Anal. Chem. 1997, 69, 887-893
Electrochemical/Piezoelectric Dual-Response Biosensor for Heme Ligands Tetsu Tatsuma*
Department of Applied Chemistry, Faculty of Technology, Tokyo University of Agriculture and Technology, Naka-cho, Koganei, Tokyo 184, Japan Daniel A. Buttry*
Department of Chemistry, University of Wyoming, Laramie, Wyoming 82071
A heme peptide-modified quartz crystal oscillator is fabricated as a dual-response biosensor, giving electrochemical and piezoelectric responses simultaneously. Binding of a ligand to heme peptide causes inhibition of its catalytic activity, which is observed as the electrochemical response, and a mass increase is monitored as the piezoelectric response. The dual-response sensor can give two independent pieces of information at the same time. That is, qualitative (identification of the species) and quantitative (determination of its concentration) analyses can be made simultaneously. Simultaneous quantitative analysis for two components is also possible. These two modes are theoretically explained and then experimentally demonstrated employing imidazole and histidine as interferants. Usually, a sensor gives a single response so that one can obtain one piece of information from a single measurement, namely a concentration of a specific substance. If one can get two independent responses from a sensor, two pieces of information can be given. The two pieces of information may be the concentrations of two species, or identification of a species and quantitation of its concentration. Although there are many sensors for multicomponent analysis, most of them are sensor arrays, each component of which has a different sensing element but the same kind of transducer. Okawa et al.1 fabricated a tin oxide-based sensor modified with glucose oxidase and urease that gives amperometric and conductometric responses. Although the sensor can measure the concentrations of glucose and urea simultaneously, the sensing part for glucose (glucose oxidase-based amperometric sensor) and that for urea (urease-based conductometric sensor) were geometrically separated. In that sense, the sensor is an array of two independent sensors. In the present work, we fabricate an electrochemical/piezoelectric dual-response sensor consisting of a single sensing element. Deakin and Byrd2 used a Prussian Blue-coated quartz crystal resonator as a cation sensor giving amperometric and piezoelectric resonses. Although the sensor can identify a cation and determine its concentration, twocomponent analysis was not discussed in their work. To demonstrate the mechanism and potential usefulness of the dual-response sensor, we use heme peptide (so-called micro(1) Okawa, Y.; Yoshida, S.; Watanabe, T. Proc. MRS Int. Meeting 1989, 14, 165-170. (2) Deakin, M. R.; Byrd, H. Anal. Chem. 1989, 61, 290-295. S0003-2700(96)00750-0 CCC: $14.00
© 1997 American Chemical Society
peroxidase, obtained from cytochrome c, Figure 1) as a sensing element. Tatsuma and Watanabe3 have reported that a heme peptide-modified electrode works as a reagentless H2O2 sensor, though the amperometric response is inhibited by strong ligands for heme. This interference effect was exploited for sensing of imidazole derivatives.4 There are some other interference-based biosensors employing cytochrome oxidase (EC 1.9.3.1),5,6 peroxidase (EC 1.11.1.7),7,8 or tyrosinase (EC 1.14.18.1),9 which are inhibited from catalyzing reduction of oxygen (cytochrome oxidase and tyrosinase) or H2O2 (peroxidase) by strong ligands coordinating to a metal ion as the catalytically active site. However, sometimes, such an interference-based biosensor is not very selective toward one specific substrate. The sensor with cytochrome oxidase5 responds to (i.e., is inhibited by) cyanide, azide, hydrogen sulfide, and carbon monoxide. The sensor with heme peptide3,4 responds to imidazole derivatives, namely