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In SituMonitoring of Adsorption of Lysozyme onto Quartz Using an Electrode-Separated Piezoelectric Sensor
MICROCHEMICAL JOURNAL ARTICLE NO.
60, 1–7 (1998)
MJ981620
In Situ Monitoring of Adsorption of Lysozyme onto Quartz Using an Electrode-Separated Piezoelectric Sensor D. Z. Shen,*,1 Y. H. Xue,† Q. Kang,† Q. J. Xie,‡ and L. X. Chen* *Chemistry College, Shandong University, Jinan 250100, People’s Republic of China; †Shandong Institute of Mining and Technology, Taian 270019, People’s Republic of China; and ‡Chemistry Department, Hunan Normal University, Changsha 410081, People’s Republic of China Received January 12, 1997; accepted March 28, 1998 The adsorption of lysozyme from phosphate-buffered saline (pH 7.0) onto a quartz crystal interface was investigated in situ using an electrode-separated piezoelectric sensor (ESPS). Increasing amounts of lysozyme adsorbed onto a quartz crystal surface resulted in linearly decreasing oscillating frequencies of the ESPS. The lysozyme adsorbed is partially desorbed after rinsing with the phosphate-buffered saline. The amounts adsorbed reversibly and irreversibly were estimated. The influence of the surface roughness of the quartz crystal on the adsorption density measurement is discussed. © 1998 Academic Press
INTRODUCTION Since the 1980s, the quartz crystal microbalance (QCM) has been extensively employed in the chemistry of solutions (1–3). Usually, a QCM comprises a thin vibrating AT-cut quartz crystal disc sandwiched between two metal film electrodes that provide an alternating electric field, which induces a shear vibration of the quartz crystal with resonant frequency in the megahertz region. Recently, a new type of electrode-separated piezoelectric sensor (ESPS for short) has been reported (4 –7). In the ESPS, the excitation electrode is separated from the quartz disc by a liquid layer. The high-frequency alternating electric field is applied to the quartz crystal disc by the conductance of the liquid layer. This configuration offers the advantage of long life of the ESPS. In this paper, an ESPS is applied to monitor the adsorption of a protein onto the quartz surface. Proteins have an amphiphilic nature and they therefore have a strong tendency to adsorb at interfaces. The adsorption of protein at solid surfaces constitutes a research area which receives increasing attention. This interest often originates from the importance of the interfacial behavior of proteins in a variety of applications in medicine, biotechnology, diagnostics, and food technology. Lysozyme is a globular protein with lytic and bactericidal activity. It is charged positively in solutions with pH lower than its isoelectric point. The isoelectric point of lysozyme is around 11. In solutions of pH , 11, the cationic lysozyme will adsorb onto negatively charged surfaces. However, adsorption of lysozyme onto hydrophilic silicon oxide surface (8), onto silica, and onto methylated silica (9) has been investigated by ellipsometry, which needs an expensive instrumentation. In this paper, the adsorption of lysozyme onto a quartz surface was monitored in situ using the ESPS. The rationale of the ESPS method is simple. In the ESPS, the bare quartz 1
To whom correspondence should be addressed. 1 0026-265X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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SHEN ET AL.
