Anal. Chem. 1993, 65,3586-3590
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Flexible Conductimetric Sensor Kohji Mitsubayashi,?Kenji Yokoyama, Toshifumi Takeuchi, Eiichi Tamiya,*and Isao Rarube* Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1, Komaba, Meguro-ku, Tokyo 153, J a p a n
A flexible sensor constructed in a sandwich configuration with a hydrophilic poly(tetrafluoroethylene) membrane placed between two gold deposited layers was evaluated for use as a conductimetric sensor in biologic fluids. The conductivity was measured using the device at frequencies ranging from 100 Hz to 100 kHz, and the device was calibrated at 100 kHz against sodium chloride solutions over the range of 0.1-50.0 g/L, which includes physiologic ion concentrations. Attached to a contact lens, the flexible conductimetric sensor can be placed directly onto the surface of the rabbit eye to monitor the electrical conductivity of tear fluids. Osmolarity obtained from the sensor data was consistently lower than previously reported values, perhaps indicating the effects of protein and lipid deposition. Tear flow with a mean turnover rate of 64.9 9%/min was elicited by eye drops of high concentration sodium chloride and distilled water.
INTRODUCTION In living organisms, the ion composition of tears, sweat, and saliva is influenced not only by basal secretion but also by secretion due to conditioned reflexes and more generally by physical condition. Fluid tonicity' and changes in ion ratios have been regarded as indexes of the altered hydration levels which frequently occur during exercise, and also of certain disease states including disorders of transcellular electrolyte transport.* In cystic fibrosis, for example, the usual function of the intracellular ion pumps is disrupted. In general, it is difficult to analyze the contents of these biologic fluids directly. Current techniques require the removal of sample fluid from the body using a filter Daper1s3.3 or capillary tube.5 Freezing point depression analysis can be used to determine the osmolarity of the fluid, while C1-, Na+, and K+ ion activities are quantified by coulometry and flame photometry. However, in carrying out these procedures, it is necessary to collect enough fluid for the analyses, to transfer it several times, and to dilute the samples, increasing evaporation and thus the apparent concentration of the ions. In biologic fluids, if the ion concentrations change, not only does the fluid
osmolarity change but the electrical conductivity changes as we11.1,6,7 Conductivity is considered to be an indirect function of electrolyte activity, or osmolarity.8 The fluid-specific conductivity is the sum of the contributions from all charged species. At very low concentrations, the specific conductivity will vary nearly linearly with concentrati~n.~ The conductivity measurement would not distinguish among the types of ions present. But comparison between the conductivity values for several physical conditions and monitoring of fluctuations in fluid conductivity are possible,s because the relative proportions of each ion are known for biologic fluids such as sweat, tears, saliva, and airway fluid.'O-14 Microfabrication technology for the manufacture of integrated circuits can produce planar interdigitated electrode arrays with areas on the order of a few square millimeters,'j decreasing the sample volume required to evaluate the conductivity of body fluid. But the solid (brittle) device substrates commonly used for such electrode arrays, such as glass plate or silicon wafer, limit the range of environments into which the sensor can be successfully introduced. Polyimide has been used as a substrate to achieve flexibility, but toxicity problems remain because of the use of substances such as chromium and nitric acid in the preposition and etching stages of