imidazole, methylimidazole, histamine, and histidine. As discussed above, given only a single experimental measurement, such as the current response, it is impossible to know to which substrate the sensor is responding from the interference response alone. To solve this problem, one may be able to identify the interfering species if one can get another kind of response on the basis of another mechanism. Here we adopt a frequency response of a quartz crystal oscillator as the second response. Coordination of a ligand to the active site results not only in a decrease of the catalytic current but also in an increase in the weight of the catalyst. Therefore, if the catalyst is immobilized on the quartz crystal oscillator, the oscillation frequency will decrease upon the coordination of a ligand. Different ligands cause different frequency decreases if the molecular weights of the ligands are different. Quartz crystal microbalance (QCM) on the basis of the Sauerbrey equation10 is a well-known method to measure a minute mass change, thus it has been exploited for affinity-based chemical sensing in liquid phases.11-14 Additionally, since the QCM method has been used for in situ microgravimetry (3) Tatsuma, T.; Watanabe, T. Anal. Chem. 1991, 63, 1580-1585. (4) Tatsuma, T.; Watanabe, T. Anal. Chem. 1992, 64, 143-147. (5) Albery, W. J.; Cass, A. E. G.; Mangold, B. P.; Shu, Z. X. Biosens. Bioelectron. 1990, 5, 397-413. (6) Amine, A.; Alafandy, M.; Kauffmann, J.-M.; Pekli, M. N. Anal. Chem. 1995, 67, 2822-2827. (7) Smit, M. H.; Cass, A. E. G. Anal. Chem. 1990, 62, 2429-2436. (8) Tatsuma, T.; Oyama, N. Anal. Chem. 1996, 68, 1612-1615. (9) Smit, M. H.; Rechnitz, G. A. Anal. Chem. 1993, 65, 380-385. (10) Sauerbrey, G. Z. Z. Phys. 1959, 155, 206. (11) Nomura, T.; Sakai, M. Anal. Chim. Acta 1986, 183, 301-305.
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Figure 1. Molecular structure of heme undecapeptide.
Figure 2. Cross-sectional diagram of a heme peptide-coated quartz crystal oscillator as a dual-response sensor.
to analyze electrochemical processes,15-18 it is easy to monitor the interfering effect as an electrochemical response and the ligand binding as a piezoelectric response simultaneously. In the present work, we fabricate a heme peptide-modified quartz crystal oscillator as a dual-response sensor exhibiting an electrochemical response and a piezoelectric response. We measure the responses of the sensor to imidazole and histidine independently and verify that the sensor can make qualitative (identification of the species) and quantitative (determination of its concentration) analyses simultaneously. Furthermore, the dualresponse sensor is subjected to measurements of solutions containing imidazole and histidine at the same time, and simultaneous determination of the two components is performed. THEORY Mechanism of Responses. The present sensor is a quartz crystal oscillator, one of the two electrodes of which is coated with carbon paint and then with a heme peptide layer (Figure 2). The heme peptide layer is in contact with an aqueous electrolyte solution to which H2O2 and analytes (heme ligands) are added. Heme peptide reacts with H2O2 as shown in Figure 3a-c. First, the heme peptide in the ferric state (Figure 3A) is oxidized by H2O2, producing a π-cation radical of heme-oxene (so-called (12) Thompson, M.; Arthur, C. L.; Dhaliwal, G. K. Anal. Chem. 1986, 58, 12061209. (13) Muramatsu, H.; Kajiwara, K.; Tamiya, E.; Karube, I. Anal. Chim. Acta 1986, 188, 257-261. (14) Okahata, Y.; Matsunobu, Y.; Ijiro, K.; Mukae, M.; Murakami, A.; Makino, K. J. Am. Chem. Soc. 1992, 114, 8299-8300. (15) Nomura, T.; Iijima, M. Anal. Chim. Acta 1981, 131, 97-102. (16) Bruckenstein, S.; Shay, M. J. Electroanal. Chem. 1985, 188, 131-136. (17) Kaufman, J. H.; Kanazawa, K. K.; Street, G. B. Phys. Rev. Lett. 1984, 53, 2461-2464. (18) Buttry, D. A.; Ward, M. D. Chem. Rev. 1992, 92, 1355-1379.