surface is in a direct contact with the liquid phase, which offers convenience in investigating the mass change on the surface of the quartz crystal. According to Fuerstenau (10), the quartz surface is charged negatively in solution at pH .2. When lysozyme was adsorbed on the quartz crystal surface in the ESPS, the mass load of the oscillating quartz crystal increased, which caused a decrease in the oscillation frequency. The adsorption mass of lysozyme onto quartz surface is monitored in situ from the frequency shift. According to the Sauerbrey equation (11), the adsorption density can be calculated from the frequency decrease in the adsorption process, Г 5 Dm/A 5 2DF/2.26 3 1026 F 2o,
(1)
where Г is the adsorption density in g.cm22, Dm is the mass of lysozyme adsorbed onto quartz surface and A is the geometric area of the quartz crystal. DF is the frequency shift in Hz and Fo the fundamental frequency of the quartz crystal in Hz. MATERIALS AND METHODS The construction of the ESPS used in this paper was similar to that described in Ref. (12). An AT-cut 9-MHz piezoelectric quartz crystal (diameter 12.5 mm with silver electrodes) was purchased from Beijing Manufactory No. 707. One of the silver electrodes on the quartz disc was dissolved in aqua regia. The bare quartz surface was polished with fine abrasive paper and washed with acetone, 10% HF (for 2 min), and water. The quartz disc was attached to a detection cell made of Teflon with silicone resin. The bare quartz side was in contact with the solution. A platinum electrode with diameter 12 mm was used as a separated electrode. The distance between the separated electrode and the quartz crystal disc was ca. 10 mm. The ESPS served as the feedback network of a home-made TTL oscillator. A universal frequency counter (7201, Shijiazhuang Radio Factory No. 4) was used to record the oscillating frequency of the ESPS. A conductivity meter (DDS11A, Shanghai Analytical Instrument Manufactory) was employed to measure the solution conductivity. The experiments were performed at a room temperature of 25 6 1°C. Lysozyme (from chicken egg white) was purchased from Sigma Chemical Company. The water used was passed through an ion exchanger and then distilled twice in a quartz still. All chemicals were of analytical grade. Phosphate-buffered saline (PBS, pH 7.0, 0.01 mol.121 phosphate and 0.15 mol.121 NaCl) was employed. The stock solutions of lysozyme were prepared with the PBS. The conductivity of the stock solutions was carefully adjusted to be the same as that of the PBS by adding NaCl or water. Before the determination, the quartz crystal surface was cleaned carefully with NH4OH: H2O2:H2O (1:1:5, v:v:v) followed by HCl:H2O2:H2O (1:1:5, v:v:v), then rinsed repeatedly with water and acetone. Next, 40 ml of the PBS was added into the detection cell of the ESPS. Under mild stirring, the stable oscillating frequency, F0, was recorded. Then an accurate amount of lysozyme solution was added into the cell with a microsyringe. The oscillation frequency, F1, was recorded with a time window of 10 s. After an adsorption time of 20 min, the decrease in oscillation frequency, DF1 5 F1 2 F0, was used for the calculation of adsorption mass according to Eq. (1). Then the lysozyme solution was replaced by the PBS and the stable oscillation frequency, F2 was recorded. The increase in frequency, DF2 5 F2 2 F1, was used to calculate the desorption mass of lysozyme
PIEZOELECTRIC MONITORING OF ADSORPTION
3
rising by the PBS. Each experiment was carried out three times and the average value was reported. RESULTS AND DISCUSSION Influence of Solution Properties on the Adsorption Experiment in the ESPS Method As shown in Eq. (1), the adsorption density is in linear correlation with the frequency decrease in adsorption process. The adsorption mass can be monitored in situ by the oscillating frequency of the ESPS. It should be emphasized that the frequency shift must be due only to the mass load variation on the quartz crystal surface. However, the oscillating frequency of the ESPS is also related to the conductivity, permitivity, viscosity, and density of the bulk liquid (6, 7). Clearly, we must be certain that, under our conditions, the change in frequency can be ascribed to the change in surface mass load. In dilute lysozyme solutions, the permittivity, viscosity, and density of the solutions remain the same as those of the PBS. So their influence on the measurement is negligible. In