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Anal. Chem. 1987,59,2760-2763
Piezoelectric Crystal Biosensor Modified with Protein A for Determination of Immunoglobulins Hiroshi Muramatsu Research and Development Department, Seiko Instruments Inc., Takatsuka-sinden, Matsudoshi, Chiba 271,Japan J o n a t h a n M. Dicks Biotechnology Centre, Cranfield Institute of Technology, Cranfield, Bedford MK43 OAL, United Kingdom Eiichi Tamiya and Isao Karube* Research Laboratory of Resources Utilization, Tok.yo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama 227, Japan
Determlnatlon of lmmunoglobullns was performed by uslng a piezoelectric crystal bhensor system. AT-cut crystals, wlth a baslc resonant frequency of 0 YHr, were modlfled by Immobillring Protein A onto the surface of crystal electrodes wlth (y-am1nopropyl)trlethoxysllane. The crystal was positloned lnslde a thennostated How cell and measurement of the resonant frequency was performed In constantly flowlng delonlzed/dlstilled water. The resonant frequency shlft resultlng from the afflnlty reactlon of Protein A and human IgG correlated wlth a concentration of human IgG In the range 10a-10-2 mg-mL-'. The analysis pattern of mouse IgG subclasses, obtalned by uslng a stepped gradient of buffer solution, ldentlfied three dlstlnctlve peaks of IgG,, IgG,,, and IgG2b.
It is well-known that the resonant frequency of an oscillating piezoelectric crystal can be affected by a change in mass at the crystal surface. This property has been applied to monitoring vapor deposition rates and for the chromatographic detection of gasses. It has also been reported that modification of the crystal surface with organic compounds or enzymes that will bind a particular gaseous substrate can provide enhanced detection specificity (1-3). The properties of piezoelectric devices immersed in water or organic solvents have recently been studied by various authors and several theoretical equations governing their behavior in liquids have been derived (4-6). Methods employing a coupled gravimetric/electrochemical assay have also been used for the determination of electrodeposited metal ions and anions (5, 7) and have been used for electrochemical analysis (8-10). The established immunoassay techniques of radioimmunoassay (RIA), fluoroimmunoassay (FIA), enzyme immunoassay (EIA), and latex immunoassay have been widely applied. They still suffer, however, from the drawbacks of complex, time-consuming procedures and potentially hazardous or expensive materials. Surface acoustic wave (SAW) devices used for microgravimetric immunoassay of human IgG have been reported (11), as well as AT-cut piezoelectric crystals modified with antihuman IgG, which exhibit a change in resonant frequency on binding to human IgG, but not IgA (12). A previous report by our laboratory also showed that a piezoimmuno device modified with anti-Candida antibody could be successfully applied to the detection of a pathogenic microbe (13). The separation and purification of the various IgG subclasses can be achieved by an affinity chromatographic method 0003-2700/87/0359-2760$0 1.50/0
employing a Protein A-sepharose column (14,15). It was our wish, therefore, to apply the piezoelectric detection system to the analysis of IgG subclasses, by monitoring the preferential binding affinity of each subclass to immobilized Protein A, in a stepped gradient of buffer solution. The system therefore would function in a similar manner to the chromatographic method but would require a considerably reduced sample volume. In this paper, the response of AT-cut piezoelectric crystals was studied under various operational conditions and was also applied to the determination of human IgG concentration. EXPERIMENTAL SECTION Apparatus and Materials. Affinity purified Protein A was obtained from UCB-Bioproducts, S.A. Human IgG and human ?-globulin were obtained from Miles Scientific. Mouse y-globulin and human albumin were obtained from Cooper Biomedical, Inc. (Cappel). Piezoelectric crystals used in this work were AT cut with a basic resonant frequency of 9 MHz. An Ag electrode was vapor deposited onto the crystal surface and further coated with a Pd layer by electrochemical plating. Prior to modification the electrodes were anodically oxidized at constant current (4 mA.cm-2) in 0.5 M NaOH (13). The crystal electrodes were first modified with (y-aminopropy1)triethoxysilane (7-APTES, 5% in acetone) for 1h, 25 "C. They were then air-dried and placed into glutaraldehyde solution (GA, 5 % , pH 7 ) for 3 h. Protein A (1mg.mL-l, pH 7 , 0.05 M phosphate buffer) was immobilized onto the electrode via the surface aldehyde and, after immobilization (1h), the remaining unreacted aldehyde