Anal. Chem. 1002, 64, 2985-2990
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Development of Submicron Chemical Fiber Optic Sensors Weihong Tan, Zhong-You Shi, and Raoul Kopelman' Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109
Optical fiber sensors have several advantages over eiectricaibased sensors in many appilcations in chemlstry and biology, but not with respect to minaturlratlon and response tlme. Here we describe a near-field optical technique that enables the development of submlcron-sired optical fiber sensors. The technology Is based on nanofabrlcated optical flber tips and near-field photoinitlated polymerization. Multimode or slnglemode optical fibers are drawn into submlcron optical fiber tips and then coated with aluminum to form submicron optlcai flber light sources. Submicron pH sensors, as an example for small sensors, have been prepared by incorporating fiuorescelnamine Into an acryiamide-methyienebls(acry1amide) copolymer that is attached covalently to a siianlzed fiber tip surface by photoinitlated polymerization. The sensors have demonstrated their spatlal resolving abilities in measuring the pH of buffer solutions inside micron-size holes in a polycarbonate membrane. These submicron pH sensors have millisecond response times due to their extremely small sires.
polymerization or by other direct covalent attachment to the distal end of an activated fiber surface has solved this pr~blem.~v~,g In the photoinitiated polymerization approach, the polymer serves to increase the surface area which results in multiple reagent immobilization sites. Since there is no need to use a membrane or other means of mechanical confinement, the response is faster and the manufacturing is easier. The other difficulty in fabricating small optical fiber sensors is that there are no small-size optical fibers commercially available. The smallest optical fiber sold commercially is still in the range of 80 pm (cladding diameter). However,just as micropipettes are manufactured from 1-mm glass tubes and used as microelectrodes, optical fibers can also be manufactured to small diameters.lOJ1 We have prepared the first submicron optical fiber chemical sensor by combining the above new developments. We formed fiber tips and then incorporated apH-sensitivedye into a copolymer which is attached covalently to a silanized fiber tip surface via photoinitiated polymerization. The sensor's excellent detection limit, response time, and spatial resolution are demonstrated below.
INTRODUCTION
EXPERIMENTAL SECTION Chemicals. Most of the chemical reagents used in our experiments were purchased from Aldrich Chemical Co., except for the fiber surface silanization reagents [y(methacry1oxy)propyl]trimethoxysilane and (aminopropyl)triethoxysilane,obtained from Sigma. All chemicals were used without further purification. Fiber Tip Fabrication. Opticalfiber tips were f i t fabricated by Betzig et al.l0 and used as scanning probes in the near-field scanning optical microscopy (NSOM)loJ1and molecular exciton microscopy (MEM)." The apparatus used for fiber tip forming consists of a P-87 Micropipette Puller from Sutter Instrument Co. and a 25-W COz infrared laser from Synrad Co. Multimode all-silicafiberswere purchased from General Fiber Inc. with core diameters ranging from 80 to 200 pm. Single-modefibers were purchased from Newport Research Corp. and 3M Co. with core diameters of a few microns and cladding diameters around 100 pm. All fibers were terminated at a length of 1-2 m. The jacket was removed from the fiber where the fiber tip will be formed. The fiber tip is produced by drawing an optical fiber in the puller while heating it with the COz infrared laser. By using appropriate program parametersand laser power, optical fibers can be tapered to 0.1-1 pm tips, hereafter referred to as pulled tips. A specially built high vacuum chamber is employed for coating these pulled fiber tips. Only the fiber tip sides are coated with aluminum, leaving the end face as a transmissive aperture. To make it in@ a light source, a laser beam is coupled to the opposite end of the pulled fiber tip. Surface Activation by Silanization. Two different kinds of fiber surface silanization have been used in our experimenta. The first is the [y-(methacryloxy)propylltrimethoxysilane,and the second with (aminopropy1)triethoxysilane. Both are successful in our present work. We prefer the first one because it is simpler than the second. As these activation methods have been described previously:J2 only a brief description is presented here.
