Anal. Chem. 2007, 79, 2762-2770
Method for Label-Free Detection of Femtogram Quantities of Biologics in Flowing Liquid Samples David Maraldo, Kishan Rijal, Gossett Campbell, and Raj Mutharasan*
Department of Chemical and Biological Engineering, Drexel University, Philadelphia, Pennsylvania 19104
Rapid (∼10 min) measurement of very low concentration of pathogens (∼10 cells/mL) and protein (∼fg/mL) has widespread use in medical diagnostics, monitoring biothreat agents, and in a broader context as a research method. For low-level pathogen, we currently use culture enrichment methods and, thus, rapid analysis is not possible. For low protein concentration, no direct method is currently available. We report here a novel macrocantilever design whose high-order resonant mode near 1 MHz exhibits mass detection sensitivity of 10 cells/mL for cells and 100 fg/mL for protein. The sensor is 1 × 3 mm and uses a piezoelectric layer for both actuation and sensing resonance. Sample is flowed (∼1 mL/min) past the antibody-immobilized sensor, and as antigen binds to the sensor, resonance frequency decreases in proportion to antigen concentration. The sensor showed selectivity to the pathogen even though copious nonpathogenic variant was simultaneously present. Rapid and single-step label-free direct detection of proteins in flowing liquid samples at a concentration of 100 fg/mL has not been reported to date. Neither has there been any report on detecting pathogens at 10 cells per mL from samples of tens of milliliters under flow conditions. Both measurements have significant applications in medicine (for biomarkers in body fluids),1 environmental monitoring (pathogens in drinking water), food safety (Listeria,2 Cyrptosporidium, Giardia,3 and Escherichia coli poisoning4), and biodefense (biothreat agents). In this paper, we show designs of millimeter-sized cantilever sensors that exhibit mass change sensitivity of femtograms under liquid flow conditions and that are potentially useful in the applications mentioned above. Three types of experiments are reported to demonstrate the high detection sensitivity. Cantilever biosensors have attracted considerable interest in the past decade for label-free detection of proteins and pathogens because of their promise of very high sensitivity.5,6 Excellent * Corresponding author. Phone: 215-895-2236. Fax: 215-895-5837. E-mail:
[email protected]. (1) Cheng, M. M.-C.; Cuda, G.; Bunimovich, Y. L.; Gaspari, M.; Heath, J. R.; Hill, H. D.; Mirkin, C. A.; Nijdam, A. J.; Terracciano, R.; Thundat, T.; Ferrari, M. Curr. Opin. Chem. Biol. 2006, 10, 11-19. (2) Gray, K. M.; Bhunia, A. K. J. Microbiol. Methods 2005, 60, 259-268. (3) Szewzyk, U.; Szewzyk, R.; Manz, W.; Schleifer, K.-H. Annu. Rev. Microbiol. 2000, 54, 81-127. (4) Tims, T. B.; Lim, D.V. J. Microbiol. Methods 2003, 55, 141-147. (5) Craighead, H. G. Science 2000, 290, 1532-1536. (6) Ilic, B.; Craighead, H.G.; Krylov, S.; Senaratne, W.; Ober, C.; Neuzil, P. J. Appl. Phys. 2004, 95, 3694-3703.
