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Electrodeless Piezoelectric Quartz Crystal Sensor for Determination of Total Urinary Reducing Sugar
Microchemical Journal 62, 328 –335 (1999) Article ID mchj.1999.1722, available online at http://www.idealibrary.com on
Electrodeless Piezoelectric Quartz Crystal Sensor for Determination of Total Urinary Reducing Sugar Shi-Hui Si 1 and Ke-Long Huang Department of Chemistry, Central South University of Technology, Changsha 410083, People’s Republic of China
Chang-Yin Lu 2 and Shou-Zhuo Yao New Material Research Institute, Hunan University, Changsha 410081, People’s Republic of China Received December 8, 1998; accepted February 10, 1999 A glucose-sensing system was developed in which an electrodeless piezoelectric quartz crystal was used to measure mass changes on the surface of a quartz plate during the reduction of Ag(NH 3) 21 by glucose. A satisfactory correlation was obtained between the frequency shift and the glucose concentration in the range 1.0 –25 mM, and the total reducing sugar in urine was determined. Treatment with 8 M nitric acid after each measurement was effective for cleaning the quartz surface, and the electrodeless piezoelectric quartz crystal sensor possessed excellent reproducibility and reusability during repeated use over 400 times. For the determination of total reducing sugar in urine (or blood), the present method can avoid the interferences occurring in a colorimetric method such as the color and turbidity of clinical specimens. © 1999 Academic Press Key Words: electrodeless piezoelectric crystal; glucose sensor; urinary reducing sugar.
INTRODUCTION Piezoelectric quartz crystals (PQC) are important sensing devices for monitoring changes in electrode mass and liquid properties (1–3). Usually the PQC has evaporated metal electrodes on either side of the quartz crystal, by which the excitation electric field is applied to the quartz crystal. An electrodeless PQC system was first reported by Nomura et al. (4). In their work, one or both electrodes of the normal PQC were dissolved with aqua regia, and both sides of the quartz plate were separated with a barrier. The electrodeless PQC oscillates in the same way as the normal PQC when the gaps between the excitation electrodes and each side of the quartz plate are filled with the electrolyte solutions. The frequency of the electrodeless PQC varies with changes of the viscosity, density, and conductivity of the solutions and the mass of trace material deposited on the quartz plate (5–7). Compared with the lifetime of the normal PQC with two evaporated metal electrodes, the lifetime of the electrodeless PQC can be greatly prolonged owing to the excellent chemical stability of the quartz plate. The determination of glucose in blood and urine in useful for clinical diagnosis. Some glucose oxidase electrode sensors have been developed on the basis of the high selectivity of glucose oxidase enzyme to glucose (8 –10). However, the lifetime of the glucose oxidase electrode is limited, and its response is affected by dissolved oxygen, pH and 1 2
To whom correspondence should be addressed. E-mail: [email protected]. On leave from Hengyang Medical College. 328
0026-265X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
TEST FOR URINARY REDUCING SUGAR
329
FIG. 1. Schematic diagram of the electrodeless PQC measuring cell. The distance between the quartz surface and the separated electrode is enlarged in the figure.
temperature of the medium, and other factors. Thus it is necessary to develop more practical, low-cost glucose assays for actual application. It is known that the main reducing sugar in urine is glucose, with only very small amounts of other reducing chemicals. Thus the total amount of reducing sugar in urine can be simply expressed as the content of glucose. In clinical laboratories, colorimetric methods based on oxidation– reduction reactions are often used to determine the concentration of reducing sugar in urine or blood (11). Additionally, a simple method for the determination of reducing sugar in urine has been the visual measurement of precipitates resulting from the reduction of Cu 21 or Ag 1 by the reducing sugar, but it is only a semiquantitative method. In this paper, the electrodeless PQC sensor was used to monitor the silver deposition on the surface of a quartz plate during the reduction of Ag(NH 3) 21 by glucose, and a quantitative method was developed for the determination of total reducing sugar in urine. EXPERIMENTAL Apparatus AT-cut quartz crystal with one evaporated silver electrode (basic frequency of 10 MHz) was prepared from the normal silver-plated quartz crystal by dissolving one electrode with aqua regia. As shown in Fig. 1, the quartz plate was fixed on the bottom of the cell (internal diameter of 8 mm) with silicone resin. The face without the silver electrode was allowed to contact the liquid phase, and the other face was set in air and sealed to avoid interferences from humidity and dust. A platinum wire (0.6-mm diameter) immersed in the solution was used as an excitation electrode, herein called the separated electrode, and the distance was about 1 mm between the separated electrode and the quartz plate. The temperature of the detector cell was controlled at 35 6 0.1°C by a thermostatic bath except where otherwise stated. An IC-TTL oscillator was designed to drive the quartz crystal at its resonant frequency
330
SI ET AL.