the microfabrication techniques used to obtain the desired interdigitated electrode p a t t e r n ~ . ~ J ~ If these toxicity problems could be overcome, flexible conductimetric sensors could be placed directly onto the desired measurement surface to measure the conductivity of biologic fluids. The steps leading to possible sample evaporation would be eliminated by placing the actual measuring device into the fluid. Results would be immediately available, and continuous measurement could be made over a period of time. Such measurements could be of use in monitoring altered disease states, and the effects of therapeutic agents could be monitored dynamically. In this work, we have constructed and evaluated a flexible, nontoxic conductimetric sensor designed for direct contact with body surfaces and continuous measurement of conductivity in biologic fluid. As a physiological application, a contact lens sensor using the flexible conductimetric sensor has been constructed and used to monitor the conductivity of tear fluids on the surface of the rabbit eye. (6) Lundgren, N. P.; Ramanathan, N. L.; Gupta, A. S.: Chakravarti, H.S.Indian Res. ~ 1955.,~ 43. ~.~ J. Med. ~.~~ - - ,157-164. ~-. . ~-~ ( 7 ) Helhing, A. R. Clin. Chem. 1963, 8 , 227-234. (8) Fouke, J. M.; Wolin, A. D.; Saunders, K. G.; Neuman, M. R.; McFadden, E. R., Jr. IEEE Trans. Biomed. Eng. 1988,35, 877-881. (9) Evans, D. F.;Matesich, M. A. The measurement andinterpretation ~~
+ Presentaddress: ResearchLaboratories, NippondensoCo., Ltd., 5001, Minamiyama, Komenoki, Nisshin-cho, Aichi-gun,Aichi 4 7 0 1 , Japan. 1 Present address: Japan Advanced Institute for Science and Technology, Hokuriku (JAIST), 15, Asahidal, Tatsuguchi-cho, Nomi-gun, Ishikawa 923-12, Japan. (1) Gram, H. C. J. Biol. Chem. 1923, 1, 593-624. (2) Shwachman, H.; Antonowicz, I. Ann. N.Y. Acad. Sei. 1962, 933, 6-20. (3) Boucher, R. C.; Stutts, M. J.; Bromberg, P. A,: Gatzy, J. T. J . Appl. Physiol.: Respir. Enuiron.Exercise Physiol. 1981, 50, 613-621. (4) Sunderman, F. E. J. Biol. Chem. 1942, 143, 185-190. (5) Haeringen, N. J.; Glasius, E. Albrecht uon Graetes Arch. Klin. Exp. Ophthalmol. 1977, 202, 1-7.
~
~
~~~~
of electrolytic conductance. In Techniques ofElectrochemistry,Yeager,
E., Salkind, A. J., Eds.; Wiley: New York, 1973; pp 1-59. (10) Shwachman, H. Pediatrics 1963, 32, 85. (11) McKendrick, T. Lancet 1962, I , 183. (12) Lobeck, C. C.; Huebner, D. Pediatrics 1962, 30, 172. (13) Botelho, S.E'. Sci. Am. 1964, 211, 78. (14) Thysen, J. H.; Thorn, N. A,: Schwartz, I. L. Am. J.Physiol. 1954, 178, 155. (15) Sheppard, N. F., Jr.; Tucker, R. C.; Wu, C. Anal. Chem. 1993,65, 1199-1202.
0003-2700/93/0365-3586$04.00/0 0 1993 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, MCEMBER 15. I993
electrical terminal area A
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hydrophilic polytetrafluoroeihylenemembrane (thickness :80 prn, pore size : 0.2 pm) Figure 1. Structwe of flexible conductimetric sensor. EXPERIMENTAL SECTION Construction of Flexible Conductimetric Sensor. The structure of the flexibleconductimetricsensorisshown inFigure 1. Goldelectrcdes wereformeddirectlybyuseofvapordeposition onto both sides of a hydrophilic poly(tetrduoroethy1ene) (HPTFE) membrane (JGWP14225, thickness 80 sm, pore size 0.2 sm; Nihon Millipore Lid., Tokyo, Japan); this membrane offers chemicalstability,tear resistance,and flexibility. The thickness of each gold electrode layer was 2000 A. Electrodes on both sides were kept at equal intervals across the H-PTFE membrane. The membrane was shaped by knife into a 3-mm-wide strip. In order to isolate a sensitive area (length 5 mm), a cyanoacrylate adhesive for medical use (Aron Alpha A Sankyo, Sankyo Co., Lid., Tokyo, Japan) was applied to the middle part of the narrow strip membrane and dried at room temperature for 1h. Because H-PTFE membrane is