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reviews have appeared that summarize progress.7,8 Briefly, the binding of an antigenic target to an antibody-immobilized cantilever surface changes the cantilever’s surface stress resulting in a deflection response.9,10 Cantilever biosensors11,12 have been successfully used in DNA hybridization studies,9,13 detection of known cancer proteins,14 environmental and foodborne pathogens,15,16 biomarkers,17,18 and explosives.19 In dynamic mode, the attachment of antigen causes a resonant frequency decrease because of increase in mass.7 Magnitude of bending deflection can be monitored by various transduction mechanisms.7,20-22 Because significant damping occurs in the dynamic mode, static deflection method is preferred when continuous measurement under liquid immersion is needed. When measurement in liquid flow condition is required, the bending mode becomes noisy and less trustworthy because of fluctuating hydrodynamic forces. It is well established23 that for the dynamic method to provide reasonable signals, cantilever Reynolds number (Re) should be large, preferably greater than 105. Since Re is proportional to the square of cantilever width, responses of microcantilevers whose widths are of a few micrometers are highly damped in liquids, (7) Lavrik, N. V.; Sepaniak, M. J.; Datskos, P. G. Rev. Sci. Instrum. 2004, 75, 2229-2253. (8) Ziegler, C. Anal. Bioanal. Chem. 2004, 379, 946-959. (9) Hansen, K. M.; Ji, H-F.; Wu, G.; Datar, R.; Cote, R.; Majumdar, A.; Thundat, T. Anal. Chem. 2001, 73, 1567-1571. (10) Hansen, K. M.; Thundat, T. Methods 2005, 37, 57-64. (11) Yang, M.; Zhang, X.; Vafai, K.; Ozkan, C. S. J. Micromech. Microeng. 2003, 13, 864-872. (12) Raiteri, R.; Grattarola, M. Butt, H.-J.; Skladal, P. Sens. Actuators, B 2001, 79, 115-126. (13) McKendry, R.; Zhang, J.; Arntz, Y.; Strunz, T.; Hegner, M.; Lang, H. P.; Baler, M. K.; Certa, U.; Meyer, E.; Guntherodt, H.-J.; Gerber, C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9783-9788. (14) Wu, G.; Datar, R. H.; Hansen, K. M.; Thundat, T.; Cote, R. J.; Majumdar, A. Nat. Biotechnol. 2001, 19, 856-860. (15) Campbell, G. A.; Uknalis, J.; Tu, S.-I.; Mutharasan, R. Biosens. Bioelectron. 2007, 22, 1296-1302. (16) Campbell, G. A.; Mutharasan R. Biosens. Bioelectron. 2006, 22, 78-85. (17) Carrascosa, L. G.; Moreno, M.; Alvarez, M.; Lechuga, L. M. Trends Anal. Chem. 2006, 25, 196-206. (18) Drukier, A. K. O.; N.; Schors, E.; Krasik, G.; Grigoriev, I.; Koenig, C.; Sulkowski, M.; Holcman, J.; Brown, L. R.; Tomaszewski, J. E.; Schnall, M. D.; Sainsbury, R.; Lokshin, A.E.; Godovac-Zimmermann, J. J. Proteome Res. 2006, 5, 1906-1915. (19) Pinnaduwage, L. A.; Gehl, A.; Hedden, D. L.; Muralidharan, G.; Thundat, T.; Lareau, R. T.; Sulchek, T.; Manning, L.; Rogers, B.; Jones, M.; Adams, J. D. Nature 2003, 425, 474. (20) Britton, C. L.; Jones, R. L.; Oden, P. I.; Hu, Z.; Warmack, R. J.; Smith, S. F.; Bryan, W. L.; Rochelle, J. M. Ultramicroscopy 2000, 82, 17-21. (21) Marie, R.; Jensenius, H.; Thaysen, J.; Christensen, C. B.; Boisen, A. Ultramicroscopy 2000, 91, 29-36. (22) Shekhawat, G.; Tark, S.; Dravid, V. P. Science 2006, 311, 1592-1595. (23) Sader, J. E. J. Appl. Phys. 1998, 84, 64-76. 10.1021/ac0621726 CCC: $37.00
© 2007 American Chemical Society Published on Web 02/20/2007
Figure 1. Schematic of experimental apparatus. A reagent reservoir manifold containing four cylindrical chambers was connected via a fourport manifold into the inlet of the sample flow cell (SFC). A peristaltic pump, connected to the SFC outlet, maintained constant flow rate between 0.4 and 1.0 mL/min. The experimental apparatus allows for a single pass through the SFC as well as for recirculation during antibody immobilization. For the E. coli detection experiments, a fifth reagent reservoir (not shown) was added for hydroxylamine or Protein G.
while millimeter-wide cantilevers provide relatively less damped response and can be used in liquid flow environments.16 For mechanical robustness, cantilever sensors should be much thicker than the thickness of microcantilevers which are typically ∼1 µm. Thick cantilevers exhibit such a weak bending response that the only useful approach is to use the dynamic or resonant mode for measurement. Sensors and detectors that are both mechanically robust and very highly sensitive will enjoy widespread use and will serve as aids in biodefense and national security. We report here that millimeter-sized cantilever sensors exhibit dominant high-order resonance near ∼900 kHz and show detection sensitivity comparable to recently reported microcantilever results.22 Unlike microcantilevers, they are mechanically robust and can be used under full liquid immersion and flow. The piezoelectric lead zirconate titanate (PZT) films give sensitive response to small stresses because of the direct piezoelectric effect and generate high strain via the inverse piezoelectric phenomena. PZT film is bonded to a base glass cantilever forming a composite cantilever. Electrical excitation of PZT causes it to expand and contract which induces bending, twisting, and buckling oscillations of the composite cantilever. The natural frequency of the cantilever depends on the flexural modulus and mass density of the composite cantilever.24 At resonance, the cantilever undergoes significantly higher stresses when the exciting electric field is at resonant frequency. At resonance, the PZT layer exhibits a sharp change in electrical impedance, and the resonance state can be measured by the phase angle.25 That is, the PZT is used both to excite the cantilever and (24) Kirstein, S.; Mertesdorf, M.; Schonhoff, M. J. Appl. Phys. 1998, 84, 17821790. (25) Campbell, G. A. Mutharasan, R. Sens. Actuators, A 2005, 122, 326-334.