via the silver electrode coated on the quartz surface and the separated electrode. The frequency of the sensor was monitored by a universal counter (Iwatsu SC-7201), and the data readings were collected and transferred to a microcomputer. Materials All of the solutions used were prepared from analytical-grade chemicals. A stock glucose solution was prepared by dissolving 19.817 g of glucose in 500 ml of 0.5 M KNO 3 solution, and the working standard glucose solutions were obtained by diluting it with 0.5 M KNO 3 solution. Ag(NH 3) 21 solution was prepared as follows: ten volumes of 0.5 M AgNO 3 were mixed with two volumes of 1.2 M NaOH, and then 3 M NH 3 z H 2O was added while stirring until the AgOH sediment was completely dissolved. Urine samples were collected from healthy volunteers, diluted to two times the original volume with 1 M KNO 3 solution, and then heated in boiling water for 10 min. After being centrifuged at 1000g for 10 min, the upper solutions were collected and then preincubated at 35°C in a water bath before the determination. Procedures Before analysis of the urine samples, the electrodeless PQC system was calibrated by using the glucose standard solution as follows: 200 ml of the Ag(NH 3) 21 solution was added to the detector cell while the frequency of the sensor was monitored. When the frequency became stable, 200 ml of the glucose solution preincubated at 35°C was injected into the cell with a microsyringe. The frequency shifts between the frequency at the third minute and at later times were used to construct a calibration graph for the glucose concentration. In the same way, the frequency responses to the urine samples were obtained, and the amounts of reducing sugar in the samples were estimated from the calibration graph. The colorimetric method for the determination of total urinary reducing sugar with o-methylaniline was described in (11). After each measurement, 8 M nitric acid was added to the cell to clean the quartz surface. When the frequency did not increase, the cell was washed five times with distilled water after the removal of nitric acid. RESULTS AND DISCUSSION Response of Electrodeless PQC Sensor to Reduction of Ag(NH 3) 21 by Glucose It is known that the oscillating frequency of the PQC immersed in a liquid phase is affected by the properties of the medium such as density, viscosity, and conductivity (3). Our experimental results showed that the oscillating frequency of the electrodeless PQC became unstable when the solution conductivity was below 0.2 S/m. Over the range 0.2–2 S/m, the frequency of the electrodeless PQC decreased with increasing medium conductivity, but the frequency of the sensor changed slightly when the conductivity of the medium was more than 2 S/m (corresponding to about 0.2 M KNO 3 solution; see Fig. 2). In this study, a concentrated electrolyte solution (0.5 M KNO 3) was used as the reaction medium to avoid the interference of medium conductivity variation. Figure 3 shows the typical response of the electrodeless PQC sensor upon the addition
TEST FOR URINARY REDUCING SUGAR
331
FIG. 2. Dependence of the oscillating frequency of the PQC sensor with a separated electrode on the conductivity of the solution. Temperature 5 20°C.
of glucose solution to the Ag(NH 3) 21 solution at 35°C. At the initial stage (about 2 min), the oscillating frequency changes slowly, which suggests that it takes some time for the silver colloidal particles to aggregate and deposit on the surface of the quartz plate. The frequency decreases rapidly when the silver particle deposition starts. The amplitude of the frequency change depends on the concentration of glucose in the reaction medium when the concentration of Ag(NH 3) 21 is much higher than the glucose concentration (10 times higher in this study). The frequency shifts between the frequency at the third minute and at later times were used to construct a calibration graph for the glucose concentration (Fig. 4). For glucose concentration (C in mM) ranging from 1.0 to 25 mM, a satisfactory correlation was obtained between the value of C and the frequency shift (Df in Hz; Df 5 f 3min 2 f 8min, where f 3min and f 8min are the frequencies at the third and eighth minutes after the addition of the glucose solution, respectively): Df 5 74.7C 1 1058
~n 5 9, r 5 0.998!
