity of its surface. The conditions for each are: ŽA. methanol at room temperature; ŽB. pH 12 solution containing 33% acetonitrile at room temperature; ŽC. pH 12 solution containing 33% acetonitrile at 65⬚C; and ŽD. pH 2 solution containing 33% acetonitrile at 65⬚C.
Fig. 1 shows a series of immobilization events performed after incubating a single C-18-derivatized fiber under conditions designed to test the resilience of the hydrophobic surface towards hydrolysis. The conditions depicted in Fig. 1 correspond to situations where RP-HPLC octadecylsilane layers are known to degrade. The conditions are low pH ŽpH 2., high pH ŽpH 12., the presence of organic solvent Ž33% acetonitrile ., and stability at elevated temperature Ž65⬚C.. Previous studies in our laboratory have noted a correlation between the amount of octadecylsilane on the surface and the amount of hydrophobic probe molecule associated with the surface. If the octadecylsilane surface of the C-18-derivatized fiber was being hydrolyzed, then the amount of the fluorescent hydrophobic probe, octylriboflavin, should decrease from immobilization to immobilization. However, the amount of octylriboflavin does not show this behavior, thereby demonstrating that the C-18-derivatized fiber ex-
hibits substantial immunity from hydrolysis under a variety of extreme conditions. The protocol for each panel depicted in Fig. 1 was as follows: incubation of the fiber for 20 min under the test conditions; immobilization of octylriboflavin; measurement of fluorescence; and finally, removal of the octylriboflavin by a methanol rinse prior to the start of the next measurement sequence. Each process was repeated a total of three times. By the end of the sequence depicted in each panel of Fig. 1, the fiber had been exposed to the incubation solution for a total of 60 min. The control experiment, depicted in panel A, was performed by incubating the fiber in roomtemperature pH 7 buffer for 20 min. Panel B represents incubation of the OF in a pH 12, 33% acetonitrile Žvrv. solution for 20 min; and panel C represents incubation in a pH 12, 33% acetonitrile Žvrv. solution at 65⬚C for 20 min. Finally, panel D represents incubation in a pH 2, 33% acetonitrile Žvrv. solution at 65⬚C for 20 min.
D.R. Fry, D.R. Bobbitt r Microchemical Journal 69 (2001) 123᎐131
Other incubation times were investigated, for example up to 1-h incubations were evaluated, and 2-propanol was also used as the organic modifier. Although there is a slight difference, approximately 10% of the signal from panel to panel, there is no gross decrease in signal, and therefore the amount of octylriboflavin immobilized on the fiber is, within experimental error, invariant. Again, these results suggest that the C-18-derivatized fiber is immune to hydrolysis in the pH range 2᎐12, at elevated temperatures, and in the presence of organic modifiers. Other groups have found similar results; long-chain stationary phases, such as octadecylsilane, slow the rate of hydrolysis w11,14x. In our case, no hydrolysis was observed. In RP-HPLC separations, care is taken to limit the pH range of the mobile phase to 2᎐8. Under conditions outside of this range, the stationary phase, octadecylsilane, is found to be ‘stripped’ from the column. The retention time of a hydrophobic species thereby decreases dramatically due to the interaction with the reduced amount of octadecylsilane on the stationary phase. In contrast, the C-18-deri¨ atized fiber, also modified with octadecylsilane, retains the same amount of octylriboflavin, even after an extensive series of incubations. One possible explanation for the observed ruggedness concerns the amount of excess derivatizing reagent used in the synthetic procedure. The OF surface being modified is extremely small, 1 mm in diameter, as compared to high surface-area silica stationary phases, which have surface areas of the order of 400᎐600 m2rg w15x. Even under conditions where 10᎐20 fibers are simultaneously derivatized, the amount of octadecylsilane reagent exceeds the available surface SiOH groups by approximately nine orders of magnitude. Thus, a very large excess of derivatizing reagent is possible for chemical modification of the optical fiber. Theoretically, the maximum coverage of an octadecylsilane monolayer is limited by the coverage of silanol groups on the surface, which has been determined to be of the order of 8 molrm2 w15x. In comparison, most RP-HPLC stationary phases based on octadecylsilane have a surface coverage of between 1 and 3 molrm2 of octadecylsilane w15x. These results suggest that the dynamic modification protocol
