Bovine Spongiform Encephalopathy Surveillance by Real-Time Biosensing of Meat-and-Bone Meal in Animal Feeds Using Microcantilever Sensors

NON-TECHNICAL SUMMARY: In this work, a surface chemist and materials engineer combine their unique backgrounds and experience in a multidisciplinary effort towards developing and demonstrating the feasibility of cantilever-based microsensors in the detection of meat-and-bone meal (MBM) biomarkers in animal feed towards preventing bovine spongioform encephalopathy (BSE) contamination of U.S. cattle. BSE continues to remain a potential global health crisis that requires aggressive precautionary measures. This work will involve examining the combination of microcantilevers with piezoresistive transduction elements to record nanoscale changes in the cantilever\\\'s behavior resulting from biomass attachment. Using numerical simulations we will investigate the optimum cantilever material and geometrical parameters to obtain the configurations that increase sensor sensitivity to minute additions of mass and also allow in-situ solution measurements. The developed platform will be combined with an immunochemical assay for a MBM biomarker towards detection and quantification of of these prohibited residues in animal feed. An attachment chemistry will be developed to functionalize and fix the antibody to the cantilever surface allowing direct detection of the BSE biomarker. Through batch processing, using standard microfabrication techniques, numerous devices can be produced in a compact and cost effective manner. The ability to inspect animal feed for prohibited MBM matter would allow the USDA to have more confidence in claims that the U.S. <P>

APPROACH: The approach to accomplishing the objectives of the work follows three streams. The first involves development and fabrication of the microsensor platform. This begins with numerical simulation of the cantilever structure that includes varying the cantilever geometry as well materials selection for determining the optimum cantilever characteristics that enable high performance and sensitivity. By this method, several platform configurations can be realized to minimize microfabrication time and costs. After fabrication, these devices will characterized by electron microcopy and load response behaviors probed by a nanoindenter. The second stream involves the development of a surface attachment chemistry for the MBM antibody. The PIs will employ Self-assembled monolayers (SAMs) to attach the biological molecules to the device\\\'s surfaces. This scheme uses specialized linker molecules that are adept at attaching the antibody to gold surfaces. The effectiveness of this immobilization chemistry will be evaluated using atomic force microscopy (AFM) to evaluate the surface bound antibodies exactly as they exist on the microcantilever sensors. The final stream is integrating the first two to arrive at a complete sensor. The final devices will be fabricated with the appropriate gold surfaces for the immobilization chemistry. They will then be exposed to solutions containing the linker molecules and then finally to antibody solutions. The device will be tested in a null solution to obtain the baseline frequency signature of the device. This frequency signature will be constantly monitored for changes as the device is exposed to an MBM containing solution with significant deviations corresponding to positive confirmation of MBM presence.
<P>
PROGRESS: 2003/08 TO 2007/07<BR>
OUTPUTS: Invited lecture, 7th Annual Electrochemistry Days, Department of Chemistry, Hacettepe University, Ankara, Turkey. \\\'New Materials for Analytical Chemistry\\\', June 29, 2006. Case Western Reserve University, Cleveland, OH. Departmental colloquium: \\\"Electrochemical Routes to Materials and Devices: Semiconductors, Electrocatalysis and Biosensors\\\", October 14, 2004. John Carroll College, Cleveland, OH. Departmental colloquium: \\\'Electrochemical Routes to Materials and Devices: Semiconductors, Electrocatalysis and Biosensors\\\', October 13, 2004. Brooklyn College, Brooklyn, NY. Department of Chemistry, March 26, 2004. Colloquium: \\\'Building Materials and Sensors from the Ground Up: A Surface Electrochemistry Approach\\\'. Pittsburgh Conference, National Meeting, New Orleans, LA, March 21, 2002. Invited talk: \\\'Surface Science Approaches to Chemical Sensing With the AFM\\\'. Results and devices features in Auburn Universities Engineering Day, which is a high school recruitment day where approximately 3000 to 5000 students from across Alabama and Georgia visit campus and view research and demonstrations by Auburn academic departments. <BR>PARTICIPANTS: Barton C. Prorok (PI): Dr. Prorok worked on designing and fabricating the devices. Curtis Shannon (PI): Dr Shannon worked on the attachment scheme of the specified antibody to the sensor surface. Shakib Morshed (Graduate Student): Mr. Morshed was the graduate student responsible for designing and fabricating the devices. Serdar Abaci (Graduate Student): Mr. Abaci worked on the attachment scheme of the specified antibody to the sensor surface. <BR>TARGET AUDIENCES: Auburn University\\\'s Engineering Day (2006) - viewed by some 3000-5000 students. Short Course on \\\'MEMS and Nanotechnology,\\\' given by Dr. Prorok at The Mechanical Engineering Conference at Auburn University (2004, 2006 and 2007). <BR>PROJECT MODIFICATIONS: The main alteration was the difficulty in fabricating the proposed devices. Fully functioning devices with adequate sensitivity above electronic noise were not able to be obtained by the end of the project time line. Thus the direct detection with the devices was not able to be performed.
<BR><BR>
IMPACT: 2003/08 TO 2007/07<BR>
During the course of this project, three schemes to covalently modify the surfaces of microcantilever sensors were developed and evaluated. The three methods were: 1) mixed self-assembled monolayers (SAMs), random antibody attachment, 2) mixed SAMs, oriented antibody attachment; 3) conducting polymers, antibody fragments. Method 1 involved forming a two-component SAMs consisting of both non-reactive (methyl) and reactive (carboxylic acid) terminal groups. Acid catalyzed amidation was used to attach antibodies to the surface-bound carboxyl groups through pendant lysine residues. The resulting surface bound antibodies were randomly oriented, rendering many of them biologically inactive. Method 2 overcame this limitation by first oxidizing the antibody chemically so as to generate reactive aldehyde groups in the hinge region of the antibody. These activated antibodies were then covalently linked to the SAM surface using chemistry similar to that of Method 1. Method 3 allows molecular recognition units such as antibody fragments to be covalently attached to a thin polymer film that is pre-deposited on a sensor surface. The oxidized form of conducting polymers such as polyaniline can be modified by nucleophilic addition, giving polymers containing different moieties linked to the polymer backbone. Each method was found to have strengths and weaknesses. Specifically, methods 1 and 2 were easy to implement, but the monolayers were not as robust as those formed using method 3. On the other hand, method 3 resulted in somewhat thicker films with unique mechanical properties. Method 3 was felt to hold greater promise for future device applications. The device aspect of this work included a combined numerical and experimental study to assess the influence of microcantilever geometry on mass sensitivity in order to improve these devices for better detection of hazardous biological agents in liquid environments. Modal analysis was performed on microcantilevers of different geometries and shapes using ANSYS software and compared to the basic rectangular shaped microcantilever structures employed by most researchers. These structures all possessed a 50 um length, 0.5 um thickness and 25 um width where the cantilever is clamped to the substrate, and were analyzed for their basic resonance frequency as well as the frequency shift for the attachment of a 0.285 picograms of mass attached on their surfaces. These numerical results indicated that two parameters dominate their behavior, (1) the effective mass of the cantilever at the free end and (2) the clamping width at the fixed end. The ideal geometry was a triangular shape, which minimized effective mass and maximized clamping width, resulting in an order of magnitude increase in mass sensitivity over rectangular shaped cantilevers of identical length and clamping width. The most practical geometry was triangular shaped cantilever with a square pad at the free end for capturing the agent of interest. This geometry resulted in a 4-fold increase in performance over their rectangular counterparts.