QCM Operation in Liquids: Constant Sensitivity during Formation of

Anal. Chem. 1997, 69, 1441-1448

QCM Operation in Liquids: Constant Sensitivity during Formation of Extended Protein Multilayers by Affinity Jan Rickert,* Andreas Brecht, and Wolfgang Go 1 pel

Institute for Physical and Theoretical Chemistry and ‘Center of Interface Analysis and Sensors’ University of Tu¨ bingen, Auf der Morgenstelle 8, D-72076 Tu¨ bingen, Germany

The quartz crystal microbalance (QCM) is a well-established tool in mass-sensitive detection. Due to recent improvements in experimental procedures, QCMs are finding increasing attention for applications in liquids. One important application is bioaffinity measurements for analytical or research purposes. The effect of the formation of solid films at a QCM surface, especially in gases or vacuum, is well understood. However, the situation is more complex in bioaffinity applications due to the comparably high viscosity of the liquid and the softness of the biological overlayer. Typically frequency responses found for protein layers exceed the values expected from simple models. The use of a hydrogel extending several hundred nanometers from the transducer surface as interacting matrix is common in bioaffinity applications and further increases complexity. Pure mass-related effects as well as viscosity-mediated effects may contribute to the overall frequency response observed experimentally. To improve our understanding of the effects during the formation of extended biological overlayers we have investigated systematically the formation of protein multilayers with a QCM in situ. The attenuation of the QCM oscillation by the liquid leads to a broadening of the resonance frequency. We have overcome this limitation by frequency-dependent admittance analysis and by curve fitting of the resulting admittance. A time resolution of 5 s and a noise of 0.2 Hz has been achieved with 6-MHz AT-cut quartz crystals operating in liquids. Protein multilayers were formed by successive incubations with a biotin-albumin conjugate and streptavidin. Frequency responses for dry protein layers in air were in agreement with mass changes estimated from the Sauerbrey equation. However, in water, the corresponding frequency decrease was increased by a factor of 4, thereby indicating that significant amounts of water are embedded in the hydrated protein layer. Unexpectedly a constant frequency decrease per layer was found during the successive formation of up to 20 protein layers (∼400 nm). Neither noise nor drift increased with the number of protein layers. These results indicate that, despite the high hydration of the protein layers, viscosity-induced effects play a negligible role and that the frequency decrease reflects primarily mass changes at the surface. Sauerbrey’s observation1 about the mass sensitivity of AT-cut quartz crystals led to their application as a mass-sensitive device S0003-2700(96)00875-X CCC: $14.00

© 1997 American Chemical Society

in various applications in vacuum, air, and liquids. The fundamental theory describing the relationship between frequency change and mass change was derived by Sauerbrey:

∆f ) -

fR2 fR ∆m ) ∆m FQdA NA

(1)

where ∆f is the frequency change, fR is the resonant frequency, FQ is the density of the crystal, d is the thickness of the crystal, A is the area of the electrodes, ∆m is the mass change, and N is the frequency constant of the quartz. This theory is limited to the assumption that the mass deposition forms a thin, rigid film and that the mass sensitivity is uniform over the entire surface. Experiments showed that a mass loading up to 0.05% of the crystal mass is acceptable to stay within this mode of operation.2 Quartz crystal microbalances (QCMs) are particularly suitable transducers for selective sensors in the gas phase.3-5 Applications for detection of CO, SO2, NH3, hydrocarbons, and organophosphorus compounds have been developed.4,5 Several publications dealing with measurements on biological compounds describe experiments where the recognition of the analyte and signal transduction are divided in time.6-11 The dip-and-dry method used here in QCM based affinity sensing is not very reliable because of the complexity in sample handing, sources of error, and lack of automated or continuous operation. It has been shown that a bulk acoustic wave (BAW) piezoelectric quartz crystal, oscillating in the thickness shear wave (TSM) mode, can be operated in the liquid phase.12-14 The (1) Sauerbrey, G. Z. Phys. 1959, 155, 206-222. (2) Lu, C.-S.; Czanderna, A. W. Applications of Piezoelectric Quartz Crystal Mircrobalances; Elsevier: Amsterdam, 1984. (3) Grate, J. W.; Frye, G. C. In Sensors Update Vol. 2. Sensor Technology: Techniques; Markets. Acoustic Wave Sensors; Baltes, H., Go¨pel, W., Hesse, J., Eds.; VCH: Weinheim, Germany, 1996; pp 38-88. (4) Snow, A.; Wohltjen, H. Anal. Chem. 1984, 56, 1411-1416. (5) Ballantine, D.; Wohltjen, H. Anal. Chem. 1989, 61, 705-712. (6) Ito, K.; Hashimoto, K.; Ishimori, Y. Anal. Chim. Acta 1996, 327, 29-35. (7) Yokoyama, K.; Ikebukuro, K.; Tamiya, E.; Karube, I.; Ichiki, N.; Arikawa, Y. Anal. Chim. Acta 1995, 304, 139-145. (8) Ariga, K.; Okahata, Y. Langmuir 1994, 10, 3255-3259. (9) Suri, C. R.; Raje, M.; Mishra, G. C. Biosens. Bioelectron. 1994, 9, 535-54. (10) Guilbault, G. G.; Hock, B.; Schmid, R. D. Biosens. Bioelectron. 1992, 7, 411-419. (11) Prusak-Sochaczewski, E.; Luong, J. H. T. Enzyme Microb. Technol. 1990, 12, 173-177. (12) Schumacher, R. Angew. Chem. 1990, 102, 347-361. (13) Thompson, M.; Kipling, A. L.; Duncan-Hewitt, W. C.; Rajakovic, L. V.; CavicVlasak, B. A. Analyst 1991, 116, 881-890. (14) Buttry, D. A.; Ward, M. D. Chem. Rev. 1992, 92, 1355-1379.