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can be applied to measurement problems involving harsh experimental conditions that were not previously amenable to optical fiber-based sensors. Hydrophobic derivatives of fluorescein were chosen as probe molecules for a number of reasons. Fluorescein is a well-characterized, high quantum yield, pH-sensitive dye w16᎐18x. In addition, hydrophobic derivatives of fluorescein are commercially available. A Hitachi F-2500 fluorescence spectrometer was used to generate a pH vs. florescence intensity plot for each derivative. Each data point is an average of three measurements. A fluorescence intensity vs. pH plot for C-18F is given in Fig. 2a; from this figure, the p K a is estimated to be 9. A spline line was added to emphasize the shape of the curve. The range of each replicate measurement is smaller than the size of the solid circles. The fluorescence intensity does reach a maximum at a pH of approximately 10, and the fluorescence decreases at higher pH values. For the other fluorescein derivative, C-15 = 2F, its p K a is reported to be approximately 6.2 w19x. Reversible association Žimmobilization. of a hydrophobic probe molecule with the C-18-derivatized fiber is key to the dynamic modification sensing scheme. Furthermore, measurement of pH in mixed solvent systems is of general interest. In the case of dynamic modification, the minimum hydrophobicity required of a probe molecule in order to assure efficient association with the fiber surface and allow sensing in a mixed solvent system is of interest. For the experiments summarized in Table 1, a 5 mM solution of each derivative was incubated with a hydrophobic fiber for 5 min. The fiber was removed, air dried and rinsed with pH 12 buffer. The fiber was then immersed in the test solvents listed in Table 1 at pH 9 for 10 min. Finally, the fiber was immersed in methanol. Each entry was repeated at least five times. Immobilization of the probe molecule can be judged in a number of ways; however, the simplest is a two-part question: does the test solvent contain fluorophore after the fiber has been removed, and does the methanol rinse contain any fluorophore? As Table 1 shows, the C-15 = 2F derivative does not leak from the fiber, even in a 15% methanol
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Fig. 2. Ža. Fluorescence vs. pH for C-18F in solution. Žb. Fluorescence vs. pH for C-18F immobilized on a C-18-derivatized optical fiber.
solution. In contrast, the C-18F derivative partially dissociates from the fiber, even in a 5% methanol solution. In both cases, it was found
that the hydrophobic derivatives of fluorescein can be reversibly associated with a single fiber in an aqueous buffer. Fig. 3 shows four ‘sensing’ events in pH 9 buffer for C-18F. The most obvious result is the lack of difference between the blocked signal and the signal from immobilized C-18F. After the ‘sensing’ event in pH 9 buffered solution, the C-18-derivatized fiber was rinsed with methanol. As the non-fluorescent fiber was rinsed with methanol, the fluorescent C-18F was clearly observed diffusing away from the C-18-derivatized fiber. In addition, no C-18F was found in the aqueous solution. C-15= 2F gave similar results Žnot shown.. Fluorescein is known to form a nonfluorescent inner lactone, as depicted in Fig. 4, in hydrophobic solvent systems w17,18x. Furthermore, Dutta and Salesse incorporated C-18F into a palmitic acid Langmuir᎐Blodget film w20x. They found that the fluorescence of C-18F was quenched as the surface pressure, and thus the hydrophobicity of the film, was increased. They also performed spectroscopic measurements on the film and in a variety of different solvent systems. These results implied that the lactone form was found in the film and in non-polar hydrophobic solvents, while the zwitterionic form was found in polar solvents. The dense hydrophobic portion of a C-18-derivatized fiber closely resembles the hydrophobic Langmuir᎐ Blodget film of palmitic acid. The literature cited, along with the visual observation of the C-18F and C-15 = 2F being removed by methanol from the C-18-derivatized fiber, strongly suggests that C-18F is associated with the fiber in the lactone non-fluorescent form.
