Anal. Chem. 1994,66, 3323-3327
Capillary Optical Sensors Bernhard H. Welgltl* and Otto S. Wolfbeis’** Joanneum Research, Institute for Sensor Interfaces, Steyrergasse 17, A-80 10 Graz, Austria, and Institute for Organic Chemistry, Analytical Division, Karl Franzens University, Heinrich St. 28, A-80 10 Graz, Austria
Class or plastic tubings with a chemically sensitive coating on the inner surface are used for optical chemical sensing. Such devices possess several attractive features in that they (a) can act as a mechanical support for optically sensitive materials (coatings), (b) represent an optical waveguide structure and enable various methods of optical interrogation, (c) can serve as a sample cavity of well-defined volume, and (d) are suitable for direct sampling. The performance of such “capillary optrodes” is exemplified with a fast-responding and fully reversible carbon dioxide sensor chemistry. Its response time to gases ( t ~varies ) from 100 to 300 ms. We also give arrangementsfor optical readout devices and show that capillary sensors can overcome some of the problems of optrode membranes mounted in flow cells or on fiber tips. Numerous solid supports for the immobilization of sensitive layers for optical (fiber) chemical sensors have been reported. For example, sensor materials have been deposited on fibers by dip-coating’ and on planar supports by spin-coating,2 photop~lymerization~ or covalent i m m o b i l i ~ a t i o n .Others ~~~ describe the spreading of chemically sensitive layers on polyester which paves the way for mass manufacturing of sensor spots which, eventually, may be attached to fiber tips. Alternatively, they may be used in disposable (singleshot) sensors. Coated polyester membranes may also be obtained by ~pin-coating.~-~ Planar optical sensors usually are placed in flow cell type modules comprising light sources and detectors, sometimes along with optical fibers which guide light from the source to the cell with the membrane and back to an optoelectronic readout device. A sample (such as blood) is passed through the cell where it changes the optical properties of the sensor membrane. Such cells, frequently made from steel or durable plastic, are robust, bulky, expensive, and have relatively large internal volumes. Due to the nature of the measurement, the sensor membrane has to be fixed on one side of the cell with O-rings, springs, screws, or the like, or glued to the surface of a transparent cell wall. The cell-membrane interfaces, + Joanneum Research.
Karl Franzens University. (1) Munkholm, C.; Walt, D. R. Talanfa 1988, 35, 109. (2) Seiler, K.; Simon, W. Anal. Chim. Acfa 1992, 266, 73. (3) Tan, W.; Zhong-You, S.;Smith, S.;Birnbaum, D.; Kopelman, R. Science 1992,258, 778. (4) Harper, B. G. Anal. Chem. 1975,47, 348. ( 5 ) Wolfbcis, 0. S.;Offenbacher, H.; Kroneis, H.; Marsoner, H. Mikrochim. Acta (Vienna) 1984, I, 153. (6) Wolfbeis,O. S.;Weis, L. J.; Leiner,M. J. P.;Ziegler, W. E. Anal. Chem. 1988, 60, 2028. (7) Wolfbeis, 0. S.,Ed. Fiber Opfic Chemical Sensors and Biosensors; CRC Press: Boca Raton, FL, 1991; Vol. I, Chapters 7 and 8. (8) Jones, T. P.; Porter, M. D. Anal. Chem. 1988.60, 404. (9) Weigl, B. H.; Holobar, A.; Rodriguez, N. V.; Wolfbeis, 0. S . Anal. Chim. Acta 1993, 282, 335-343. 0003-2700/94/0366-3323$04.50/0 0 1994 American Chemical Society
however, are a notorious source of error due to gas permeation, leakage, swelling, scratching, or delamination. Alternatively, chemically sensitive optical materials also have been mounted at the tip of an optical fiber. Such devices, in principle, can be placed directly in the sample (such as blood in an artery). Hence, both sampling and transportation of the sample become unnecessary. However, this approach suffers from several shortcomings including (a) the fiber bending effect, (b) mechanical disrupture, (c) interferences by false light because of inadequate optical isolation and protection from both ambient light and sample fluorescence, and (d) the poor reproducibility of the thickness of the coatings on fiber tips. The most serious difficulty we encountered is the reproducible efficiency in coupling light into and out of the tip coating (or a tip membrane) which requires rather sophisticated optical alignments. In addition to the use of optical fiber sensors, schemes have been reported in which capillaries have been used as waveguides. Giuliani et al.1° and Reisfeld et al.” use a small glass capillary tube covered with a dye to sense ammonia in air. Another approach was reported12 in which a glass tube was filled with porous sol-gel silica powder doped with a complexing agent which gave a color with the analyte. The length of the stained section of the tube was found to be a measure of the concentration of the analyte. The device resembles commercially available test tubes. More recently, another configuration has been reported in which the chemically sensitive material was deposited at the tip of a micropipet using near-field photop~lymerization.