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Figure 3. Reactions of heme peptide with H2O2 (a-c) and with an imidazole derivative (d). The heme moiety and the imidazole ring of proximal histidine are illustrated.
compound I), (Figure 3B). Compound I accepts one electron from the carbon electrode, producing compound II (Figure 3C), which then accepts another electron to produce the original ferric heme peptide. The observed current density i can be written as
1/i ) 1/ia + 1/ib + 1/ic + 1/id
(1)
where ia, ib, ic, and id are limiting currents for reactions a, b, and c (kinetic process, see Figure 3) and the diffusion process, respectively. These currents are given as follows:
ia ) 2FkaΓC
(2)
ib ) FkbΓ
(3)
ic ) FkcΓ
(4)
id ) 2FDC/d
(5)
where F is the Faraday constant, ka is the second-order rate constants for reaction a (in M-1 s-1), kb and kc are the heterogeneous rate constants for reactions b and c, respectively (in s-1), Γ is the surface coverage of active heme peptide, C is the H2O2 concentration, D is the diffusion coefficient of H2O2, and d is the diffusion layer thickness. If the H2O2 concentration is so small that kaC , kb and kc, reaction a determines the rate of the kinetic process, and eq 1 can be rewritten to give
i)
2FC d/D + 1/kaΓ
(6)
If kaC is comparable with kb and/or kc (i.e., reaction a does not determine the rate of the kinetic process), the observed current will not be proportional to the H2O2 concentration. However, the experimentally observed current was proportional to the H2O2 concentration under our experimental conditions (see below), so that eq 6 holds. As can be seen from eq 6, the current density should be a function of the surface coverage of active heme peptide, Γ. In the presence of a strong ligand for heme, heme peptide forms a complex which does not react with H2O2 (Figure 3D). Γ is a function of the concentration of the ligand CL:
Γ)
Γ0 CL/K + 1
(7)
where β is the mass-to-frequency proportionality constant, which is specific for an oscillator, and M is the molecular weight of the ligand. If the heme peptide layer is not completely elastic and/ or thin enough, the correlation between the response and the mass increase per unit area () M(Γ0 - Γ)) will not be linear. Also, if the hydrophilicity of the layer is changed by the complex formation (this changes the acoustic coupling between the transducer and the solution), the β value should be different from that in the case where the hydrophilicity is unchanged, and vice versa. On the assumption that those undesired effects are negligible, the response RP can be rewritten as a function of the ligand concentration CL as follows:
where K is the dissociation constant (K ) [heme peptide][ligand]/ [heme peptide-ligand]) and Γ0 is the total surface coverage of heme peptide. Therefore, eq 6 can be rewritten into the following equation:
i)
2FC d/D + (CL/K + 1)/kaΓ0
(8)
Here we define the electrochemical response to the ligand RE as
RE ) 1 - i/i0
(9)
where i0 ) i(CL ) 0). Thus, RE is given by4,8
RE )
1 RK/CL + 1
(10)
where R is given by
RP )
(13)
[(
) ]
RP 1 1 -1 +1 βΓ0 R RE
(11)
If the H2O2 reduction is controlled not by diffusion but by the enzymatic reaction (namely kaΓ0d/D , 1) in the absence of ligand, R equals unity. On the other hand, if the H2O2 reduction is controlled by diffusion in the absence of ligand, R depends on the parameters ka, Γ0, d, and D. When CL equals RK, RE ) 0.5. Thus, RK, the apparent dissociation constant, defines how low a concentration of a ligand the sensor can determine, and one can control the R value by changing Γ0 and d.4,8 The sensor is the most sensitive to the ligand when R equals unity. The RK value can be determined experimentally from the response-concentration curve (RK ) CL when RE ) 0.5), and the R value can be determined by measuring K by spectroscopic means.4 Complex formation of heme peptide with a heme ligand results not only in the catalytic current suppression but also in a mass increase of the electrode. Typically, the mass increase causes a resonance frequency decrease of the quartz crystal oscillator. If the heme peptide layer is completely elastic and thin enough, and the complex formation does not change the interaction between the layer and the solution (i.e., hydrophilicity of the layer does not change), the frequency decrease -∆F should be proportional to the mass increase. This frequency decrease accompanying the complex formation is defined as the piezoelectric response RP. Thus, the piezoelectric response should be given by
βMΓ0 K/CL + 1