Optical fiber sensors have been widely used in recent years in analytical chemistry, biology, physiology, and other areas.'* They have demonstrated several advantages over electrical based sensor^,^,^ but not with respect to size or response time. The smallest optical fiber sensors reported to date are about 100pm, with most being in the 150-1000-pm range. Existing optical fiber sensors have response times of seconds or minutes, while microelectrodes have subsecond response time.* This has been a problem for many years, which greatly limits the applications of optical fiber sensors in intracellular and intercellular physiology and in many other microscopic studies and protocols related to biology, biotechnology, medicine, materials science, etc. In this paper we describe a new technique with the potential of making nanometer optical fiber sensors with extremely short response times. There are two major difficulties in making submicron optical fiber chemical and biological sensors. One is the limitation of most of the present techniques in attaching the chemicals or biological reagents to the fiber surface. The attachment of chemical or biological reagents by membranes or tubing or any other mechanical method makes only very limited use of the fiber tip surface, which translates into very low signals and very slow response time. For a given optical fiber sensor, the smaller the sensing area, the weaker the signal. The immobilization of reagents by photoinitiated (1)Wolfbeis, 0.S.Fiber Opt. Chem. Sens. Biosens. 1991, I, 413 pp. (2)Seitz, W. R.Anal. Chem. 1984,56,16A-34A;CRC Crit. Reu. Anal. Chem. 1988,19, 135. (3)Narayanaswamy, R.Biosensors Bioelectron. 1991, 6960,467-75. (4)Barnard, S.M.; Walt, D. R. Nature 1991,353, 338-340. (5) Arnold, M. A.;Meyerhoff, M. E.CRC Crit. Reu. Anal. Chem. 1988, 20 (3),145-96. (6)Schultz, J. S. Sci. Am. 1991, 265 (2),64-9. (7)Madou, M. J.;Otagawa, T. AIChE Symp. Ser. 1989,85, (267,Process Sens. Diagn.), 7-13. Armstrong, W. McD.; Garcia-Dim, J. F. Fed. h o c . 1980, 39, (ll),2851-9. (8) Fleet, B.; Bound, G. P.; Sandbach, D. R. Bioelectrochem.Bioenerg. 1976, 3, 158-68.
(9) Munkholm, C.; Walt, D. R. Anal. Chem. 1987,58, 1427-30. (10)Betzig, E.;Troutman, J. K.; Harris, T. D.; Weiner, J. S.; Koertelak, R. L. Science 1991,251, 1468. (11) Kopelman, R.; Smith, S.; Tan, W.; Zenobi, R.; Lieberman, K.; Lewis, A. SPIE 1992, 1637, 33-40.
0003-2700/92/0364-2985$03.00/0 0 1902 American Chemical Society
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Preparation of Acryloylfluorescein. Before being used for photopolymerization, fluoresceinamineis converted into its amide derivative, acryloylfluorescein.13The derivative is formed after reaction of 2:l to 3:l stoichiometric quantitiesof acryloylchloride with fluoresceinamine in dry acetone, which was obtained by drying HPLC-grade acetone in silicagel. The mixture was stirred for about 1 h until most of the product precipitated. The product was collected by filtration and then washed by both acetone and by CHzClz a few times. It was dried in air by evaporation and stored in the dark. Polymer Solution Preparation. The solution for polymerization on the fiber surface is a mixture which consists of three solutions. The methods of preparation of these solutions have been described p r e v i ~ u s l y . ~Modifications J~J~ made for this work will be briefly discussed. Acryloylfluorescein, prepared as described above, was dissolved in ethanol, with concentrations around 15 mM. This replaces the solution 3 in Munkholm and Walt’sprocedure.9 Solutions 1 and 2 are pH = 6.5 buffer solutions of acrylamide and N,N-methylenebis(acrylamide),respectively. They were prepared according to the procedures of Updike and Hicks.’