Use of Piezoelectric-Excited Millimeter-Sized Cantilever Sensors To

Anal. Chem. 2006, 78, 2328-2334

Use of Piezoelectric-Excited Millimeter-Sized Cantilever Sensors To Measure Albumin Interaction with Self-Assembled Monolayers of Alkanethiols Having Different Functional Headgroups Gossett A. Campbell and Raj Mutharasan*

Department of Chemical Engineering, Drexel University, 32nd and Chestnut Streets, Philadelphia, Pennsylvania 19104

In this paper, we describe a new modality of measuring human serum albumin (HSA) adsorption continuously on CH3-, COOH-, and OH-terminated self-assembled monolayers (SAMs) of C11-alkanethiols and the direct quantification of the adsorbed amount. A gold-coated piezoelectric-excited millimeter-sized cantilever (PEMC) sensor of 6-mm2 sensing area was fabricated, where resonant frequency decreases upon mass increase. The resonant frequency in air of the detection peak was 45.5 ( 0.01 kHz. SAMs of C11-thiols (in absolute ethanol) with different end groups was prepared on the PEMC sensor and then exposed to buffer solution containing HSA at 10 µg/ mL. The resonant frequency decreased exponentially and reached a steady-state value within 30 min. The decrease in resonant frequency indicates that the mass of the sensor increased due to HSA adsorption onto the SAM layer. The frequency change obtained for the HSA adsorption on CH3-, COOH-, and OH-terminated SAM were 520.8 ( 8.6 (n ) 3), 290.4 ( 6.1 (n ) 2), and 210.6 ( 8.1 Hz (n ) 3), respectively. These results confirm prior conclusions that albumin adsorption decreased in the order, CH3 > COOH > OH. Observed binding rate constants were 0.163 ( 0.003, 0.248 ( 0.006, and 0.381 ( 0.001 min-1, for methyl, carboxylic, and hydroxyl end groups, respectively. The significance of the results reported here is that both the formation of selfassembled monolayers and adsorption of serum protein onto the formed layer can be measured continuously, and quantification of the adsorbed amount can be determined directly. Adsorption of proteins on solid surfaces and the evaluation of their characteristics for comparison to the native protein are of general interest in biomedical1-3 and biomaterials area and in efforts to design biocompatible surfaces.4,5 Protein adsorption on synthetic material is a complex process that involves conforma* Corresponding author. Tel.: (215) 895-2236. Fax: (215) 895-5837. E-mail: [email protected]. (1) Yan, J.; Dong, S. J. Electroanal. Chem. 1997, 440, 229-238. (2) Ostuni, E.; Yan, L.; Whitesides, G. M. Colloids. Surf., B 1999, 15, 3-30. (3) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714-10721.

