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Nanomechanical Resonators for Prion Protein Detection

Philip S. Waggoner, Madhukar Varshney, Christine P. Tan, and Harold Craighead

Abstract

In this study, arrays of nanomechanical resonators were employed to detect small molecules using specifically bound nanoparticle mass labels to amplify the frequency shift associated with chemical binding. As a model we used established anibody-based binding of prion protein (PrP). The detection limit of the system was improved by the use of antibodies and nanoparticles as mass labels, both specific for the presence of PrP.

Research Summary

Figure 1. Resonant frequency shifts for the detection of PrP, (a) with secondary antibody mass labels, and (b) with nanoparticle mass labels.

The resonators are fabricated from a 200 nm thick layer of low-stress silicon nitride deposited on a 1.5 µm thick layer of silicon dioxide. Devices were patterned using optical photolithography and released from the substrate by removing the underlying sacrificial oxide with hydrofluoric acid. They were loaded into a small vacuum chamber mounted on a motorized stepper stage and the resonant frequencies were measured using optical techniques [1]. A 405 nm diode laser was modulated in intensity and used to periodically heat and excite the resonators. Thermal expansion mismatch between the silicon nitride and oxide is ultimately responsible for sensor actuation. Resonant frequencies were determined interferometrically by measuring the reflectance variation from an incident HeNe laser (632.8 nm) focused at the free end of the resonator. Computer control of the stage and spectrum analyzer allowed easy measurement of large arrays of resonators in a short amount of time. For each concentration of PrP, 2 arrays of resonators (24 devices) per chip were chosen randomly for use. The average values and errors were calculated based on frequency measurements from 24 resonators.

The primary antibodies were coated on the surface using amino-propyltriethoxysilane and gluteraldehyde [2].  The resonators were exposed to PrP alone and subsequently with secondary antibodies and nanoparticles for mass labeling. For the detection of PrP alone, the resonant frequency of each device was measured before and after exposing the resonators to PrP.  The frequency shifts from the control (no PrP) were then subtracted from that of the sample (with PrP).  It was observed that PrP could not be detected at concentrations of 20 µg/mL and below (data not shown). Therefore, mass labeling was required in order to detect PrP at these concentrations. Because both the secondary antibodies and nanoparticles specifically bind to PrP, mass labeling corresponds to the presence of PrP on the device surface. The frequency shifts due to secondary antibody binding for different concentrations of PrP (20 ng/mL, 200 ng/mL, 2 µg/mL and 20 µg/mL) are shown in Figure 1a. This sandwich assay improved the detection limit for PrP to 2 µg/mL.  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 1b. The results indicate that the nanoparticles improved the detection limit to 2 ng/mL of PrP.  Figure 2 shows an SEM image of a resonator functionalized with nanoparticles for a PrP concentration of 2 µg/mL.

Figure 2. SEM image of a resonator with nanoparticle mass labels for the detection of 2 µg/mL PrP.

References

  1. Ilic, B.; Krylov, S.; Aubin, K.; Reichenbach, R.; Craighead, H. G. Appl. Phys. Lett. 2005, 86, 193114-193116. Abstract
  2. Varshney, M.; Waggoner, P.; Tan, C. P.; Aubin, K.; Montagna, R.; Craighead, H. G. Anal Chem. 2008, 80, 2141-2148. Abstract