this paper, the variation in conductivity arising from the change in lysozyme concentration was eliminated by using a stock solution of lysozyme with the same conductivity as that of the PBS. Thus the conductivity of the test solutions is not affected by the concentration of lysozyme. Furthermore, the PBS with high background conductivity was chosen. As discussed previously (7), the oscillating frequency of the ESPS is insensitive to variation in conductivity in the case of high background conductivity. Hence, the influence of conductivity on the determination of adsorption density may also be neglected under experimental conditions chosen. The frequency shift in the adsorption experiment was assumed to be from the variation in mass load and used to calculate the adsorption density according to Eq. (1). In addition, high background conductivity strengthens the oscillation ability of the ESPS (13). In fact, the behavior of an ESPS in a highly conductive solution is close to that of a normal piezoelectric quartz crystal with one side facing the liquid phase (6). Influence of Surface Roughness on Adsorption Density in the ESPS Method In the ESPS method, the adsorption density was calculated using the frequency shift according to the Sauerbrey equation. It should be pointed out that the geometric area instead of the true surface area of the quartz crystal disc is used in Eq. (1). Because of surface roughness, the true surface area of the quartz crystal disc is greater than its geometric area. As a result, the adsorption density in the ESPS method (ГE) will be greater than that true adsorption density (ГT). Hence, a calibration coefficient is needed to obtain the true adsorption density in the ESPS method. Physically, the calibration efficient is the ratio of the geometric area to the true surface area of the quartz crystal. Because of the difficulty in the measurement of true surface area of the quartz crystal disc, the calibration coefficient was obtained by comparing the adsorption densities of butyl-rhodamine B in the ESPS method and in a solution-depletion method. Butyl-rhodamine B is used, as its cation can adsorb onto the negatively charged quartz surface. In the ESPS method, the density of butyl-rhodamine B adsorption on the quartz surface is estimated from the frequency shift according to Eq. (1). In the solution-depletion method, finely ground quartz powder with size ca. 5–20 mm was prepared by crushing
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FIG. 1. Adsorption isotherm of butyl-rhodamine B in PBS (pH 7.0, 0.01 phosphate, and 0.15 mol.121 NaCL): (1) on quartz crystal by ESPS method; (2) on quartz powder by solution depletion method.
large, hand-picked Brazilian quartz crystals according to the method in Ref. (14). The specific surface of the fine quartz powder was determined by the BET krypton adsorption method to be 0.48 m2.g21. Adsorption measurements were made by agitating 2.5 g fine quartz powder with a 50-ml butyl-rhodamine B solution prepared by the PBS in polyethylene bottle until equilibration of the system was reached. After centrifugation, the concentration of butyl-rhodamine B in the clear supernatant was determined by a fluorometric method. Adsorption densities were evaluated from the decrease in the concentration of butyl-rhodamine B after agitation with quartz powder. Then an accurate amount of butyl-rhodamine B was added in the equilibrium system and the total adsorption density was calculated in the new equilibration concentration of butyl-rhodamine B solution. Figure 1 shows the adsorption densities obtained by the ESPS method (ГE in curve 1) and the solution-depletion method (ГT in curve 2). In curve 1 the initial concentration was used as the equilibrium solution concentration, as the amount of butyl-rhodamine B adsorbed on quartz crystal surface is much less than the total amount. It can be seen that the values of ГE are greater than those of ГE in the same equilibrium solution concentration. A regression relation between the ГE and ГT in the same equilibrium solution concentration of butyl-rhodamine B was obtained: Г E 5 1.73Г T 1 0.012 ~n 5 10, r 5 0.991).
(2)
The good linearity of the regression relation between ГE and ГT reveals that the difference in the adsorption densities in the two methods is mainly resulted from the
PIEZOELECTRIC MONITORING OF ADSORPTION
5
FIG. 2. Frequency shifts of ESPS in the adsorption process of 4 mg/ml of lysozyme.