to sense resonance.16 Such a method of excitation and measurement has been termed self-excitation by previous researchers.26-31 The macrocantilevers used in this study were fabricated by adhesive-bonding a piezoelectric layer to a nonpiezoelectric layer (borosilicate glass or fused silica) and then anchoring either the PZT alone (Design A, Figure 2) or PZT and glass (Design B, Figure 2).32-35 Design A is of unconventional design and is a new resonating cantilever structure that exhibits high-order mode that shows mass change sensitivity of fg/mL. Design B was shown earlier to have low-order resonant mode that exhibits 20 pg/Hz sensitivity34 and has been used to detect pathogens at a few hundred per mL.33 Here, we examine its high-order mode for a more sensitive detection at 10 cells/mL. EXPERIMENTAL SECTION Chemicals. Radiation-killed E. coli samples (1 × 109 cells/ mL) were a contribution from the USDA-ERRC (Wyndmoor, PA). Goat polyclonal anti-Escherichia coli O157:H7 antibody was purchased from KPL (Gaithersburg, MD). Bovine serum albumin and antibovine serum albumin were purchased from Sigma-Aldrich (Allentown, PA). Protein G was purchased from Pierce Biotech(26) Lee, C.; Itoh, T.; Suga, T. Sens. Actuators, A 1999, 72, 179-188. (27) Lee, Y.; Lim, G.; Moon, W. Sens. Actuators, A 2006, 130-131, 105-110. (28) Campbell, G. A.; Mutharasan, R. Sens. Actuators, A 2005, 122, 326-334. (29) Zhou, J.; Li, P.; Zhang, S.; Huang, Y.; Yang, P.; Bao, M.; Ruan, G. Microelectron. Eng. 2003, 69, 37-46. (30) Itoh, T.; Suga, T. Sens. Actuators, A 1996, 54, 477-481. (31) Lee, S. S.; White, R. M. Sens. Actuators, A 1996, 52, 41-45. (32) Campbell, G. A.; Mutharasan R Biosens. Bioelectron. 2005, 21, 462-473. (33) Campbell, G. A.; Mutharasan R Biosens. Bioelectron. 2006, 21, 1684-1692. (34) Campbell, G. A.; Mutharasan, R. Biosens. Bioelectron. 2006, 22, 35-41. (35) Campbell, G. A.; Mutharasan R. Langmuir 2005, 21, 11568-11573.
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Figure 2. Resonance spectra of Designs A and B in air, vacuum, and PBS. Main dimensions and details of construction of Designs A and B are given above panel A. The PZT layer and the glass layers were bonded and then embedded in a nonconductive epoxy to mechanically fix one end of the structure. Solder connection to the PZT layer with 30 gage wire was then connectorized to BNC couplers. Panel A: Resonance spectra of a PZT-anchored cantilever (Design A). The spectra, a plot of phase angle versus excitation frequency, showed one resonant peak in air at 941.5 ( 0.05 kHz. In vacuum (50 mTorr), the resonant frequency increased to 944.50 ( 0.05 kHz, and when the sensor was submerged in PBS at 0.4 mL/min, the resonant frequency decreased to 902.5 ( 0.05 kHz. Also, the peak height decreased in liquid by ∼40% because of damping. The dissipative losses due to viscous effects are measured by the quality of the peak (peak sharpness). Peak quality (Q-factor) decreased from 43 to 23 in going from vacuum to liquid flow conditions. Panel B: Typical resonance spectra of a PZT/glass-anchored cantilever (Design B) in the frequency range of 0.8-0.9 MHz in vacuum, air, and PBS at 0.7 mL/min. The quality of the resonant peak in air was significantly better than the PZT-anchored cantilever. However, the resonant peak showed significant damping under liquid, a change of 74% in Q. For both sensors, the large change observed in resonant frequency in going from air to liquid is an indication that both peaks are very sensitive to mass changes. The inset in each panel shows the general construction of cantilever Design A and Design B, respectively.
nology (Rockford, IL). All other chemicals and buffers were purchased from Sigma-Aldrich (Allentown, PA) or Fisher Scien2764 Analytical Chemistry, Vol. 79, No. 7, April 1, 2007
tific. Deionized water used was from a Milli-Q plus ultrapure water system (18.2 MΩ-cm).