Analytical Chemistry, Vol. 69, No. 7, April 1, 1997 1441

detection of components eluted during liquid chromatography,15 the determination of inorganic species,16 the electrochemical QCM,17 the detection of phase transitions in liquid crystals and lipid layers,18 and the indirect measurements of affinity reactions19 are applications in the liquid phase showing the ability of this analytical tool. In the latter case a latex immunoassay,20-23 and an enzyme-amplified reaction24,25 were reported. In both cases, the measured effects were changes in viscosity and/or density of the test solution. In most analytical applications using the highly specific interactions between antigen and antibody, the affinity reaction and the measurement of the resonant frequency take place at different times. The coated quartz crystal is incubated with the analyte and dried. Thereafter, the resonance frequency is determined and compared with the resonance frequency before the incubation with analyte.26-28 Some papers present in-situ measurements of specific immunoreactions.29 First results were published by Roederer and Bastiaans.30 Their analyte was human IgG, and they worked with a surface acoustic wave (SAW) device. IgG as analyte was also detected using a BAW crystal in the TSW mode.31-34 Other antibodies as analyte were detected using synthetic peptides as immobilized antigens.35-37 Quartz crystals coated with fluorescein embedded in LB layers allowed the masssensitive detection of anti-fluorescein IgG down to concentrations of 2 nM.38 The RNA homopolymers were immobilized on the surface of a QCM, and the sequence-specific hybridization could be detected.39 The frequency shifts of a QCM were used to determine the immunological activity of different immobilized antibody layers.40 (15) Nomura, T.; Yanagihara, T.; Mitsui, T. Anal. Chim. Acta 1991, 248, 329335. (16) Yao, S.; Nie, L. Anal. Proc. 1987, 24, 336-337. (17) Gordon, J. S.; Johnson, D. C. J. Electroanal. Chem. 1994, 365, 267-274. (18) Okahata, Y.; Ebato, H. Anal. Chem. 1989, 61, 2185-2188. (19) Muramatsu, H.; Tamiya, E.; Suzuki, M.; Karube, I. Anal. Chim. Acta 1988, 215, 91-98. (20) Kurosawa, S.; Tawara, E.; Kamo, N.; Ohta, F.; Hosokawa, T. Chem. Pharm. Bull. 1990, 38, 1117-1120. (21) Muratrugu, M.; Kurosawa, S.; Kamo, N. Anal. Chem. 1992, 64, 2483-2487. (22) Ghourchian, H. O.; Kamo, N.; Hosokawa, T.; Akitaya, T. Talanta 1994, 41, 401-406. (23) Ghourchian, H. O.; Kamo, N. Anal. Chim. Acta 1995, 300, 99-105. (24) Ebersole, R. C.; Ward, M. D. J. Am. Chem. Soc. 1988, 110, 8623-8628. (25) Ebersole, R. C.; Miller, J. A.; Moran, J. R.; Ward, M. D. J. Am. Chem. Soc. 1990, 112, 3239-3241. (26) Guilbault, G. G.; Suleiman, A. Am. Biotechnol. Lab. 1990, 8 (4), 28-32. (27) Ngeh-Ngwainbi, J.; Suleiman, A. A.; Guilbault, G. G. Biosens. Bioelectron. 1990, 5, 13-26. (28) Suleiman, A. A.; Guilbault, G. G. Anal. Lett. 1991, 24, 1283-1292. (29) Ward, M. D.; Buttry, D. A. Science 1990, 259, 1000-1007. (30) Roederer, J. E.; Bastiaans, G. J. Anal. Chem. 1983, 55, 2333-2336. (31) Davis, K. A.; Leary, T. R. Anal. Chem. 1989, 61, 1227-1230. (32) Muramatsu, H.; Dicks, J. M.; Tamiya, E.; Karube, I. Anal. Chem. 1987, 59, 2760-2763. (33) Thompson, M.; Dhaliwal, G. K.; Arthur, C. L.; Calabrese, G. S. IEEE Trans. Ultrasonics, Ferroelectrics, Frequency Control 1987, UFFC-34, 127-135. (34) Thompson, M.; Arthur, C. L.; Dhaliwal, G. K. Anal. Chem. 1986, 58, 12061209. (35) Rickert, J.; Weiss, T.; Kraas, W.; Jung, G.; Go¨pel, W. Biosens. Bioelectron. 1996, 11, 591-598. (36) Ko ¨sslinger, C.; Drost, S.; Aberl, F.; Wolf, H. Fresenius J. Anal. Chem. 1994, 349, 349-354. (37) Ko ¨sslinger, C.; Drost, S.; Aberl, F.; Wolf, H.; Koch, S.; Woias, P. Biosens. Bioelectron. 1992, 7, 397-404. (38) Ebato, H.; Gentry, C. A.; Herron, J. N.; Mu ¨ ller, W.; Okahata, Y.; Ringsdorf, H.; Suci, P. A. Anal. Chem. 1994, 66, 1683-1689. (39) Su, H.; Chong, S.; Thompson, M. Langmuir 1996, 12, 2247-2255. (40) Caruso, F.; Rodda, E.; Furlong, D. N. J. Colloid Interface Sci. 1996, 178, 104-115.