was blocked with 0.1 M glycine (10 mL of stirred solution) (13). The piezoelectric crystal was positioned inside a Perspex flow cell leaving the entire surface of the crystal exposed within the cell (1-mL volume). An oscillator circuit was constructed from a transistor-transistor logic integrated circuit (TTL-IC),and the crystal frequency was monitored with a universal counter (Iwatsu SC-7201). A microcomputer (Nippon Electric Co. 9801E) was used to record resonant frequency data, to control the flow rate and flow direction of the peristalic pump (Atto Co. Ltd. AC-2120), and to operate miniature solenoid valves (The Lee Co. 121618H) during the measurement or rinsing steps. The cell was enclosed in a constant-temperature water jacket and either deionized/ distilled water, glycine-HC1 buffer (pH 2.4, 0.1 M), or NaCl solution (0.5 M) was pumped into the cell by selectively controlling the state of the solenoid valves. Samples and other test solutions were injected into the cell through a separate injection port (Figure 1). Procedure. Measurement of Human IgG Concentration. The
Protein A modified piezoelectric crystal was placed inside the flow cell and first rinsed with glycine-HC1 buffer (pH 2.4, 0.1 M) t o remove any adsorbed substances. The cell was then washed with doionized/distilled water and the resonant frequency was mon8 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 8926550
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Flgure 1. Schematic diagram of experimental system: (A) piezoe-
lectric crystal in the cell; (B) oscillator: (C) frequency counter: (D) miaocomputer; (E)peristaiic pump; (F) solenoid valves; (0)thermostatic bath; (H) distllled water supply; (I) glycine-HCI buffer supply: (J) 0.5 M NaCi supply: (K) sample injection port.
itored at a constant flow rate (0.7 rnl-min-') and temperature (30 "C). The steady resonant frequency (FJ was obtained after 5-10 min. The water was drained off and solutions of human IgG (1 mL, 10-1-104 mgmL-') in phosphate buffer (pH 7,0.05 M) were then injected into the cell. After incubating for various periods of time, the crystal was rinsed with 0.5 M NaCl solution to remove any nonspecifically adsorbed IgG, followed by remeasurement of the steady resonant frequency (F,) in constantly flowing deionized/distilled water (0.7 mL-min-'). IgG bound to Protein A was removed with glycine-HC1 buffer (pH 2.4,O.l M), allowing the successive measurement of different human IgG concentrations. Human albumin solution was also analyzed by the same procedure. Determination of ZgG Subclasses. Mouse y-globulin (pH 8.0, 0.2 M) or human y-globulin (pH 7.0, 0.05 M) solutions (1 mL, 0.1 mgmL-') were injected into the cell and the steady resonant frequencies,Fl and F2,were measured as above. The crystal was then rinsed with a stepped gradient of phosphate-citric acid buffer (0.1 M) from pH 7 to pH 2.5, each step in pH being 0.5 pH units. After the cell was rinsed at each step, the steady resonant frewas remeasured in flowing water. quency (FZpH) In addition to these operations, measurement of the resonant frequency was performed in air, ethanol, NaCl solution, and deionized/distilled water at various temperatures, to analyze the characteristics of the sensor system.
RESULTS AND DISCUSSION Resonant Frequency Response of Piezoelectric Crystals in Various Liquids. The resonant frequency shift of piezoelectric crystals in various liquids has been previously studied by several authors. Nomura and Minemura derived an equation, which related the conductivity and density of the liquid, based on their experimental results compared with an equivalent circuit of a piezoelectric crystal (5). Nomura and Okuhara derived an equation that related the viscosity and density of the liquid from experimental results in various organic solvents (6). Kanazawa and Gordon suggested an equation that related the viscosity and density of the liquid. This was obtained from a physical model of shear waves and damped shear waves setup in the quartz crystal and fluid, respectively, during resonation, as follows ( 4 ) : where fo is the basic resonant frequency of the crystal, TL and pL are the absolute viscosity and density of the liquid, respectively, qQ and p~ are the elastic modules and density of the quartz, respectively. It is clear that the resonant frequency is affected by the density, viscosity, temperature, and conductivity of the liquid, but as they stand, each of the equations described above cannot predict all these factors. Indeed, the magnitude of each parameter may possibly be affected by the characteristics of each resonating crystal. These effects were not described and cannot be described by a unification of the above equations.