Figure 3. Detection of bovine serum albumin using Design A. Resonant frequency change as BSA binds to an antibody-functionalized PZT-anchored cantilever. In all experiments, the 902.5 kHz peak at sample flow rate of 0.5 mL/min was used. Panel A shows the sequential attachment of a-BSA at 100 µg/mL, BSA detection at 1 pg/mL, and the release of the bound BSA using a pH 2 solution. The a-BSA attachment caused a frequency change of 6954 ( 21 Hz. The PBS rinse that followed did not cause any significant change in the resonant frequency, indicating that nonspecific binding, if any, was small. Upon exposure to an aliquot of 10 mL of 1 pg/mL BSA, the sensor responded with an immediate decrease in frequency and reached a steady-state change (2022 ( 25 Hz) in ∼20 min. The PBS rinse that followed shows a slight increase in frequency, which may be a result of the removal of nonspecifically adsorbed BSA. A pH 2 release solution caused the resonant frequency to increase by 413 ( 10 Hz above the value prior to BSA attachment. After stabilization, PBS was again pumped in to obtain final resonant frequency for comparison. In PBS, the frequency increased further by 186 ( 5 Hz. The higher value obtained may be due to some release of antibody. Panel B shows the frequency response of BSA at 100 fg/mL, 1 pg/ mL, and 10 pg/mL with the same sensor freshly prepared each time. Panel C shows the sensor response to 100 fg/mL in three different experiments. The positive control was a silanylated cantilever exposed to flowing PBS and the negative control was an unsilanylated clean sensor in the presence of 10 pg/mL BSA solution. The signal-to-noise (S/N) ratio was a minimum of 15.
Cantilever Fabrication. The cantilevers were fabricated from a piezoelectric (lead zirconate titanate, PZT; Type 5A, d31 ) -190 × 10-12 m/V; 127-µm thick) layer bonded to borosilicate glass or fused silica layer (160-µm thick). Two cantilever sensors of Design A were fabricated having free-end dimensions of 1.95 ( 0.005 × 1.00 ( 0.005 × 0.127 ( 0.005 mm3 (L × W × t) PZT and 2.00 ( 0.005 × 1.00 ( 0.005 × 0.160 ( 0.005 mm3 borosilicate glass and 2.54 ( 0.005 × 1.00 ( 0.005 × 0.127 ( 0.0005 mm3 (L × W × t) PZT and 1.78 ( 0.005 × 1.00 ( 0.05 × 0.160 ( 0.005 mm3 fused silica, respectively. One cantilever sensor of Design B was fabricated with free-end dimensions of 1.26 ( 0.25 × 1.00 ( 0.05 × 0.127 ( 0.005 mm3 (L × W × t) PZT and 2.03 ( 0.05 × 1.00 ( 0.05 × 0.160 ( 0.005 mm3 fused silica. The glass (or fused silica) layer provides the surface for antibody functionalization. For cantilevers of Design A, all exposed PZT was polyurethane coated (15-30 µm) for liquid contact. For cantilevers of Design B, the fused silica portion of the cantilever was first cleaned with piranha solution (70%:30% volume ratios of H2SO4 and 30% H2O2), followed by DI water and 99.8% ethanol, and was dried in air. The entire cantilever was coated with SU-8 (MicroChem Corp., MA) and then was cured as per vendor’s protocol. Next, both sides of the fused silica were sputtered with 100 nm gold (99.9%) in a Denton Desk II Sputtering System (Denton Vacuum, New Jersey). The goldsensing area was 2.8 mm2 and X-ray diffraction confirmed that surface was greater than 90% Au〈111〉. Experimental Apparatus. The experimental setup consisted of fluid reservoirs, peristaltic pumps, and a sensor flow cell (SFC) shown schematically in Figure 1. The sensor flow cell (SFC) was constructed of Plexiglas (diameter 7.0 mm, hold-up volume 120 µL) with inlet and outlets located at the bottom and on the side, respectively. The experimental apparatus consists of four fluid reservoirs (one each for PBS buffer pH 7.4, antibody, target antigen, and release solution (10 mM PBS/HCl, pH 2.0 or 100 mM glycine-HCl with 1% (v/v) ethylene glycol, pH 2.4)) connected to the SFC via a four-entrance port manifold with a single outlet. The outlet was connected to a peristaltic pump that controls the flow of the desired fluid into and out of the SFC. In some experiments (e.g., Figure 4), a fifth fluid reservoir containing 10 mM hydroxylamine was added to the manifold. Hydroxylamine was added to ensure that activated carboxyl groups on the antibody that did not participate in the immobilization reaction were deactivated to carboxylic groups. This step was found to be unnecessary when immobilization was carried out over 1 h. For the experiments detailed in Figure 