(1)
In the five experiments, the variation of the frequency was not more than 18 Hz during the 20-min observation period when 200 ml of 0.5 M KNO 3 was added to the Ag(NH 3) 21 solution, but the frequency shift for the addition of 0.10 mM glucose was 76 Hz under the same conditions. The electrodeless PQC sensor is sensitive enough for the clinical determination of total urinary reducing sugar (4.5– 8.9 mM glucose for the normal value (11)).
332
SI ET AL.
FIG. 3. Frequency responses of the electrodeless PQC sensor upon addition of glucose solution to Ag(NH 3) 21 solution. Glucose concentration in the reaction medium: (a) 7 mM; (b) 3 mM. Curve c is for 0.5 M KNO 3. The Ag(NH3)21 concentration is 0.2 M. The downward arrow shows the injection of glucose solution. Temperature 5 35°C.
Reproducibility and Reusability It was found that the volume of the reaction solution affected the reproducibility of the sensor response. When the glucose concentration in the reaction solution was controlled at 3.0 mM, the standard deviation of Df for five runs using 4 ml of the reaction solution was 224 Hz (relative deviation 12.7%), 133 Hz (9.1%) for 1 ml of the reaction solution, and 57 Hz (5.2%) for 0.3 ml of the reaction solution. Herein, 0.4 ml of the reaction solution (200 ml of Ag(NH 3) 21 solution 1 200 ml of the glucose solution) was chosen, and the standard deviation for 3.0 mM glucose was 53 Hz (rd 5 4.1%, n 5 6). Over the range 10 – 80°C, the effect of temperature on the sensor response was also investigated. As seen in Table 1, the amplitude of Df increases with increasing reaction temperature; however, the reproducibility of the sensor response becomes poor when the reaction temperature is more than 50°C, and so the reaction temperature was optimized at 35°C in our experiments. After each measurement, the quartz plate was treated with 8 M nitric acid to remove the formed silver and then washed with distilled water five times. No change in the properties of the quartz plate was found after it was repeatedly used over 400 times within 2 months, which means the electrodeless PQC sensor has an excellent reusability and long lifetime. The present method is feasible and practical for the determination of glucose in clinical tests. Determination of Total Reducing Sugar in Urine Clinically, the main reducing sugar in urine is glucose, and the amounts of other reducing chemicals in urine are very low. The total amount of reducing sugar in urine can
TEST FOR URINARY REDUCING SUGAR
333
FIG. 4. Correlation between glucose concentration (C) and frequency shift (Df ): (■) Df 5 f 3min 2 f 6min, r 5 0.991, n 5 9; (●) Df 5 f 3min 2 f 8min, r 5 0.998, n 5 9; (Œ) Df 5 f 3min 2 f 10min, r 5 0.993, n 5 9. f 3min is the frequency at 3 min after the addition of glucose solution, f 6min that at 6 min, f 8min that at 8 min, and f 10min that at 10 min. Temperature 5 35°C.
be simply expressed as the content of glucose and is usually determined by using colorimetric methods based on oxidation–reduction reactions (11). To reduce the effect of the urine matrix on the sensor response, the urine samples were heated in boiling water for 10 min, and the sediment (mainly proteins) was removed after centrifugation at 1000g for
TABLE 1 Effect of Temperature on the Response of the Electrodeless PQC Sensor Observed during Reduction of Ag(NH 3) 21 by Glucose Temperature (°C)
Df a (Hz)
Relative deviation (%)
10 20 30 35 45 56 65 78
368 864 1034 1506 1932 2135 2445 2956
7.1 7.9 6.1 4.2 7.7 9.5 12 13.4
a
Average of five experiments obtained by measuring the frequency shift between the frequency at 3 and 8 min after addition of 200 ml of 14 mM glucose solution to 200 ml of Ag(NH 3) 21 solution.
