Prion Protein Detection Using Nanomechanical ... - ACS Publications

Feb 14, 2008 - Nanomechanical resonators have shown potential ap- plication for mass sensing and have been used to detect a variety of biomolecules. I...
6 downloads 4 Views 490KB Size
Download PDF
Anal. Chem. 2008, 80, 2141-2148

Prion Protein Detection Using Nanomechanical Resonator Arrays and Secondary Mass Labeling Madhukar Varshney,† Philip S. Waggoner,† Christine P. Tan,‡ Keith Aubin,† Richard A. Montagna,§ and Harold G. Craighead*,†

School of Applied and Engineering Physics, Department of Biomedical Engineering, Cornell University, Ithaca, New York 14853, and Innovative Biotechnologies International, Incorporated, Grand Island, New York 14072

Nanomechanical resonators have shown potential application for mass sensing and have been used to detect a variety of biomolecules. In this study, a dynamic resonance-based technique was used to detect prion proteins (PrP), which in conformationally altered forms are known to cause neurodegenerative diseases in animals as well as humans. Antibodies and nanoparticles were used as mass labels to increase the mass shift and thus amplify the frequency shift signal used in PrP detection. A sandwich assay was used to immobilize PrP between two monoclonal antibodies, one of which was conjugated to the resonator’s surface while the other was either used alone or linked to the nanoparticles as a mass label. Without additional mass labeling, PrP was not detected at concentrations below 20 µg/mL. In the presence of secondary antibodies the analytical sensitivity was improved to 2 µg/mL. With the use of functionalized nanoparticles, the sensitivity improved an additional 3 orders of magnitude to 2 ng/mL. Prion proteins (PrP) are transmissible infectious particles, devoid of nucleic acid, that cause fatal neurodegenerative diseases known as bovine spongiform encephalopathy (BSE) in bovine, scrapie (SC) in sheep, and Creutzfeldt-Jakob disease (CJD) in humans.1 These diseases are caused by genetic, infectious, or sporadic disorders where host-encoded, noninfectious cellular prion proteins (PrPc) are converted into a conformationally altered, infectious forms designated as PrPsc, PrPCJD, and PrPBSE.2,3 PrPc is present in abundance in most tissues of the central nervous system. However, posttranslational processes convert PrPc into PrPsc, which leads to altered physiochemical and biochemical properties such as aggregation, insolubility, protease digestion resistance, and a β-sheet-rich secondary structure. One such altered property of PrPsc, namely, protease digestion resistance, forms the basis of several diagnostic biochemical tests. * To whom correspondence should be addressed. Phone: +1 607-255-8707. Fax: +1 607-255-7658. E-mail: [email protected]. † School of Applied and Engineering Physics, Cornell University. ‡ Department of Biomedical Engineering, Cornell University. § Innovative Biotechnologies International, Inc. (1) Triantaphyllidou, I. E.; Sklaviadis, T.; Vynios, D. H. Anal. Biochem. 2006, 359, 176-182. (2) Prusiner, S. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 13363-13383. (3) Birkmann, E.; Scha¨fer, O.; Weinmann, N.; Dumpitak, C.; Beekes, M.; Jackman, R.; Thorne, L.; Riesner, D. Biol. Chem. 2006, 387, 95-102. 10.1021/ac702153p CCC: $40.75 Published on Web 02/14/2008