hydrophilic and gold deposited layers are porous, the liquid cyanoacrylate adhesive could infiltrate through the membrane, so application to one side was sufficient to coat and insulate electrically both gold electrode layers, allowing the dipped membrane area to function as an electrical lead. Cyanoacrylate adhesive infiltration was used to separate the narrow strip membrane into three areas: sensitive area, lead area, and electrical terminal area. This integral three-area structure is convenient for application directly to body surfaces such as skin, airway,cornea, andconjunctiva, becauseconnection toaseparate electrical lead is not necessary. After inspection of electrical shorts and washing using 80% ethanol solution, the flexible conductimetric sensor was stored at room temperature except when in use for measurement. Measurement System. Behavior ofthesensor wascalibrated using test solutionsof distilled waterorsodiumchloride standard solutionsof varying concentrations ([NaCIl 0.1,0.5,1.0,5.0,10.0, 50.0, and 100.0 g/L). A 50.0-mL measuring cell was filled with these test solutions. Electrical conductivity can be measured using either alternating current or direct current methods.* A four-probe method using alternating current was used to avoid problems resulting from electrode polarization. The flexible conduetimetricsensor wasconnected to themeasurement system using a grip-type connector. A personal computer-controlled Multifrequency LCR Meter (Model 4214A, Yokogawa-Hewlett-Packard Ltd., Tokyo, Japan) system using the four-probe method at frequencies between 100 Hz and 100 kHz was used to measure the output signals at the flexible conductimetric sensor; impedance (0)and phase angle (degree) were measured to calculate the electrical conductivity (siemens), which represents the reciprocal of the electrical resistance ( Q ) . These values were monitored graphically on a continuous computer display and saved on floppy disks for later analysis. The effect of varying frequencies applied to the flexible conductimetric sensor was investigated using each standard solution. In addition, the variance in the sensor properties and reproducihility of the sensor were assessed Monitoringof Rabbit Tear Fluid Conductivity. JapaneseWhite rabbits [grade healthy (Kbs); Oriental Kobo Co. Ltd., Tokyo, Japan] were used for monitoringoftear fluid conductivity with the sensor using a frequency of 100 KHz and an applied signal amplitude of 0.5 V. During the monitoring procedure, the rabbit was restrained by a special apparatus [Oshida-type fixer
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NaCl CONCENTRATION (g/L) Figure 2. Calibration c w e s of the flexible conductimetric sensor in sodium chloride solutions of varying concentrations at ac frequencies ranging from 100 Hz to 100 kHz.
(KN-311), Furuya Co. Ltd., Kanagawa, Japan]; no anesthetic or other treatment was required. Although the flexible conductimetric sensor itself can be applieddirectlyto theeye,theusefulnessofthesensorwas further improved by bonding it with cyanoacrylate adhesive to a contact lens (Acuvue, Vistakon, a division of Johnson & Johnson Vision Products, Inc., Jacksonville, FL) which was shaped to suitable size (approximately 5 mm X I mm). After sterilization using 80% ethanol solution and drying, versed by direct observation of the increase in phase angle, the sensor was applied to the rabbit eye. Use of the contact lens minimized the loss of sensor contact due to eye blink. The contact lens-type sensor was calibrated in NaCl standard solutions at 35 OC before and after use in monitoring. Thecharacteristicsofthesensor wereaeaessedand thecleaning functionoftear flow wasinvestigated bymonitoringconductivity change following application of eye drops (20.0 rL) of distilled water or NaCl solution (10.0,20.0, and 40.0 g/L) using a syringe.
