Impact of Nano- and Mesoscale Particles on the Performance of

Microcantilever-based sensors comprise an emerging class of chemomechanical sensors. The crucial challenge for every new and promising sensing platfor...
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Anal. Chem. 2004, 76, 5685-5689

Impact of Nano- and Mesoscale Particles on the Performance of Microcantilever-Based Sensors Lawrence A. Bottomley,*,† Mark A. Poggi,† and Shanxiang Shen‡

School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, and Protiveris, Inc., 15010 Broschart Road, Rockville, Maryland 20850

Microcantilever-based sensors comprise an emerging class of chemomechanical sensors. The crucial challenge for every new and promising sensing platform lies in its performance in complex mixtures. Since most biofluids are rich in particulates, we assessed the impact of particles in the liquid stream on the performance of microcantilever sensors operated in both deflection and resonance modes. For both detection modes, sensor response depends on the particle diameter, concentration, and velocity as well as the composition of a thin-film coating. The presence of particles in the fluid stream produce substantial scattering of the laser beam used to measure cantilever deflection. Thus, prior removal of particulate matter from biofluids is required for optimal performance of microcantilever-based biosensors. Microcantilever-based sensors comprise an emerging class of chemomechanical sensors.1-6 Molecular adsorption on a cantilever produces a shift in its resonance frequency. Adsorption onto one surface of a microcantilever results in a differential stress between this and the opposite surface thus inducing bending.7-10 Cantilever bending can be measured with angstrom resolution using optical reflection, piezoresistive, capacitance, and piezoelectric measurement methods commonly used in atomic force microscopy. A compelling feature of microcantilever-based sensors is that they can be operated in air, vacuum, or liquid.11 Although the damping effects of a liquid medium reduces the resonance response of a * To whom correspondence should be addressed. e-mail: [email protected]. Phone: 404-894-4014. Fax: 404-894-7452. † Georgia Institute of Technology. ‡ Protiveris, Inc. (1) Gimzewski, J. K.; Gerber, C. H.; Mayer, E.; Schlitter, R. R. Chem. Phys. Lett. 1994, 217, 589. (2) Barnes, J. R.; Stephenson, R. J.; Welland, M. E.; Gerber, C.; Gimzewski, J. K. Nature 1994, 372, 79. (3) Wachter, E. A.; Thundat, T. Rev. Sci. Instrum. 1995, 66, 3662-3667. (4) Thundat, T.; Warmack, R. J.; Chen, G. Y.; Allison, D. P. Appl. Phys. Lett. 1994, 64, 2894. (5) Chen, G. Y.; Thundat, T.; Wachter, E. A.; Warmack, R. J. J. Appl. Phys. 1995, 77, 3618. (6) Thundat, T.; Chen, G. Y.; Warmack, R. J.; Allison, D. P.; Wachter, E. A. Anal. Chem. 1995, 67, 519-521. (7) Headrick, J. J.; Sepaniak, M. J.; Lavrik, N. V.; Datskos, P. G. Ultramicroscopy 2003, 97, 417-424. (8) Tipple, C. A.; Lavrik, N. V.; Culha, M.; Headrick, J.; Datskos, P.; Sepaniak, M. J. Anal. Chem. 2002, 74, 3118-3126. (9) Lavrik, N. V.; Tipple, C. A.; Sepaniak, M. J.; Datskos, P. G. Chem. Phys. Lett. 2001, 336, 371-376. (10) Raiteri, R.; Butt, H.-J. J. Phys. Chem. 1996, 99, 15728. 10.1021/ac049111x CCC: $27.50 Published on Web 08/27/2004