© 2008 American Chemical Society

To differentiate between PrPc and PrPsc, the sample is pretreated with protease K. Since PrPsc is digestion resistant and PrPc is easily digested by protease K, pretreatment results in a sample that is rich in PrPsc as compared to PrPc.4,5 Because neither the sensitivity of PrPc nor the resistance of PrPsc to digestion is absolute, the “protease sensitivity assay” cannot definitively measure the presence or absence of PrP. Current prion detection methods employ post mortem analysis after suspicious animals manifest one or more symptoms of the disease. These post mortem tests include gel electrophoresis and western blot, direct-binding and sandwich ELISA, and conformational-dependent immunoassay.4-6 To improve food safety it would be beneficial to screen all the animals for prion disease using ante mortem testing, regardless of the presence of symptoms. Because ante mortem tests could be performed on presymptomatic animals, they would therefore be required to detect extremely small amounts of PrP circulating in blood samples and would have to differentiate PrPc and PPsc.7-9 Some recent approaches for ante mortem diagnosis include applying newly developed ligands against PrPsc,10,11 conformational-dependent immunoassays,12,13 spectroscopic techniques,14,15 and PrPsc amplification.16,17 Efforts to avoid protease digestion and use of other means to differentiate PrPsc and PrPc are the most desirable, but these methods are under trial and no practical application has been suggested so far. Fischer et al. reported the use of plasminogen as a ligand (4) Soto, C. Nat. Rev. Microbiol. 2004, 2, 809-819. (5) Lourenco, P. C.; Schmerr, M. J.; MacGregor, I.; Will, R. G.; Ironside, J. W.; Head, M. W. J. Gen. Virol. 2006, 87, 3119-3124. (6) Kim, J. I.; Wang, C.; Kuizon, S.; Xu, J.; Barengolts, D.; Gray, P. C.; Rubenstein, R. J. Neuroimmunol. 2005, 158, 112-119. (7) Brown, P.; Cervenakova, L.; Diringer, H. J. Lab. Clin. Med. 2001, 137, 5-13. (8) Collins, S.; Boyd, A.; Fletcher, A.; Gonzales, M. F.; McLean, C. A.; Masters, C. L. J. Clin. Neurosci. 2000, 7, 195-202. (9) Ingrosso, L.; Vetrugno, V.; Cardone, F.; Pocchiari, M. Trends Mol. Med. 2002, 8, 273-280. (10) Kornblatt, J. A.; Marchal, S.; Rezaei, H.; Kornblatt, M. J.; Balny, C.; Lange, R.; Debey, M. P.; Hui Bon Hoa, G.; Marden, M. C.; Grosclaude, J. Biochem. Biophys. Res. Commun. 2003, 305, 518-522. (11) Negredo, C.; Monks, E.; Sweeney, T. BMC Biotechnol. 2007, 7, 43-49. (12) Thackray, A. M.; Hopkins, L.; Klein, M. A.; Bujdoso, R. J. Virol. 2007, 81, 12119-12127. (13) Korth, C.; Streit, P.; Oesch, B. Methods Enzymol. 1999, 309, 106-122. (14) Fujii, F.; Horiuchi, M.; Ueno, M.; Sakata, H.; Nagao, I.; Tamura, M.; Kinjo, M. Anal. Biochem. 2007, 370, 131-141. (15) Beekes, M.; Lasch, P.; Naumann, D. Vet. Microbiol. 2007, 123, 305-319. (16) Jones, M.; Peden, A.; Prowse, C.; Gro¨ner, A.; Manson, J.; Turner, M.; Ironside, J.; Macgregor, I.; Head, M. J. Pathol. 2007, 213, 21-26. (17) Klohn, P. C.; Stoltze, L.; Flechsig, E.; Enari, M.; Weissmann, C. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 11666-11671.

Analytical Chemistry, Vol. 80, No. 6, March 15, 2008 2141

that interacts specifically with PrPsc and not with PrPc.18 However, others have suggested that the plasminogen also has some affinity for PrPc,19 which might compromise its application for diagnosis. In another effort to develop conformation-specific antibodies, antiDNA antibodies called OCD4 were shown promising with PrP from brains affected by prion diseases in both humans and animals but not from unaffected controls.20 OCD4 immunoreacts with DNA (or DNA-assisted molecule) that forms a conformational-dependent complex with PrPsc in prion disease. PrP immunoprecipitated by OCD4 is often protease resistant, but some complexes are digestible by protease, thus preventing OCD4 as an exclusive antibody for the precipitation of PrPsc. Micro- and nanoelectromechanical systems (MEMS and NEMS) are being developed as sensitive chemical and biological sensors capable of detecting small amounts of analytes.21 In general, sensors built with this technology are operated in either the static or dynamic sensing mode. The static mode of sensing utilizes device structures functionalized on one side for binding of a specific analyte. Unbalanced surface stresses resulting from analyte binding cause the structures to deflect, signifying detection. Dynamic mode sensors are excited at natural resonant frequencies, and shifts in resonant frequency as a result of analyte binding signify detection. Static deflection-based sensors are suitable for in situ detection of analytes, whereas in most cases, dynamic sensors require measurements performed in air or vacuum to improve sensitivity, which is strongly limited by viscous damping effects in fluids. However, there has been some concern that the drying process and transport to and from solution may result in increased noise and nonspecific binding. Recently, Burg et al. demonstrated a novel approach for operation of dynamic resonators in the solution while maintaining the advantage of high quality factor by working in vacuum.22 They designed a suspended cantilever with built-in microfluidic channel that was used for all binding events and flow of solution. The measurement was done in the vacuum while the solution was flowing through the microchannels. Resonant mechanical devices are commonly modeled as harmonic resonators with the resonant frequency, f0, given by