* The monomer solution, referred to as solution 4 hereafter, is formed by combining 10 mL of solution 1, 40 mL of solution 2, and 2 mL of solution 3. Solution 4 can be stored in a refrigerator for about 1 month without evident polymerization. Thermal and Photopolymerization. The reactor used for both polymerizations is a small glass bottle with three holes in its cover,through which the fiber tip is inserted into the monomer solution, nitrogen is introduced through a glass tube, and a small glass rod is introduced to test the gelation of the monomer solution. An oil bath heater is used to control the reaction temperature between 50 and 80 OC. For most of our submicron sensor work, photoinitiated polymerization was used while thermal initiation has also been tried. The key difference between them is that a catalyst has to be added to solution 4 for thermal polymerization, while light is employed for initiation in the photopolymerization. The catalyst is riboflavin or persulfate of either potassium or ammonium. We have noticed that the thermal reaction can not occur without both heating and catalyst. The disadvantages of the thermal polymerization are as follows: first, there is not much control over where the polymer grows; hence polymers are formed anywhere along the portion of the tip immersed in the solution; second, once the reaction is initiated, all the solution inside the whole reactor will be polymerized quickly; thus miniaturization is not easily realized; third, it is relatively difficult to optimize the reaction conditions for the thermal polymerization. For photopolymerization, light initiation and heating were combined, but no catalyst was used. The fiber tips were first silanized for about 1 h and then dried in air for another hour. Before being placed in solution 4 for polymerization, fiber tips were first sensitized by soaking in a benzophenonelcyclohexane solution16for about 15 min. They were then put into solution 4 for photopolymerization. Solution 4 was bubbled with nitrogen for about 20 min before laser light was directed into the fiber tip and the nitrogen atmosphere was maintained during the reaction. Laser light of 442 nm from a He/Cd laser or 488 nm from an Ar ion laser was coupled into the fiber and transmitted to the tip where the photopolymerization was initiated directly on the silanized surface. Usually some heating is needed for the reaction, and 10-30 min will be spent in order to get good sensors, which gave us good control over the thickness of the polymers on the sensor tips. The polymerization reaction rate depends on the concentrations of reactants, the reaction temperature and the light intensity emitted from the fiber tip. We managed to make the thickness of the sensor prepared by photopolymerization close to the size (12) Masbach,K. Methods inEnzymol0gy;Academic Press: New York, 1976; Vol. XLIV, p 139. (13) Munkholm, C.; Parkinson, D.-R.; Walt, D. R. J. Am. Chem. SOC. 1990,112,2608-12. (14) Hicks, G. P.; Updike, S. J. Anal. Chem. 1966, 38, 726-30. (15) Rempp, P. Polymer Synthesis, 2nd, rev. ed.; Huthig & Wepf: Heidelberg, 1991; p 419-459. (16) Tazuke, S.; Kimura, H. Makromol. Chem. 1978, 179, 2603-12.
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Fbure 1. Schematic diagram of the submicron optical fiber mnsor apparatus.