1442

Analytical Chemistry, Vol. 69, No. 7, April 1, 1997

A QCM operating in liquid does often not behave as predicted by the Sauerbrey equation (1), as a number of factors may influence the oscillating behavior. The resonance frequency and its changes depend on the interfacial liquid properties (i.e., density, viscosity, conductivity, and dielectric constant),41-43 thin-film stiffness,44 electrode morphology,45-47 and mechanism of acoustic coupling.48,49 The dependence of the QCM frequency on density, viscosity, and viscoelasticity stems from the shear motion of the crystal surface, which generates a shear wave that penetrates into the liquid. This shear wave is damped by energy dissipation associated with the viscosity of the liquid. The characteristic decay length is ∼1 µm.50 For the simple model of a semiinfinite Newtonian liquid, the frequency shift compared to vacuum can be derived as51

∆f ) -fR3/2

x

Flηl πµQFQ

(2)

where ∆f is the frequency change, fR is the resonant frequency, Fl and ηl are the density and viscosity of the liquid, and FQ and µQ are the density and the shear modulus of the quartz crystal, respectively. This relation has been proven for various liquids.43 In protein-binding studies, various authors observed for protein mass loading a frequency shift higher than theoretically expected from the Sauerbrey equation,40,52-56 In comparison to results obtained independently by the radioisotope method, the frequency decrease was increased by a factor of 2.1-3.9.56 Comparing the frequency shifts in water and air, a factor of 2.2-3.7 was found54 for IgG molecules. For β-globulin, a frequency enhancement factor of 1.5 was observed.52 Hybridization of DNA to complementary single strands immobilized on the sensor led to frequency changes, exceeding by 18 times the values predicted by the Sauerbrey equation.55 Several theories have been derived to explain interfacial phenomena, and several models have been proposed to describe simultaneous mass loading and interfacial liquid effects.50 Most theoretical approaches are based on empirical equations.43,57 The effect of mass and liquid loading on the resonance of an AT-cut QCM has been calculated by Martin et al. using a continuum electromechanical model.58 Although the potential of the QCM application to affinity-based analytical techniques in liquids is obvious from work already (41) Shana, Z. A.; Zong, H.; Josse, F.; Jeutter, D. C. J. Electroanal. Chem. 1994, 379, 21-33. (42) Zhou, T.; Nie, L.; Yao, S. J. Electroanal. Chem. 1990, 293, 1-18. (43) Yao, S.-Z.; Zhou, T.-A. Anal. Chim. Acta 1988, 212, 61-72. (44) Noel, M. A. M.; Topart, P. A. Anal. Chem. 1994, 66, 484-491. (45) Urbakh, M.; Daikhin, L. Langmuir 1994, 10, 2836-2841. (46) Martin, S. J.; Frye, G. C.; Ricco, A. J. Anal. Chem. 1993, 65, 2910-2922. (47) Yang, M.; Thompson, M. Langmuir 1993, 9, 1990-1994. (48) Duncan-Hewitt, W. C.; Thompson, M. Anal. Chem. 1992, 64, 94-105. (49) Muramatsu, H.; Tamiya, E.; Karube, I. Anal. Chem. 1988, 60, 2142-2146. (50) Thompson, M.; Kipling, A. L.; Duncan-Hewitt, W. C.; Rajakovic, L. V.; CavicVlasak, B. A. Analyst 1991, 116, 881-890. (51) Kanazawa, K. K.; Gordon, J. G., II Anal. Chem. 1985, 57, 1170-1171. (52) Ebara, Y.; Okahata, Y. Langmuir 1993, 9, 574-576. (53) Caruso, F.; Serizawa, T.; Furlong, D. N.; Okahata, Y. Langmuir 1995, 11, 1546-1552. (54) Geddes, N. J.; Paschinger, E. M.; Furlong, D. N.; Ebara, Y.; Okahata, Y.; Than, K. A.; Edgar, J. A. Sens. Actuators 1994, B 17, 125-131. (55) Su, H.; Kallury, K. M. R.; Thompson, M.; Roach, A. Anal. Chem. 1994, 66, 769-777. (56) Muratsugu, M.; Ohta, F.; Miya, Y.; Hosokawa, T.; Kurosawa, S.; Kamo, N.; Ikeda, H. Anal. Chem. 1993, 65, 2933-2937. (57) Nomura, R.; Okuhara, M. Anal. Chim. Acta 1982, 142, 281. (58) Martin, S. J.; Granstaff, V. E.; Frye, G. C. Anal. Chem. 1991, 63, 22722281.

Figure 1. Cross section of a QCM loaded with a surface layer on one side facing the liquid. Shear displacement in the x direction is shown schematically in the direction y normal to the layer for the time at which quartz displacement is maximum. The left side shows a rigid layer on the quartz coupled in which the shear wave is propagated without losses. It decays in the surrounding liquid. On the right, a viscoelastic layer is shown in which the shear wave decays exponentially already in the layer. Here the surrounding liquid will has no influence on the resonance behavior.