f0 )

1 2π

xmk

(1)

where k is the spring constant and m is the mass of the resonator. For rectangular cantilevers, the spring constant is given by Ewt3/ 4l3, where E is the material stiffness or Young’s modulus and w, l, and t are the width, length, and thickness of the cantilever, respectively.23 It is apparent from these expressions that resonant frequency is a function of more than just mass and that several different parameters can potentially be used in sensing applications. (18) Fischer, M. B.; Roeckl, C.; Parizek, P.; Schwarz, H. P.; Aguzzi, A. Nature 2000, 408, 479-483. (19) Shaked, Y.; Engelstein, R.; Gabizon, R. J. Neurochem. 2002, 82, 1-5. (20) Zou, W. Q.; Zheng, J.; Gray, D. M.; Gambetti, P.; Chen, S. G. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 1380-1385. (21) Waggoner, P. S.; Craighead, H. G. Lab Chip 2007, 7, 1238-1255. (22) Burg, T. P.; Godin, M.; Knudsen, S. M.; Shen, W.; Carlson, G.; Foster, J. S.; Babcock, K.; Manalis, S. R. Nature 2007, 446, 1066-1069. (23) Sader, J. E.; Chon, J. W. M.; Mulvaney, P. Rev. Sci. Instrum. 1999, 70, 3967-3969.

2142

Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

However, the majority of cantilevered sensors are long and thin (l > w . t) such that analyte binding will most strongly change device thickness, and changes in width and length can be neglected. For small changes in stiffness, thickness, or mass the change in the frequency, ∆f ) f - f0, can be approximated to the first order by

∆t ∆m 1 ∆E +3 ∆ f ) f0 2 E t m

(

)

(2)