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microcantilever to a value ∼1 order of magnitude smaller than that in air, subnanometer bending of the microcantilever can be readily measured. Microcantilever-based detection of airborne components is the most common application of this sensor platform.12-17 Mass sensitivities in the picogram to femtogram range are commonplace with optical reflection as the measurement mode.18-21 Recent interest has shifted to detection of fluid-borne components where high sensitivity in response is achievable through incorporation of thin-film coatings on one side of the cantilever. High selectivity in response is provided by incorporation of biomolecular recognition elements into these coatings. Innovative applications of this concept include microcantilever-based immunoassays and detection of nucleic acid hybridization.15,22-32 (11) O’Shea, S. J.; Welland, M. E.; Brunt, T. A.; Ramadan, A. R.; Rayment, T. J. Vac. Sci. Technol., B 1996, 14, 1383. (12) Baller, M. K.; Lang, H. P.; Fritz, J.; Gerber, C.; Gimzewski, J. K.; Drechsler, U.; Rothuizen, H.; Despont, M.; Vettiger, P.; Battiston, F. M. R., J. P.; Fornaro, P.; Meyer, E.; Guntherodt, H. J. Ultramicroscopy 2000, 82, 1-9. (13) Fritz, J.; Baller, M. K.; Lang, H. P.; Strunz, T.; Meyer, E.; Guntherodt, H. J.; Delamarche, E.; Gerber, C.; Gimzewski, J. K. Langmuir 2000, 16, 96949696. (14) Fritz, J.; Baller, M. K.; Lang, H. P.; Rothuizen, H. V., P.; Meyer, E.; Guntherodt, H.-J.; Gerber, C.; Gimzewski, J. K. Science 2000, 288, 316318. (15) McKendry, R.; Zhang, J.; Arntz, Y.; Strunz, T.; Hegner, M.; Lang, H. P.; Baller, M. K.; Certa, U.; Meyer, E.; Guntherodt, H.-J.; Gerber, C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9783-9788. (16) Oden, P. I.; Chen, G. Y.; Steele, R. A.; Warmack, R. J.; Thundat, T. Appl. Phys. Lett. 1996, 68, 3814-3816. (17) Thundat, T.; Oden, P. I.; Warmack, R. J. Proc.-Electrochem. Soc. 1997, 97-5, 179-187. (18) Ji, H. F.; Hansen, K. M.; Hu, Z.; Thundat, T. Sens. Actuators, B: Chem. 2001, B72, 233-238. (19) Ji, H.-F.; Dabestani, R.; Brown, G. M.; Britt, P. F. Chem. Commun. (Cambridge) 2000, 457-458. (20) Ji, H.-F.; Thundat, T. Biosens. Bioelectron. 2002, 17, 337-343. (21) Ji, H.-F.; Thundat, T.; Dabestani, R.; Brown, G. M.; Britt, P. F.; Bonnesen, P. V. Anal. Chem. 2001, 73, 1572-1576. (22) Dutta, P.; Tipple, C. A.; Lavrik, N. V.; Datskos, P. G.; Hofstetter, H.; Hofstetter, O.; Sepaniak, M. J. Anal. Chem. 2003, 75, 2342-2348. (23) Grogan, C.; Raiteri, R.; O’Connor, G. M.; Glynn, T. J.; Cunningham, V.; Kane, M.; Charlton, M.; Leech, D. Biosens. Bioelectron. 2002, 17, 201-207. (24) Kim, B. H.; Mader, O.; Weimar, U.; Brock, R.; Kern, D. P. J. Vac. Sci. Technol., B 2003, 21, 1472-1475. (25) Stevenson, K. A.; Mehta, A.; Hansen, K. M.; Thundat, T. G. Proc.Electrochem. Soc. 2002, 2002-6, 218-225. (26) Tamayo, J.; Humphris, A. D. L.; Malloy, A. M.; Miles, M. J. Ultramicroscopy 2001, 86, 167-173. (27) Thundat, T. G.; Jacobson, K. B.; Doktycz, M. J.; Kennel, S. J.; Warmack, R. J. Lockheed Martin Energy Research Corp., U.S. Patent 6,289,717 Bi, Sept. 18, 2001. (28) Hansen, K. M.; Ji, H. F.; Wu, G. H.; Datar, R.; Cote, R.; Majumdar, A.; Thundat, T. Anal. Chem. 2001, 73, 1567-1571.