Thus, both the electrochemical and piezoelectric responses depend on the ligand concentration, but the dependencies are manifested in completely different ways. This means one can obtain two independent pieces of information simultaneously from those two responses. Mode A: Simultaneous Qualitative-Quantitative Analysis. In one mode of operation, the two pieces of informtion which are obtained from the two responses can be the species of analyte (ligand) and its concentration. In other words, one can make qualitative and quantitative analyses simultaneously. The molecular weight of a ligand contained in a sample solution can be calculated from RE and RP on the basis of eqs 10 and 13:
M) R ) kaΓ0d/D + 1
(12)
RP ) -∆F ) βM(Γ0 - Γ)
(14)
Thus, the analyte is identified. Then, the concentration CL can be determined from M and K, which is unique for the analyte, as follows:
CL )
(15)
RK 1/RE - 1
However, eqs 10 and 13-15 may not hold for practical systems due to viscosity of the heme peptide film and so forth, which lead to nonideal variations in the piezoelectric response. In such a case, one should obtain RP vs RE curves over a concentration range of interest before analysis. If the RP vs RE curves of two or more analytes do not overlap each other, simultaneous qualitativequantitative analysis is possible. Note that a sample solution should not contain more than one ligand. Mode B: Simultaneous Quantitative Analysis for Two Components. In this mode of operation, one can determine the concentrations of two ligands from the two responses if one knows what species are contained in the sample solution. In this case, RE and RP are given as follows:
(
RE ) 1/
)
R +1 CL1/K1 + CL2/K2
Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
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M1CL1/K1 + M2CL2/K2 RP ) βΓ0 CL1/K1 + CL2/K2 + 1
(17)
where subscripts 1 and 2 correspond to two different ligands. The concentrations can be calculated from these equations as follows:
CLm )
[(
)(
) ]
Km RP R +1 - Mn + Mn Mm - Mn 1/RE - 1 βΓ0
(18)
where subscripts m and n are 1 or 2. Again, these equations do not hold under conditions where eqs 10 and 13 are not valid. In such a case, three-dimensional calibration graphs of RE-CL1-CL2 and RP-CL1-CL2 should be obtained for two-component analysis. As an example of this approach, a few values of CL1 and CL2 will be determined from the electrochemical and piezoelectric responses on the basis of such calibration graphs (discussed below in detail). In this case, the sample solution should not contain more than two ligands. EXPERIMENTAL SECTION Materials. Heme undecapeptide and glutaraldehyde (grade II) were obtained from Sigma. Imidazole, histamine, and histidine were purchased from Aldrich. Sørensen phosphate buffer (1/15 M, pH 7.4) was used as an electrolyte solution. Quartz crystal plates (5 MHz, 25-mm diameter) were obtained from ValpeyFisher. Carbon paint BP-333 (Nippon Kokuen, Japan) was diluted 10-fold with butyl acetate before use. Preparation of Heme Peptide-Modified Quartz Crystal. A quartz crystal plate was modified with (3-mercaptopropyl)trimethoxysilane by a method described by Goss et al.19 Gold films (keyhole-shaped, 6.2-mm diameter, 200 nm thick) were deposited on both sides of the quartz plate by evaporation. A 1.5 µL aliquot of the carbon paint was spread on one of the gold electrodes. We used the carbon paint to obtain larger surface area and better adhesion of heme peptide. After the solvent was evaporated, a 2-µL aliquot of an aqueous solution containing 10 g/L of heme peptide and 5% of glutaraldehyde was cast on the carbon-coated electrode using a precise micropipet (Ulster Scientific) with a yellow tip and left overnight at room temperature. The obtained heme peptide-modified quartz crystal was thoroughly rinsed with water and then immersed in the phosphate buffer (pH 7.4) for several minutes to remove mobile heme peptide molecules and finally rinsed again with water. Measurements. Measurements were performed at room temperature. The modified electrode of the crystal was in contact with the electrolyte solution (20 mL) and used as a working electrode (Figure 2). The quartz crystal was connected to a feedback oscillator (built in-house), and the oscillation frequency was measured with a frequency counter (Philips PM-6654). Measured frequency was transferred to a computer for the following data processing. The modified (working) electrode was connected also to the ground and to a Wenking-style potentiostat (built in-house) to control the potential. Reference and counter electrodes were a Ag/AgCl/NaCl (saturated) and platinum wire, respectively. During a typical measurement, the working electrode was polarized at +150 mV vs Ag/AgCl, at which the (19) Goss, C. A.; Charych, D. H.; Majda, M. Anal. Chem. 1991, 63, 85-88.