2328 Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

tional changes, orientation and rearrangement, and protein detachment, which affects the protein activity.6,7 Therefore, there is a need for a rapid real-time protein adsorption technique to characterize the adsorption process on various surfaces. The wellcharacterized self-assembled monolayer (SAM) of alkanethiol with different terminal function groups on gold surface is a general model system for studying the adsorption of proteins and has been used by several researchers for biointerface design.8-11 The functional end groups provide proper orientation and molecular organization when SAM is formed on a gold surface. Several realtime techniques have been used to measure protein adsorption on solid surfaces via an alkanethiolate monolayer. They include acoustic plate mode sensors,12 chronopotentiometry,13 and piezoelectric quartz crystal sensors.14,15 Although these methods offer excellent platforms for characterizing protein adsorption on surfaces, their sensitivity per unit area is low.29 On the other hand, (4) Andrade, J. D. Surface and Interfacial Aspects of Biomedical Polymers: Protein Adsorption; Plenum: New York, 1985. (5) Tang, L.; Jiang, W.; Welty, S. E. J. Biomed. Mater. Res. 2002, 62, 471-477. (6) Mrksich, M.; Whitesides, G. M. Annu. Rev. Biophys. Biomol. Struct. 1996, 25, 55-78. (7) Mrksich M.; Whitesides G. M. Trends Biotechnol. 1995, 13, 228-235. (8) Silin, V.; Weetall, H.; Vanderah, D. J. Colliod Interface Sci. 1997, 185, 94103. (9) Kalltorp, M.; Carle´n, A.; Thomen, P.; Olsson, J.; Tengvall, P. J. Mater. Sci.: Mater. Med. 2000, 11, 191-199. (10) Wadu-Mesthrige, K.; Amro, N. A.; Liu, G. Scanning 2000, 22, 380-388. (11) Margel, S.; Vogler, E. A.; Firment, L.; Watt, T.; Haynie, S.; Sogah, D. Y. J. Biomed. Mater. Res. 1993, 27, 1463-1476. (12) Dahint, R.; Seigel, R. R.; Harper, P.; Grunze, M.; Josse, F. Sens. Actuators, B 1996, 35-36, 497-505. (13) Martins, M. C. L.; Fonseca, C.; Barbosa, M. A.; Ratner, B. D. Biomaterials 2003, 24, 3697-3706. (14) Shen, D.; Wu, X.; Liu, X.; Kang, Q.; Chen, S. Microchem. J. 1999, 63, 322332. (15) Shen, D.; Huang, M.; Chow, L.; Yang, M. Sens. Actuators, B 2001, 77, 664670. (16) Campbell, G. A.; Mutharasan, R. Biosens. Bioelectron. 2005, 21, 597-607. (17) Campbell, G. A.; Mutharasan, R. Langmuir 2005, 21, 11568-11573. (18) Naik, T.; Longmire, E. K.; Mantell, S. C. Sens. Actuators, A 2003, 102, 240254. (19) Campbell, G. A.; Mutharasan, R. Biosens. Bioelectron. 2005, 21, 462-473. (20) Cass, T.; Ligler, F. S. Immobilized Biomolecules in Analysis: A Practical Approach; Oxford University Press Inc.: New York, 1998; p 56. (21) Jin, Q.; Rodriguez, J. A. Surf. Sci. 1999, 425, 101. (22) Kapur, R.; Rudolph, A. S. Exp. Cells Res. 1998, 244, 275-285. (23) Faucheux, N.; Schweiss, R.; Lu ¨ tzow, K.; Werner, C.; Groth, T. Biomaterials 2004, 25, 2721-2730. 10.1021/ac0517491 CCC: $33.50

© 2006 American Chemical Society Published on Web 02/28/2006

a piezoelectric-excited millimeter-sized cantilever sensor (PEMC) offers high sensitivity and has been used successfully to monitor the self-assembly of 1-hexadecanethiol17 and detection of protein.16 The electromechanical resonator operates on the converse piezoelectric effect where the application of an electric field creates a strain. The sensor is a composite structure of two layers: a piezoelectric (lead zirconate titanate, PZT) layer and a gold coated stainless steel layer, a few millimeters in length. The piezoelectric layer attached to the sensor’s base acts as both an actuating and a sensing element. Resonance of the oscillating sensor depends on its geometric properties. The mass change of PEMC sensors is due to adsorption of target analyte to the sensor surface. Once the target of interest binds to the sensing surface, the effective mass of the sensor increases, which decreases its resonant frequency. Therefore, by monitoring the resonant frequency change a quantitative measure of adsorption can be done. Recently Martins et al.,13 used chronopotentiometry techniques to determine the adsorption of human serum albumin (HSA, 10 µg/mL) on SAMs of various functional end groups: carboxyl (COOH), hydroxyl (OH), and methyl (CH3). They concluded that the adsorption of HSA increased in the following order: CH3 > COOH > OH. In this paper, we report the adsorption behavior of HSA on a self-assembled monolayer of the same set of end groups using a new modality: a PEMC sensor. We have used the same reagents, namely, alkanethiols and HSA, from the same source as did Martins et al.13 CANTILEVER PHYSICS PEMC sensors are constructed from a piezoelectric layer (PZT) and a stainless steel layer of different lengths to provide bending in the structure as it resonates. The PZT layer is ferroelectric, and therefore, the application of an ac electric field across its thickness strains the layer, a phenomenon known as the converse piezoelectric effect. The piezoelectric effect is a result of charge displacement within the PZT layer that results in a net dipole moment. The bottom surface of the PZT is bound to the nonpiezoelectric stainless steel layer, and as a result, the stainless steel layer undergoes flexural deformation. The higher than normal deformation that occurs at resonance generates a potential difference across the PZT, due to the converse piezoelectric effect. As the effective mass of the sensor changes (adsorption of analyte), the response is reflected in the electrical signal due to the electromechanical coupling. The adsorption of analyte increases the cantilever’s effective mass, which decreases the resonant frequency. The resonant frequency is proportional to the inverse square root of the sensor’s effective mass, and thus, the change in resonant frequency with time can be used to provide quantitative measures of mass change. The resonant frequency of an oscillating rectangular PEMC sensor in air can be expressed by the classical expression18