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The crucial challenge for every new and promising sensing platform lies in its performance in complex mixtures. Since most biofluids are rich in particulates, the purpose of this study was to evaluate the impact of particles in the liquid stream on the performance of microcantilever sensors. We report herein that the response of the cantilever depends on the particle diameter, concentration, and velocity as well as the composition of a thinfilm coating. EXPERIMENTAL SECTION Materials. Latex particles of various diameters were obtained from the following suppliers: 0.093-, 0.304-, and 0.482-µm diameter (Ted Pella, Inc, Redding, CA); 1.1- and 1.9-µm diameter (Interfacial Dynamics Corp, Portland, OR). Particle-containing solutions were diluted to a final concentration of 0.1% (w/v) with deionized water (E-pure grade). The gold surface modifier, 1-mercaptoundecanoic acid, was used as received from the supplier (Sigma-Aldrich, Milwaukee, WI). Standard silicon nitride cantilevers (Veeco Metrology, Santa Barbara, CA) with spring constants ranging from 0.30 to 0.01 N/m were used in all experiments. Surface contamination was removed by exposure of microcantilevers for 20 min to an ozone-enriched atmosphere generated by a UVO cleaner Model 342 (Jelight Corp., Irvine, CA). Methods. Selective application of a protective thin film to the gold surface of the microcantilever was achieved by self-assembly. Microcantilevers were treated with a 0.1% (w/v) aqueous solution of 1-mercaptoundecanoic acid for 1-12 h either at ambient or refrigerator temperatures. Next, the microcantilever was repetitively rinsed in deionized water, placed in the fluid cell, and subsequently mounted in the microscope. The injection loop was filled with the particle-containing solution to be examined. Deionized water was pumped through the cell until a stable baseline was obtained. Cantilever deflection was measured before, during, and after injection of precisely known volumes of particlecontaining solutions into the flowing stream using either a PicoSPM (Molecular Imaging, Phoenix, AZ) or a Veeco Instruments (Santa Barbara, CA) NanoScope model IIIA scanning probe microscope. Experiments conducted on the PicoSPM employed an open top fluid cell (volume ∼100 µL) purchased from Molecular Imaging. A Masterflex C/L 77120-52 pump (Cole-Parmer Instrument Co. Vernon Hills, IL) was used to deliver fluids to and from the cell. Known aliquots of particle-containing solutions were injected into the flowing stream with a six-port injection valve (Upchurch Scientific, Oak Harbor, WA) interchanging injection loops of defined volume as required. Narrow-bore Tygon tubing was employed to provide a fluid flow path between the solvent reservoir and the pump, the pump and the injector, and the injector and the cell. The pump, injector, and microscope were housed in a thermal isolation chamber (Igloo Corp., Houston, TX). The injector switch was mounted on the outside of the thermal isolation chamber, enabling injection of solutions into the flow stream without exposure of the system to an external heat source. (29) Marie, R.; Jensenius, H.; Thaysen, J.; Christensen, C. B.; Boisen, A. Ultramicroscopy 2002, 91, 29-36. (30) Stevenson, K. A.; Mehta, A.; Sachenko, P.; Hansen, K. M.; Thundat, T. Langmuir 2002, 18, 8732-8736. (31) Su, M.; Li, S.; Dravid, V. P. Appl. Phys. Lett. 2003, 82, 3562-3564. (32) Zhang, J.; Grubb, M.; Hansen, A. G.; Kuznetsov, A. M.; Boisen, A.; Wackerbarth, H.; Ulstrup, J. J. Phys.: Condens. Matter 2003, 15, S1873S1890.

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Figure 1. Cantilever deflection versus time following injection of 0.304-µm-diameter particles into fluid cell containing an uncoated microcantilever. Particle concentrations (w/v): A ) 0.01%; B ) 0.10%. Fluid flow rate, 2 mL/h.