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electrochemical response of a bare electrode (without heme peptide) to H2O2 is minimal (in the absence of ligand). RESULTS AND DISCUSSION Modification with Heme Peptide. Accompanying the modification of the carbon-coated quartz crystal with heme peptide, the resonance frequency measured in air decreased about 0.47 ( 0.05 kHz (mean ( standard error, n ) 6). The small scatter in this value probably arose because the temperature and humidity were not completely controlled during the overnight modification. The noise level in the frequency measurement during sensing was much smaller (∼2 Hz), due to the short time scale of the measurement. The surface coverage of heme peptide estimated from the frequency decrease is about 4.4 × 10-9 mol/cm2, on the assumption that heme peptide has no counterions (i.e., has free acid and base) (MW ) 1881). Since amino and carboxyl residues of heme peptide must have counterions, the intrinsic coverage must be lower. Lower coverage resulted in too low sensitivity in piezoelectric response, and much higher coverage suffered from unstable oscillation. Direct attachment of heme peptide on the gold surface did not occur under the present immobilization condition. Response to H2O2. The heme peptide-modified quartz exhibited a cathodic current response to H2O2 in a 1/15 M phosphate buffer (pH 7.4) at +150 mV vs Ag/AgCl. The cathodic current was proportional to the H2O2 concentration from 1 up to 20 µM. In this range, the sensitivity was about 1.5 nA/µM. A quartz crystal oscillator which was not modified with heme peptide (carbon paint alone was coated on the gold surface) did not respond electrochemically to H2O2. Therefore, the electrochemical response observed for the modified oscillator was ascribed to the catalytic activity of the heme peptide. The linear relationship between the current and the concentration means that reaction a in Figure 3 determines the rate of the kinetic process (reactions a-c). Therefore, almost all of the heme peptide molecules should exist as ferric heme peptide (Figure 3A), and eq 6 holds. Based on these results, we chose 10 µM as the H2O2 concentration for interference measurements. Under these conditions, we do not need to consider the mass difference between ferric heme peptide and compound I or II (Figure 3B and C, respectively). As expected, no frequency change larger than the normal noise fluctuation ((2 Hz) was observed upon the addition of H2O2 (concentration, 10 µM). The buffer concentration of 1/15 M is far above the H2O2 concentration of 10 µM. Thus, changes in proton concentration during the redox catalysis of H2O2 by the immobilized heme peptide are negligible. Cathodic current response of the heme peptide-modified quartz to 10 µM H22 was 15.3 ( 1.6 nA (n ) 6). The reproducibility of the response (i.e., reproducibility of the catalytic activity) is almost the same as that of the amount of immobilized heme peptide. It is worth noting that the frequency noise of an unmodified quartz crystal oscillator (with carbon paint coated on the gold surface) was higher than that of the oscillator modified with carbon paint and heme peptide. The frequency fluctuation of the latter was (2 Hz, while that of the former was up to (5 Hz. We infer that the carbon paint surface of the modified oscillator was more stable because of adsorbed heme peptide molecules linked each other via glutaraldehyde, though the mechanism for this change in noise levels is not clear. Frequency changes of the unmodified oscillator to H2O2 were not significant compared to the normal noise fluctuation.