5, a fifth fluid reservoir containing 10 µg/mL of Protein G was added. The Protein G binds IgG antibody via the Fc region, thus orienting the antigen-binding Fab region away from the sensor surface.36,37 The functionalized sensor, installed vertically into SFC, was connected to an impedance analyzer (Agilent, HP 4192A or HP 4294A) interfaced to a PC with LabVIEW application for obtaining impedance and phase-angle measurements in the frequency range of 40 kHz to 1.5 MHz at 100 mV excitation. The SFC was maintained at 30 ( 0.3 °C and 28 ( 0.2 °C for the experiments given in Figures 3 and 4, respectively, by circulating (17 mL/ min) constant temperature water (38 ( 0.1 °C for Figure 3 and (36) Neubert, H.; Jacoby, E. S.; Bansal, S. S.; Iles, R. K.; Cowan, D. A.; Kicman, A. T. Anal. Chem. 2002, 74, 3677-3683. (37) Bae, Y. M.; Oh, B. K.; Lee, W.; Lee, W. H.; Choi, J. W. Biosens. Bioelectron. 2005, 21, 103-110.
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Figure 4. Design A detection of E. coli. Panel A: Transient response of resonant frequency change for antibody (anti-EC) attachment, followed by detection of EC at 100 cells/mL from a 70mL sample solution, and the release of the bound EC with a pH 2 solution. Throughout the experiment, the flow rate was kept constant at 1 mL/min. Panel B is a magnified version of the EC attachment and release frequency response. The frequency response for EC attachment was 2999 ( 130 Hz and the release solution gave an increasing response of 2272 ( 50 Hz. However, the PBS flush after the release step increased further the resonant frequency to approximately a value at the beginning of detection. Panel C shows the resonant frequency change caused by the binding of EC at various concentrations as a function of time. In each experiment, 70 mL of EC sample was flowed once through the SFC at 1 mL/min. All experiments shown in panel B were conducted with the same sensor and a fresh antibody immobilization was done each time. Each experiment was repeated three times at 0, 100, 1000, and 10 000 EC/mL. The sensor response is repeatable and is a strong function of antigen concentration. The S/N was greater than 40. The control was an antibody-functionalized cantilever exposed to PBS buffer in the same fashion as the EC sample.
35 ( 0.1 °C for Figure 4) through a jacket surrounding the SFC. The experiments in Figure 5 were conducted at room temperature (23.6 ( 0.2 °C) and no circulation of constant temperature water 2766 Analytical Chemistry, Vol. 79, No. 7, April 1, 2007
was used. The temperature directly influences kinetics of antigenantibody binding, and thus careful and close control of temperature of the sample flow cell was maintained. Cantilever Functionalization. For cantilevers of Design A, the sensing glass surface was cleaned (sequentially with methanolhydrochloric acid solution (1:1 volume ratio), concentrated sulfuric acid, hot sodium hydroxide, and finally boiling water; CAUTION: corrosive and dangerous) and silanylated with 0.4% 3-aminopropyltriethoxysilane (APTES) in DI water at pH 3.0 (adjusted by hydrochloric acid, 0.1 N) and 75 °C for 2 h.38 Approximately 1600 µg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 4400 µg of sulfo-N-hydroxysuccinimide (Sulfo-NHS)39 were added to 40 µg of antibody and were contacted with the sensor surface for 2 h at 28 ( 0.2 or 30 ( 0.3 °C. After each detection experiment, the sensor surface was renewed starting with the cleaning protocol noted above. This ensured that there were no carryover effects from one experiment to the next. For cantilevers of Design B, the sensor was used immediately after gold coating. The gold sensor surface was exposed to Protein G and antibody solution in succession at 0.7 mL/min in a recirculation mode. After each detection experiment, both sides of the sensor surface were cleaned and reused. After three such reuses, the sensor surface was recoated with 100 nm of gold. Antigen Preparation. Both BSA samples (1 × 10-11, 1 × 10-12, to 1 × 10-13 g/mL) and E. coli samples (10, 100, 1000, and 10 000 cells/mL) were prepared in PBS (10 mM, pH 7.4) starting with stock solutions of known concentration. E. coli concentration was verified by microscopic examination at 105 cells/mL. Detection Experiments. All valves were manipulated manually and are shown schematically in Figure 1. The detection experiments were carried out at flow