of its diameter. The thickness of the polymer on the fiber tip is basically controlled by reaction time allowed for the polymerization on the fiber tip. Since the sizes of our submicron fiber tips are very small and cannot be resolved well by a conventional microscope, we did systematic thickness monitoring experiment by using unpulled single-mode fibers and calculated the relationship between the thickness of the polymer and the polymerization reaction time under specific reaction conditions. This relationship serves as a good guideline for us to optimize the reaction time on submicron fiber tips in such a way that the thickness of the polymer will be close to its diameter. The longer the sensor stays in the monomer solution, the thicker the sensor will be. In the submicron sensor preparation process, we strictly controlled the depth of the fiber tip inserted into all of the solutions either for pretreatments or for the polymerization. A three-way XYZ stage with 2 resolution of 0.07 pm was used to position the fiber tip; thus, only the fiber tip end surface will come in contact with all of the solutions. For some cases, we noticed that there is a liquid rise at the interface between the liquid surface and the fiber tip due to surface tension. This may leave somemonomer solution on the Al-coated aidesurface. This, however, will not affect the operation of this sensor since internal illumination was used and no light is emitted from the sides of the probes. Furthermore, we believe that the unpolymerized dye deposited on the aluminum was later washed off. Measurements. We used two different kinds of signal detection apparatus. The first one is for large-sizedsensors and similar to those described in the review by Seik2 For smaller diameter sensors, a new apparatus was designed and built. An inverted Olympus microscope-based optical fiber sensor set-up was employed, as shown schematically in Figure 1. In thie apparatus, the inverted frame fluorescence microscope is connected with a spectrometer and a photomultiplier tube (PMT). A high-precision single-mode fiber coupler from Newport Research Corp. is used to direct the laser beam into the fiber sensor tip. The reagent is excited by the incident beam, and the fluorescence light, together with the light of the incident beam, is collected by an objective lens. The higher the numerical aperture of the objectives used for collection, the stronger the signal collected. The fluorescence is transmitted by a dichroic mirror and subsequentlydirected into either a PMT with a bandpass filter or into a J Y spectrometerfor analysis. The submicron fiber sensor is mounted on a three-way XYZ translational stage, with X, Y resolutions of 0.5 pm and Z resolution of 0.07 pm. The entire set-up is on an air table for vibration isolation. Allsamplea are mounted on the viewing stage of the microscope for observation before data collection. A 0.3-W 488-nm Argon ion laser beam was used for excitation of the polymer. The spectra were taken from 490 to 650 nm
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992 0 2887
Flgure 2. Photographsof optlcal fiber pH sensors. Left, a sensor prepared from an unpulled multimode optlcal fiber. Center, a sensor prepared from an unpulled slngle-mode optical fiber. Right, a sensor prepared from a pulled submicron optlcal fiber tlp. In each case the scale bar represents 10 pm.
while the sensor was immersed inside a pH = 7 buffer.solution. The data for the pH response of the sensor tip were collected at 540 nm by using a combination of a dichroic mirror and a bandpass fiiter. Using the apparatus shown in Figure 1, intensities of the fluorescence of the polymer at the submicron sensor tip under different pH environments were recorded by a photon counting system (EG&G photon counter Model 1109 and Discriminator Control Unit Model 1121) with a Hamamatsu R1529 photomultiplier tube controlledby an IBM PC computer. Response Times of Submicron pH Sensors. Response times of the submicron sensor have been determined by the microscope-based sensor apparatus shown in Figure 1. Ten milliseconds is used as the data acquisition time in order to get time-resolveddata. The submicronpH sensor is immersed inside a 10-mL glass container with distilled water as the test sample. The sensor is positioned toward the microscope objective and light collection is optimized. While data acquisition is going on, a tiny drop of 0.1 M HC1 or NaOH is added to the distilled water in the glass container. A curve of the signal changing from one level to another is recorded over time, giving an upper limit for the response time of the submicron pH sensor. We note that no stirring took place and thus, most likely, this “responsetime” is determined by both the diffusion to the sensor and that inside the sensor.
RESULTS Submicron Optical Fiber Sensors. After pulling the multimode or single-mode fiber, the end structure tapers uniformly from the original fiber to a submicron tip with a flat end-surface perpendicular to the fiber axis (see Figure 3, SEM picture of the tip). Figure 2 has three sensors: on the left there is a 125-pm sensor, in the middle a micron-sized sensor with a thicker polymer, and in the right a submicron pH sensor. The one in the right shows how the fiber tapers to form a submicron tip. After the sides are coated with aluminum, the flat end of the tip isleft bare and a tiny aperture is formed. When a 488-nm laser beam is coupled into the aluminum-coated fiber tip, a very bright spot at the tip could be seen under the microscope. This probe delivers light very efficiently to the aperture since all the radiation remains bound to the core until a few microns from the tip. The signal emerging from a randomly chosen 0.2-pm fiber tip (blank)was 1012photonsper second measured using neutral
Figure 3. SEM picture of submicron optlcal fiber tips (magnhtkm of about 12 000).