fn ) knxK/Me

(1)

the first four eigenvalues for a rectangular cantilever. The parameter K is the effective composite structure spring constant and depends on the thickness (t), width (w), length (L), and the Young’s modulus (E) of the cantilever material, namely, both stainless steel and PZT.17

K)

3w2(Ep2tp4 + Es2ts4 + 2EpEstpts(2tp2 + 2ts2 + 3tsts)) 12Lp3(Eptp + Ests)

where subscripts p and s refer to PZT and stainless steel, respectively. Me is the effective mass of the composite structure in air and can be approximated as16

Me ) 0.236(Fptp + Fsts)wLp + Fstsw(L - Lp)

(3)

where L is the overall length of the cantilever and F is the density. In the above, we have approximated the bilayer region (PZT and stainless steel) as a concentrated mass located at Lp from the fixed end of the cantilever. The stainless steel overhang is approximated as a concentrated mass at the same position. One notes that the bending modulus or flexural rigidity (D ) KLp3/3w) of the twolayer composite is significantly larger than the single stainless steel layer. The resonant frequency of the oscillating PEMC sensor in liquid-phase detection is written to include both the added mass of liquid and the mass of adsorbed analyte as

x

f′nf ) kn

K Me + mae + ∆m

(4)

where f′nf is the resonant frequency of the nth mode in fluid when analyte of mass, ∆m, adsorbs at the sensor tip. The term mae is the effective added oscillating mass of fluid at the cantilever tip and can be calculated, from experimentally determined resonant frequency in air (fn) and the resonant frequency in fluid (fnf) when no analyte has been adsorbed. That is

mae ) (fn2/f′nf2 - 1)Me

(5)

eq 4 can be rearranged as

1 ∆m fnf - f′nf ) fnf 2 Me + mae

(6)

where (fnf - f′nf) is the change in resonant frequency of the nth mode in liquid due to analyte attachment. The change in resonant frequency represented by the left-hand side of eq 6 is linearly dependent on the change in mass of analyte, ∆m, at the resonant mode of interest. In this paper, we measure the change in resonant frequency (fnf - f′nf) under various measurement conditions. Equation 6 can be rearranged to

where kn ) 0.1568, 0.9827, 2.7517, and 5.3923 corresponding to (24) Blawas, A. S.; Reichert, W. M. Biomaterials 1998, 19, 9, 595-609. (25) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 3464-3474. (26) Hederos, M.; Konradsson, P.; Liedberg, B. Langmuir 2005, 21, 2971-2980. (27) Holmlin, R. E.; Chen, X.; Chapman, R. G.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 2841-2850.