Table 1 Summary of association measurements for C-18F and C-15 = 2F a Probe
Buffer
MeOH rinse after buffer
5% MeOH buffer
MeOH rinse after 5% MeOH buffer
10% MeOH buffer
MeOH rinse after 10% MeOH buffer
15% MeOH buffer
MeOH rinse after 15% MeOH buffer
C-18F C-15 = 2F
No No
Yes Yes
Yes No
Yes Yes
Yes No
No Yes
Yes No
No Yes
a Immobilization is judges by a two-part question: does the solvent contain fluorophore after the fiber has been removed; and does the methanol ŽMeOH. rinse contain fluorophore?
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Fig. 3. Immobilization of C-18 on C-18-derivatized optical fiber. Immobilized C-18F, a, and pH 9 blank, b, are indistinguishable; g represents residual light with the laser blocked. The C-18F immobilization was confirmed by immersing the probe in methanol, where the fluorophore could be seen diffusing away from the probe tip Žnot shown..
From these observations, a model of how C-18F and C-15= 2F interact with a C-18-derivatized fiber can be proposed. A model, describing the decay of the signal in Fig. 3, can be proposed based on the following premises: Ž1. the solvent environment of the octadecylsilane chains affects the degree of extension of the chains, and there-
Fig. 4. Solvent-dependent equilibrium of fluorescein-free acid form and non-fluorescent inner lactone form.
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fore the local environment of the probe molecule; and Ž2. the C-18-derivatized fiber is a very densely packed layer of octadecylsilane. First, in an organic solvent the octadecylsilane chains are extended, and the probe molecule selectively partitions from the organic solvent into the chains. In addition, a portion of the organic solvent also partitions into the chains. The volatile organic solvent is easily removed by application of a stream of warm air to the fiber, leaving the probe molecule behind. As the C-18-derivatized fiber is rinsed with aqueous buffer, the outside chains, which are exposed to the aqueous environment, collapse onto the inside chains. Finally, as the C-18-derivatized fiber is placed in an aqueous solution, the probe molecules are in contact with the aqueous environment. As time progresses, the chains collapse onto themselves, thereby changing the local environment experienced by the probe. In this extremely hydrophobic environment, it is reasonable to expect the probe molecule to form the lactone, which is non-fluorescent. In an effort to open the chains so that probe molecules can be more effectively exposed to the surrounding solution, a small % of methanol was added to the buffer. These results are summarized in Table 1. At 5% methanol, the C-18F did fluoresce; however, it was also not immobilized onto the fiber. In contrast, the C-15= 2F was immobilized in the inner lactone form with up to 15% methanol; however, above 15%, C-15 = 2F was not immobilized on the fiber. Returning to the model explained above, it appears that if the hydrophobicity of the OF surface could be reduced by decreasing the amount of octadecylsilane on the surface, the fluorescein derivatives would be less likely to form the non-fluorescent lactone form, since this process is so clearly tied to the hydrophobicity of the environment surrounding the probe. The hydrophobic derivatization of the OF does provide several mechanisms for producing reduced-coverage fibers. One possibility is to stop the nitrogen purge gas 3 h into the reaction, so that the moisture-sensitive octadecylsilane reagent will react with the moisture in the atmosphere, thereby decreasing the amount of octadecylsilane and therefore the surface coverage of octadecylsilane
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Fig. 5. The two figures, A and B, represent immobilization events of C-8R on two different C-18-derivatized optical fibers. Panel A depicts the amount of C-8R immobilized on a low-coverage C-18-derivatized optical fiber, while B depicts an increased amount of C-8R immobilized on a normal-coverage C-18-derivatized optical fiber. The blocked signal is represented by a, the blank signal by b, and immobilization of C-8R by i.