The effects of stiffness and thickness on resonant frequency have been experimentally demonstrated and analyzed in recent work.24-26 It has also been shown that the location of the bound analyte on the sensor can also determine to what extent changes in mass or stiffness affect the resonant frequency. Tamayo et al. have shown that adsorption of bacteria at the free end of a cantilever, where the motion is maximum, results in negative frequency shifts due to mass-related effects, whereas adsorption near the clamped end, where the motion is minimum, gives way to positive frequency shifts due to increased stiffness.24 Resonant sensors for mercury vapor have also shown positive and negative shifts depending on how the mercury is adsorbed on the sensor.27 For cantilevers entirely coated in gold, the frequency is observed to increase, whereas if gold was coated only on the tip of the resonator, the same mercury vapor will cause the frequency to decrease. With proper experimental design, one or more of these effects can be neglected, facilitating data analysis and interpretation of results. Although unbalanced surface stress induced by analyte binding to cantilevers causes bending, there has been some debate concerning how surface stress affects resonant frequency, if at all. In several experiments unexpected frequency shifts, both positive and negative, have been attributed to surface stress affects.28,29 Typically, a one-dimensional model is used to convert surface stresses into an axial load that then alters the spring constant. Recent work, however, has pointed out that this simplified model is incorrect and violates Newton’s third law.30 They also come to an expression for the change in frequency due to strain-independent surface stresses and state that in general this effect is relatively small and cannot account for the unexpected resonant frequency shifts seen in the literature. Resonant sensors are exquisitely sensitive for mass detection, demonstrating analytical sensitivities on the order of femtograms or less.31-33 They are of diagnostic interest because of their small size, high sensitivity, and suitability for integration into miniaturized analytical systems. Furthermore, fabrication capabilities allow (24) Tamayo, J.; Ramos, D.; Mertens, J.; Calleja, M. Appl. Phys. Lett. 2006, 89, 224104-224107. (25) Ramos, D.; Tamayo, J.; Metens, J.; Calleja, M.; Zaballos, A. J. Appl. Phys. 2006, 100, 106105-106108. (26) Gupta, A. K.; Nair, P. R.; Akin, D.; Ladisch, M. R.; Broyles, S.; Alam, M. A.; Bashir, R. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 13362-13367. (27) Thundat, T.; Wachter, E. A.; Sharp, S. L.; Warmack, R. J. Appl. Phys. Lett. 1995, 66, 1695-1697. (28) Chen, G. Y.; Thundat, T.; Wachter, E. A.; Warmack, R. J. J. Appl. Phys. 1995, 77, 3618-3622. (29) Dorignac, J.; Kalinowski, A.; Erramilli, S.; Mohanty, P. Phys. Rev. Lett. 2006, 96, 186105. (30) Lachut, M. J.; Sader, J. E. Phys. Rev. Lett. 2007, 99, 206102. (31) Ilic, B.; Yang, Y.; Aubin, K.; Reichenbach, R.; Krylov, S.; Craighead, H. G. Nano Lett. 2005, 5, 925-929. (32) Ilic, B.; Yang, Y.; Craighead, H. G. Appl. Phys. Lett. 2004, 85, 2604-2606.

the creation of large arrays of devices that can improve sensitivity, reduce false positives and negatives, and support highly multiplexed systems for parallel detection of several analytes.21 Although the absolute mass sensitivity is a strong point for resonant sensors, sensitivity to a very small concentration of analyte in a biologically relevant medium can be more important for early detection of disease or trace constituent analysis. One method which may overcome some limitations of resonant sensors at low analyte concentrations is secondary mass labeling for signal amplification. If additional mass could be added to only those devices where the analyte has bound, then the frequency shift would be enhanced and the detection limit improved.34 Also, at low analyte concentrations, relatively large mass labels that are sparsely bound to resonators can potentially negate stiffness and thickness effects and result in a strictly mass-dependent frequency shift. In this work we use microfabricated biosensor chips consisting of arrays of low-stress silicon nitride resonators as a sensing platform for PrP detection. Device surfaces were functionalized via silanization and glutaraldehyde chemistry. We used dynamic resonance-based techniques for the detection of PrP, with and without additional mass labels. The resonator arrays were operated in vacuum during frequency measurement. A sandwich assaybased mass labeling technique was employed to enhance the frequency shift for the detection PrP proteins. The use of two antibodies in the sandwich assay not only enhances the specificity of the sensor for PrP protein but also provides a means for linking heavier nanoparticle mass tags to the immobilized PrP in order to amplify resonator response to low concentrations. We will also assess the ability of nanoparticle-based mass labeling to limit resonant frequency shifts to mass-related effects. EXPERIMENTAL SECTION Chemicals and Materials. Recombinant prion protein (Chemicon, Temecula, CA), primary monoclonal antibodies against amino acids 23-237 of bovine prion protein (Chemicon, Temecula, CA), secondary monoclonal antibodies against amino acids 123-136 and 140-160 of bovine prion protein (Abcam Inc., Cambridge, MA), bovine serum albumin (BSA, Sigma), phosphate-buffered saline (PBS, Sigma), streptavidin-conjugated nanoparticles (R&D Systems, Minneapolis, MN), 3-aminopropyl triethoxysilane (APTES, Sigma), gluteraldehyde (GA, Sigma), anhydrous toluene (Sigma), and glycine (Sigma) were all commercially obtained. Magnetic nanoparticles of 150 nm diameter have more than 85% of oxide as Fe3O4, approximately 80% w/w of magnetite. The estimated mass of one nanoparticle is 2.4 fg based on the density of 1.34 g/cm3. A solution of 1% BSA in PBS was used for blocking between different steps of the methodology. PBS was used for the dilution of PrP protein and antibodies. A 50 mM solution of glycine in deionized (DI) water was used for quenching. Blocking solutions were filtered through 2 µm filters prior to use. Secondary antibodies were biotin conjugated to attach 5-7 biotin molecules per antibody molecule by using NHS-PEO4-biotin (Pierce Chemicals). The biotin per antibody was determined by using EZ biotin quantification kit (Pierce Chemicals). (33) Forsen, E.; Abadal, G.; Ghatnekar-Nilsson, S.; Teva, J.; Verd, J.; Sandberg, R.; Svendsen, W.; Perez-Murano, F.; Esteve, J.; Figueras, E.; Campabadal, F.; Montelius, L.; Barniol, N.; Boisen, A. Appl. Phys. Lett. 2005, 87, 043507043509. (34) Su, M.; Li, S.; Dravid, V. P. Appl. Phys. Lett. 2003, 82, 3562-3564.