(2)

σn )

2(Me + mae) ∆m ) fnf - f′nf fnf

(7)

where σn is the mass change sensitivity of the nth mode. The left-hand side of eq 7 represents the mass change that causes Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

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unit frequency shift, generally expressed in g/Hz. Note that smaller values of Me and higher frequency, fnf, provides for a sensitive measurement. For instance, if the mass of PEMC is in the order of 1 mg and the frequency is 50 kHz, then the mass sensitivity achievable is 20 ng/Hz. If the sensor is miniaturized, the parameter Me in eq 7 decreases resulting in a more sensitive sensor. Miniaturization also compromises measurement under liquid immersion conditions.18,30 MATERIALS AND METHODS Cantilever Fabrication. The PZT-stainless steel cantilever was fabricated using two main components: a 127-µm-thick PZT single sheet (Piezo Systems Inc., Cambridge, MA) and a 50-µmthick stainless steel foil (Alfa Aesar, Ward Hill, MA). The PZT and stainless steel foil were cut to 5 mm × 2 mm (length × width) and 10.5 mm × 2 mm, respectively. The stainless steel foil was bonded to the PZT layer with a conductive epoxy (GC Electronics, Rockford, IL) such that 3.5 mm of the stainless steel layer protrudes out at one end and 2 mm at the other end. A LCR meter was used to ensure electrical integrity of the PZT layer as indicated by its impedance (typically 0.13 MΩ at 1 kHz), capacitance value (typically 1.45 nF at 1 kHz), and dissipation factor (typically 0.015 at 1 kHz). The sensing stainless steel end (3.5 mm) of the bilayer cantilever was cleaned and coated with chromium (∼5 nm thick) followed by the vapor deposition of a 10-nm gold layer, at the University of Pennsylvania microfabrication laboratory. The gold film yielded a polycrystalline, predominantly (111) surface. The top nickel surface of PZT, which serves as the top electrode, and the non-gold-plated stainless steel end (2 mm), which serves as the bottom electrode, were connectorized using 30-gauge copper wire soldered to BNC couplers. The electrode end of the cantilever was encapsulated in a glass tube by a nonconductive epoxy. A schematic illustration of the sensor and the experimental arrangement used are given in Figure 1. In Figure 1 (panel A) it can be seen that the gold-plated stainless steel layer at the cantilever free end is longer than the PZT layer to allow for self-assembled monolayer adsorption. Reagents. 1-Decanethiol (SH-(CH2)9CH3, 96%), 11-mercapto1-undecanol (SH-(CH2)11OH, 97%), 11-mercaptoundecanoic acid (SH-(CH2)10COOH, 95%), concentrated sulfuric acid, hydrogen peroxide, and ethanol (99.8%) were purchased from Sigma-Aldrich (Allentown, PA). Deionized water used was from a Milli-Q plus ultrapure water system (18.2 MΩ‚cm). Gold Substrate, Monolayer Formation, and HSA Interaction. The gold-coated sensing stainless steel surface was cleaned with a freshly prepared piranha solution (1:3 H2O2 (30%) to concentrated H2SO4) for 2 min (Caution: this reaction is exothermic, and the solution reacts violently with many organic materials and, thus, should be handled with great care.). The sensing gold surface was then rinsed three times in deionized water and absolute ethanol and then air-dried and used immediately. One-milliliter stock solutions (1-decanethiol, 11-mercapto-1undecanol, 11-mercaptoundecanoic acid) were diluted with absolute ethanol to a final concentration of 1 mM. The samples were (28) Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 6336-6343. (29) Kirstein, S.; Mertesdorf, M.; Schonhoff, M. J. Appl. Phys. 1998, 84, 17821790. (30) Sader, J. E. J. Appl. Phys. 1998, 84, 64-76.

2330 Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

Figure 1. Panel A: Two-dimensional cross sectional schematic of the PEMC sensor. The cantilever free end consists of three main components: a stainless steel layer 50 µm thick, adhesive 10 µm thick, and PZT 127 µm thick. The stainless steel layer was gold coated with a 100-Å-thick layer. Panel B: Schematic illustration of the experimental setup.

kept in sealed containers at 4 °C until use. Care was taken to minimize evaporative loss of ethanol by conducting each experiment in a temperature-controlled chamber, saturated with ethanol, humidity 92%, maintained at 25 ( 0.2 °C. During a typical 1-h experiment, the liquid level in the sample container (1 mL) decreased slightly (∼4 µm/min). The linear change in liquid level with time resulted in a linear change in resonant frequency. Thus, immediately before and after an experiment, the rate of change of resonant frequency due to liquid level change was measured and an average value of the two was used to correct the peak position in the detection experiment. Prior to each measurement, the sample container (1 mL) was rinsed with the solution of interest. A self-assembled thiol monolayer was formed on the sensor surface by immersion of the clean sensor 1.5 mm into the alkanethiol solution (1 mL of 1 mM thiol solution) for 1 h. The formation of the thiol monolayer on the cantilever gold surface was measured by the resonant frequency response.