TlMEhn)
Figure 2. Resonant frequency response in (A) air, (B) ethanol, and (C)
deionized/distiiied water at 30 OC.
891490' 0'
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Figure 3. Resonant frequency response in (A) deionized/distiiled water, (B) 0.5 mM NaCi, and (C) 2.5 mM NaCi at 30 OC.
Figure 2 shows the resonant frequency response of the crystal from the start of oscillation in air, ethanol, and distilled water (1X lo6 Dcm-l). The steady resonant frequency shifts from air to ethanol and from air to water were 11235 and 13848 Hz, respectively. The theoretical results of frequency shift ratio obtained by using eq 1 predicted 1:1.005. The discrepancy between the theoretical and experimental frequency shifts for a crystal in water can be explained as a result of the conductivity or polarity of water, which is not assessed in eq 1. A difference in relaxation curves for ethanol and water is also observed in Figure 2 and a greater time-dependent frequency response was observed in water. Figure 3 shows the resonant frequency response in distilled water and NaCl solutions. Both the oscillation relaxation period and the decrease in resonant frequency a t relaxation is greater for higher NaCl concentrations. The steady resonant frequency is lower in high NaCl concentrations and this value is much lower compared with the difference in density and viscosity of the solution. The effect of temperature on the frequency shift was also measured. The crystal showed a characteristic 30 Hz/OC shift with a change in temperature (Figure 4), indicating the necessity for temperature control during measurement of the resonant frequency. Both the density and viscosity of water and the oscillation of the quartz are affected by a change in temperature; however the relaxation curves did not alter from one temperature to another. Thompson et al. described a time-dependent frequency response of a quartz crystal obtained when using hydrophobic and hydrophilic treatment of the surface (12). They proposed that the frequency response is mediated by interfacial interaction of the crystal/electrode interface with water. The greater time to frequency stabilization for the hydrophilic experiments, compared to the hydrophobic case, was ascribed
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Flgure 6. Relationship between reaction time and steady resonant 1X fre uency shift for human IgG concentrations of 1 X lo-' (0), 10-9 (A),and 1 X (0)mg-mL-'.
Figure 4. Resonant frequency response in deioniredldistilled water at (A) 21.5, (B) 26, (C) 30, (D) 35, and (E) 40 'C.
TIME
Figure 5. Resonant frequency response after the successive (A) rinsing with glycine-HCI buffer (pH 2.4), (B) reaction with human IgG (1 X lo4 mg-mL-'), (C) rinsing with glycine-HCI buffer (pH 2.4), and (D) reaction with human IgG (1 X mgemL-').
to equilibration of the boundary structure. This information, therefore, indicated the possibility that the ionic structure of the crystal/electrode interface to water was concerned with the resonant frequency shift. Furthermore this view can be applied to explain the resonant frequency shift that occurs after protein binding. In our experimental system, in 10 mM NaCl the oscillation was unstable. This was because both side electrodes of the crystal were immersed in the cell and a parallel circuit formed through the highly conductive solution, which electrically influenced the oscillating circuit. Although this limited the oscillation in highly conductive solutions, our system, in which the whole crystal is immersed, has an advantage in that it gives no mechanical stress to the crystal and gives stable oscillations in comparison witn the single-sided immersed type. This is because the crystal and the cell are suppressed within the single-sided immersed type. Determination of H u m a n IgG Concentration. Figure 5 shows typical resonant frequency shifts during the determination of human IgG concentration. Curves A and B show the resonant frequency response before and after reaction of human IgG (1X mgmL-l) with Protein A immobilized onto the piezoelectric crystal surface. The decrease in steady resonant frequency from curve A to B was caused by a quantity of human IgG binding to the Protein A Iayer. Curve C is the frequency response after rinsing the crystal with glycine-HC1 buffer (pH 2.4,O.l M) and curve D is the response curve after reaction with a second sample of human IgG (1 X mgmL-'). These results conclude that the sensor could be used repeatedly, since after washing, the frequency shifts responded proportionally to the human IgG concentration. The sensor was capable of being used approximately 10 times in succession; however, at higher sample concentrations aging of the sensor became more rapid. The relationship between reaction time and the resonant frequency shifts, obtained by using several human IgG sample
CONCENTRATIONhqd?