~ The method lends itself to the fabrication of miniature fiber-optic pH sensors with an extremely small sample volume and very rapid response times. Due to the minute size of the fiber tip, these sensors are excellent research tools but fragile and unlikely ever to be used in an adverse environment or by unskilled personnel. We describe an attractive alternative for making “integrated (in a that they can act as sampling device, sampling vessel, and optical element) by making use of small capillaries. We show how this has led to a new immobilization technique (referred to as “reversed pumping”), and that such “integrated capillary sensors” have attractive novel features iincluding (a) a new sampling format (by making the sensor the sample cuvette), (b) the use of capillaries as a new support for optical sensor materials, and (c) the use of capillaries as disposable sampling vessels of defined volume. (10) Giuliani J. F.; Wohltjen H.; Jarvis N. L. Opt. Lett. 1983, 8, 54. (1 1) Chernyak V.; Reisfeld R.; Gvishi R.; Venezky D. Sens. Mater. 1990,2,117. (12) Kuselman I.; Lev 0. Tulanfa 1993, 40, 749.
Analytical Chemistry, Vol. 66, No. 20, October 15, 1994 3323
EXPERIMENTAL SECTION Preparation of the Capillary Sensor. It was found that most sensor materials can be deposited inside glass or plastic tubes by pumping or sucking the sensor cocktail through the tubes. Disposable glass micropipets with an inner diameter of 0.7 mm (Brand, Wertheim, FRG) were used in this particular case. The capillaries were connected to a peristaltic pump and then dipped into the cocktail solutions (i.e., the mixtures prepared for immobilization). The pump speed was adjusted such that the solution was moving up at a rate of 5 mm/s. After the capillary was filled about half, the rotation of the pump was reversed. The pump speed depended largely on the viscosity and temperatureof the immobilizationsolution. After the sensor material was pumped out, the capillary was dried by drawing air through the capillary using a water jet aspirator. After several minutes, the inner coating is completely dry. The capillary was then inserted into a suitable optical monitoring device (see below), or stored in clean ambient air until used. For the experiments described in the following, carbon dioxide sensitive layers were used as an inner coating. The sensor cocktail comprises an ion pair consisting of an organic quaternary cation and a pH indicator anion.13J4 The color change of the indicator dye was monitored. The following cocktail was employed: Solution A was prepared by dissolving 5.0 g of ethyl cellulose (Aldrich) in 45 g of toluene at room temperature. Solution B was prepared by stirring 1.368 g of tetraoctylammoniumbromide (TOABr) (Aldrich) with 1.16 g of silver oxide (Aldrich) and 5 mL of methanol for 5 h at room temperature; the solid phase (silver bromide) was then removed. Solution CI was prepared by dissolving 13.77 mg of cresol red (Aldrich) in 22.5 mL of 0.1 M NaOH. Solution C2 was prepared by dissolving 37.39 mg of TOABr in 3 mL of toluene. Solutions CI and C2 were mixed and stirred for 5 min, and then the organic phase was removed and washed two times with 3 mL of 0.1 M NaOH. The resulting organic phase (solution C) contained the pure ion pair [cresol red-/ TOA+] dissolved in toluene. One gram of solution A , 2 19 pL of solutionB, 200 p L of solution C, 400 p L of tributyl phosphate (Aldrich), and 400 pL of toluene were mixed and homogenized by stirring for 5 min. This cocktail was used to coat the capillaries as described above. In one series of experiments, an additional coating was applied, in that a moisture-curing basic silicone (PS250, from Huels-Petrarch; obtained from ABCR, Karlsruhe,Germany) was deposited on the first layer, again by the “reversed pumping” technique. When applied at room temperature, the cocktail deposited on the inner wall, after drying, has a thickness of around 2 pm, as estimated at a section of the capillary with a microscope and by comparison with steel spacers of defined thickness. The additional silicone coating applied in one series of experiments had an estimated thickness of about 3 pm. In order to obtain thinner coatings, the coating solution may be further diluted with toluene. Figure l a shows a cross section of a capillary with one single sensor layer. However, several layers may be deposited consecutively by this technique, as it may become necessary in case of COz sensors with an additional hydrophobic (proton-impermeable) layer of silicone ~
~~
(13) Mills, A.; Chang, Q.; McMurray, N. Anal. Chem. 1992, 64, 1383. (14) Raemer, D. B.; Walt, D. R.; Munkholm, C. US.Pat. 5,005.572, 1991.