published, theoretical and practical aspects of this application are still by far less developed than for the operation of QCM in gases. This paper aims at a better understanding of QCM devices for liquid sensing. The operation of a QCM in conducting liquids is possible, provided that one side of the quartz is shielded against the liquid. However, the change from air to liquid inevitably introduces a broadening of the resonance curve. This leads to a less stable operation of the oscillator and to increased noise. For high mechanical damping, high interaction with the liquid due to electrode morphology, and low signal quality due to limited electrode area, the stable operation of a QCM with an oscillator circuit usually becomes difficult to achieve. We therefore utilized admittance spectroscopy, whereby the response of the QCM is assessed at fixed frequencies, as a robust alternative to conventional oscillator circuits. This allows the investigation of frequency changes even for highly damped systems. QCMs operating in liquids cause a transverse shear wave in the liquid with a decay length of ∼1 µm. Viscosity changes within this region influence the device resonance frequency according to eq 2. For a viscous layer extending from the surface of the QCM, the frequency response will therefore decay exponentially with increasing layer thickness. In contrast, thin solid layers with acoustic impedances comparable to the quartz material itself will rigidly couple to the oscillation of the QCM and give a “Sauerbreytype” frequency response correlating linearly with the amount of mass deposited. Figure 1 illustrates these two theoretical models. In the present study, we used a self-assembling protein multilayer structure that allows for building up layers over several hundred nanometers to investigate empirically the type of response caused by protein multilayers of high thickness. We have carried out comparative investigations on protein layers in air and in liquid to get insight into the mechanisms affecting QCM response in affinity investigations in liquids. EXPERIMENTAL SECTION AT-cut 6- and 12-MHz quartz crystals with a diameter of 14 mm (Kristallverarbeitung Neckarbischofsheim, Germany) were coated with gold electrodes under UHV conditions. The electrodes with a diameter of 7 mm contain lateral contacting pads. The area of these electrodes was 38.5 mm2. The gold films (150 nm) were prepared by thermal evaporation under ultrahigh vacuum conditions (base pressure 4 × 10-10 mbar) subsequent to the deposition of titanium (∼2 nm) as adhesion layer. During evaporation of Ti the chamber pressure was 3 × 10-9 mbar, and of Au 7 × 10-7 mbar. The evaporation rate was 0.1 nm s-1 for Au.

Figure 2. Schematic illustration of the experimental setup for the QCM experiments.

The QCM experimental setup is illustrated in Figure 2. The quartz crystals were mounted by concentric rubber seals into a flow-through cell to provide contact with only one side of the quartz crystal to the liquid. The flow-through cell was made of Plexiglas (Perspex). The cell volume was 100 µL. The flowthrough cell was combined with a pump system (Ismatec, Glattbrug-Zu¨rich, Switzerland). Solutions were supplied through a six-way valve. The cell was shielded with a high-grade steel box and thermostated to 30 ( 0.1 °C. Measurements in air were performed with a spring holder. Phosphate-buffered saline (PBS), pH 7.4, was used for the preparation of all solutions (0.01 M K2HPO4/KH2PO4, 0.1 M NaCl). All reagents were of analytical grade. Water (resistance of 18 MΩ cm) was obtained from a Milli-Q unit (Millipore Inc.). The magnitude of the admittance (|Y|) and the phase angle (θ) were monitored using a HP 4192A impedance analyzer.59 A personal computer was used for the collection (via an IEEE interface), evaluation, and interpretation of the data. The data acquisition program is written in Turbo Pascal. A typical resonance curve of a QCM illustrates the evaluation scheme (Figure 3). For the determination of the resonance frequency (fR), the HP 4192A was scanned over a given frequency domain and 1240 data points were collected within 0.1-2 kHz by measuring the phase angle (Figure 3 lower part). The resonance frequency [with fR ) f(θ ) 0)] was determined by linear regression (Figure 3, lower part) and recorded as a function of time. The impedance analyzer instead of a conventional oscillator circuit was chosen for determining the resonance frequency, because it is more sensitive and less critically influenced by electrical and mechanical disturbances of the QCM. A two-component bioaffinity system was used to prepare selfassembled protein multilayers at the QCM electrodes. The system is based on the high-affinity interaction of biotin and streptavidin. One molecule of the tetrameric streptavidin (∼60 000 g mol-1) binds four molecules of biotin.60 Thermally treated biotinylated bovine serum albumin with 15 mol of biotin/mol of albumin [t-BSA-bi(15)] and 5 mol of biotin/mol of albumin [t-BSAbi(5)] served as biotin-containing compounds. The effect of the thermal treatment is a slight denaturation of the BSA. This makes the compound more sticky and leads also to a limited aggregation of protein molecules. Covalently cross-linked streptavidin (pSA) was used as biotin-binding compound. Both compounds were obtained from Boehringer Mannheim and prepared according to (59) Soares, D. M.; Kautek, W.; Frubo¨se, C.; Doblhofer, K. Ber. Bunsenges. Phys. Chem. 1994, 98, 219-228. (60) Green, N. M. Methods Enzymol. 1970, 18A, 418.

Analytical Chemistry, Vol. 69, No. 7, April 1, 1997

1443

Figure 3. Evaluation scheme illustrating the generation of the resonance frequency signal by linear regression. The magnitude of admittance |Y| and the phase angle θ are plotted as a function of frequency f. fm denotes the frequency of maximum admittance, fR the series resonance frequency, and fP the parallel resonance frequency.