The thermal isolation chamber was placed on top of a MOD-1 antivibration device (HWL Scientific Instruments, Ammerbuch, Germany) to reduce vibrational noise. Experiments conducted on the NanoScope employed a Multimode Head and a commercial AFM fluid cell (volume ∼75 µL) supplied by Veeco Instruments. Solutions were delivered to the cell with a Yale Apparatus Multiphaser model YA-12 syringe pump and PEEK tubing of varying diameters (Upchurch Scientific). Particle-containing solutions were injected into the flow stream using an Upchurch Scientific six-port injection valve. To minimize vibrations, the microscope was placed on a TMC vibration isolation table whereas the pump and injector were placed on an adjacent laboratoru bench. Frequency spectra were acquired with a Stanford Research Systems model SR-785 dynamic signal analyzer. Raw sum and deflection signals were taken directly off the preamplifier board on the base of the multimode microscope and routed directly into the dynamic signal analyzer and a LeCroy model 9310CM dual 400-MHz digital oscilloscope through coaxial cable and a tee connector. No signals were taken from the signal access module to bypass low-pass filters incorporated by the manufacturer into the circuitry of the Multimode base. Postrun data treatment included baseline flattening to remove slow bending of the cantilever induced by temperature fluctuations in the laboratory. The sensitivity of the position-sensing device to changes in cantilever deflection was calibrated using the force curve method. This value was used to convert output voltages from the position-sensing device to microcantilever deflections in nanometers. RESULTS AND DISCUSSION The response of a cantilever to a bolus of solution containing latex particles passing through the cell depends on the velocity of the flow stream, the concentration and diameter of particles in the stream, and the affinity of the particles for the gold surface of the cantilever. Uncoated Microcantilever. Figure 1 depicts a typical response profile when latex particles were injected into the fluid cell containing an uncoated microcantilever. Cantilever response

Figure 2. Cantilever deflection versus time following injection of 1.9-µm-diameter particles past a microcantilever coated with 1-mercaptoundecanoic acid as a function of flow rate (trace A, 7.0 mL/h; trace B, 2.0 mL/h).

Scheme 1. Chemisorption of Particles onto the Microcantilever Surface

is divided into three regimes. In the first few hundred seconds after injection of particle-laden solution, corresponding to the transit time of the bolus through the tubing from the injector to the cell, little change in cantilever deflection is observed. The duration of the first regime is inversely proportional to flow stream velocity. During the second regime, the cantilever deflects as a function of time. A downward deflection is observed consistent with physi- or chemisorption of particles to the gold surface and a proportionate increase in cantilever mass. This is shown schematically in Scheme 1. In the third regime, the deflection stabilizes, holding constant even when the particle bolus has long passed through the cell. The magnitude of cantilever deflection (measured from the point of injection to the plateau) is proportional to particle concentration. The chemisorption of latex particles to the clean gold surfaces has been previously documented.33,34 Coated Microcantilever. When the microcantilever surface is covered with a thin film that inhibits either physi- or chemisorption of particles, a significantly different response profile is obtained. Figure 2 depicts data typical of that obtained when latex particles were injected into the fluid cell containing a microcantilever coated with a self-assembled, carboxyl-terminated, alkanethiol monolayer. Cantilever response is again divided into three regimes. No change in cantilever deflection is observed in the first regime. Similar to when an uncoated cantilever is used, the duration of this regime depends only upon flow stream velocity. In the second regime, transient upward and downward deflection of the cantilever is observed; however, the average (33) Xu, H.; Schlenoff, J. B. Langmuir 1994, 10, 241-245. (34) Fu, T. Z.; Stimming, U.; Durning, C. J. Macromolecules 1993, 26, 32713281.

Figure 3. Cantilever deflection versus time following injection of 1.9-µm-diameter particles past a microcantilever coated with 1-mercaptoundecanoic acid at a flow rate of 7.0 mL/h. Particle concentrations (%, w/v): A, 0.0; B, 0.0125; C, 0.025; D, 0.05; E, 0.10.