3324 Analytical Chemistry, Vol. 66, No. 20, October 15, 1994
glass or plastic tubing
nner sample volume, or
glass or plastic tubing
otective hydrophobic
sample volume, or
Figure 1. Cross section through typical capillary sensors. I n a only one coating is deposited on the inner wall of the capillary, and the resulting sensor is preferably used for analyzing gaseous samples. I n b two layers are depositedon the inner wall by the “reversed pumping” technique. The first layer is the carbon dioxide chemistry (polystyrene based), and the second a gas-permeable but proton-impermeable silicone layer. A third layer of black silicone may optionally be placed on layer 2 as an optical isolator.
rubber (such as the PS 250, from Huels-Petrarch). The additional coating prevents dye leaching and diffusion of protons into the sensing layer. Hence, the sensor becomes applicable to aqueous samples. Finally, an optical isolation may be placed on the chemistries so as to prevent ambient light from interfering. Figure l b shows a cross section of a capillary having three sensor layers. Optical Arrangement. Figure 2 shows a conventional arrangement for monitoring the changes in the absorption or fluorescence of the tube sensors. A 1.5” diameter hole was drilled through an ST optical fiber coupler (Hirschmann, Vienna), into which the sensor capillary was inserted. Optical glass fibers (core diameter 125 pm) were attached to each side of the coupler so that the springs of the fiber connectors pressed the fibers against the capillary. One fiber was then attached to a yellow LED (Amax 590 nm), and the other to a Hewlett-Packard 8 153Alightwave multimeter. The capillary was connected to either the gas line with a short piece of polyethylene tubing or to a flow of buffered sample solutions which were equilibrated with gas mixtures of defined carbon dioxide partial pressure. The flow rate was 1 mL min-l. In the optical arrangement where the capillary acted as a waveguide for the 543.3-nm He-Ne laser line, an input and an output coupler, respectively, were created at each end of the capillaries. In essence, a 0.5- X 0.8-mm coupler spot was ground onto the outer surface using conventional optical grinding paper. The resulting, highly scattering surface acts
I
n coated wmor
\L detector 0
T
gas or liquid sample
Figure 2. Optical capillary sensor with fiber coupler for absorption measurements. Note that the light beam passes through the sensing layer (the inner coating of the capillary) twice.
2
4
6
8
IO
12
14
t i m e (min)
Figure 3. Response curves of a capillary sensor (this work) and a planar carbon dioxide sensor (with the chemistry placed on a planar polyester support), toward gaseous carbon dioxide. The same sensor layer chemistries and thicknesses were applied in both experiments. (1 hPa equals 1 mbar and 0.76 Torr).
as a very efficient coupler, and the capillary with its index of refraction of 1.54 acts as the waveguide. The refractive index of the C02-sensitive coating (composed of mainly ethyl cellulose and acting as the “cladding” of the glass waveguide) is 1.35.