HSA solution was prepared in phosphate-buffered saline (PBS, pH 7.4) to a final concentration of 10 µg/mL. Upon formation of the alkanethiolate monolayer on the sensor surface, the sensing area was rinsed in PBS and then exposed to the HSA (10 µg/ mL) solution until equilibrium was achieved as indicated by constant resonant frequency of PEMC. The SAM-protein interaction was monitored by tracking the measured resonant frequency of the sensor. Experimental Arrangement. The cantilever sensor was mounted in a clamp between two vertical parallel aluminum plates. The clamp was attached to an XYZ-position stage (Optosigma Corp., Santa Ana, CA) placed on a level vibration-free table so that the sensor was positioned perpendicular to the liquid sample level. The XYZ manipulator permitted vertical positioning of the cantilever into the liquid sample at 1.5-mm immersion depth with (10 µm accuracy. The translation of the cantilever tip was always made in a downward direction, without reversing, to avoid formation of residual liquid film on the cantilever detecting gold surface. The cantilever electrodes were connected to an impedance analyzer (Agilent, HP4192A) that was interfaced to a PC for continuous measurements of impedance, phase angle, and amplitude ratio with an excitation voltage of 100 mV; see Figure 1, panel B. PEMC Sensor Calibration. The mass change sensitivity of the PEMC sensor in air was determined by adding a known point mass of paraffin wax at the cantilever tip. A small fragment of the paraffin wax (0.23 mg) was dissolved in 4 mL of hexane solution. A 0.5-µL aliquot was dispensed into 10 weighing dishes (the weight of each dish was recorded before dispensing the solution), and the dishes were left to dry in a fume hood for 30 min, after which each dish was reweighed and the mass of wax dispersed was computed from the difference between the dish and dish plus wax mass measurement. The frequency of the resonant mode used in the detection experiment was measured in air. The mass change sensitivity of the sensor was determined by dispensing 0.5 µL of the paraffin-hexane solution on the sensor tip, dried for 30 min in the fume hood, and the resonant frequency of the detection mode was measured immediately. The procedure was repeated for six different mass changes and five times for each mass change. The resonant frequency changes resulting from these specific paraffin mass changes were plotted to determine experimental measure of the mass change sensitivity of the PEMC sensor used in this study. RESULTS AND DISCUSSIONS Characterization of the PEMC Sensor. The resonance characteristics of the sensor are presented in Figure 2. The resonant spectrum, a plot of phase angle versus excitation frequency, exhibits resonance modes in air at 12.5, 27.5, and 45.5 kHz in the frequency range 1-100 kHz. Several repeat experiments showed that these resonant frequencies were within (50 Hz of each other when measured at different times. In any single experiment, the resonant frequency remained constant within (5 Hz, suggesting that the basic features of the sensor were stable. The day-to-day variations ((5 Hz) are thought to be due to changes in the local temperature. The resonant peaks are followed by a sharp change in phase angle from -90°. The first peak is the fundamental mode, and the higher modes follow consecutively. Upon immersion of the sensor in liquid ethanol, the resonant

Figure 2. Resonance spectrum of PEMC sensor in air (solid line) and in ethanol (broken line). The spectrum is a plot of phase angle versus excitation frequency. The fundamental (first), second, and third modes in air occurred at 12.5 ( 0.05, 27.5 ( 0.05, and 45.5 ( 0.05 kHz and in ethanol were 10.5 ( 0.05, 25 ( 0.05, and 39.5 ( 0.05 kHz, respectively. Resonance is followed by a sharp change in the phase angle. The sensor was excitated with 100 mV.