rates of 0.4 mL/min (Figure 3), 1.0 mL/min (Figure 4), and 0.7 mL/min (Figure 5). Following antibody immobilization, PBS solution was recirculated through the SFC until the sensor’s resonant frequency reached a constant value. This typically took 5-15 min and was completed to ensure that the tubing and SFC were flushed prior to a detection experiment. During the experiment in Figure 4, a 10 mM hydroxylamine solution was flowed past the sensor surface prior to the PBS flush. A detection experiment was initiated by introducing the target antigen solution until steady state was reached. The flow circuit was rinsed by flowing PBS followed by a release buffer (pH 2.0). Finally, a PBS flow was carried out until the resonant frequency value reached steady state. Calibration Experiments. Two dilute solutions of paraffin were prepared in hexane or toluene, starting from a stock solution at 1 mg/100 mL. Resonance frequency in vacuum (50 mTorr, 23 °C) was determined. Carefully measured 0.5 or 1 µL of the wax solution was dispensed on the glass surface ensuring no spills occurred or overflow onto the PZT part. The sensor was air-dried for 15 min in a dust-free enclosure, followed by resonance frequency determination at 50 mTorr and 23 °C. The resonance frequency increased and reached steady-state value in 15-30 min as the solvent from the wax film evaporated leaving behind the nonvolatile paraffin wax. The sensor was then removed and an additional 0.5 or 1 µL of wax solution was added and dried and (38) Immobilized Biomolecules in Analysis, A Practical Approach; Cass, T., Ligler, F. S., Eds.; Oxford University Press: New York, 1998. (39) Hermanson, G. T. Bioconjugate Technique; Elsevier: San Diego, CA, 1996.
Figure 5. Design B detection of E. coli and sensor selectivity. Panel A: Transient response of resonant frequency change for the detection sequence of E. coli O157:H7 at 10, 100, 1000, and 10 000 cells/mL in 30 mL of PBS using an anchored nonpiezoelectric cantilever (Design B). The frequency change during the release of the bound E. coli equals approximately the change because of attachment, and no loss in antibody activity was observed for at least two regeneration cycles. Panel B is an illustration of the sensor’s selectivity. The sensor was exposed to various concentrations of EC/JM101 mixed samples; each sample contained 100 EC/mL. JM101 is a nonpathogenic strain and is, therefore, not recognized by the antibody. The sensor responded with a decrease in resonant frequency as the concentration of pathogenic EC was increased. When exposed to 100% JM101, no significant change in the frequency response was observed. In addition, the results suggest that nonspecific binding, if any, was negligible. Unlike the experiment presented in Panel A, the antibody activity appears to have decreased slightly after the first regeneration and remains constant for the second and third regeneration.
resonance frequency was again measured in 50 mTorr. Repeated measurements provide a plot of change in resonance frequency with added paraffin mass and resulted in a straight line whose slope gave mass change sensitivity. For the various cantilevers fabricated during the current study, mass change sensitivity ranged from 0.3 to 2 fg/Hz. RESULTS AND DISCUSSION Cantilever Characterization. Cantilevers fabricated exhibited dominant modes in the region 0-200 kHz and 700 kHz to 1.0 MHz with Q-values ranging from 20 to 110. In Figure 2, design dimensions of Designs A and B and their resonance spectra (phase angle versus excitation frequency) in air, vacuum, and PBS are given. Impedance and phase angle versus excitation frequency for the dominant 941.5 kHz resonant peak were measured. The impedance response (data now shown) shows the classic reso-
nance-antiresonance characteristic. Depending on the dimension and construction of the composite cantilever, resonance frequencies were observed in the range of 0.8-1.2 MHz. Design B was previously characterized by the authors for detecting low concentration of pathogens (300 per mL),33 alkanethiol self-assembly at nM levels,35 and protein interaction40 using PZT-borosilicate glass macrocantilevers which exhibited resonant bending modes below 100 kHz in liquid.34 When borosilicate glass was replaced with fused silica of same thickness, a high-order resonant mode near 1 MHz became prominent. When excited at various frequencies (