RESULTS AND DISCUSSION The CO2-sensitive capillaries, which show the blue color of cresol red in its base form,do not require further conditioning and can be tested immediately after being dried for several minutes at room temperature. They reversibly change color from blue via green to yellow when exposed to increasing levels of carbon dioxide. The sensing mechanism is based on the reversible reaction between the blue [cresol red-/TOA+] ion pair and carbon dioxide, which results in the formation of a new ion pair consisting of the quaternary cation and a bicarbonate anion, thereby releasing a proton which converts the blue indicator anion into its yellow (protonated) form.* The process is fully reversible, and in the absenceof C02 the blue color is obtained again. Expired air contains 2-5% C02. The response time of this kind of sensor to gases is less than 0.3 s (t90, the time of 90% of the total signal change to occur) because of the very thin coating. This makes it possible to instrumentally monitor the concentration of carbon dioxide in respiratory air (Figure 2), but also visually by observing the continuous color change (from blue to yellow and back) while breathing through the capillary. In order to increase the resolution of the sensor, the capillary was inserted into the readout device shown in Figure 2. The measured response to varying concentrations of carbon dioxide in a gas mixture is shown in Figure 3. The gas mixtures also contained nitrogen and oxygen at atmosphericpressure. Also shown is a response curve of the same material, but immobilized on a polyester support and mounted in a planar flow-through cell arrangement. It clearly can be seen that response times are much shorter and the relative signal changes larger in case of the capillary sensor.
0
10
20 30 time (min)
40
50
Figure 4. Responseof the capillarysensor to carbondioxide dissolved in water. (1 hPa equals 1 mbar and 0.76 Torr.)
In an experiment with aqueous samples, a capillary optrode with an additional hydrophobic coating (Figure lb) was employed. The response to samples tonometered (gasequilibrated) with gases of different pC02 is shown in Figure 4. Again, the sensor responds fully reversibly although response times are much slower, being in the order of 3 min for the forward response, and 4-7 min in the reverse direction. This is only partially due to the second coating which is required to prevent interferences by changes in pH but also acts as a diffusional barrier. The major reason for the slower response is the low concentration of COz in the water sample which requires a substantial quantity of water to pass the membrane until it is equilibrated with carbon dioxide. To document insensitivity to pH, the capillary sensor was alternately exposed to 0.1 M buffer solutions of pH 4 and pH 9, both purged with nitrogen. No cross sensitivity to pH was found. However, when exposing it to 0.1 M HCl, the coating irreversibly turned yellow after less than 1 h. Not unexpectedly, ammonia interfered when present along with carbon dioxide. The reproducibility of the insertion of the capillary into the optical arrangement (Figure 2) was tested by inserting and removing it 10 times, while the membrane was exposed to pure nitrogen. In a second experiment, 10 different AnalyticalChemistry, Vol. 66, No. 20, October 15, 1994
3325
“‘UJ
I
I
photodiode. light output sample “ow in
0,45
20
40 60 time (sed
80
100
Flgure 5. Response time, relative signal change, and reversibilityof the capillary sensor for carbon dioxide, alternately exposed to 0 and 80 hPa carbon dioxide in dry nitrogen.
I gating coupler for fiber or LED. light input
Figure 6. Capillary sensor used as a “tubular waveguide”. The tubing is supplied with an input grating coupler for light from an optical fiber or directly from an LED and an output coupler from where light is directed to a photodiode. The optical changes are monitoredvia the evanescent field of the waveguide which hits the chemically sensitive inner coating.
green line of an He-Ne ion laser (A 543.3 nm) was coupled into the capillary waveguide at one end, and attenuated light left the waveguide at the other end. Light was focused, via a standard lens, onto a silicone photdiode. When exposed to several levels of carbon dioxide in air (0-20%) by incorporating the tube into a gas line, a substantial and fully reproducible change in absorption was observed in all experiments. In addition to absorptionmeasurements,the capillary may as well be used to monitor changes in the refractive index of the sensor layer, because the depth of the evanescent field-and, consequently, the efficiency of the absorption-is a function of the ratio between the refractive indices of tube (“core”) and sensor coating (“cladding”). The capillary then acts as sort of input/output coupler.16 Without any coating, it is the refractive index of the sample passing the capillary which is measured. Finally, the capillary can be considered as a useful sampling vessel and, at the same time, acts as a sensor, so that there is no need for sample transfer. Samples may be drawn by capillary action, and this is considered to be particularly promising in case of blood and other physiological liquids. Such a sampling technique is especially useful when only scarce amounts of sample are available.