surface roughness of the quartz crystal. Accordingly, a calibration coefficient of k 5 1/1.73 was used to correct the influence of the surface roughness on the determination of adsorption density in the ESPS method under our experimental conditions G T 5 kG E 5 2 DF/1.73 3 2.26 3 10 26 F 2o
(3)
Determination of Lysozyme Absorbed onto the Quartz by ESPS As shown in Fig. 2, the oscillation frequency of the ESPS decreases upon the addition of lysozyme. As discussed above, the decrease in the frequency is due to the increase in mass load by the adsorption of cationic lysozyme on quartz crystal. According to the frequency decrease value, the adsorption density can be monitored in situ by the ESPS method. In Fig. 2, the adsorption density was calculated according to Eq. (3). It can be seen that the adsorption rate decreases with adsorption time. After an adsorption time of 20 min, the increase in adsorption density was small. Part of the lysozyme adsorbed onto the quartz crystal surface is desorbed upon rinsing with the PBS. According to the increase in the frequency, the amount of lysozyme desorbed after rinsing can be estimated (Г3), which may be considered as the part that is reversibly adsorbed. By subtracting the reversible part from the total adsorption density before rinsing (Г1), the mass of lysozyme adsorbed irreversibly on quartz surface was obtained (Г2 5 Г1 2 Г3). Adsorption Density of Lysozyme on the Quartz Crystal Surface Figure 3 shows the densities of lysozyme adsorption onto the quartz crystal surface. Curves 1 shows the total adsorption density in 20 min without rinsing (Г1). Curve 2
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FIG. 3. Adsorption density of lysozyme as a function of solution concentration of lysozyme: (1) total adsorption density before rinsing; (2) adsorption density after rinsing; (3) desorption density by rinsing.
represents the adsorption density after rinsing (Г2), which may be considered as the part of the lysozyme adsorbed irreversibly. Curves 3 gives the desorption densities after the rinsing (Г3), i.e., the part of reversible adsorption for lysozyme. It can be seen that the values of Г1, Г2, and Г3 increase with increasing concentration of lysozyme. After a concentration of 2 mg/ml is attained, there is a slight increase in Г3; the slopes of curves 1 and 2 decrease at higher concentration of lysozyme. The dependence of adsorption density on solution concentration of lysozyme is explained below. Lysozyme is a globular protein of slightly ellipsoidal shape with the dimensions of 45 3 30 3 30 Å. Its molar mass is approximately 14,000 (15). The maximal adsorption densities are 0.207 and 0.310 mg.cm22 for the hexagonal packing of monolayer molecules adsorbed side-on and end-on, respectively. The adsorption densities in Fig. 3 reveal bilayer adsorption for lysozyme. In the case of low surface coverage, electrostatic attraction plays a major role in governing adsorption. The lysozyme adsorbed by electrostatic attraction can easily be desorbed by rinsing with PBS. As the surface coverage increases, the original negative charge on quartz surface is neutralized by the adsorption of cationic lysozyme and eventually reversed. When the surface is positively charged, the adsorption of lysozyme cation must overcome electrostatic repulsion between the oncoming lysozyme and the surface. Hence, the slope of curve 1 reduces in higher adsorption density. The increase in adsorption density is mainly due to the interaction of the hydrophobic chains of oncoming lysozyme cations with those of previously adsorbed
PIEZOELECTRIC MONITORING OF ADSORPTION
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lysozyme. Hence, the irreversibly adsorbed amount increases while the reversibly adsorbed part increases little after a lysozyme concentration of 2 mg/ml. ACKNOWLEDGMENT This work is supported by the National Science Foundation of China and the Go Beyond the Century Foundation of Shandong University.