RESULTS AND DISCUSSION Evaluation of Flexible Conductimetric Sensor. The calibration curves (log-log plot) of the flexihle conductimetric sensor in sodium chloride solutions ofvarying concentrations at frequencies spanning from 100 Hz to 100 kHz, with temperature maintained at 25 "C, are shown in Figure 2. At low applied frequencies, the slope of the calibration curve gradually declined with increasing concentration of sodium chloride. This broad tendency was eliminated and the range of the calibration was improved gradually by increasing the applied frequency. The reason for the decreased slope of the curve with increasing NaCl concentration is not only the decrease in ion mobility at higher ion concentrationss but also the effect of ac frequencies applied to the sensor. Figure 3 illustrates the effect of frequency on the phase angle of conductivity measurement, indicating that the phase angles in the test solutions were high. The decreased slope is explained by electrode polarization, which leads to a n increase in sensor reactance. Indistilled water, the increase of phase angle with increasing frequency is believed to reflect the effect of permittivity of water. As Figure 3 indicates, phase angles were less than 20° and electrode polarization effects could be reduced substantially if ac excitation at a 100-kHz frequency was used. At 100-kHz applied frequency, the conductivity was linearly related to the NaCl concentration on a log-log plot over the range of 0.1-50.0 g/L (Figure l), with a correlation coefficient of 0.998 deduced by exponential regression analysis as shown by conductivity (S)= 0.00253[NaC1 (g/L)l"lSz The decreased slope of the curve at high concentrations, [NaClI 2 50 g/L, is not of concern since the measurement at
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, DECEMBER 15, 1993 i
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to adhere to the surface of the flexible sensor, it could alter the measured conductivity between gold deposited electrodes. The protein concentration of tear fluids has been reported to fall within the range of 6.99-8.00 g/L.16 Accordingly, we conducted in vitro studies a t 35 "C using bovine serum albumin added to the NaCl test solutions at concentrations of up to 8.0 g/L. With the addition of protein we observed a 7.7 94 decline in conductivity in NaCl concentrations over the range of 1.0-20.0 gIL. As protein adsorption by H-PTFE membrane is only 4.0 wg/cm2,1iwe believe the reason for the observed decline is protein deposition on the gold electrode layers. The sensor device was constructed using gold deposited H-PTFE membrane without chemical treatment and predeposition using dangerous substances such as nitric acid or chromium, thus gaining structural simplification and nontoxicity. Deposited gold adhered to the membrane and could not be peeled off by mechanical stress such as bending. The gold deposited membranes retain their flexibility even after vacuum evaporation. I t follows from these results that the optimum range of sodium chloride concentration for conductivity measurement using the flexible conductimetric sensor at 100-kHz ac frequency is from 0.1 to 50.0 g/L which includes the physiological range of interest. In this connection, we note that the concentration of physiologic saline solution is 0.9% NaC1. Thus, the sensor is well-suited for use in conductivity sensing applications involving body fluids. Monitoring of Rabbit Tear Fluid Conductivity, As a physiological application of the flexible conductimetric sensor, we examined its properties in the monitoring of rabbit tear fluid conductivity. The sensor was supple and constructed using nontoxic materials and methods as described previously. It could be applied directly to eye surfaces such as cornea and conjunctiva. We have verified the absence of injury from such application by medical microscope inspection. As noted above, to improve blink resistance we carried out our conductimetric monitoring of rabbit eye using a contact lenstype sensor in which part of the flexible sensor was affixed to the contact lens using cyanoacrylate adhesive. Figure 5 shows the typical response for conductivity measurement of rabbit tear fluid. As the figure indicates, continuous and stable conductimetric monitoring of rabbit tear fluid was possible using the sensor. The mean value of conductivity over a period of 4 min was 5.74 mS. The inset figure shows the calibration curves for the contact lens-type conductimetric sensor before and after use in monitoring.
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NaCl CONCENTRATION (glL) Effect of the concentration of NaCl on the coefficient of variation for sensor reproducibility (filled circles; n = 5)and variance (opensquares: n = 20). The applied frequencyand the signalamplitude were 100 kHz and 0.5 V, respectively. Figure 4.