Figure 7. Correlation between human IgG and the steady resonant and , 15 rnin (A),30 frequency shift after reaction time of 30 min (0) OC.
concentrations, is shown in Figure 6. Figure 6 shows that the progress of the reaction had virtually reached equilibrium after 30 min. Therefore, a reaction time of 30 min was used in all further experiments. Figure 7 shows the log-log plot of human IgG concentration and the resonant frequency shift. A frequency shift was affected only by human IgG concentration and no response was obtained for the addition of human albumin sample solutions. For the case of a 30-min reaction time, linear correlation of log (AF) = 0.40 log (AC) + 3.4 was given in the range 104-10-3 mgmL-'. The resonant frequency shift saturated gradually for human IgG concentrations above mgmL-l. The reason for this occurrence is the binding limit of immobilized Protein A. For the case of a 15-min reaction time, linear correlation of log (AF) = 0.49 log (AC) + 3.2 was given in the range 10-4-10-2 mgmL-l. Consequently, human IgG concentration in the range 104-10-2 mgmL-' can be determined by the system, and higher concentration of human IgG can be determined with shorter reaction time. Estimation of t h e Amount of Immobilized Protein A and Bound Human IgG. In previous experiments, Protein A was immobilized onto the quartz crystal prior to placing it into the flow cell for IgG analysis. For estimation of the amount of immobilized Protein A, however, immobilization was performed inside the flow cell and the steady resonant frequencies before and after binding of Protein A were measured. A solution of Protein A (1mL, l mgmL-') in phosphate buffer (pH 7.0,0.05 M), equivalent to that used in previous immobilizations,was added to the cell and the steady resonant frequency was measured after 1h. Protein A immobilization gave resonant frequency shifts of 180 and 200 Hz for two replicate measurements.
ANALYTICAL CHEMISTRY, VOL. 59, NO. 23, DECEMBER i, 1987
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Flgure 9. Resonant frequency difference of each rinse after reaction with human y-globulin (0.1 mg.mL-').
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Flgure 8. (a) Steady resonant frequency (A) before and (6)after reaction with 0.1 mg-mL-' mouse y-globulln and after rinsing with a stepped gradient of phosphate-citric acid buffer, pH 7-3. (b) Resonant frequency difference of each rinse shown in part a.