334
SI ET AL. TABLE 2 Content of Total Urinary Reducing Sugar in Samples Content of urinary reducing sugar (mM) (mean 6 SD, n 5 5) Sample
PQC a
CM b
1 2 3 4 5 6 7
6.4 6 0.3 5.9 6 0.2 7.8 6 0.3 7.2 6 0.2 7.8 6 0.3 5.7 6 0.2 8.2 6 0.4
6.2 6 0.3 5.8 6 0.2 7.5 6 0.4 7.1 6 0.3 7.6 6 0.4 5.3 6 0.2 7.9 6 0.3
a Obtained by the electrodeless PQC method on the basis of Eq. (1). b Obtained by colorimetric method using o-methylaniline and 4.5– 8.9 mM glucose for the normal value (11).
10 min. The purified urine sample was added to the detector cell, and the frequency of the sensor was monitored for 0.5 h. The experimental results showed that the nonspecific adsorption of materials in the purified urine sample on the quartz surface was not strong (mean 6 SD, 76 6 5 Hz, n 5 8). In the following test, this matrix effect was taken into account. Herein, the healthy urine specimens were analyzed by the present method and the conventional colorimetric method using o-methylaniline (11). From Table 2, it can be seen that the results obtained by the present method are in accordance with those obtained by the colorimetric method. Additionally, various amounts of glucose were added to a fixed volume of normal urine, and the glucose contents (above the normal value) in those synthetic samples was determined by the electrodeless PQC method (see Table 3). The results show that this method is effective for clinical inspection of the high glucose content in urine. TABLE 3 Results for Determination of Glucose in Synthetic Samples Glucose added to normal urine (mM)
Found a (mM)
Recovery (%)
0 4.2 5.6 6.4 8.4
6.2 10.2 11.9 12.4 14.4
95.2 102 96.8 97.6
a Average of three determinations using the electrodeless PQC method.
TEST FOR URINARY REDUCING SUGAR
335
In conclusion, the proposed method requires only a sample size of 200 ml and gives a reliable result for the total urinary reducing sugar. The sensitivity and the simplicity of this method are comparable to those of the colorimetric method. Since the present method is based on the piezoelectric mass response, some interferences such as the color and turbidity of clinical specimens occurring in colorimetric methods can be avoided. A single-drop method (sample size of 5 ml) for testing the glucose content in blood is being pursued using a small-size quartz crystal. REFERENCES 1. Sauerbrey, G. Z. Phys., 1959, 155, 206. 2. Alder, J. F.; McCallum, J. J. Analyst, 1983, 108, 1169. 3. Thompson, M.; Kipling, A. L.; Duncan-Hewitt, W. C.; Rajakovic, L. V.; Cavic-Vlasak, B. A. Analyst, 1991, 116, 881. 4. Nomura, T.; Tanaka, F.; Yamada, T.; Itoh, H. Anal. Chim. Acta, 1991, 243, 273. 5. Nomura, T.; Yanagihara, T.; Mitsui, T. Anal. Chim. Acta, 1991, 248, 329. 6. Nomura, T.; Ohno, Y.; Takaji, Y. Anal. Chim. Acta, 1993, 272, 187. 7. Zhu, W. H.; Wei, W. Z.; Mo, Z. H.; Nie, L. H.; Yao, S. Z. Anal. Chem., 1993, 65, 2568. 8. Bartlett, P. N.; Cooper, J. M. J. Electroanal. Chem., 1993, 362, 1. 9. Moody, G. J.; Sanghera, G. S.; Thomas, D. R. Analyst, 1986, 111, 1235. 10. Ye, L.; Hammerle, M.; Olsthoorn, A. J. J.; Schuhmann, W.; Schmidt, H. L.; Duine, J. A.; Heller, A. Anal. Chem., 1993, 65, 238. 11. Ye, Y. W.; Li, Z. J.; Wang, Y. S. Clinic Detection and Diagnosis. People’s Hygiene Publishing House, Beijing, 1989 (in Chinese).