density fiiters and the setcup shown in Figure 1. The light spot defiies the size of the light source, and hence the size of the sensor. Sizes of the fiber tips were determined by scanning electron microscopy in this work and are from 0.1 to 1pm, with most of the sensors prepared around 0.5 pm 88 shown in Figure 3. We have made smaller tips in our NSOM and MEM research,ll and the smallest fiber tip liiht source reported so farlo is about 200 This opens the possibility
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for developing nanometer-sized optical fiber sensors. For the fabrication Of the submicron sensor, reaction temperature, polymerhation time, laser power, and coupling efficiency had to be optimized in order to grow submicronsize polymers on thefiber tip surfaces. Pictures of these sensors were also taken, but, just as shown in the right side of Figure 2, no fine structure of the polymer at the fiber tip could be seen due to the resolution limitations of our optical microscopes. As only a small light spot is seen a t the tip of the sensor when viewing the sensor with internal excitation under the microscope (Olympus inverted fluorescence microscope, 600 times magnification); this illustrates that a submicron size sensor has been fabricated. The photograph shown here, right side in Figure 2, was taken by external illumination when the sensor was horizontally placed on a microscope slide. By doing this, the whole probe is shown clearly. When internal illumination was used, one can only see a small fluorescence spot, much smaller than that shown in the right of Figure 2, at the end surface of the fiber tip. Its size is below 1 pm, and within the resolution limit of our microscope. We noticed that the biggest advantage of photoinitiated polymerization is that the size of the sensor is defined by the size of the probe. In the middle of Figure 2, we present a picture of the polymer formed by photopolymerization on an unpulled single-mode fiber. The fiber end surface includes core and cladding. Both the fiber core and cladding were subjected to identical reaction conditions, but the polymer only formed on the light-emitting core. It is clear that the diameter of the polymer, i.e. the size of our sensor, is defined by the size of the light source,the core, where light is emitted. It is also clear that the diameter does not expand as the polymer grows; in fact, it appears that the diameter decreases. All this clearly shows that the sensor size can be maintained by photopolymerization. We thus believe that the photopolymerization only happened in the near-field region. Experiments to prove this are in progress. pH Testing in Membrane Holes. The submicron pH sensorswere tested using UNucleopore"porous polycarbonate membranes. The samples are very similar to biological cells in size and shape. The different hole sizes available in these membranes17 range from 0.02to 10 pm, and the hole depths are about 6 pm for a 10-pm hole, deep enough to hold pH buffer solutions inside. Figure 5 gives a picture of a polycarbonate membrane. Before testing, the membranes were first immersed inside a pH buffer solution and then taken out and put on the microscope viewing stage where the sensor was aligned with a specific hole using the microscope. By driving the Z translational stage, the sensor could be inserted into one of the holes, just as shown by the blue dot in Figure 5. The position of the sensor can be controlled easily. The sizes of the membrane pores used in our experiments are from 2 to 10 pm.
Flgure 5. Channei-pore polycarbonatemembrane. Pore size 10 pm, thickness about 6 pm (magnification of 300). Blue light emanates from the hole where the sensor (invisible) is inserted. T ~ Wscale bar represents 10 pm.
Nine different buffers were used in the pH tests of the submicron pH sensors in our experiments. Measurements were cycled several times from pH 4 to 9 and then back to 4. The data of photon counts shown in Figure 4 are the averages of a few measurements in different cycles. Replicate results at each pH measurement were always slightly lower than the previous one since there was some photobleaching of the dye inside the copolymer. The bleaching is superlinear with intensity. We note that with the highest intensity (up to about lo3 W/cm2), the sensor output drops about 10% after 40 min of continuous excitation (see below). Routinely one can easily work with 5% of this power so that the photobleaching becomes much less significant. Furthermore, the fluorescence intensity, recorded with a simple photomultiplier tube and shown in Figure 4 for the submicron sensor, is from about 15 OOO counts s-l at pH = 4 to about 50 OOO counts s-l at pH = 9. This fluorescence intensity is one of the highest among other reported pH sensors, even though the sensors used here are the smallest ever reported. One major reason behind this fact is simply that there is a much higher efficiency of signal collection in our microscopebased apparatus. The other is the very high excitation efficiency of our submicron optical fiber sensor.