on the surface. Fig. 5 shows two immobilizations of octylriboflavin on a normal fiber and two immobilizations on a reduced-coverage fiber. As is evident, the normal fiber holds significantly more octylriboflavin than the reduced-coverage fiber. On the latter, C-18F was immobilized, and its fluorescence proprieties changed with pH. Fig. 2b shows the calibration curve for four immobilizations of C-18F. A spline line was added to emphasize the shape of the curve. Comparison of the fluorescence response versus pH for C-18F in solution, Fig. 2a, and C-18F immobilized on a reduced-coverage fiber, Fig. 2b, confirms that the immobilized probe is responsive to pH. The shapes and slopes of the curves are nearly identical. Furthermore, the probe was immobilized on a renewable surface. Although the reduced coverage fibers can immobilize C-18F and C-15= 2F in a fluorescent and pH-sensitive form, they do not show the same resilience to hydrolysis. For instance, after several w8x immobilizations and measurement in pH 10 or 11 buffered solutions, the reduced-coverage fibers failed to hold the immobilized C-18F. The fluorphore could be seen diffusing away from the OF surface. However, other OF sensors have shown similar inadequacies. For example, sol᎐gel matrices are known to leak probe molecule if not stored and handled carefully. An extensive list of fluorescent pH indicators is available in most
chemistry reference books w21,22x. Obviously, one consideration of any potential probe molecule should be its fluorescent properties in hydrophobic solvents. We are currently pursuing several other probe molecules for pH sensing. Previous studies in our laboratory have shown that placement of the C-18-derivatized fiber in the sensing solution alone leads to approximately 5᎐7% R.S.D. in measurements. For the experiments summarized in Fig. 2b, the R.S.D. was approximately 10%. When the R.S.D. of the each pH measurement was subtracted from the R.S.D. of the blank signal, the result was nearly zero. This implies that fiber placement within the sensing vessel is a limiting factor in the sensing protocol. To overcome this limitation, a pH-insensitive dye with a larger Stokes shift could be immobilized concurrently with the pH-sensitive dye. The ratio of the pH-insensitive dye to the pHsensitive dye would be invariant to placement. We immobilized octylriboflavin four times and monitored its fluorescence intensity at two wavelengths, 525 and 535 nm, without moving the fiber. Although the R.S.D. was 7% from immobilization-to-immobilization, the ratio of the response at the two wavelengths had an R.S.D. which was, within experimental error, f 0%. This shows that the dynamic modification event can be used to prepare a suitable mixed-probe surface, which can respond to a specified analyte while
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providing immunity from measurement-to-measurement variations.
4. Conclusions The dynamic modification protocol is suitable for pH sensing at the extremes of the pH scale, as the high-density octadecylsilane surface provides significant immunity from hydrolysis, and the surface provides a renewable sensing surface. Two pH-sensitive derivatives of fluorescein were immobilized on this platform. Fluorescein is known to form a lactone, which is non-fluorescent and non-pH sensitive in hydrophobic solvents. The derivatives of fluorescein were shown to be nonfluorescent and non-pH sensitive when immobilized on a dense C-18-derivatized fiber. Renewable pH sensing was achieved by reducing the density of the octadecyl chains attached to the fiber; however, the octadecyl chains were more easily hydrolyzed, causing the sensor to degrade. The sensor responds over the pH range 7.5᎐10. These results provide the basis for future applications involving pH sensing. First, the development of specific hydrophobic, pH-sensitive dyes for use in pH sensing protocols is currently being explored. Second, new strategies for controlling the hydrophobicity of the OF surface, while providing immunity to hydrolysis, are under investigation. For example, the use of a mixture of monomeric short- and long-chain hydrocarbon phases and a polymeric surface are both being explored.
Acknowledgements Support from the National Science Foundation for portions of this work through grant no. EPS9977778 is acknowledged.
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