Berger and Maler.61 Photon correlation spectroscopy carried out by the supplier indicated a mean diameter of 86 nm/protein aggregate. The use of polymeric and highly multivalent compounds has important consequences with regard to the resulting layer structure. The affinity interaction of the compounds leads to crosslinking of individual protein aggregates present at the transducer surface. This improves the stability of the layers. The high molecular weight of the compounds ensures an excess of binding sites after each incubation step. This leads to a very stable and reproducible formation of protein multilayers. Measurements were performed with QCMs subsequent to the evaporation of the gold electrodes or to their cleaning with Piranha solution (7:3 concentrated H2SO4/H2O2 v/v). Caution: Piranha solution should be handled with extreme care, and only small volumes should be prepared at any time. The quartz crystals were immersed in freshly prepared, therefore, hot piranha solution for 10-15 s. Afterward they were carefully rinsed with high-purity water and dried in an argon stream. Impedance spectra were measured after mounting the crystals in the flow-through cell to check the performance of the equipment. With one side exposed to the liquid, a maximum in the magnitude of admittance of at least 2 mS and a dip in the phase angle to -45° occurred. This indicated negligible damping of the QCM which otherwise may be caused by an extraneous element in the flow cell or by incorrect mounting of the quartz crystal. Both BSA derivatives and the pSA were dissolved in PBS at a concentration of 50 µg mL-1. Solutions with 100 µg mL-1 were used only for building of the first physisorbed layer. In Figure 4 the flow scheme is shown, which was used for the deposition of the protein layers. When protein solution was injected, the flow rate was increased to 0.4 mL min-1 for 4 min to ensure a quick purging of the flow cell with protein solution. For additional 26 min, the flow rate was decreased to 0.07 mL min-1, allowing the complete buildup of the monolayer. After this incubation time, the flow cell was rinsed with pure PBS at an average flow rate of (61) Berger, M.; Maler, J. Eur. Pat. 0331127, 1993.

1444 Analytical Chemistry, Vol. 69, No. 7, April 1, 1997

Figure 4. Resonance frequency f and flow rate νfl of the peristaltic pump as a function of time t during the preparation of two protein layers. The proteins were present for 30 min in the flow cell as indicated by the hatched areas in the lower part. Also the areas of data evaluation (to calculate the step height, noise, and drift) are marked. The enlarged inlay in the upper right shows the resonance frequency with better resolution to estimate the drift and noise.

0.2 mL min-1. Data evaluation took place during this rinsing time as indicated in Figure 4. A linear regression curve was determined over a time interval of 20 min. The resonance frequency decrease caused by each protein layer was calculated by the last value of one regression curve and the starting value of the next. The noise values (in rms) were calculated from the deviation of the measured data to the regression curve and the drift was calculated from the slope of the regression curve. RESULTS AND DISCUSSION Initial experiments showed that the radial mounting of the quartz crystal leads to less mechanical damping than the conventional mounting technique with O-ring seals. Impedance analysis gave a 50% lower value for the resistance as a measure of the mechanical losses of the vibrating quartz.62 Therefore, the mount was finally chosen as indicated in Figure 2. Each point of the impedance measurements (|Y| and θ) requires a time of 0.2 s. A minimum time resolution of 3 s could be reached, because 12 measurement points were the minimum to obtain a reliable linear regression line. The precision and reproducibility of a single measurement point is better than 0.02° for frequencies around 6 MHz. Therefore, the theoretical frequency resolution is better than 10-2 Hz as the slope of the linear regression is quite small (typically -10 to -20 Hz/deg expressed in frequency as a function of the phase angle) and the resonance frequency is given by the intersection with the y axes. The frequency resolution obtained with measurement in air was 0.03 Hz (no drift, with rms, values better than 0.01 Hz). In this approach, we reached a frequency resolution of 0.2 Hz for measurements in liquid, As compared to a conventional oscillator circuit with a frequency resolution of 1 Hz, provided that the damping caused by the liquid did allow for a stable operation at all. The chosen affinity system consists of multivalent compounds. Typically, not all binding sites of the compound binding from solution will be saturated during an incubation step. Therefore, the formation of multilayers is possible by alternating incubation (62) Frubo¨se, C.; Doblhofer, K.; Soares, D. M. Ber. Bunsenges. Phys. Chem. 1993, 97, 475-478.

Figure 5. Resonance frequency as a function of time during the buildup of a protein multilayer with 10 single layers. The oddnumbered layers consist of t-BSA-bi(5), the layers in between of pSA.

Figure 6. Comparison of the frequency decreases per layer during the deposition of 20 protein layers. The odd-numbered layers consist of t-BSA-bi(15), the even-numbered of pSA. The value for layer no. 10 is missing as problems with the flow system prevented the correct data evaluation.

with t-BSA-bi and pSA. This allows us to determine the influence of an increasing layer thickness on the frequency response of the QCM. The frequency change during the buildup of such a multilayer system with t-BSA-bi(5) is shown in Figure 5 as a function of time. After the first incubation step [with t-BSA-bi(5)], which shows a relatively small frequency change, a stable pattern is established. Figure 6 shows the frequency changes per layer for a multilayer of t-BSA-bi(15) and pSA. Above three to four single layers, the frequency response becomes quite reproducible. During deposition of layer 10, problems were caused by air in the flow system. They did prevent a data evaluation but had no influence on the following layer preparation. With the exception of this incubation step, an average frequency decrease of 111 ( 5 Hz for the pSA layers (6, 8, 12-20) and of 54 ( 3 Hz for the t-BSA-bi(15) layers (5-19) was found. This indicates that the deposition process is fairly reproducible, once a dense layer of protein has been established and masks the individual surface properties of the transducer. Deviations during the first layers reflect surface properties that influence the nonspecific adsorption properties of the QCM surface. The frequency response is highly stable, and no decreasing frequency responses with increasing number of layers could be observed (see Figure 6). Typical frequency responses during subsequent incubations of t-BSA-bi(15) and pSA are given in Figure 4. To achieve a quick rise of the protein concentration, the flow rate was increased at