SensorResponseTimes,Reversibility,andSensitivity. The miniaturization of the sensor results in very fast response times. The size of the sensor is between 0.1 and 1 pm, and no mechanical confinement is used; thus the analytes have immediate access to the dye on the sensor tip. This gives our sensorsthe shortest response times among any other reported optical fiber sensors. Usually, the response time for a 200pm-sized pH sensor is from 40 to 120 s. Using photopolymerization to fabricate the sensor, this could be reduced to 9 s . ~However, it is still too long for many applications such as measuring fast biological and chemical changes, especially induced transient response experiments. Our sensors have response times shorter than 500ms. The curve obtained from the addition of a tiny drop of HCl or NaOH to the distilled water inside a glass container where the sensor is immersed gives a good estimation of the response time of the submicron pH sensor. Depending on the size of the sensor and the experimentalconditions,the response times ranged from less than 100 to about 500 ms. Without stirring, the 1040% response time is around 3001118 (most probably controlled by diffusion to the sensor). For a stirred or flowing sample, the theoretical diffusion time should be about 105 times shorter than those for sensors around 100pm (based on the Einstein diffusion relation). (17)Prasad, J.; Kopelman, R. Phys. Rev. Lett. 1987,59,2103-06.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
Table I. Sensor Fluorescence Intensity vs Excitation Power laser power intensity laser power (mW) (counts 8-1) (mW) 0.3 900 3 1.5
3100
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0.96
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The reversibility of the submicron pH sensor is good. The history, i.e. the order of how data are taken, does not affect the fluorescence intensity for a specific pH buffer solution. This greatly enhances its ability to deal with biological cells where an abrupt change often happens (Le., upon cell death) and in the applications as scanning tips. Also,the submicron pH sensor has a good detection limit. We notice that the smallest volume that gave definite pH measurements had less than 3000 hydrogen ions (at pH = 8). With all the above performance values and considering the highest fluorescence intensity recorded in our work (105 counts s-l), we believe that there should be no problem to push the size down to tens of nanometers. Power Characteristicsand Sensor Stability. Different laser powers have been used to excite the dye on the submicron pH sensor tip. With sensor immersed inside a pH = 7 buffer solution, the excitation power has been changed from 30 to 0.3 mW with the same coupling efficiency. The fluorescence of the sensor is monitored by the apparatus shown in Figure 1. Table I gives the results. Table I clearly shows that lower power sources (e.g. microscope lamps, tiny lasers) could be used for our sensor operation. A high-power laser is not necessarily needed. This opens many ways for the excitation of the dye on the submicron pH sensor tip. Down to about 1mW, the relation is linear within the experimental uncertainties. The submicron pH sensor has reasonable stability even when high power (30 mW, collected fluorescence intensity around lO5counts8-1) is used. In Figure 6 we show the stability results of the sensor recorded when it is immersed inside a pH = 7 buffer solution. The same experiment was done with the sensor tip out in the air. Over the same period of time, a 70% signal loss was recorded compared to a 10% loss for the sensor immersed inside the buffer. This indicates that the photobleachingis either oxygen related or heating related or both. Removal of oxygen and/or efficient stirring may thus significantlyreduce photobleaching. Furthermore, the bleaching is practically imperceptible at the lower laser power operating conditions.