Electrodeless Piezoelectric Quartz Crystal Sensor for Determination of Total Urinary Reducing Sugar Shi-Hui Si 1 and Ke-Long Huang Department of Chemistry, Central South University of Technology, Changsha 410083, People’s Republic of China
Chang-Yin Lu 2 and Shou-Zhuo Yao New Material Research Institute, Hunan University, Changsha 410081, People’s Republic of China Received December 8, 1998; accepted February 10, 1999 A glucose-sensing system was developed in which an electrodeless piezoelectric quartz crystal was used to measure mass changes on the surface of a quartz plate during the reduction of Ag(NH 3) 21 by glucose. A satisfactory correlation was obtained between the frequency shift and the glucose concentration in the range 1.0 –25 mM, and the total reducing sugar in urine was determined. Treatment with 8 M nitric acid after each measurement was effective for cleaning the quartz surface, and the electrodeless piezoelectric quartz crystal sensor possessed excellent reproducibility and reusability during repeated use over 400 times. For the determination of total reducing sugar in urine (or blood), the present method can avoid the interferences occurring in a colorimetric method such as the color and turbidity of clinical specimens. © 1999 Academic Press Key Words: electrodeless piezoelectric crystal; glucose sensor; urinary reducing sugar.
INTRODUCTION Piezoelectric quartz crystals (PQC) are important sensing devices for monitoring changes in electrode mass and liquid properties (1–3). Usually the PQC has evaporated metal electrodes on either side of the quartz crystal, by which the excitation electric field is applied to the quartz crystal. An electrodeless PQC system was first reported by Nomura et al. (4). In their work, one or both electrodes of the normal PQC were dissolved with aqua regia, and both sides of the quartz plate were separated with a barrier. The electrodeless PQC oscillates in the same way as the normal PQC when the gaps between the excitation electrodes and each side of the quartz plate are filled with the electrolyte solutions. The frequency of the electrodeless PQC varies with changes of the viscosity, density, and conductivity of the solutions and the mass of trace material deposited on the quartz plate (5–7). Compared with the lifetime of the normal PQC with two evaporated metal electrodes, the lifetime of the electrodeless PQC can be greatly prolonged owing to the excellent chemical stability of the quartz plate. The determination of glucose in blood and urine in useful for clinical diagnosis. Some glucose oxidase electrode sensors have been developed on the basis of the high selectivity of glucose oxidase enzyme to glucose (8 –10). However, the lifetime of the glucose oxidase electrode is limited, and its response is affected by dissolved oxygen, pH and 1 2
To whom correspondence should be addressed. E-mail: [email protected]. On leave from Hengyang Medical College. 328
0026-265X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
TEST FOR URINARY REDUCING SUGAR
329
FIG. 1. Schematic diagram of the electrodeless PQC measuring cell. The distance between the quartz surface and the separated electrode is enlarged in the figure.
temperature of the medium, and other factors. Thus it is necessary to develop more practical, low-cost glucose assays for actual application. It is known that the main reducing sugar in urine is glucose, with only very small amounts of other reducing chemicals. Thus the total amount of reducing sugar in urine can be simply expressed as the content of glucose. In clinical laboratories, colorimetric methods based on oxidation– reduction reactions are often used to determine the concentration of reducing sugar in urine or blood (11). Additionally, a simple method for the determination of reducing sugar in urine has been the visual measurement of precipitates resulting from the reduction of Cu 21 or Ag 1 by the reducing sugar, but it is only a semiquantitative method. In this paper, the electrodeless PQC sensor was used to monitor the silver deposition on the surface of a quartz plate during the reduction of Ag(NH 3) 21 by glucose, and a quantitative method was developed for the determination of total reducing sugar in urine. EXPERIMENTAL Apparatus AT-cut quartz crystal with one evaporated silver electrode (basic frequency of 10 MHz) was prepared from the normal silver-plated quartz crystal by dissolving one electrode with aqua regia. As shown in Fig. 1, the quartz plate was fixed on the bottom of the cell (internal diameter of 8 mm) with silicone resin. The face without the silver electrode was allowed to contact the liquid phase, and the other face was set in air and sealed to avoid interferences from humidity and dust. A platinum wire (0.6-mm diameter) immersed in the solution was used as an excitation electrode, herein called the separated electrode, and the distance was about 1 mm between the separated electrode and the quartz plate. The temperature of the detector cell was controlled at 35 6 0.1°C by a thermostatic bath except where otherwise stated. An IC-TTL oscillator was designed to drive the quartz crystal at its resonant frequency
330
SI ET AL.