Resonator Fabrication and Functionalization. The resonators used in this experiment were fabricated using standard photolithographic techniques. A 200 nm thick layer of low-stress silicon nitride was deposited on a thermally oxidized silicon wafer using low-pressure chemical vapor deposition. The silicon nitride device layer was then patterned using an anisotropic reactive ion etch. Device chips were dipped in hydrofluoric acid in order to remove the 1.5 µm thick sacrificial silicon dioxide layer under the resonators and release them from the substrate, allowing them to move freely. Typical bare resonators exhibited resonant frequencies of approximately 4.6 MHz, with quality factors of about 7000. Each chip had approximately 1000 devices configured in an arrayed block format, providing on-chip redundancy that can give statistically significant results measured from a large number of sensors experiencing identical experimental conditions. Resonators used in this work are cantilevered beams with a 3 µm × 10 µm paddle at the free end, as shown in Figure 1. A detailed schematic of device dimensions is also shown in Figure S1 in the Supporting Information. These devices are excited in their fundamental mode, which is analogous the fundamental mode in rectangular cantilevers, where motion is in and out of plane. The paddle has been added in order to improve device sensitivity to secondary mass labels. As previously discussed, masses bound near the free end of cantilevered structures have the greatest effect on resonant frequency; these paddles are meant to increase the sensing area at this most sensitive region of the resonator.25,35 Before functionalization, the resonators were cleaned by soaking in piranha etchant (mixtures of 98% sulfuric acid and 30% hydrogen peroxide in a volume ratio of 2:1) for 30 min followed by washing with a large amount of DI water and finally an oxygen plasma treatment for 30 min. For silanization, the devices were immersed in 10% APTES in dry toluene in a controlled nitrogen atmosphere glovebox for 14 h at room temperature (RT). This treatment provides reactive amine groups on the nitride surface. After removal of resonators from APTES, they were washed with toluene, isopropyl alcohol, and DI water. They were then soaked in DI water for 15 min to remove excess APTES. After silanization, the resonators were soaked in 5% GA solution in borate buffer (pH 8.0) for 2 h at RT. GA is a homobifunctional cross-linker between amine groups on the APTES and primary amines on the antibodies. After thorough washing with DI water, the chips were incubated with 10 µL of 50 µg/mL of primary antibodies for 1 h at RT followed by a washing and a quenching necessary to saturate any free amine groups so that unreacted GA does not subsequently bind with secondary antibodies which also have addressable primary amine groups. This was done by immersing resonators in a 50 mM glycine solution for 30 min. After quenching, blocking was performed for 20 min using a blocking solution to prevent nonspecific binding of prion protein to the surface. Following the blocking step, the resonators were incubated with different concentrations of PrP protein (ranging from 200 pg/mL to 20 µg/mL) by incubating with 10 µL of PrP samples for 1 h at RT. Another blocking step of 10 min was performed in order to prevent the nonspecific binding of secondary antibodies to the device surface, followed by a washing step. After blocking (35) Dohn, S.; Sandberg, R.; Svendsen, W.; Boisen, A. Appl. Phys. Lett. 2005, 86, 233501-233504.

Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

2143

Figure 1. Schematic of the mass labeling with nanoparticles to detect prion protein. Schematic of laser interferometric optical setup (adopted from ref 24) and the mass labeling with nanoparticles to detect prion protein. The resonators were operated in the fundamental mode of vibration, and the total active surface area of the resonator was 56 µm2 as shown by the broken lines. The sizes for the base, neck, free end, and etch holes are 10 µm × 10 µm, 1 µm × 2 µm, 2 µm × 10 µm, and 1 µm × 1 µm, respectively (see Supporting Information Figure S1).

and washing, the resonators were incubated with 10 µL of biotinylated secondary antibodies (50 µg/mL) for 1 h at RT followed by a washing step. For nanoparticle mass labeling, the resonators were incubated with 10 µL of streptavidin-conjugated nanoparticles (1:75 dilution in blocking solution) for 30 min at RT immediately after functionalization with biotinylated secondary antibodies. The resonators were thoroughly cleaned with DI water to remove unbound nanoparticles and were dried under stream of nitrogen gas before frequency measurement. The washing in all the above steps was done by squirting DI water from a wash bottle for 1 min followed by a 2 min at 80 rpm wash using an orbital shaker. Frequency Measurement. Resonators were both excited and sensed in vacuum using optical techniques (Figure 1).36 A 405 nm diode laser was modulated in intensity and used to excite the resonators by focusing the beam near its clamped end. Due to the higher thermal conductivity of the silicon nitride layer as compared to the underlying silicon dioxide layer, a temperature gradient is induced that is responsible for the inhomogeneous thermal expansion of the device. Thermal expansion mismatch between the silicon nitride and oxide is ultimately responsible for actuation of the sensor. Resonant frequencies were determined interferometrically by measuring the reflectance variation from an incident HeNe red laser focused at the free end of the resonator. As the sensor moves in and out of plane, the gap between the wafer and device layers changes in thickness. This dynamic film stack changes the degree of optical interference and modulates the intensity of reflected light at the mechanical resonant frequency of the device. The reflected signal is collected by a photodetector, and the resonant spectrum of the device is extracted using a spectrum analyzer. For the resonators in this work, the lowest frequency mode observed was the 4.6 MHz mode, and the reflected signal was highest when focused at the center of the paddle. As a result, we believe that this mode is the

fundamental mode since torsional, wing-flapping, or in-plane modes would not produce the out-of-plane motion at the center of the paddle that is required for this optical interference technique. The excitation and detection laser powers were kept at a minimum, on the order of 1 and 100 µW, respectively, since at higher powers the resonance was observed to drift in frequency over short time periods. Because silicon nitride absorbs light more strongly at shorter wavelengths, it is important to use a low diode laser power. At these low powers, peak frequencies were found to be stable for several minutes, giving sufficient time to measure the resonant frequency of a device. The time taken to measure resonant frequencies of 24 devices was 10 min. Resonators chips were dried under the stream of nitrogen gas before frequency measurements. They were loaded into a small vacuum chamber that was mounted to a motorized stepper stage, and the resonant frequencies were measured. Computer control of the stage and spectrum analyzer allowed easy measurement of large arrays of resonators in a short amount of time. The chip consists of blocks of arrays of the resonators, each containing 12 devices. All devices on the chip were alike, and they gave similar response in terms of the resonant frequency and quality factor. For each concentration of PrP, two arrays of resonators (24 devices) per chip were chosen randomly on the chip, and they were used for frequency measurement in a single test. The average values and errors were calculated based on frequency measurements from 24 resonators. When mass is added uniformly across resonant devices, such as in antibody immobilization or analyte binding, it is instructive to consider a surface mass density, σ, rather than ∆m in eq 1. Assuming that binding occurs on both sides of the resonator and any stiffness or thickness changes are negligible, the frequency shift signal becomes

(36) Ilic, B.; Krylov, S.; Aubin, K.; Reichenbach, R.; Craighead, H. G. Appl. Phys. Lett. 2005, 86, 193114-193116.

