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Successful completion of this multidisciplinary project will enhance the protection and safety of the Nation's agriculture and food supply. Asymptomatic, prion-infected, cows represent a more serious threat to the human food supply than those that outwardly display symptoms. In order to improve the safety of the food supply, we will exploit the exquisite sensitivity of resonating mechanical biosensors already capable of detecting single microbes (bacteria and virions) and single DNA molecules. The resonating mechanical biosensors detect analytes by registering decreases in their resonant frequency upon binding of target analytes to their surface. <BR><BR>Although masses as low as 0.39 attograms (0.39 x 10-18 grams) can presently be detected, new advances will be required to permit direct detection of low numbers of circulating prions in asymptomatic cows. Since a single prion has a mass approximately ten times less than the current analytical threshold of the resonating mechanical biosensor, methods will be developed to first concentrate and then add mass to circulating prions so that once they bind to the resonating structures they will register an easily detectable change in resonant frequency. <BR><BR>Furthermore, since the concentration of circulating prions in asymptomatic cows is expected to be in the low attomolar to femtomolar range, it may be necessary to reduce the dissociation constants (Kd) of the biorecognition elements to achieve reproducible binding of low concentrations of target analytes that will be necessary for detection. Successful completion of this project will provide an approach for the rapid screening of all cows prior to slaughter. Furthermore, similar technology could also be employed to screen human blood donations to reduce the risk of transmission of prion diseases from one human to another.
NON-TECHNICAL SUMMARY: Current evaluation of cows for Bovine Spongiform Encephalopathy (BSE) relies upon post mortem testing of suspicious animals. The ability to directly detect infectious prions in the blood of all cows prior to slaughter will dramatically improve the safety of the human food supply. Using modern nanotechnology methods, this project will focus on the development of an ultrasensitive nanobiosensor for the direct detection of prions in the blood of cows prior to slaughter.
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APPROACH: At the request of the reviewers, we have modified the originally proposed 3 year program to complete it in 2 years. We will determine if resonating mechanical biosensors (RMBs) can be used to detect low concentrations of prions in buffered solutions. We will focus the initial efforts on developing the fundamental science required to detect the lowest possible concentration of prions in buffered solutions. Subsequent efforts be directed at optimizing the methods developed to directly detect prions in whole blood samples to permit the ante mortem detection of prions in the blood of cattle.
<p>These objectives will be addressed in the following manner: <ol>
<li> Resonating cantilever beams will be fabricated and specific antibodies applied as described in the proposal. In addition, making use of position dependent thiol based linking chemistry, gold regions may be patterned on the cantilever using a second lithography step and standard lift-off techniques. If necessary, this will permit the use of gold chemistry to specifically bind the biorecognition elements (i.e., antibodies) to the most sensitive, terminal portion of the cantilever. Using methods described in the proposal, we will determine the resonant frequency of the RMBs, both before and after introduction of prion containing solutions, to generate a dose response curve. <li> It is anticipated that the detection limit determined above will be higher than the low quantity of prion proteins anticipated in circulating blood of infected cows. Therefore, we will strive to increase the mass of the bound prion proteins. To accomplish this we will develop methods employing secondary antibodies (recognizing a different prion protein epitope than that used as the primary capture antibody on the surface of the cantilever beam) that will be conjugated to one of several different types of nanoparticles. Such particles as nanogold, quantum dots, latex particles or liposomes can be conjugated to the secondary antibodies. When these conjugated antibodies bind to the prion proteins, the added mass will increase the change in resonant frequency and thereby permit the detection of lower quantities of prion proteins. <li> Because the final volume of the assay is expected to be in the range of 50 to 100 microliters, it will probably be necessary to preconcentrate the blood sample prior to exposure to the resonating mechanical biosensor. Since the Kd of both the primary and secondary antibodies is in the range of 0.1 - 1 nM, it is anticipated that a preconcentration step may be required to detect prion proteins expected to be found at concentrations less than this in circulating blood. Although a number of different methods will be employed, we have had success with immunomagnetic concentration for other organisms. Therefore, this method will be investigated first. While investigating the use of IMS beads to concentrate the prions from the buffered solutions, we will also determine if the IMS beads can serve the function of not only concentrating the prion particles, but also provide the additional mass we expect to be required to enhance the resonant frequency shift. </ol>
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PROGRESS: 2007/01 TO 2007/12<BR>