the beginning of the incubation for an interval of 4 min. The system exhibits a delay time of ∼2 min before a substantial change in the resonance frequency indicates binding of protein. An initial, but slight, change in resonance frequency may be due to a change in fluid pressure caused by the change in flow rate. The binding of t-BSA-Bi(15) is rapid (∆f/∆t ≈ -24 Hz min-1, average value for eight binding curves) and the frequency change is almost linear with time, indicating a diffusion-controlled binding process. A final frequency change of ∼45 Hz is reached within 5 min. For the rest of the incubation time, the resonance frequency decreases only slightly. After the incubation, the flow rate is restored to its initial value and the protein is washed out from the flow cell. The final frequency change, drift, and noise were determined over a period of 20 min, as indicated in Figure 4. Over this interval, the baseline was stable with a drift of less than 3 Hz h-1. Thus, the washout of bound protein appeared to be negligible. The noise was not changed if compared with the baseline before layer formation. The time course of the frequency change during the following incubation with pSA was clearly different from the preceding incubation. Again the change in flow rate at the beginning of the incubation time interval leads to a small frequency decrease followed by a more significant effect after the dead time of 2 min. The overall frequency decrease amounts to 100 Hz, but the initial rate of change is only ∼-14 Hz min-1, which is nearly half the value as observed with t-BSA-bi(15). Since the primary binding event is the same for both incubations, and since the concentrations (expressed in mass per volume) of the reactants are the same, this effect must be attributed to differences in the diffusion rate of the reacting compounds. This is expected from the increased molecular weight of the cross-linked pSA molecules. In contrast to the almost linear binding curve of t-BSA-bi(15), the time course of the frequency change for pSA shows strong saturation effects. After the first few minutes at a frequency change of ∼40 Hz, the binding rate begins to decrease reaching almost saturation at the end of the incubation time (∆f/∆t < -0.5 Hz min-1 after 30 min). As the diffusion of the pSA to the QCM surface is constant during the incubation, the decrease in binding rate indicates specific surface processes occur. A likely explanation is that the steric requirements for access of the large pSA molecules to the surface and subsequent binding increase rapidly with increasing surface coverage. Due to the high affinity constant, the binding process can be assumed to be kinetically controlled. This indicates that streptavidin-bound biotin residues will remain bound and dissociation and subsequent rebinding will not happen. Consequently, with increasing surface coverage, the binding process is changed from diffusion controlled to binding rate controlled, where rearrangements within the protein layer are the rate-limiting steps. The time course of the binding reaction indicates that slightly more pSA may be bound for prolonged incubation time. Large effects are not expected. The characteristics of the baseline after rinsing of the flow cell were essentially the same as those after binding of t-BSA-bi(15). A further set of experiments was carried out with another type of biotinylated bovine serum albumin. For this purpose, we chose t-BSA-bi(5) with a reduced amount of biotin residues per protein molecule. The binding of t-BSA-bi(5) was slower than with t-BSAbi(15) with an initial rate of frequency change of ∆f/∆t ≈ -15 Hz min-1. Saturation effects were observed with decreasing binding rate with time (see Figure 5). The overall frequency Analytical Chemistry, Vol. 69, No. 7, April 1, 1997

1445

change was ∼160 Hz for t-BSA-bi(5). The reduced initial binding rate indicates a higher average molecular weight for t-BSA-bi(5). However, the saturation characteristics of the binding curve again are expected to reflect surface processes. We assume that the reduced amount of biotin present on the t-BSA-bi(5) is not sufficient to cover all streptavidin-binding sites within its “footprint”. Therefore, a higher amount of t-BSA-bi(5) may be bound per surface area, which leads to increased sterical requirements for “packing” of the BSA-conjugate molecules. This is reflected by the increased overall frequency change as well as the saturation behavior of the binding curve. The binding of pSA led to a net frequency change of ∼120 Hz, with other wise no significant differences to the previous experiment. The higher binding capacity for pSA is attributed to the differences in the layer structure for the biotin conjugates. Comparative optical investigations on the same affinity system with spectral ellipsometry63 indicate an increase of layer thickness of ∼20 nm/protein layer. These data also show only little variation with the number of layers deposited. The estimated refractive index of the protein layers indicates a high degree of hydration in the protein film. From this optical characterization, a layer thickness of roughly 400 nm can be estimated. This value will now be compared with the acoustical characteristics of the same protein layers. The protein layer is a highly hydrated, soft protein meshwork. One likely explanation of the frequency decrease observed during buildup of the thick protein layer could be viscosity effects: The protein layer could be approximated as a fluid layer with increased viscosity but poor coupling to the QCM oscillation. The sensitivity of the QCM to such effects is expected to decay exponentially with a penetration depth of ∼250 nm in water.50 With increasing viscosity the decay length will grow with η1/2.50 If viscoelastic coupling were predominant, an exponential decay of the observed effects should be found. However, there is no evidence for any decrease in sensitivity of the device. The stable frequency response indicates a stable coupling of the protein layer to the oscillation of the QCM. At the investigated frequency, the protein layer seems to behave as “rigid” solid. This conclusion is supported by the minute changes found for the damping of the QCM. Impedance spectra taken before and after buildup of the protein layer system showed no significant broadening of the resonance curve. This indicates independently that the viscous load of the QCM is not significantly increased by the protein deposition. Additional experiments were subsequently performed with higher sensitivity by using 12-MHz quartz crystals (Figure 7). The observed values are slightly higher than predicted by eq 1. Doubling the resonance frequency of a quartz crystal should cause a 4-fold increase of the frequency shifts. Average frequency decreases of 780 Hz for t-BSA-bi(5), and 572 Hz for pSA, were found. These values systematically exceed the frequency shifts expected from theory by ∼20%. Without further investigations at higher frequencies, an interpretation cannot be given yet for these deviations. The decrease of the frequency shifts from layer 13 to 15 is remarkable. This phenomenon was reproducible in several independent runs of experiments. For thicknesses above 14 protein layers, the overall frequency shift is ∼7.5 kHz. As this frequency shift is more than 0.06% of the resonance frequency, the Sauerbrey equation is expected to be no longer suitable to (63) Spaeth, K.; Brecht, A.; Gauglitz, G. J., submitted to J. Colloid Interface Sci.