σ ∆f )f Ft

2144

Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

(3)

Figure 2. AFM images of the device surface shown after (a) surface cleaning and after functionalization with (b) APTES and GA, (c) primary antibodies, (d) prion, and (e) secondary antibodies. The z-axis bar on all images is 30 nm, and the scan size is 2 µm × 2 µm.

where F is the density of the resonator and t is the device thickness. One important implication of this relationship is that device geometry does not affect the sensitivity to a uniformly bound layer; only the device thickness is significant. RESULTS AND DISCUSSION Functionalization and Mass Labeling. Resonator surfaces during functionalization are shown by AFM images in Figure 2. These images demonstrate that chemical/biological moieties were added to the resonator surface after each functionalization step. The addition of PrP did not result in any significant visual change to the resonator surface, as the PrP is very small. Furthermore, the addition of PrP did not show any significant change in the resonant frequency (see Figure 4a). The steps of functionalization were also analyzed by measuring resonant frequencies of the resonators. The resonant frequencies were measured after each functionalization step; Figure 3a shows how the resonant frequency peak changes with each functionalization step for one particular device used to detect PrP at a concentration of 20 µg/ mL. The general trend shows a decreasing peak frequency as more substance is added to the resonator. Frequency shifts shown in Figure 3b are measured on an entire array of devices, and the values are averaged in order to determine an error based on their standard deviation. For one such array measuring 20 µg/mL of PrP, the cumulative frequency shifts for APTES + GA, primary antibodies, PrP, secondary antibodies, and nanoparticles were -22.4 ( 4.12, -51.86 ( 4.28, -50.71 ( 1.68, -53.67 ( 0.79, and -74.02 ( 5.56 kHz, respectively. The silanization process was found to have a strong impact on the success of the subsequent functionalization steps and the

ability to ultimately detect the analyte. We have optimized the silanization process by studying incubation times of 20 min, 80 min, and 14 h. The efficacy of the silanization was quantified in terms of the frequency shift for the detection of 20 µg/mL PrP using mass labeling with nanoparticles. Only the incubation time during silanization was varied; all other steps during functionalization were kept the same. The frequency shifts for the control sample (with no PrP) and the sample (20 µg/mL PrP) for 20 min, 80 min and 14 h of incubation were -2350 ( 783 Hz, -3067 ( 1717 Hz; -6310 ( 2038, -13291 ( 3745 Hz; -8107 ( 3348 Hz, -25298 ( 4231 Hz, respectively (see Supporting Information Table S1). While the frequency shift for control as well as sample scaled with the incubation time, the PrP shift was amplified by a greater amount. We attribute this behavior to improved functionalization in terms of a higher number of binding sites per unit area. Howarter and Youngblood,37 showed that the increase in the incubation time during silanization increases the surface roughness as well as surface coverage, resulting in an increased surface area for the subsequent functionalization steps. It was observed that the frequency shifts for the control and sample increased by a factor of approximately 2.7 and 4.3, respectively, for the first 60 min increase in incubation time (from 20 to 80 min), and they increased by 1.3 and 1.9 for the 14 h incubation time compared to the 80 min incubation. The frequency change due to the presence of PrP is represented by the “differential signal” calculated by subtracting control (no PrP) frequency shift from that of the sample (with PrP). The differential signal improved greatly with increase in silanization time. The values of differential (37) Howarter, J. A.; Youngblood, J. P. Langmuir 2006, 22, 11142-11147.

Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

2145

Figure 3. (a) Resonant frequency peaks and (b) cumulative resonant frequency shifts for different steps of functionalization to detect 20 µg/mL PrP. Only one resonator was used for the frequency peak calculations, whereas 24 were used for the cumulative frequency shifts.