REFERENCES 1. Thompson, M., Kipling, A. L., Duncan-Hewitt, W. C., Rajakovic´, L., Cˇavic´-Vlasak, B. A. Analyst, 1991, 116, 881. 2. Minunni, M., Mascini, M., Guilbault, G. G., Hock, B. Anal. Lett., 1995, 28, 49. 3. Buttry, D. A., Word, M. D. Chem. Rev., 1992, 92, 1355. 4. Nomura, T., Tanaka, F., Yamada, T., Itoh, H. Anal. Chim. Acta, 1991, 243, 273. 5. Shen, D. Z., Nie, L. H., Yao, S. Z. J. Electroanal. Chem., 1993, 360, 71. 6. Shen, D. Z., Lin, S., Kang, Q., Nie, L. H., Yao, S. Z. Analyst, 1993, 118, 1143. 7. Shen, D. Z., Nie, L. H., Yao, S. Z. J. Electroanal. Chem., 1994, 371, 117. 8. Bos, M. A., Kleijn, J. M. Biophys. J., 1995, 68, 2566. 9. Bos, M. A., Werkhoven, T. M., Kelijn, J. M. Langmuir, 1996, 12, 3980. 10. Fuerstenau, D. W. J. Phys. Chem., 1956, 60, 981. 11. Sauerbrey, G. Z. Phys., 1959, 155, 206. 12. Shen, D. Z., Kang, Q., Zhang, X. L., Wang, L. Z., Ma, C. S. Anal. Commun., 1997, 34, 97. 13. Shen, D. Z., Kang, Q., Yao, S. Z. Chem. Res. Chin. Univ. 1996, 12, 22. 14. Li, H. C., De Bruyn, P. L. Surf. Sci. 1966, 5, 203. 15. Imoto, T., Johnson, L. N., North, A. C. T., Phillips, D. C., Rupley, J. A. in ‘‘The Enzyme’’ (P. D. Boyer, Ed.), p. 665. Academic Press, New York, 1972.
60, 1–7 (1998)
MJ981620
In Situ Monitoring of Adsorption of Lysozyme onto Quartz Using an Electrode-Separated Piezoelectric Sensor D. Z. Shen,*,1 Y. H. Xue,† Q. Kang,† Q. J. Xie,‡ and L. X. Chen* *Chemistry College, Shandong University, Jinan 250100, People’s Republic of China; †Shandong Institute of Mining and Technology, Taian 270019, People’s Republic of China; and ‡Chemistry Department, Hunan Normal University, Changsha 410081, People’s Republic of China Received January 12, 1997; accepted March 28, 1998 The adsorption of lysozyme from phosphate-buffered saline (pH 7.0) onto a quartz crystal interface was investigated in situ using an electrode-separated piezoelectric sensor (ESPS). Increasing amounts of lysozyme adsorbed onto a quartz crystal surface resulted in linearly decreasing oscillating frequencies of the ESPS. The lysozyme adsorbed is partially desorbed after rinsing with the phosphate-buffered saline. The amounts adsorbed reversibly and irreversibly were estimated. The influence of the surface roughness of the quartz crystal on the adsorption density measurement is discussed. © 1998 Academic Press
INTRODUCTION Since the 1980s, the quartz crystal microbalance (QCM) has been extensively employed in the chemistry of solutions (1–3). Usually, a QCM comprises a thin vibrating AT-cut quartz crystal disc sandwiched between two metal film electrodes that provide an alternating electric field, which induces a shear vibration of the quartz crystal with resonant frequency in the megahertz region. Recently, a new type of electrode-separated piezoelectric sensor (ESPS for short) has been reported (4 –7). In the ESPS, the excitation electrode is separated from the quartz disc by a liquid layer. The high-frequency alternating electric field is applied to the quartz crystal disc by the conductance of the liquid layer. This configuration offers the advantage of long life of the ESPS. In this paper, an ESPS is applied to monitor the adsorption of a protein onto the quartz surface. Proteins have an amphiphilic nature and they therefore have a strong tendency to adsorb at interfaces. The adsorption of protein at solid surfaces constitutes a research area which receives increasing attention. This interest often originates from the importance of the interfacial behavior of proteins in a variety of applications in medicine, biotechnology, diagnostics, and food technology. Lysozyme is a globular protein with lytic and bactericidal activity. It is charged positively in solutions with pH lower than its isoelectric point. The isoelectric point of lysozyme is around 11. In solutions of pH , 11, the cationic lysozyme will adsorb onto negatively charged surfaces. However, adsorption of lysozyme onto hydrophilic silicon oxide surface (8), onto silica, and onto methylated silica (9) has been investigated by ellipsometry, which needs an expensive instrumentation. In this paper, the adsorption of lysozyme onto a quartz surface was monitored in situ using the ESPS. The rationale of the ESPS method is simple. In the ESPS, the bare quartz 1
To whom correspondence should be addressed. 1 0026-265X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
2
SHEN ET AL.