physiological ionic concentrations has adequate sensitivity. The calibration characteristics of the flexible conductimetric sensor at 100 kHz allowed measurement of the conductivity and estimates of the osmolarity of biologic fluids, and this frequency was selected for general use. The reproducibility and variance of the flexible conductimetric sensor in test solutions are shown in Figure 4. Sensor performance was reproducible over multiple measurements, showing a coefficient of variation of less than 4 % (n = 5 ) in test solutions covering the range of 0.1-100.0 g/L. The maximum coefficient of variation across multiple sensors was 10.3% (n = 20). The effect of NaCl concentration for both coefficients of variation can be characterized by a parabola with its lowest point a t a concentration of 1.0 g/L. The reproducibility and variance of the sensor are attributable to the effects of electrode polarization at 100-kHz ac frequency a t high concentrations of NaCl and to the permittivity of water a t low concentrations, as discussed above. These results are consistent with the linearity of the calibration curves at 100-kHz ac frequency in Figure 2. Varying the temperature of the solution in the range of 25-40 "C by use of the flexible conductimetric sensor yielded approximately a 0.8% change in conductivity per degree centigrade. This was predicted because of the known effect of temperature on ionic mobility.8 The effect of varying the sensitive area within the range of 3-25 mm2 was a 0.48-mS change in conductivity per square millimeter. Protein in the physiologic fluids could have been expected to affect the conductivity measurement. If the protein were
(16)McClellan, B. H. A m . J. Ophthalmol. 1973, 76, 89. (17) Millipore Catalogue, Nihon Millipore Ltd., Tokyo, Japan, 1990; p 14.
ANALYTICAL CHEMISTRY, VOL. 65,NO. 24,DECEMBER 15, lQ93 S568
conductivityvalue of 7.12 mS and an apparent sodium chloride concentration of 9.57 g/L, respectively. The apparent con12.0 centration of NaCl was in agreement with the eye drop concentration of 10.0 g/L. In the same way, the calculated conductivitiesand apparent concentrations of sodium chloride for eye drops of 20.0 and 40.0 g/L NaCl and distilled water were 8.29 mS and 15.1 g/L, 10.6 mS and 34.7 g/L, and 2.50 mS and 1.71 g/L, respectively. The normal volume of rabbit tear is -2.7 pL (estimated by dilution methods), which is much smaller than the eye drop volume of 20.0 pL. Most of the tear fluid was replaced, accounting for our observation that the apparent concentration is consistently lower than the concentration of the eye drops. I I I In several studies,20-22 quantitative assessments of tear flow 0.0 0 5 10 15 have been reported. Tear flow can be evaluated reliably by TIME @in) determination of tear turnover using a f l u o r o p h ~ t o m e t e r . ~ ~ ~ ~ Tear turnover rate is defined as the percentage decrease of Figure 6, Effect on tear conductivity of 20.0-pL eye drops (arrows; fluorescein concentration in tear film per minute as a result 10.0.20.0,and 40.0g/L NaCl and dlstllled water). Inset: enlargement of the response curve to eye drops of 10.0 g/L NaCl and regressbn of tear flow. We calculated tear turnover rates by regression CUNe. analysis of data from each of a series of measurement runs; the mean value of the calculated rates was 64.9 % /min (SD The sensor's behavior can be characterized using the follow = 4.3%/min; n = 9). This value is higher than the fluoroequations: photometer-determined value for tear flow in the human of 40.6 ?6/min (range 7.7-80.5 % /min) reported by Mishima et before application The difference in the turnover rate values may perhaps conductivity (S)= 0.00120[NaCl(g/L)1°~'s7 be accounted for by the development of a physiologic cleaning function in rabbits to compensate for a low blink rate of 1/ (coefficient of correlation l.OO0,O.l g/L < NaCl < 10.0 g/L) 10 min. The effects of temperature on the mobilities of ions and after application on sensor sensitivity are known. Because the purpose of this conductivity (S)= 0.001 13[NaCl(g/L)l0.763 experiment was to construct a new device which could be applied to body surfaces to measure the conductivity of (coefficient of correlation 0.999,O.l g/L < NaCl < 10.0 g/L) biologic fluids, a temperature-sensitive resistor such as an Comparison of the calibration curves revealed a slight decline interdigitated conductimetric aensor15 was not used due to in sensitivity, which we attribute to the effect of tear protein the toxic substances required for its fabrication. Since it has on the adhesive. However, the size of the effect was