Interferants. We chose imidazole (MW ) 68.08) and histidine (MW ) 155.16) as heme ligands (i.e., interferants). Histidine is an amino acid with an imidazole ring, and it often controls the catalytic activity of enzymes and acts in maintaining the higher structure of proteins. Additionally, histidine and its derivatives are indexes of histidinemia.20 Since the pKa value of imidazole is 7.0,21 28% of imidazole molecules are protonated at pH 7.4. The protonated imidazole cannot coordinate to heme peptide because no lone electron pair is available for the coordination. For histidine, pKa values for the amino, carboxyl, and imidazole groups are 9.2, 1.8, and 6.0.21 Therefore, its amino group is protonated while its carboxyl group is deprotonated at pH 7.4, and they should have counterions. Additionally, amino and carboxyl groups as well as the imidazole ring (-NH- site) of imidazole and histidine can form hydrogen bonds with water molecules. These interactions may increase the effective molecular weights (i.e., molecular weights reflected by frequency changes) of those compounds. Although quantitative evaluation of these effects (counterions and hydrogen bonding or solvation) has not yet been established, it is obvious that the effective molecular weight of histidine should be substantially larger than that of imidazole in these measurements. Mode A: Simultaneous Qualitative-Quantitative Analysis. Measurements were performed in a 1/15 M phosphate buffer (pH 7.4) at +150 mV vs Ag/AgCl. H2O2 was added (concentration, 10 µM) to the buffer, and after a steady state was reached, imidazole or histidine was added. As a result, a decrease in the cathodic current was observed as well as a resonance frequency decrease. This reflected the facts that the ligand coordinated to heme peptide and that the catalytic activity of heme peptide was, therefore, inhibited while its mass was increased. Figure 4a,b shows electrochemical responses (RE from eq 9) and piezoelectric responses (RP from eq 12), respectively, as functions of the ligand concentration (open symbols). The error of the electrochemical responses is (0.05. For the piezoelectric responses, the error for imidazole is (2 Hz, and that for histidine is (3 (e5 mM histidine) or (10 Hz (g10 mM histidine). Anodic currents of an unmodified quartz (coated by the carbon paint alone) in the presence of 50 mM imidazole and 50 mM histidine were about 4 and 2 nA, respectively. Therefore, the suppression of the cathodic current (∼15 nA), which ranged up to 100%, cannot be explained by those anodic currents. Piezoelectric responses of the unmodified quartz are shown in Figure 4b for comparison (closed symbols). They are clearly smaller than those of the modified quartz. Adsorption of imidazole and histidine onto the carbon and/or gold (the gold surface may not be coated completely with the carbon paint in the molecular level) or changes in density and/or viscosity of fluid22,23 (especially that trapped within the roughness features at the surface)24 must be responsible for the piezoelectric responses of the unmodified quartz. Although coordination of the ligands to heme peptide is reversible, denaturation and/or detachment of heme peptide occur. Thus, the sensor can be used repeatedly 3-5 times. (20) Dorland’s Illustrated Medical Dictionary, 27th ed.; Taylor, E. J., Ed.; W. B. Saunders: Philadelphia, PA, 1988. (21) Dictionary of Organic Compounds, 5th ed.; Chapman and Hall: New York, 1982; Vol. 3. (22) Kanazawa, K. K.; Gordon, J. G., II. Anal. Chim. Acta 1985, 175, 99-105. (23) Bruckenstein, S.; Shay, M. Electrochim. Acta 1985, 30, 1295-1300. (24) Beck, R.; Pittermann, U.; Weil, K. G. J. Electrochem. Soc. 1992, 139, 453461.
Figure 4. Typical electrochemical (RE, a) and piezoelectric (RP, b) responses (obtained simultaneously) of the heme peptide-modified dual-response sensor to imidazole (Im, O) and histidine (His, 4). See eqs 9 and 12 for the definitions of RE and RP, respectively. Electrode potential is +150 mV vs Ag/AgCl. Electrolyte is 1/15 M phosphate buffer (pH 7.4). Piezoelectric responses of an unmodified quartz are also plotted for comparison (b and 2 for imidazole and histidine, respectively). Theoretically expected responses calculated from eqs 10 and 13 are also shown (see text for detail).