via the silver electrode coated on the quartz surface and the separated electrode. The frequency of the sensor was monitored by a universal counter (Iwatsu SC-7201), and the data readings were collected and transferred to a microcomputer. Materials All of the solutions used were prepared from analytical-grade chemicals. A stock glucose solution was prepared by dissolving 19.817 g of glucose in 500 ml of 0.5 M KNO 3 solution, and the working standard glucose solutions were obtained by diluting it with 0.5 M KNO 3 solution. Ag(NH 3) 21 solution was prepared as follows: ten volumes of 0.5 M AgNO 3 were mixed with two volumes of 1.2 M NaOH, and then 3 M NH 3 z H 2O was added while stirring until the AgOH sediment was completely dissolved. Urine samples were collected from healthy volunteers, diluted to two times the original volume with 1 M KNO 3 solution, and then heated in boiling water for 10 min. After being centrifuged at 1000g for 10 min, the upper solutions were collected and then preincubated at 35°C in a water bath before the determination. Procedures Before analysis of the urine samples, the electrodeless PQC system was calibrated by using the glucose standard solution as follows: 200 ml of the Ag(NH 3) 21 solution was added to the detector cell while the frequency of the sensor was monitored. When the frequency became stable, 200 ml of the glucose solution preincubated at 35°C was injected into the cell with a microsyringe. The frequency shifts between the frequency at the third minute and at later times were used to construct a calibration graph for the glucose concentration. In the same way, the frequency responses to the urine samples were obtained, and the amounts of reducing sugar in the samples were estimated from the calibration graph. The colorimetric method for the determination of total urinary reducing sugar with o-methylaniline was described in (11). After each measurement, 8 M nitric acid was added to the cell to clean the quartz surface. When the frequency did not increase, the cell was washed five times with distilled water after the removal of nitric acid. RESULTS AND DISCUSSION Response of Electrodeless PQC Sensor to Reduction of Ag(NH 3) 21 by Glucose It is known that the oscillating frequency of the PQC immersed in a liquid phase is affected by the properties of the medium such as density, viscosity, and conductivity (3). Our experimental results showed that the oscillating frequency of the electrodeless PQC became unstable when the solution conductivity was below 0.2 S/m. Over the range 0.2–2 S/m, the frequency of the electrodeless PQC decreased with increasing medium conductivity, but the frequency of the sensor changed slightly when the conductivity of the medium was more than 2 S/m (corresponding to about 0.2 M KNO 3 solution; see Fig. 2). In this study, a concentrated electrolyte solution (0.5 M KNO 3) was used as the reaction medium to avoid the interference of medium conductivity variation. Figure 3 shows the typical response of the electrodeless PQC sensor upon the addition
TEST FOR URINARY REDUCING SUGAR
331
FIG. 2. Dependence of the oscillating frequency of the PQC sensor with a separated electrode on the conductivity of the solution. Temperature 5 20°C.
of glucose solution to the Ag(NH 3) 21 solution at 35°C. At the initial stage (about 2 min), the oscillating frequency changes slowly, which suggests that it takes some time for the silver colloidal particles to aggregate and deposit on the surface of the quartz plate. The frequency decreases rapidly when the silver particle deposition starts. The amplitude of the frequency change depends on the concentration of glucose in the reaction medium when the concentration of Ag(NH 3) 21 is much higher than the glucose concentration (10 times higher in this study). The frequency shifts between the frequency at the third minute and at later times were used to construct a calibration graph for the glucose concentration (Fig. 4). For glucose concentration (C in mM) ranging from 1.0 to 25 mM, a satisfactory correlation was obtained between the value of C and the frequency shift (Df in Hz; Df 5 f 3min 2 f 8min, where f 3min and f 8min are the frequencies at the third and eighth minutes after the addition of the glucose solution, respectively): Df 5 74.7C 1 1058
~n 5 9, r 5 0.998!