deflection remains constant and identical to that observed in the first regime. The duration of the second regime is proportional to injection loop volume and flow stream velocity. The third regime commences when the particle bolus has left the fluid cell. Transient response of the cantilever diminishes, soon becoming identical to that found in the first regime. Constant average deflection over the three regimes indicates that the thin-film coating prevents chemisorption of particles on the gold surface of the cantilever. Figure 3 depicts the response of the coated microcantilever to repeated injections of increasing particle concentration. While the duration of random upward and downward deflections of the microcantilever is independent of particle concentration, the magnitude of oscillation is proportional to particle concentration and flow rate (data not shown). To determine the impact of particles on cantilever resonance, thermal noise spectra were acquired prior to, during, and after particles entered the flow cell. This was accomplished by tracking cantilever deflection over time and converting the time-dependent data into the frequency domain using the Fourier transform.35 Figures 4 and 5 present thermal noise spectra acquired during each of the three regimes of coated cantilever response following injection of particle-laden solutions into the flowing stream. As 1.9-µm-diameter particles enter the fluid cell (Figure 4), the 1/f noise increases in proportion to the concentration of particles in the fluid stream. For both concentrations presented in Figure 4, the level of 1/f noise masks the cantilever resonance. In contrast, as 93-nm-diameter particles enter the fluid cell (Figure 5), the increase in 1/f noise level is measurable only at high concentration and no apparent shift in cantilever resonance frequency is observed. Two plausible explanations are put forth to account for the observed dependence of cantilever deflection on particle concentration, diameter and fluid flow rate as well as the increase in the level of 1/f noise with particle concentration and diameter. The first, illustrated in Scheme 2, suggests that the particles are colliding with the cantilever surface with sufficient momentum to cause momentary deflection and an increase in its Brownian motion.36,37 (35) Roters, A.; Johannsmann, D. J. Phys.: Condens. Matter 1996, 8, 7561-7577.

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Scheme 3. Scattering of Light off Particles

Figure 4. Amplitude versus frequency acquired on a coated microcantilever prior to entry (blue) and during (red) and after clearance (green) of 1.9-µm-diameter particles in the flow cell. The lower set depicts the spectra obtained at a particle concentration of 0.0125% (w/v). The upper set, offset for clarity, depicts the spectra obtained at a particle concentration of 0.10% (w/v). The spring constant of the cantilever used in this experiment was 0.010 N/m.

Figure 5. Amplitude versus frequency acquired on a coated microcantilever prior to entry (blue) and during (red) and after clearance (green) of 93-nm-diameter particles in the flow cell. The lower set depicts the spectra obtained at a particle concentration of 0.0125% (w/v). The upper set, offset for clarity, depicts the spectra obtained at a particle concentration of 0.10% (w/v). The spring constant of the cantilever used in this experiment was 0.010 N/m.

Scheme 2. Collision of Particles with the Microcantilever Surface

This explanation is consistent with the observed dependence of cantilever deflection on particle concentration (Figure 3) and flow velocity (Figure 2) and the dependence of the 1/f noise level on particle diameter and concentration (Figures 4 and 5). An alternative explanation is that the presence of particles in the fluid (36) Mehta, A.; Cherian, S.; Hedden, D.; Thundat, T. Appl. Phys. Lett. 2001, 78, 1637-1639.