1446 Analytical Chemistry, Vol. 69, No. 7, April 1, 1997

Figure 7. Frequency decreases during the deposition of 15 protein layers on a 12-MHz quartz crystal. The odd-numbered layers consist of t-BSA-bi(5), the even-numbered of pSA.

describe the frequency shift of such highly mass loaded quartz crystals.2 The added mass loading is no longer negligible as compared to the mass of the quartz crystal itself. In comparison, the overall frequency shift of the 6-MHz quartz crystal in Figure 6 is less than 0.03% of the resonance frequency. The examination of noise and drift during multilayer formation allows characterization of the analytical performance of the system, including the minimum detectable change in resonance frequency and the systematic changes of QCM performance with increasing layer thickness. The noise was determined as the rms deviation of the baseline from a linear regression curve. The maximum noise was 0.5 Hz, which is due to a systematic deviation of the baseline from the linear approximation used. A mean value of 0.2 Hz rms characterizes the performance of the QCM. No systematic change of the noise values was found during layer formation. The other performance parameters also did not change. Assuming a noise of 0.2 Hz and negligible drift, the minimum detectable change in frequency amounts to be 0.6 Hz. This corresponds to a coverage of less than 1/100 of a protein monolayer. The drift was determined from the difference between the first and the last value of the linear regression curve. The maximum drift observed was (3 Hz h-1. The mean value of the magnitude of drift was 1.6 Hz h-1. There is no systematic change in the drift values during the deposition of the 20 protein monolayers. We assume that this drift is caused by small deviations in the temperature and flow rate. With the assumption that the buildup of one monolayer takes 20 min, and assuming a noise value of 0.2 Hz, the overall minimum detectable change in resonance frequency is 1.1 Hz. This corresponds to 1/40-1/150 of a protein layer, depending on the molecular weight and covered area per molecule. We found comparable performance values for the 12MHz quartz crystals. The noise value was in the order of 1 Hz and the magnitude of drift around 5 Hz h-1. This indicates no decrease in the limit of detection for higher frequencies. The limited precision and reproducibility of our impedance analyzer is with its frequency limit of 13 MHz may be the reason for no gain in sensitivity at higher frequencies. The frequency response observed per protein layer can be converted to an increase in the mass loading by assuming Sauerbrey-type behavior of the system. From eq 1 an average mass protein coverage of 10 ng [for the first experiment with t-BSA-bi(15)] and 17 ng mm-2 [for the second measurement with t-BSA-bi(5)] results. These values clearly exceed the values for

Table 1. Comparison of Frequency Decreases ∆f and Corresponding Mass Changes ∆m of 6-MHz Quartz Crystalsa no. of layers ∆f in PBS (Hz) ∆f in air (Hz) (∆f in PBS)/(∆f in air) ∆m per mass and area in PBS according to the Sauerbrey equation (ng/mm2) ∆m in air per layer and area calculated from the described calibration (ng/mm2)

10 1342.0 326.6 4.11 16.49

4 616.0 148.0 4.16 18.91

5.05

5.73

a Measured on-line in PBS and calculated from the resonance frequency measured before and after the protein multilayer’s preparation in air.

Figure 8. Frequency decrease as a function of deposited mass of protein. The measured data are average values of 4-10 single measurements. The standard deviation is indicated by error bars. The linear regression curve is used as calibration curve; its slope is 0.1678 Hz ng-1 (corresponding 6.465 Hz ng-1 mm2).

typical protein monolayer coverages found in a variety of other studies (∼5 ng mm-2 for protein monolayers). From the abovementioned optical studies,59 a nominal protein coverage of less than 10 ng mm-2 was estimated. The recorded frequency changes clearly exceed the expected values. To elucidate this difference, experiments were carried out in which the frequency response of the QCM in air and in buffer were recorded comparatively during protein multilayer formation. After recording the frequency change upon layer formation in buffer, the layers were rinsed with bidistilled water. This treatment reduces artifacts due to retention of buffer compounds (salts) within the protein layer, but does not affect the biotin-streptavidin interaction. Afterward, the protein layer was dried carefully in the flow cell in an argon stream until the frequency remained stable and the net frequency change in air was determined (see Table 1). The Sauerbrey theory is based on the assumption that mass loading attached to the QCM shows ideal acoustic coupling to the crystal surface and the crystal is an infinite plane. A simple calibration of the QCM with protein solutions can therefore be made, which is based on this theory. For this purpose, known amounts of protein in bidistilled water were deposited on a QCM and dried until a stable resonance frequency was obtained. Due to this simple experimental procedure, the data show considerable scatter. However, the calibration graph (Figure 8) is linear, and a response of ∼0.168 ( 0.015 Hz ng-1 was found (or 6.47 ( 0.58 Hz ng-1 mm2 if related to the unit area). Based on this calibration, mass loading values were determined (Table 1). As indicated