frequencies decreased (peaks shifted to the left) due to the increase in the effective mass of the sensor caused by the added mass of ethanol. Also, the peak heights decreased due to mass damping. The change in resonant frequency upon liquid immersion is a measure of peak sensitivity; the larger the change in resonant frequency the more sensitive the sensor. The change in resonant frequency from air to ethanol for the first, second, and third modes were 2, 2.5, and 6 kHz, respectively; see Figure 2, broken lines. In this study, the third resonant mode, 39.5 kHz in ethanol, was used due to the sharpness of the peak under liquidimmersed conditions and the greater response upon immersion. The sharpness of the peaks is defined by the quality factor (Q value) determined from the ratio of the resonant frequency to the peak width at half the peak height. The larger the Q value, the more suitable a resonant peak is for detection. The Q values in air were 25.4 ( 0.1, 39.2 ( 0.1, and 50.9 ( 0.2 and in ethanol 17.5 ( 0.1, 27.7 ( 0.1, and 43.9 ( 0.2 for the first, second, and third modes, respectively. SAM Formation. In Figure 3, we show how the third flexural mode resonant frequency changes as a function of time, during the self-assembled monolayer formation of 1-decanethiol, 11mercapto-1-undecanol, and 11-mercaptoundecanoic acid from a 1-mL, 1 mM solution in absolute ethanol. Each experiment was repeated at least twice, and the data shown are typical of the results obtained. The resonant frequency showed an exponential decrease before reaching a constant value. The steady-state resonant frequency change observed suggests that a stable monolayer was formed on the gold-coated sensing surface. 11Mercaptoundecanoic acid showed the largest change in resonant frequency followed by 11-mercapto-1-undecanol, and then 1-decanethiol. The total resonant frequency change obtained for SH(CH2)10COOH, SH-(CH2)11OH, and SH-(CH2)9CH3 were 885 ( 21 (n ) 2), 590 ( 14 (n ) 2), and 383 ( 10 (n ) 2) Hz, respectively. These results suggest that the monolayer formation depends on the functional headgroup of the alkanethiolate. Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

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Table 1. Calculated Parameters of the Second Flexural Mode Resonant Frequency spring constant, K × 10-3 (N/m) (eq 2) effective mass Me × 106 (kg) (eq 3) mae, 1.5-mm immersion in ethanol 107 (kg) (eq 4) σ3a × 109 (g/Hz) exptl σ3f × 109 (g/Hz) (eq 7)

Figure 3. Resonant frequency change for the adsorption of the various alkanethiols on the gold-coated PEMC sensor surface. The terminal end groups were CH3, OH, and COOH. Control response was in thiol-free ethanol solution.

corresponding resonant frequency change, Figure 4 panel B, of the detection peak yielded a straight line whose slope is the mass change sensitivity in air, 6.49 ( 1.40 ng/Hz. This suggests that if resonant frequency resolution is 1 Hz, then mass changes in the range 5.09-7.89 ng are discernible. The mass change sensitivity in liquid was then calculated from the sensitivity in air using a model equation developed in our earlier work.19

( )

σnf ) σna

Figure 4. Panel A: Plot of resonant peak as known mass of wax was added to the PEMC sensor’s tip. For clarity the figure shows the resonant peaks of the 47.8, 143.4, 239, 334.4, and 382.2 ng of added paraffin wax, respectively. Panel B: Plot of known mass change versus the corresponding resonant frequency change. The mass change sensitivity of the PEMC sensor in air was determined from the slope as 6.497 ng/Hz.

The mass change sensitivity of the sensor was determined using the known tip mass technique discussed in PEMC Sensor Calibration. The mass of wax in 0.5 µL of the paraffin-hexane solution was determined as 47.8 ( 12.2 ng (n ) 5). As the known mass of wax was added to the cantilever tip, the resonant frequency decreased, which is verified by the shifted in the resonant peak to the left (see Figure 4, panel A). For clarity only, the first, third, fifth, seventh, and eighth mass additions are presented in panel A, due to the small shifts in the resonant peak. A plot of the known mass change to the sensor tip against the 2332

Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

6.35 2.97 9.71 6.49 9.74

Mef fn Me fnf

(8)

where σna and σnf are the mass change sensitivity of the nth mode in air and in fluid, respectively. Mef is the effective mass of the sensor under liquid immersion, and therefore, Mef ) Me + mae. Calculated values for Me, mae, K, and σnf are given in Table 1. The mass change sensitivity of the third mode (detection peak) under liquid immersion (σ3f) was determined as 9.74 ( 1.4 ng/Hz. Using the mass change sensitivity, the mass of adsorbed thiol molecules on the sensor gold surface after 1 h were 3.73 ( 0.45, 5.75 ( 1.53, and 8.62 ( 1.38 µg corresponding to 1-decanethiol, 11mercapto-1-undecanol, and 11-mercaptoundecanoic acid, respectively. The sensor used in this investigation has a sensing area of 6 mm2. For complete monolayer coverage of the alkanethiols, 7.2 × 1013 molecules were calculated to be absorbed on the goldsensing cantilever surface. The sulfur atom at the end of each thiol molecule binds at the Au (111) crystal lattice sites with the closest member ∼5 Å.20,21 In this study, the sample volume was 1 mL and the concentration of each sample was 1 mM. Therefore, theoretically there were enough thiol molecules (6.023 × 1020 molecules) to fully cover the sensing area in each of the experiments. From the calculated value of the number of thiol molecules in a monolayer, the mass of the three monolayers were determined as 21, 24, and 26 ng corresponding to 1-decanethiol, 11-mercapto-1-undecanol, and 11-mercaptoundecanoic acid monolayer, respectively. It is clear from this calculation that the mass changes obtained from the sensitivity value are 2 orders of magnitude higher. In the literature, the surfaces of self-assembled monolayers with different terminal end groups have been reported by several authors.22,23 Faucheux et al.23 showed that a selfassembled monolayer with the following terminal headgroups, methyl, hydroxyl, and carboxyl, creates hydrophobic, wettable, and moderately wettable surfaces, respectively. Therefore, one possible explanation for the higher mass determined by the sensitivity is that the ethanol solution penetrates the monolayers and as a result adds to the mass change on the sensor. It is also suggested that the three monolayers allow different levels of penetration. The hydrophobic nature of the 1-decanethiol monolayer provides the least penetration due to the polar nature of the

Figure 5. Resonant frequency change for the adsorption of HSA on the different SAM-terminated functional end groups. The control response is typical of a SAM monolayer exposed to HSA-free PBS solution.

ethanol molecules. On the other hand, the amphipathic nature of 11-mercapto-1-undecanol and 11-mercaptoundecanoic acid allows greater penetration and more ethanol molecule in the monolayer, resulting in a larger resonant frequency change and the corresponding calculated mass change. Human Serum Albumin Adsorption. The response of the cantilever to HSA interaction to SAMs terminated with CH3, OH, and COOH end groups is presented in Figure 5. The concentration of HSA used in each of the experiments was 10 µg/mL. Upon exposure of the monolayer to the serum protein, the resonant frequency decreased immediately and reached a steady-state value within 5-30 min. For the three terminal groups, the total change in resonant frequency was 520, 290, and 210 Hz, respectively. In the case of OH-terminated SAM, the interaction was rapid and steady state was achieved in 6 min. On the other hand, for COOH and CH3 functional groups, the response was considerably longer, taking 20-30 min. All the responses were exponential (see Figure 5), suggesting binding or phase-transfer behavior. The interaction of HSA to SAM terminated with CH3 gave the largest total resonant frequency change of 520.8 ( 8.6 (n ) 3) Hz. The total resonant frequency change of the COOH- and OH-terminated selfassembled monolayer to HSA interaction was significantly less than CH3-HSA response and was slightly different from each other. The total frequency response of the COOH- and OHterminated SAM to HSA interaction were 290.4 ( 6.1 (n ) 2) and 210.6 ( 8.1 (n ) 3) Hz, respectively. It is clear from Figure 5 that the initial adsorption kinetics for the different types of HSA interaction was approximately the same. It is worth noting that the CH3-terminated SAM formation gave the lowest total resonant frequency change and, however, showed the largest frequency response upon exposure to the serum protein solution. Protein conformation on artificial surfaces is an active research area.8,24 Protein adsorption is complex and is difficult to predict.26 Monolayers have been shown to possess at least three different characteristics to prevent protein adsorption. These monolayers should be hydrophilic, contain hydrogen bond acceptors, and have an overall electrical neutrality.27,28 Hydrophobic surfaces have been shown to exhibit high albumin adsorption, while hydrophilic surfaces have low albumin affinity.13,25 Using the mass change sensitivity of the sensor in liquid, 9.74 ng/Hz, the mass changes per unit area of sensor surface to HSA adsorption on CH3-, COO-,