capillaries having the same coating were inserted into the detector. The error due to misalignment (insertion) was less than 1%, whereas the error in absorption due to variations in manufacturing was less than 5% of the relative signal change. In the arrangement shown in Figure 2, the optical damping due to absorption is twice as high as in a conventional sensor membrane placed in a cell, because the optical path passes through the membrane twice. Since the inner layers are mechanically protected by the capillary, a larger amount of plasticizer can be added to the coating solution. The addition of plasticizer is knownlS to reduce the response time by increasing the permeability for gases. However, large amounts of plasticizer cause the membrane to assume a sticky consistency which is a problem in case of planar sensor spots. In capillary sensors, in contrast, the inner coating is well protected from mechanical attack. Up to 80% tributyl phosphate (TBP) may be added to ethyl cellulose. This compares favorably to the maximally 50% TBP that can be added to ethyl cellulose when deposited on planar glass slides or polyester membranes. The response curve of this sensor, when exposed to 0 and 80 hPa carbon dioxide (Le., 0 and around 8% COz in gas mixtures at ambient pressure) is given in Figure 5 . The response time (?go) for the reaction from 0 to 80 hPa was 100 ms, the back-reaction occurred in less than CONCLUSIONS 300 ms. These are among the shortest reported response times We think that the capillary type sensor presented here has for chemical optical sensors based on a chemical r e a c t i ~ n . ~ J ~ J ~a number of interesting and novel features. These include a Capillary sensors lend themselves to several other methods simple and reproducible immobilization procedure and a of optical interrogation. The capillary tube, when connected mechanical protection of the sensor surface. It can act as to a light source and a detector via grating couplers, can act both a flow-through cell and a sampling vessel and applied to as a “tubular waveguide”. Changes in the absorption or both liquids and gases. No leakage of sample, or air fluorescence of the sensor layer within the evanescent field of permeation into the tubing, both of which are common the waveguide can be monitored. With this method, interferdrawbacks in flow-cell type arrangements (because of poor ences by color and turbidity of the sample are widely membrane-to-cell alignment), can occur. Because of its eliminated, since the coating is thicker than the penetration geometry, air bubbles do not tend to reside in the tube area. depth of the evanescent wave which is in the order of 1 pm. The sensors have extremely short response time for three Figure 6 shows the arrangement that has been used in reasons, namely because (a) the two sensing layers passed by experiments on evanescent wave absorptiometry(or fluorimthe light beam when penetrating the capillary can be very etry). In a typical experiment,two gratings were ground into thin, (b) equilibration of two thin layers is faster than that of a COz-sensitive capillary close to each end (Figure 6). The a single layer of the same total thickness, and (c) the sample (1 5) Brandrup, I., Immergut, E. H., Eds. Polymer Handbook, 3rd ed.; Wiley: New York, 1989; p VI/440.
3326
Analytical Chemistry, Vol. 66, No. 20, October 15, 1994
(16) Clerc, D.;Lukosz, W. Sens. Actuators 1993, BIZ, 461.
flow is laminar, resulting in a nonturbulent flow and a fast exchange of sample volume. Conceivably, such sensors may also act as sampling devices, e.g. when drawing whole blood. Because of the use of lowcost materials and simple manufacturing techniques, such capillary sensors can be produced at low costs which, in turn, paves the way for disposable sensors. A final function of the capillary is that of a tubular waveguide. If appropriate means for coupling light into and out of the capillary are provided (such as shown in Figure 6 ) , the evanescent field phenomena can be applied to optically interrogate the sensor. Initial experiments with this geometry are extremely promising.
The main disadvantage of capillary (“integrated”) sensors over planar sensor geometries is a more sophisticated optical geometry which is required to couple light in and out of a curved capillary surface (Figure 2). However, we think that this kind of sensor allows the construction of a variety of simple and cost-effective measurement devices, and the capillaries may be disposable and easy to change and to handle. Received for review December 1, 1993. Revised manuscript received June 15, 1994. Accepted June 16, 1994.” Abstract published in Advance ACS Abstracts, August 1, 1994.
Analytical Chemistry, Vol. 66, No. 20, October 15, 1994
3327