surface is in a direct contact with the liquid phase, which offers convenience in investigating the mass change on the surface of the quartz crystal. According to Fuerstenau (10), the quartz surface is charged negatively in solution at pH .2. When lysozyme was adsorbed on the quartz crystal surface in the ESPS, the mass load of the oscillating quartz crystal increased, which caused a decrease in the oscillation frequency. The adsorption mass of lysozyme onto quartz surface is monitored in situ from the frequency shift. According to the Sauerbrey equation (11), the adsorption density can be calculated from the frequency decrease in the adsorption process, Г 5 Dm/A 5 2DF/2.26 3 1026 F 2o,
(1)
where Г is the adsorption density in g.cm22, Dm is the mass of lysozyme adsorbed onto quartz surface and A is the geometric area of the quartz crystal. DF is the frequency shift in Hz and Fo the fundamental frequency of the quartz crystal in Hz. MATERIALS AND METHODS The construction of the ESPS used in this paper was similar to that described in Ref. (12). An AT-cut 9-MHz piezoelectric quartz crystal (diameter 12.5 mm with silver electrodes) was purchased from Beijing Manufactory No. 707. One of the silver electrodes on the quartz disc was dissolved in aqua regia. The bare quartz surface was polished with fine abrasive paper and washed with acetone, 10% HF (for 2 min), and water. The quartz disc was attached to a detection cell made of Teflon with silicone resin. The bare quartz side was in contact with the solution. A platinum electrode with diameter 12 mm was used as a separated electrode. The distance between the separated electrode and the quartz crystal disc was ca. 10 mm. The ESPS served as the feedback network of a home-made TTL oscillator. A universal frequency counter (7201, Shijiazhuang Radio Factory No. 4) was used to record the oscillating frequency of the ESPS. A conductivity meter (DDS11A, Shanghai Analytical Instrument Manufactory) was employed to measure the solution conductivity. The experiments were performed at a room temperature of 25 6 1°C. Lysozyme (from chicken egg white) was purchased from Sigma Chemical Company. The water used was passed through an ion exchanger and then distilled twice in a quartz still. All chemicals were of analytical grade. Phosphate-buffered saline (PBS, pH 7.0, 0.01 mol.121 phosphate and 0.15 mol.121 NaCl) was employed. The stock solutions of lysozyme were prepared with the PBS. The conductivity of the stock solutions was carefully adjusted to be the same as that of the PBS by adding NaCl or water. Before the determination, the quartz crystal surface was cleaned carefully with NH4OH: H2O2:H2O (1:1:5, v:v:v) followed by HCl:H2O2:H2O (1:1:5, v:v:v), then rinsed repeatedly with water and acetone. Next, 40 ml of the PBS was added into the detection cell of the ESPS. Under mild stirring, the stable oscillating frequency, F0, was recorded. Then an accurate amount of lysozyme solution was added into the cell with a microsyringe. The oscillation frequency, F1, was recorded with a time window of 10 s. After an adsorption time of 20 min, the decrease in oscillation frequency, DF1 5 F1 2 F0, was used for the calculation of adsorption mass according to Eq. (1). Then the lysozyme solution was replaced by the PBS and the stable oscillation frequency, F2 was recorded. The increase in frequency, DF2 5 F2 2 F1, was used to calculate the desorption mass of lysozyme
PIEZOELECTRIC MONITORING OF ADSORPTION
3
rising by the PBS. Each experiment was carried out three times and the average value was reported. RESULTS AND DISCUSSION Influence of Solution Properties on the Adsorption Experiment in the ESPS Method As shown in Eq. (1), the adsorption density is in linear correlation with the frequency decrease in adsorption process. The adsorption mass can be monitored in situ by the oscillating frequency of the ESPS. It should be emphasized that the frequency shift must be due only to the mass load variation on the quartz crystal surface. However, the oscillating frequency of the ESPS is also related to the conductivity, permitivity, viscosity, and density of the bulk liquid (6, 7). Clearly, we must be certain that, under our conditions, the change in frequency can be ascribed to the change in surface mass load. In dilute lysozyme solutions, the