Figure 6. Panel A: Initial adsorption kinetics for the adsorption of alkanethiols on to the gold-plated sensor surface. Correlation coefficients range from 0.98 to 0.99. Panel B: Initial kinetic analysis for the binding of HSA to self-assembled thiol monolayers having different terminal end groups. The fits are good with correlation coefficient ranged from 0.96 to 0.98. The slope of each line gives the observed characteristic binding rate kobs.

and OH-terminated SAMs were determined as 0.84 ( 0.01, 0.47 ( 0.02, and 0.34 ( 0.01 µg/mm2, respectively. Kinetics of HSA Adsorption. The adsorption of 1-hexadecanethiol17 and antibody-antigen binding19 on PEMC sensors have been shown to obey Langmuir kinetics. Here, we examine wether HSA adsorption on the various thiol monolayers with different functional end groups may obey the same kinetics. At time close to τ ) 0, HSA concentration gradient between the sensor surface and the bulk sample is small, and thus, diffusion effects may be ignored. The model is expressed as17

(∆f) ) (∆f∞)(1 - e-kobsτ)

(9)

where (∆f) is the change in resonant frequency at time (τ), (∆f∞) is the steady-state resonant frequency change, and kobs is the observed adsorption rate constant.17 Equation 9 can be rearranged as

(

ln

)

(∆f∞) - (∆f) (∆f∞)

) - kobsτ

(10)

The observed rate constant kobs during the initial time (far from equilibrium) can be determined from a plot of the left-hand side Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

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versusτ of eq 10 and is shown in Figure 6, where we have considered data obtained during the first 9 min. In Figure 6A, the observed adsorption rate constant (kobs) for the adsorption of the various alkanethiols to the sensor gold surface was determined as 0.041 ( 0.001, 0.069 ( 0.004, and 0.081 ( 0.001 min-1 corresponding to CH3, COOH, and OH terminal alkanethiols. Note that the experiments were carried out at 1 mM. These rates are similar in magnitude to the values reported by Hu and Bard for mercaptoundecanoic acid on gold surfaces as 0.045 ( 0.005 and 0.02 ( 0.003 min-1 at 0.5 and 0.05 mM, respectively.31 Modeling the HSA adsorption data presented in Figure 5 in the same fashion, kobs was determined to be 0.163 ( 0.003, 0.248 ( 0.006, and 0.381 ( 0.001 min-1 corresponding to the adsorption of HSA to the CH3, COOH, and OH self-assembly thiol monolayer functional end groups, Figure 6B. The quality of the fits was good with correlation coefficients of 0.96-0.98. This indicates that the adsorption rate

of HSA on alkanethiolate is about 1 order of magnitude more rapid than the adsorption of thiol on gold surface.

(31) Bard, J.; Hu, K. Langmuir 1998, 14, 4790-4794.

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CONCLUSION The PEMC sensor demonstrates the adsorption of HSA on CH3-, COOH-, and OH-terminated self-assembled monolayers in real time. The total resonant frequency change for the interaction of HSA with the different terminal SAM functional groups increased in the following order: OH < COOH < CH3. ACKNOWLEDGMENT The authors acknowledge the financial support of the Environmental Protection Agency Grant R8296041 and the National Institutes of Health Grant 5R01EB000720. We are also thankful to Dan Luu for the development of the data acquisition programs. Received for review September 29, 2005. Accepted February 1, 2006.