(1)
In the five experiments, the variation of the frequency was not more than 18 Hz during the 20-min observation period when 200 ml of 0.5 M KNO 3 was added to the Ag(NH 3) 21 solution, but the frequency shift for the addition of 0.10 mM glucose was 76 Hz under the same conditions. The electrodeless PQC sensor is sensitive enough for the clinical determination of total urinary reducing sugar (4.5– 8.9 mM glucose for the normal value (11)).
332
SI ET AL.
FIG. 3. Frequency responses of the electrodeless PQC sensor upon addition of glucose solution to Ag(NH 3) 21 solution. Glucose concentration in the reaction medium: (a) 7 mM; (b) 3 mM. Curve c is for 0.5 M KNO 3. The Ag(NH3)21 concentration is 0.2 M. The downward arrow shows the injection of glucose solution. Temperature 5 35°C.
Reproducibility and Reusability It was found that the volume of the reaction solution affected the reproducibility of the sensor response. When the glucose concentration in the reaction solution was controlled at 3.0 mM, the standard deviation of Df for five runs using 4 ml of the reaction solution was 224 Hz (relative deviation 12.7%), 133 Hz (9.1%) for 1 ml of the reaction solution, and 57 Hz (5.2%) for 0.3 ml of the reaction solution. Herein, 0.4 ml of the reaction solution (200 ml of Ag(NH 3) 21 solution 1 200 ml of the glucose solution) was chosen, and the standard deviation for 3.0 mM glucose was 53 Hz (rd 5 4.1%, n 5 6). Over the range 10 – 80°C, the effect of temperature on the sensor response was also investigated. As seen in Table 1, the amplitude of Df increases with increasing reaction temperature; however, the reproducibility of the sensor response becomes poor when the reaction temperature is more than 50°C, and so the reaction temperature was optimized at 35°C in our experiments. After each measurement, the quartz plate was treated with 8 M nitric acid to remove the formed silver and then washed with distilled water five times. No change in the properties of the quartz plate was found after it was repeatedly used over 400 times within 2 months, which means the electrodeless PQC sensor has an excellent reusability and long lifetime. The present method is feasible and practical for the determination of glucose in clinical tests. Determination of Total Reducing Sugar in Urine Clinically, the main reducing sugar in urine is glucose, and the amounts of other reducing chemicals in urine are very low. The total amount of reducing sugar in urine can
TEST FOR URINARY REDUCING SUGAR
333
FIG. 4. Correlation between glucose concentration (C) and frequency shift (Df ): (■) Df 5 f 3min 2 f 6min, r 5 0.991, n 5 9; (●) Df 5 f 3min 2 f 8min, r 5 0.998, n 5 9; (Œ) Df 5 f 3min 2 f 10min, r 5 0.993, n 5 9. f 3min is the frequency at 3 min after the addition of glucose solution, f 6min that at 6 min, f 8min that at 8 min, and f 10min that at 10 min. Temperature 5 35°C.
be simply expressed as the content of glucose and is usually determined by using colorimetric methods based on oxidation–reduction reactions (11). To reduce the effect of the urine matrix on the sensor response, the urine samples were heated in boiling water for 10 min, and the sediment (mainly proteins) was removed after centrifugation at 1000g for
TABLE 1 Effect of Temperature on the Response of the Electrodeless PQC Sensor Observed during Reduction of Ag(NH 3) 21 by Glucose Temperature (°C)
Df a (Hz)
Relative deviation (%)
10 20 30 35 45 56 65 78
368 864 1034 1506 1932 2135 2445 2956
7.1 7.9 6.1 4.2 7.7 9.5 12 13.4
a
Average of five experiments obtained by measuring the frequency shift between the frequency at 3 and 8 min after addition of 200 ml of 14 mM glucose solution to 200 ml of Ag(NH 3) 21 solution.