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cell results in scattering of the laser light used to track cantilever deflection (Scheme 3). From Mie scattering theory, the intensity of scattered light should be proportional to the number density of particles in the optical beam (Figure 3) as well as the level of 1/f noise (Figures 4 and 5). In a conference proceeding describing our preliminary results,38 we presented an experiment intended to discriminate between these two possibilities. In this experiment, the laser spot was positioned on the support chip rather than the microcantilever. Particles were then injected into the fluid cell, and the response of the position-sensitive detector was recorded and processed as if the beam reflected off the microcantilever. No transient signals were observed when the 0.093-, 0.304-, and 0.482-µm-diameter particles were present in the cell. However, when the 1.1- and 1.9-µm-diameter particles were injected, apparent “transient deflections” were observed. This observation appears consistent with scattering theory, which predicts that the intensity of scattered light should be negligible when the particle diameter drops below the wavelength of incident light (670 nm). We incorrectly concluded that the transient signals observed when particles flow past a coated microcantilever are predominantly due to elastic collisions with the surface. This conclusion was based on comparison of the integrated amplitude of the transient deflections of the cantilever when the beam was focused on the chip versus the microcantilever. The cantilever deflection signal available with the commercial AFMs used for these experiments is actually the ratio of the raw deflection signal over the sum signal from the position-sensitive detector to account for variations in laser diode intensity. Thus, our previous conclusion is invalid because we neglected to account for the increase in light intensity reaching the detector when the beam was reflected off the chip (compared to that reflecting off the cantilever). To account for variations in light intensity reaching the detector, raw deflection and sum signals were taken directly off the preamplifier board on the base of the multimode microscope and routed directly to a digital oscilloscope and a dynamic signal analyzer. The laser beam was focused on the end of the coated cantilever, and the detector position was adjusted for maximum light intensity at zero deflection. Figure 6 displays the individual signals as varying concentrations of 1.9-µm-diameter particles were injected into the flow stream. Introduction of particles into the fluid cell resulted in random excursions of the raw deflection signal about the zero value; the magnitude of raw deflection signal excursions were proportional to particle concentration. (37) Janata, J. Dynamic Immunochemical and Like Chemical Species Sensor Apparatus and Method. U.S. Patent 5,227,134, July 13, 1983. (38) Shen, S.; Bottomley, L. A. Thin Films: Preparation, Characterization, Applications. Proceedings of a Symposium of the American Chemical Society, San Diego, CA, Apr. 1-5, 2001; 2002; pp 349-359.

Figure 6. Detector response following injection of particles into fluid cell. The deflection (lower trace) and sum (upper trace) signals were monitored following sequential injection of 1.9-µm-diameter particles past a coated microcantilever. Particle concentrations (%, w/v) are given in the figure.

Figure 7. Decrease in sum signal magnitude as a function of particle concentration and diameter. Particle diameters: 1900 (circle), 300 (square), and 93 (diamond) nm, respectively.

Introduction of particles into the fluid cell diminished the magnitude of the sum signal. The decrease in sum signal magnitude was proportional to particle concentration and diameter. Figure 7 displays the dependence of the magnitude of sum signal decrease with particle concentration for three different diameter particles. The decrease in sum signal is directly proportional to the intensity of scattered light. The results presented in Figures 6 and 7 are consistent with significant scattering of the incident beam by particles in the fluid

cell. The magnitude of the intensity of scattered light (indirectly measured by the sum signal decrease) and its marked dependence upon particle diameter provide compelling evidence that the presence of particles in the fluid cell result in substantial scattering of the laser beam used to measure cantilever deflection and diminished signal-to-noise ratios. It is interesting to note that, for the 93-nm-diameter particles, little change in sum signal is observed yet there remains transient deflection of the cantilever. Particles with diameters less than half of the wavelength of the incident optical beam do not appreciably scatter light. Thus, the transient deflection may reflect an increase in cantilever motion induced by elastic and inelastic collisions of particles. A test of this postulate will require use of a nonoptical (e.g., piezoresistive) detection method. The results reported herein have important implications for microcantilever-based biosensing applications. Particulate matter of varying dimensions is found in most biofluids. For example, particles found in blood include erythrocytes, leukocytes, and fibrinogen assemblies. Saliva contains single-cell organisms and protein aggregates organized within micellular structures. Direct injection of biological fluids past a microcantilever without prior removal of particles (diameter >0.7 µm) may prove to be problematic. False positive detection of biomarkers will result when biofluids with particles that chemisorb to the cantilever surface are analyzed. Decreased signal-to-noise ratios will be found when biofluids laden with particles that do not chemisorb to the surface but scatter the incident beam are anylzed. Thus, for optimal performance, prior removal of particulate matter from biofluids is required for microcantilever-based biosensors operating in either deflection or resonance detection modes. ACKNOWLEDGMENT We thank Dr. Madhushree Ghosh (Stratagene, Inc.) and Mr. Jean Jarvaise (Veeco Metrology Division) for helpful discussions and acknowledge financial support by NIH (Grant 5 R21 EB 73702).

Received for review June 16, 2004. Accepted July 14, 2004. AC049111X

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