As shown in Figure 7, saturation of human IgG occurs at a resonant frequency shift of approximately 250 Hz. Since one molecule of Protein A can bind two molecules of IgG (I6), and the molecular weights of Protein A and IgG are 42 000 and 150000, respectively, the ratio of Protein A for maximum binding IgG should be approximately 1:7.1. The ratio obtained in these experiments was about 1:1.3 on the assumption that the resonant frequency shifts was proportional to the amount of protein. During immobilization it is probable that the location of Protein A active sites are obscured by the orientation of Protein A molecules binding to the crystal. If, therefore, the Protein A molecules are closely packed, then the binding of an IgG molecule will sterically hinder the binding of a second. By use of the equation (I, 2)
AF = -Am/mF
5
PH
I
/
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(2)
where A F is the resonant frequency shift (Hz), Am is the change in mass at the crystal surface (g), m is the crystal mass (g), and F is the basic resonant frequency (Hz), a resonant frequency shift of 1 Hz should correspond to about 1 ng of mass change. As shown in Figure 7, 1 mL of lo4 mgmL-' IgG solution gave 8 Hz of frequency shift and mg.rnL-' IgG solution gave 33 and 22 Hz of frequency shift. These frequency shifts are greater than the value deduced from eq 2. Similar results has been reported for work on determination of gases with coated piezoelectric quartz crystal (17,18).These results could possibly be explained by the interfacial effect of the crystal/electrode interface to water, as described above. Radioactive labeling methods for IgG will clearly reveal that these conclusions are correct. Determination of IgG Subclasses. It was previously reported that Protein A has selective affinity for each IgG
subclass; therefore we set out to examine this property using our system. In Figure 8a, the first and second points indicate the steady resonant frequency before and after the reaction with mouse y-globulin; the other points indicate the steady resonant frequency after rinsing with a stepped gradient of phosphate-citric acid buffer from pH 7 to pH 3. Figure 8b shows the overall resonant frequency shift during each of the separate rinsing steps. The pattern of this figure corresponded to the result that was obtained by affinity chromatography (14).The peaks at pH 6.5, pH 5.5-4.5, and pH 3.5-3.0 indicate the presence of IgG1, IgG2,, and IgG2b, respectively. Figure 9 shows the overall resonant frequency shift during each of the separate rinsing step for the case of bound human y-globulins. The pattern of this figure clearly shows a difference from that of Figure 8b and corresponded to the results previously reported in which IgG2 and IgG, were eluted at pH 4.7 and IgG, and IgG, were eluted at pH 4.3 (15). We have demonstrated that a piezoelectric crystal modified with an immobilized Protein A layer can be effectively applied to the determination of IgG concentrations and the various IgG subclasses. We believed that this system can be applied to various analyses, through the application of protein-protein affinity reactions.
ACKNOWLEDGMENT We thank Koji Sode and Mark E. A. Downs for useful discussion. LITERATURE CITED King, W. H., Jr. Anal. Chem. 1984, 36, 1735-1739. King, W. H.,Jr.; Corbett, L. W. Anal. Chem. 1989, 4 1 , 580-583. Guilbault, G. G. Anal. Chem. 1983, 55, 1682-1684. Kanazawa, K. K.; Gordon, J. G. Anal. Chim. Acta 1985, 775, 99-105. Nomura, T.; Minemura, A. Nippon Kagaku Kaishi 1980, 1621-1625. Nomura, T.; Okuhara, M. Anal. Chim. Acta 1982, 142, 281-284. Nomura. T.; Tsuge, K. Anal. Chim. Acta 1985, 169, 257-262. (8) Bruckenstein, S.; Swathirajan, S. Electrochim. Acta 1985, 3 0 ,
(1) (2) (3) (4) (5) (8) (7)
851-855. (9) Bruckenstein. S.:Shay, M. Electrochim. Acta 1985, 30, 1295-1300. (10) Bruckenstein, S.; Shay, M. J. Nectroanal. Chem. Interfacial Electrochem. 1985, 188, 131-136. (11) Roederer, J. E.; Bastiaans, G. J. Anal. Chem. 1983, 55, 2333-2336. (12) Thompson, M.; Arthur, C. L.; Dhaliwal, G. K. Anal. Chem. 1988, 58, 1206- 1209. (13) Muramatsu, H.; Kajiwara, K.; Tamiya, E.;Karube, I. Anal. Chim. Acta 1986, 788, 257-26 1. (14) Ey, P. L.; Prowse, S. J.; Jenkin, C. R. Immunochemistry 1978, 75, 429-436. (15) Duhamel, R. C.; Schur, P. H.; Brendei, K.; Meezan, E. J. Immunol. Methods 1979, 37,211-217. (16) Sjoquist, J.; Meioun, B.; Hjelm, H. f u r . J . Biochem. 1972, 29, 572-578. (17) Beitnes, H.; Schrder, K. Anal. Chim. Acta 1984, 758, 57-65. (18) Lai. C. S. I.; Moody, G. J.; Thomas, J. D. R. Analyst (London) 1988, 1 7 1 . 511-515.
RECEIVED for review October 1, 1986. Accepted August 5, 1987.