DISCUSSION The microscope-based apparatus used in this work has demonstrated its high efficiency in signal collectioncompared with that described in Seitz's review2 where only a small portion of the signal is collected. However, our apparatus loses the advantage of remote working ability. This is tolerable since this sensor is not designed for such applications. A high-quality microscope is desirable for this work because it is not only used for signal collection but also in alignment of the sample with the sensor and simultaneous sample observation. This requirement is very reasonable for most microscopic operations since an optical microscope is a must in such a laboratory environment. We note, however, that the resolution is only limited by the molecular probe size. The submicron pH optical fiber sensors prepared are the first of their kind. The reason a pH sensor was chosen is because of the special importance of pH in both chemistry (18)Lieberman, K.;Harush, S.;Lewis, A.; Kopelman, R. Science 1990, 247, 59-62. (19)Lewis, A,; Lieberman, K. Anal. Chen. 1991,63, 625A-635A.
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and biology. pH has been widely used and is easily calibrated by other standard techniques. The samples for a pH test are attainable in every research lab. Also a pH sensor was one of the earliest optical fiber sensors. There are many chemical reagents and very mature techniques available for their fabrication. Although we made a submicron pH sensor with these techniques, the more fundamental goal is to prove the suitability of dye immobilization by photoinitiated polymerization or other covalent bonding and the pulled fiber tips in making a variety of new small sensors. Our development of submicronpH opticalfiber sensorshas proven the feasibility of these techniques. While only a pH chemical sensor has been discussed in this work, there is little difficulty in expanding these techniques into the fabrication of other sensors. There are many new biosensors based on selective binding of an enzyme or a biological reagent on the fiber surface.1.5 We expect to be ahle to develop smaller biosensors that can be implanted in certain positions of a cell to continuously monitor the biological changes. The work reported here is only the first step of our efforts in fabricating even smaller size optical fiber sensors, aiming a t molecular sizes for nanoscopic applications in many fields. Using exciton-optical techniques,18J9 the miniaturization is only limited by the size of the molecular probe. We are also optimistic that the response time will be even shorter once smaller sensors are made. With our sensor's miniaturized sizes, this could lead to the development of scanning sensing microscopy and to many applications in monitoring fast chemical and biological reactions. Our preliminary measurements of response times of the sensorshave demonstrated the submicron pH sensor's applicability for induced transient response experiments.
CONCLUSIONS 1. The first submicron optical fiber sensor has been prepared with a pH-sensitive dye. This shows the feasibility of the combination of photoinitiated polymerization and submicron optical fiber tips in fabricating submicron sensors and opens a novel approach for fabricating new and smaller optical fiber chemical and biological sensors. 2. The sample volume of our submicron optical fiber pH sensor has been reduced a t least one million times; the response time has been shortened a t least 100 times. The submicron optical fiber pH sensor has very high fluorescence intensities and is reversible with respect to pH changes. It is reasonably stable and reproducible. It is also relatively easy to manufacture.
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3. The pH test with membrane holes has demonstrated the submicron pH sensor’s spatial resolving abilities and its potential applications for cell biology and other microscopic operations.
ACKNOWLEDGMENT Special thanks go to Dr. D. Walt of Tufts University for his discussion with us regarding his work in polymerization on optical fiber and to Mr. S. J. Smith for his cooperation, (20) Tan,W.; Shi, Z.-Y.; Smith, S.; Bimbaum, D.; Kopelman, R. Science, in press.
especially in the coatingof fiber tips. Very helpful discussions with Drs. D. Birnbaum, M. E. Meyerhoff, M. Morris, S. Parus, and Mr.M.Shortreed, all at The University of Michigan, are also appreciated. This work was funded by DOE Grant DEFG02-90ER 60984. Note Added in Proof. Our very recent work applies these sensors to in situ rat embryo pH measurements.M RECEIVEDfor review March 17, 1992. Accepted July 22, 1992. Regietry No. Fluoresceinamine, 27699-63-9; acrylamide methylenebis(acrylamide)copolymer, 25034-5&6.