above and also reported by other authors,49-52,64 the measured frequency changes in water exceed the response in air. We found a ∼4-fold increase of the response (4.13 exactly as mean value of two measurements, see Table 1). Using this calibration data, we found an average mass loading of 5 ng mm-2 per protein layer (see Table 1). Therefore, the minimum detectable protein mass loading is 0.04 ng mm-2. As our results indicate no major influence from viscosity effects on the QCM recording, we assume that this difference is due to the water content of the protein layer. From the refractive index values found in the optical characterization of the protein layers and the refractive index change with protein concentration,65 a water content in affinity bound protein layers up to 70% can be estimated. The high QCM response can therefore be ascribed to water entrapped in the protein layer. This concerns not only tightly bound “structural” water, which accounts typically for only for a few percent of the protein molecular weight,66 but also involves all water molecules present within the protein layer. We assume, therefore, that the entire highly hydrated protein layer couples rigidly to the oscillation of the QCM. The stable response of the QCM to increasing protein layer thickness rules out viscosity effects (which might also lead to a change in the response of the QCM in liquids as was discussed for QCM loaded with extended DNA chains60). SUMMARY AND CONCLUSIONS Real-time studies on biospecific interaction processes are becoming increasingly popular. Mass-sensitive devices are particularly suitable transducers in this field of research. As an example, Tom-Moy et al. showed for a mass-sensitive immunoassay the identification of atrazin with a limit of detection below 1 ppb.67 Mass changes down to a few picograms per square millimeter could be resolved with a SAW device operating in the 100-MHz range. Our results show that the simpler bulk acoustic wave QCM also can be operated in liquids with good performance. Careful mounting of the QCM sensors and evaluation of the admittance curve allow a frequency resolution (rms noise) of 0.2 Hz. Noise and drift values are comparable to QCM operation in the gas phase.68 This leads to a minimum of detection of less than 1% of a protein monolayer (for typical molecular weights in the range of 50.000-150.000). The presented data show that BAW devices are well suited as a tool in sensitive biospecific interaction measurements. We have also studied the response of the device for high overlayer thicknesses with protein loadings up to a few hundred nanometer layer thickness (∼100 ng/mm2 protein loading). A biotin-avidinbased system allowed the stable and reproducible formation of protein multilayers. No change in device sensitivity or device performance was detected with increasing layer thickness. This indicates rigid coupling of hydrated protein layers to the QCM oscillation. Viscosity effects play a negligible role in the device response. The linear response with increasing thickness makes the BAW device suitable for simple quantitative studies over a wide range of surface coverage of such biosystems. For 12-MHz (64) Su, H.; Thompson, M. Biosens. Bioelectron. 1995, 10, 329-340. (65) De Fejter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17, 1759. (66) Bone, S.; B. Bioelectronics; John Wiley & Sons Ltd.: Chichester, England, 1992; pp 89-129. (67) Tom-Moy, M.; Baers, R. L.; Spira-Solomon, D.; Doherty, T. P. Anal. Chem. 1995, 67, 1510. (68) Bodenho ¨fer, K.; Hierlemann, A.; Noetzel, G.; Weimar, U.; Go ¨pel, W. Anal. Chem. 1996, 68, 2210-2218.

Analytical Chemistry, Vol. 69, No. 7, April 1, 1997

1447

devices, a change in the simple calibration slope was found for frequency changes exceeding 0.05% of the resonance frequency. The frequency responses of our QCM in situ exceed the values expected from Sauerbrey’s equation significantly. We elucidated this effect by a comparative study of responses of protein multilayers in air and in water. A additional water content of ∼70% was found in the water if compared to the air. If rigid coupling of proteins and water in the protein film is assumed, the 4-fold increase of frequency changes between measurements in air and in water can be explained quantitatively. An additional benefit of the QCM analysis is an increased device sensitivity for operation in aqueous media, with correspondingly lower limits of detection. For rigid couplings of the layer, simple models may be applied to explain the data. This makes the use of extended polymer matrices promising as operation is possible in increased interaction volumes. This approach has successfully been used by Pharmacia Biosensors in their BIACore optical device. The versatile goldthiol chemistry, which can be readily applied to QCMs, should make this approach feasible. Further work aims at estimating the limit in thickness for the observed rigid coupling in the frequency domain. The effect of

1448

Analytical Chemistry, Vol. 69, No. 7, April 1, 1997

increased sensitivity in water will be further investigated by carefully chosen model systems, which allow the preparation of well-defined submonolayers of protein. In this case, it might be possible to distinguish between effects mediated by the water attached directly to the single protein molecules and by the water entrapped within a cross-linked protein layer. Finally, QCM devices are operated with simple hardware of limited complexity and cost and are, therefore, attractive for bioaffinity studies. ACKNOWLEDGMENT The authors thank Tilo Weiss for preparing the gold electrodes of the quartz crystals used for all experiments. We gratefully acknowledge Boehringer Mannheim (FRG) for the donation of the proteins.

Received for review August 28, 1996. Accepted January 21, 1997.X AC960875P X

Abstract published in Advance ACS Abstracts, March 1, 1997.