OUTPUTS: The main output of the above-referenced grant project during the first twelve-month period has been research efforts intended to develop a resonating mechanical biosensor (RMB) capable of directly detection prion proteins (PrP) in circulating blood in cows. Upon capture and immobilization of target analytes to the surface of the RMBs, the resonant frequency of the device structure should decrease as the mass of the bound analytes is increased. In order to exploit the exquisite sensitivity of the RMBs to detect the presence of minute quantities of added mass, several goals need to be achieved: (1) development of an appropriate resonating mechanical structure to which antibodies, specific for PrP can be bound, thus providing a means to capture/immobilize the target analytes on the surface of the device structure; (2) due to the exceedingly small mass of individual PrP particles, it is anticipated that the mass of bound PrP would have to be enhanced by secondary mass labels in order for the presence of the PrP participles to be detected; (3) because the final volume of the assay is anticipated to be in the range of 50 - 100 microliters, a means to pre-concentrate the test specimens will have to be developed and integrated into the "upfront" processing portion of the RMBs. During the first year of this two year grant project, significant progress has been made to achieve objectives (1) and (2), above (see Outcomes/Impacts below). <BR>PARTICIPANTS: In addition to the two co-PDs (Harold Craighead, Cornell University and Richard Montagna, Innovative Biotechnologies International), Madhukar Varshney and Phillip Waggoner, a postdoctoral fellow and graduate student, respectively, in Professor Craighead's group have been responsible for the majority of the bench research. Others that contributed to the ongoing effort include C. Tan, K. Aubin, as reported in the publications portion of this report. <BR>TARGET AUDIENCES: The main target audience for the current work is the scientific community, which will gain information about the use of resonating mechanical biosensors (with and without mass enhancement) for the detection of pathological analyties. In addition, IBI has sought to identify corporate partners in the meat packing industry that would either serve as customers or co-developers/marketers of the resulting diagnostic platform.
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IMPACT: 2007/01 TO 2007/12<BR>
Scientific Outcomes: During the first year of this two-year grant project, we have successfully designed a RMB structure that provides diagnostically significant benefits. Specifically, we indicated in our original grant proposal that the most sensitive portion of a resonating cantilever was its terminal end as opposed to portions of the resonating structure closest to the clamped end. Therefore, a "paddle" was designed (Figure 1 of the publication referenced below) to increase the surface area of the sensing portion of the RMB, thus improving the device sensitivity to secondary mass labels. <BR><BR>A variety of different methods of functionalization of the RMB surface were evaluated, including several blocking methods intended to reduce non-specific binding of components in the assay buffers and test specimens. Furthermore, assay parameters such as optimum time and temperature of the various steps has been elucidated. Once the various components of the structure and assay buffers were optimized, we have determined that without mass enhancement, PrP concentrations as high as 20 micrograms per milliliter did not cause statistically significant changes in resonant frequency. However, use of secondary mass labels have, indeed, enhanced the ability to detect considerably lower concentrations of PrP. Upon the use of a second set of antibodies as a secondary mass label (which were permitted to bind to PrP particles that had already been captured/immobilized by the primary antibodies on the surface of the RMB), the analytical sensitivity of the assay was improved by at least one order of magnitude to 2 micrograms per milliliter. <BR><BR>A further enhancement of analytical sensitivity was achieved by the additional use of streptavidin-conjugated nanoparticles in the presence of biotinylated secondary antibodies. Immobilization of the PrP was achieved by the immunocapture by the primary antibodies, followed by addition of biotinylated secondary antibodies and then streptavidin-conjugated nanoparticles, resulting in an improvement of the analytical sensitivity to 2 ng/ml. Taken together, we have thus far enhanced the analytical sensitivity by a factor of at least four orders of magnitude. <BR><BR>Business Development Activities<BR> While the present work has been ongoing, IBI has been seeking one or more potential commercial partners that will be able to effectively market one or more commercial products based upon this technology. Although no specific strategic relationship has thus far been established, several companies engaged in either offering either BSE test kits or other related products to the meat industry have been made aware of the developments achieved. The impact of the USDA funding has been to not only develop the underlying scientific basis for the direct detection of PrP proteins in cow's blood, but will help to foster interest in potential commercial opportunities. Furthermore, successful completion of this grant project will permit ante mortem testing of all cows prior to slaughter, thus improving the safety of the U.S. beef supply and permitting the U.S. beef industry to remain competitive on a worldwide basis.