signal were -717, -6981, and -17 191 Hz for 20 min, 80 min, 14 h, respectively. Therefore, 14 h of incubation time during silanization was used for all further experiments. Sensitivity and Detection Limit. The frequency shifts from resonators exposed to different concentrations of PrP are shown in Figure 4a. The frequency shift for each concentration was determined from frequency measurements before and after exposing the resonators to PrP. The respective differential signals were calculated by subtracting the control (no PrP) frequency shift from that of the sample (with PrP). The errors were added as per the propagation of uncertainties for the sum of two values. It was observed that the mass addition due to PrP alone was not sufficient to cause a significant frequency shift up to a concentration of 20 µg/mL. Therefore, the additional mass labels were used to amplify the change in frequency corresponding to the presence 2146

Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

of PrP on the resonator surface. The frequency shifts for different concentrations of PrP (20 ng/mL, 200 ng/mL, 2 µg/mL, and 20 µg/mL) on resonators with secondary antibodies added are shown in Figure 4b. The results showed that the detection limit for PrP with secondary antibody labeling was improved to 2 µg/mL. PrP concentrations less than 20 ng/mL were not tested for the two experiments mentioned above as no significant frequency shifts (with respect to the control) were observed below 20 µg/mL for the detection of PrP alone and 200 ng/mL with secondary antibody labeling. In order to further amplify the frequency shift and improve the detection limit, streptavidin-conjugated nanoparticles were then attached to biotinylated secondary antibodies present on the resonator surfaces. The frequency shifts due to the presence of nanoparticle mass labels for different concentrations of PrP (from

200 pg/mL to 20 µg/mL) are shown in Figure 4c. The results clearly showed that the use of nanoparticles as mass labels improved the detection limit to 2 ng/mL of PrP. Figure 5 shows SEM images of resonators functionalized with nanoparticles at each concentration of PrP measured. They clearly demonstrate that the number of bound nanoparticles decreased with the decrease in the PrP concentration. The number of particles on each resonator was determined by manually counting particles and multiplying by a factor of 2 to account for particles on the bottom device surface. An average value was obtained from several resonators and used to calculate the total mass responsible for causing the frequency shift. This calculated mass was compared with the mass obtained from experimentally observed frequency shifts using eq 3. It was observed that these two calculated masses were not significantly different (P > 0.05) at each concentration of PrP (Supporting Information Table S2). Therefore, we conclude that the frequency shift was due to mass addition only and the effect of stiffness due to the addition of nanoparticles was negligible. In addition, the agreement between these values demonstrates that the bound nanoparticles, although sparsely distributed across the resonator, still behaved as a uniform layer. The anticipated improvement in sensitivity due to the unique device geometry was not observed, which we attribute to the detection limit including a large number of nanoparticles on the device surface. Further reduction of nonspecific binding and improved resonator sensitivity may be able to shed more light on these effects.

Figure 4. Resonant frequency shifts for the detection of PrP (a) with no mass labeling, (b) with secondary antibodies mass labels, and (c) with nanoparticle mass labels. Twenty-four resonators were used for the measurement of frequency shifts.

CONCLUSIONS A detection system based on arrayed resonant sensors was successfully developed to detect PrP using secondary mass labeling. With the use of antibodies as secondary mass labels we were able to detect 2 µg/mL concentrations of PrP, while the addition of nanoparticle mass labels improved the detection limit by 3 orders of magnitude to 2 ng/mL. Owing to the high sensitivity

Figure 5. SEM images of resonators with nanoparticles as mass labels for the detection of (a) 20 µg/mL, (b) 2 µg/mL, (c) 200 ng/mL, (d) 20 ng/mL, (e) 2 ng/mL, and (f) no PrP (control). All scale bars are 2 µm in length.

Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

2147

of resonator-based detection systems, we project that they can be suitably applied to develop ante mortem tests to directly detect PrP in body fluids. ACKNOWLEDGMENT This material is based upon work supported by the Cooperative State Research, Education, and Extension Service, U.S. Department of Agriculture, under Award No. 2007-35603-17746. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture.

2148

Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

SUPPORTING INFORMATION AVAILABLE Tables showing the frequency shifts for different times of silanization and comparison of mass calculations from frequency shift measurements and counting of number of particles on the resonator; figure showing the relevant dimensions of the device. This material is available free of charge via the Internet at http:// pubs.acs.org. Received for review October 18, 2007. Accepted January 7, 2008. AC702153P