permittivity, viscosity, and density of the solutions remain the same as those of the PBS. So their influence on the measurement is negligible. In this paper, the variation in conductivity arising from the change in lysozyme concentration was eliminated by using a stock solution of lysozyme with the same conductivity as that of the PBS. Thus the conductivity of the test solutions is not affected by the concentration of lysozyme. Furthermore, the PBS with high background conductivity was chosen. As discussed previously (7), the oscillating frequency of the ESPS is insensitive to variation in conductivity in the case of high background conductivity. Hence, the influence of conductivity on the determination of adsorption density may also be neglected under experimental conditions chosen. The frequency shift in the adsorption experiment was assumed to be from the variation in mass load and used to calculate the adsorption density according to Eq. (1). In addition, high background conductivity strengthens the oscillation ability of the ESPS (13). In fact, the behavior of an ESPS in a highly conductive solution is close to that of a normal piezoelectric quartz crystal with one side facing the liquid phase (6). Influence of Surface Roughness on Adsorption Density in the ESPS Method In the ESPS method, the adsorption density was calculated using the frequency shift according to the Sauerbrey equation. It should be pointed out that the geometric area instead of the true surface area of the quartz crystal disc is used in Eq. (1). Because of surface roughness, the true surface area of the quartz crystal disc is greater than its geometric area. As a result, the adsorption density in the ESPS method (ГE) will be greater than that true adsorption density (ГT). Hence, a calibration coefficient is needed to obtain the true adsorption density in the ESPS method. Physically, the calibration efficient is the ratio of the geometric area to the true surface area of the quartz crystal. Because of the difficulty in the measurement of true surface area of the quartz crystal disc, the calibration coefficient was obtained by comparing the adsorption densities of butyl-rhodamine B in the ESPS method and in a solution-depletion method. Butyl-rhodamine B is used, as its cation can adsorb onto the negatively charged quartz surface. In the ESPS method, the density of butyl-rhodamine B adsorption on the quartz surface is estimated from the frequency shift according to Eq. (1). In the solution-depletion method, finely ground quartz powder with size ca. 5–20 mm was prepared by crushing
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FIG. 1. Adsorption isotherm of butyl-rhodamine B in PBS (pH 7.0, 0.01 phosphate, and 0.15 mol.121 NaCL): (1) on quartz crystal by ESPS method; (2) on quartz powder by solution depletion method.
large, hand-picked Brazilian quartz crystals according to the method in Ref. (14). The specific surface of the fine quartz powder was determined by the BET krypton adsorption method to be 0.48 m2.g21. Adsorption measurements were made by agitating 2.5 g fine quartz powder with a 50-ml butyl-rhodamine B solution prepared by the PBS in polyethylene bottle until equilibration of the system was reached. After centrifugation, the concentration of butyl-rhodamine B in the clear supernatant was determined by a fluorometric method. Adsorption densities were evaluated from the decrease in the concentration of butyl-rhodamine B after agitation with quartz powder. Then an accurate amount of butyl-rhodamine B was added in the equilibrium system and the total adsorption density was calculated in the new equilibration concentration of butyl-rhodamine B solution. Figure 1 shows the adsorption densities obtained by the ESPS method (ГE in curve 1) and the solution-depletion method (ГT in curve 2). In curve 1 the initial concentration was used as the equilibrium solution concentration, as the amount of butyl-rhodamine B adsorbed on quartz crystal surface is much less than the total amount. It can be seen that the values of ГE are greater than those of ГE in the same equilibrium solution concentration. A regression relation between the ГE and ГT in the same equilibrium solution concentration of butyl-rhodamine B was obtained: Г E 5 1.73Г T 1 0.012 ~n 5 10, r 5 0.991).