negligible been reported that the temperature of the corneal surface is for this application. -35 "C for both humanz5@ and rabbit:' correction of The tear NaCl concentration and osmolarity, converting conductivity values for temperature was not necessary for a mean value (5.74 mS) of monitored tear conductivity by use comparison of the values obtained in the two species. of the precalibration curve, was 7.91 g/L NaCl and 250 mOsm/ The separation of the sensor into three areas-a sensitive L, respectively. The calculated osmolarity is 12 % lower area, a lead area, and an electrical terminal area-facilitated than previously reported values of 28G300 m 0 ~ m / L . As ~~J~ the application of our sensor to the surface of the rabbit eye mentioned above, protein and lipids in tear fluids may affect and allowed continuous monitoring of tear conductivity. No the measurement, altering the conductivity between the gold injuries to the corneal or conjunctival surfaces and no other deposited electrodes. harmful effects of the sensor were observed during and after Figure 6 illustrates the effect on tear conductivity of eye its use. drops. In order to evaluate the validity of the sensor output signal and to investigate the cleaningfunction of tear secretion CONCLUSIONS and flow, the sensor was used to monitor tear conductivity following application of eye drops of distilled water and A flexible conductimetric sensor was constructed using gold sodium chloride solutions of varying concentrations. After deposited H-PTFE membrane without chemical treatment the conductivity peaks, the sensor signal decreased gradually and predeposition using harmful substances, thus gaining as a result of the tear cleaning function. For the distilled structural simplification and nontoxicity. The sensor was water, the conductivity decreased rapidlywith the application calibrated at an ac of 100-kHz frequency against sodium of the eye drops and then increased gradually to a stablechloride solutions from 0.1 to 50.0 g/L, covering the physiconductivity value, showing a symmetrical curve to that given ological range of interest. The measurement of rabbit tear by the NaCl eye drops. conductivity was carried out as a physiological application of The inset shows an enlargement of the response curve to the flexible conductimetric sensor, evaluating tear conduceye drops of 10.0 g/L sodium chloride and a regression curve drawn by the power law equation as a function of time. The (20) Sorensen, T.; Taagehoj, J. F. Acta Ophthalmol. 1979,67, 564observed decrease was well-expressed by a regression equation 581. (21) Lamberts, D. W.; Foster, C. S.; Perry, H. D. Acta Ophthalmol. with a coefficient of correlation of 0.982. 1979,97, 1082-1085. The apparent value of tear conductivity for eye drops of (22) Mishima, S.; Gasset, A,; Klyce, S. D.; Baum, J. L. Invest. 10.0 g/L sodium chloride was calculated using the regression Ophthalmol. Visual Sci. 1966,5, 264-276. (23) Occhipinti, J. R.; Moiser, M. A.; LaMotte, J.; Monji, G. T. Curr. equation and the apparent concentration of sodium chloride Eye Res. 1988, 7,995-1000. was converted by use of the calibration curve, yielding a (24) Jordan, A.; Baum, J. Ophthalmology 1980,87, 920-930.
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(18) Mastman, G. J.; Blades, E. J. Arch. Ophthalmol. 1961, 65, 509. (19) Mizukawa, T. J . Jpn. Ophthalmol. SOC. 1971, 75 (9), 1953-1973.
(25) Zeiss, E. Graejes Arch. Augenhk. 1930,102, 523. (26) Mapstone, R. Ezp. Eye. Res. 1968, 7, 237. (27) Schwartz, B. Invest. Ophthalmol. 1964, 3, 100.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, DECEMBER 15, 1993
tivity values and osmolarity. In addition, tear flow was observed by measuring the effects of eye drops of distilled water and sodium chloride solutions of varying concentrations, yielding a mean turnover rate of 64.9 % /min. The sensor is well-suited for use in conductivity sensing applications involving body fluids. Potential applications of the flexible sensor include the diagnosis of disease states such as keratoconjunctivitis sicca and the measurement of airway conductivity. Future versions of the conductimetric biosensor in which biomolecules are
coupled to the flexible sensor may permit a wider range of clinical applications.
ACKNOWLEDGMENT The authors thank Dr. Katsunori Ogasawara, Prof. Tadahiko Tsuru, and Prof. Kanjiro Masuda at the Department of Ophthalmology, University of Tokyo, for valuable advice. for review
21p
l993. Accepted August 30,
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Abstract published in Aduance ACS Abstracts, October 1, 1993.