334
SI ET AL. TABLE 2 Content of Total Urinary Reducing Sugar in Samples Content of urinary reducing sugar (mM) (mean 6 SD, n 5 5) Sample
PQC a
CM b
1 2 3 4 5 6 7
6.4 6 0.3 5.9 6 0.2 7.8 6 0.3 7.2 6 0.2 7.8 6 0.3 5.7 6 0.2 8.2 6 0.4
6.2 6 0.3 5.8 6 0.2 7.5 6 0.4 7.1 6 0.3 7.6 6 0.4 5.3 6 0.2 7.9 6 0.3
a Obtained by the electrodeless PQC method on the basis of Eq. (1). b Obtained by colorimetric method using o-methylaniline and 4.5– 8.9 mM glucose for the normal value (11).
10 min. The purified urine sample was added to the detector cell, and the frequency of the sensor was monitored for 0.5 h. The experimental results showed that the nonspecific adsorption of materials in the purified urine sample on the quartz surface was not strong (mean 6 SD, 76 6 5 Hz, n 5 8). In the following test, this matrix effect was taken into account. Herein, the healthy urine specimens were analyzed by the present method and the conventional colorimetric method using o-methylaniline (11). From Table 2, it can be seen that the results obtained by the present method are in accordance with those obtained by the colorimetric method. Additionally, various amounts of glucose were added to a fixed volume of normal urine, and the glucose contents (above the normal value) in those synthetic samples was determined by the electrodeless PQC method (see Table 3). The results show that this method is effective for clinical inspection of the high glucose content in urine. TABLE 3 Results for Determination of Glucose in Synthetic Samples Glucose added to normal urine (mM)
Found a (mM)
Recovery (%)
0 4.2 5.6 6.4 8.4
6.2 10.2 11.9 12.4 14.4
95.2 102 96.8 97.6
a Average of three determinations using the electrodeless PQC method.
TEST FOR URINARY REDUCING SUGAR
335
In conclusion, the proposed method requires only a sample size of 200 ml and gives a reliable result for the total urinary reducing sugar. The sensitivity and the simplicity of this method are comparable to those of the colorimetric method. Since the present method is based on the piezoelectric mass response, some interferences such as the color and turbidity of clinical specimens occurring in colorimetric methods can be avoided. A single-drop method (sample size of 5 ml) for testing the glucose content in blood is being pursued using a small-size quartz crystal. REFERENCES 1. Sauerbrey, G. Z. Phys., 1959, 155, 206. 2. Alder, J. F.; McCallum, J. J. Analyst, 1983, 108, 1169. 3. Thompson, M.; Kipling, A. L.; Duncan-Hewitt, W. C.; Rajakovic, L. V.; Cavic-Vlasak, B. A. Analyst, 1991, 116, 881. 4. Nomura, T.; Tanaka, F.; Yamada, T.; Itoh, H. Anal. Chim. Acta, 1991, 243, 273. 5. Nomura, T.; Yanagihara, T.; Mitsui, T. Anal. Chim. Acta, 1991, 248, 329. 6. Nomura, T.; Ohno, Y.; Takaji, Y. Anal. Chim. Acta, 1993, 272, 187. 7. Zhu, W. H.; Wei, W. Z.; Mo, Z. H.; Nie, L. H.; Yao, S. Z. Anal. Chem., 1993, 65, 2568. 8. Bartlett, P. N.; Cooper, J. M. J. Electroanal. Chem., 1993, 362, 1. 9. Moody, G. J.; Sanghera, G. S.; Thomas, D. R. Analyst, 1986, 111, 1235. 10. Ye, L.; Hammerle, M.; Olsthoorn, A. J. J.; Schuhmann, W.; Schmidt, H. L.; Duine, J. A.; Heller, A. Anal. Chem., 1993, 65, 238. 11. Ye, Y. W.; Li, Z. J.; Wang, Y. S. Clinic Detection and Diagnosis. People’s Hygiene Publishing House, Beijing, 1989 (in Chinese).