The apparent dissociation constant (RK, obtained at the concentration where RE ) 0.5, see the Theory section) for the heme peptide-imidazole complex is estimated to be 0.14 mM from Figure 4a. This roughly agrees with the reported RK value for a heme nonapeptide-modified electrode (∼0.3 mM at pH 7.4)4 and K value for heme nonapeptide solutions (0.25 mM at pH 7.4).4 Therefore, the R value may be close to unity in these measurements. On the other hand, the estimated RK value for histidine is 1.5 mM. Although the values for histidine are not available in the literature, the electrochemical responses (RE) of a heme nonapeptide-modified electrode4 to 1 mM imidazole and 1 mM histidine were 0.73 and 0.45, respectively, at pH 7.4. These values are close to the electrochemical responses of the present sensor (0.78 and 0.45, respectively). This difference between the response to imidazole and that to histidine must be due to a difference in the dissociation constant K. Note that the R value (eq 11) is independent of the ligand species. Theoretically expected electrochemical responses calculated from eq 10 and the RK values estimated from the experimental data (see above) are also shown in Figure 4a (dashed lines). In comparison with these theoretical curves, the experimentally obtained sigmoidal curves change less rapidly with ligand concentration. This behavior is common among interference-based Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
891
Figure 5. Correlations between the electrochemical response (RE) and piezoelectric response (RP) to imidazole (Im, O) and histidine (His, 4) for the dual-response sensor. Data from Figure 4.
biosensors.4,8,25 This may be because the immobilized heme peptide molecules have a range of different binding constants, depending on different microenvironment. Theoretically expected piezoelectric responses calculated from eq 13 are also plotted in Figure 4b (dashed lines). The R value was assumed to be unity (this assumption is plausible as described above). Molecular weights for free acid and base were used for heme peptide and histidine (as well as imidazole). The experimentally obtained responses which are also plotted clearly deviate from the theoretical ones. However, there is an important characteristic that is common between the theoretical and experimental responses. That is, the response to imidazole is higher than that to histamine at 1 mM. This fact clearly reflects that the K value for imidazole is smaller than that of histidine and that the effective molecular weight of the imidazole is smaller than that of histidine. The appearance of this feature in the experimental data is a strong indication that the sensor response derives from the scheme discussed above. In the low-concentration region, the experimentally obtained responses were lower than the theoretical ones. Overestimation of the surface coverage of heme peptide could be one of the reasons for this. This overestimation can arise from the underestimation of the effective molecular weight of heme peptide (see the section on Modification with Heme Peptide). Another possible reason is that all of the heme peptide molecules immobilized on the oscillator may not be reactive with the ligands due to degradation and/or steric hindrance. On the other hand, in the high concentration region, the experimentally obtained responses considerably exceeded the theoretical ones. This was chiefly caused by the adsorption of the ligands onto the carbon and/or gold surface or changes in density and/or viscosity of fluid mentioned above (see the responses from the unmodified oscillator). Although subtracting the background responses gives responses closer to the theoretical ones, this processing is not necessarily justified, because heme peptide may inhibit the adsorption of ligands onto the carbon and/or gold surface. Even after the subtraction, the experimental responses are larger than (25) Tatsuma, T.; Tani, K.; Oyama, N.; Yeoh, H. H. Anal. Chem. 1996, 68, 29462950.
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Figure 6. Typical electrochemical (RE, a) and piezoelectric (RP, b) responses (obtained simultaneously) of the dual-response sensor for imidazole-histidine mixed solutions as functions of imidazole (CIm) and histidine (CHis) concentrations. See eqs 9 and 12 for the definition of RE and RP, respectively. Iso-response curves (RE ) 0.4, 0.6, and 0.8 and RP ) 10, 20, 30, and 40 Hz) are also drawn. Electrode potential is +150 mV vs Ag/AgCl. Electrolyte is 1/15 M phosphate buffer (pH 7.4).