(2)
The good linearity of the regression relation between ГE and ГT reveals that the difference in the adsorption densities in the two methods is mainly resulted from the
PIEZOELECTRIC MONITORING OF ADSORPTION
5
FIG. 2. Frequency shifts of ESPS in the adsorption process of 4 mg/ml of lysozyme.
surface roughness of the quartz crystal. Accordingly, a calibration coefficient of k 5 1/1.73 was used to correct the influence of the surface roughness on the determination of adsorption density in the ESPS method under our experimental conditions G T 5 kG E 5 2 DF/1.73 3 2.26 3 10 26 F 2o
(3)
Determination of Lysozyme Absorbed onto the Quartz by ESPS As shown in Fig. 2, the oscillation frequency of the ESPS decreases upon the addition of lysozyme. As discussed above, the decrease in the frequency is due to the increase in mass load by the adsorption of cationic lysozyme on quartz crystal. According to the frequency decrease value, the adsorption density can be monitored in situ by the ESPS method. In Fig. 2, the adsorption density was calculated according to Eq. (3). It can be seen that the adsorption rate decreases with adsorption time. After an adsorption time of 20 min, the increase in adsorption density was small. Part of the lysozyme adsorbed onto the quartz crystal surface is desorbed upon rinsing with the PBS. According to the increase in the frequency, the amount of lysozyme desorbed after rinsing can be estimated (Г3), which may be considered as the part that is reversibly adsorbed. By subtracting the reversible part from the total adsorption density before rinsing (Г1), the mass of lysozyme adsorbed irreversibly on quartz surface was obtained (Г2 5 Г1 2 Г3). Adsorption Density of Lysozyme on the Quartz Crystal Surface Figure 3 shows the densities of lysozyme adsorption onto the quartz crystal surface. Curves 1 shows the total adsorption density in 20 min without rinsing (Г1). Curve 2
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FIG. 3. Adsorption density of lysozyme as a function of solution concentration of lysozyme: (1) total adsorption density before rinsing; (2) adsorption density after rinsing; (3) desorption density by rinsing.
represents the adsorption density after rinsing (Г2), which may be considered as the part of the lysozyme adsorbed irreversibly. Curves 3 gives the desorption densities after the rinsing (Г3), i.e., the part of reversible adsorption for lysozyme. It can be seen that the values of Г1, Г2, and Г3 increase with increasing concentration of lysozyme. After a concentration of 2 mg/ml is attained, there is a slight increase in Г3; the slopes of curves 1 and 2 decrease at higher concentration of lysozyme. The dependence of adsorption density on solution concentration of lysozyme is explained below. Lysozyme is a globular protein of slightly ellipsoidal shape with the dimensions of 45 3 30 3 30 Å. Its molar mass is approximately 14,000 (15). The maximal adsorption densities are 0.207 and 0.310 mg.cm22 for the hexagonal packing of monolayer molecules adsorbed side-on and end-on, respectively. The adsorption densities in Fig. 3 reveal bilayer adsorption for lysozyme. In the case of low surface coverage, electrostatic attraction plays a major role in governing adsorption. The lysozyme adsorbed by electrostatic attraction can easily be desorbed by rinsing with PBS. As the surface coverage increases, the original negative charge on quartz surface is neutralized by the adsorption of cationic lysozyme and eventually reversed. When the surface is positively charged, the adsorption of lysozyme cation must overcome electrostatic repulsion between the oncoming lysozyme and the surface. Hence, the slope of curve 1 reduces in higher adsorption density. The increase in adsorption density is mainly due to the interaction of the hydrophobic chains of oncoming lysozyme cations with those of previously adsorbed
PIEZOELECTRIC MONITORING OF ADSORPTION
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lysozyme. Hence, the irreversibly adsorbed amount increases while the reversibly adsorbed part increases little after a lysozyme concentration of 2 mg/ml. ACKNOWLEDGMENT This work is supported by the National Science Foundation of China and the Go Beyond the Century Foundation of Shandong University.
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