the theoretical ones at concentrations >5 mM. Solvent swelling of the heme peptide layer, which could result in an unexpectedly large frequency decrease (i.e., large piezoelectric response), might also have occurred in that concentration range. Given these discrepancies between the observed and predicted behavior, the molecular weight and concentration of the ligand cannot be unambiguously determined on the basis of eqs 14 and 15. In such a case, as described in the Theory section, one approach to allow such a determination is to plot piezoelectric responses against electrochemical responses as show in Figure 5 (RP vs RE curves). In this approach, one would obtain a set of values of the electrochemical response, x, and the piezoelectric response, y, for a sample containing imidazole or histidine. Then one would plot the point (x,y) on Figure 5, and if the point appears on the curve for imidazole (or histidine), one would know that imidazole (or histidine) was contained in the sample. Because the two curves overlap each other for e0.1 mM imidazole and e1 mM histidine, qualitative analysis is not possible in this concentration range. However, it should be possible in the higher concentration region. Then, once one knows the species contained in the sample, one can determine its concentration from the calibration curve for electrochemical (Figure 4a) or piezoelectric (Figure 4b) responses. In this way, qualitative and quantitative analyses can be made simultaneously. As discussed above, this method is suitable only for cases in which the sample
to the latter (at the same concentrations, data not shown). The present sensor can, therefore, distinguish histamine from histidine. This may be important due to histamine’s pharmacological activity.20 Mode B: Simultaneous Quantitative Analysis for Two Components. In this mode, we measured responses to solutions containing both imidazole and histidine under the same conditions as in mode A. Figure 6a,b shows three-dimensional calibration graphs for the electrochemical and piezoelectric responses, respectively. Since the experimental RP values were not in quantitative agreement with theoretical expectations in mode A, this should also be the case for mode B. However, the two-component analysis is still possible using the calibration graphs via the following procedure. Assume again that one obtained a set of an electrochemical response, x, and a piezoelectric response, y, for a sample containing imidazole and/or histidine. One takes iso-response curves (Figure 7a,b) for RE ) x and RP ) y from Figure 6a,b, respectively, and projects them down to the same zero response surface (Figure 7c for the case in which RE ) 0.8 and RP ) 20 Hz). The obtained intersection of the two curves gives the concentrations of imidazole and histidine. Thus, one can make quantitative analysis for two components simultaneously. As discussed above, this method is suitable only for cases in which the sample solution contains two known ligands (at the most). CONCLUSIONS We fabricated a heme peptide-modified quartz crystal oscillator as an electrochemical/piezoelectric dual-response biosensor. We demonstrated that simultaneous qualitative-quantitative analysis (mode A) and simultaneous quantitative analysis for two components (mode B) are possible with the dual-response sensor. The demonstrated principle may be able to be extended to another dual-response systems including the electrochemical/piezoelectric system, and even to multiresponse systems, though these are beyond the scope of the present work. Figure 7. Iso-response curves for electrochemical (RE, a) and piezoelectric (RP, b) responses (data from Figure 6). The intersection of the curve for obtained electrochemical response and the curve for piezoelectric response gives the concentrations of imidazole and histidine (c, for the case in which RE ) 0.8 and RP ) 20 Hz).
solution contains only one ligand with a reasonable affinity for the complex. This method can be extended to other ligands unless they have almost the same effective molecular weights. For instance, the electrochemical response to histamine (MW ) 111.15), which also has an imidazole ring, was similar to that to histidine, while the piezoelectric response to the former was much smaller than that
ACKNOWLEDGMENT This work was supported by the Ministry of Education, Science, Sports and Culture of Japan (T.T.) and Office of Naval Research (D.A.B.). The authors are grateful to Prof. N. Oyama (TUAT, Japan) for discussion, K. Ariyama (TUAT, Japan) for his help with preliminary experiments, and W. Walker and A. R. Faxon (UW) for their help with QCM preparation. Nippon Kokuen, Japan, is acknowledged for supplying the carbon paint. Received for review July 26, 1996. Accepted December 20, 1996.X AC960750K X
Abstract published in Advance ACS Abstracts, February 1, 1997.
Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
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