INTELLECTUAL MERIT: It is proposed to investigate the M13 bacteriophage virus as a biological platform that will provide amplified, optically-detectable responses to the binding of biological analytes. The bacteriophage will be engineered to bind antigenic species with very high specificity through its pIII protein peptide tails; the correct sequence to obtain specific analyte binding will be found using phage display. Moreover, because the body of each M13 bacteriophage contains over 2700 pVIII coat proteins, each of which can either be genetically engineered or chemically modified to react with a given set of nanomaterials, it is possible to obtain a large amplification in signal detection in response to a single binding event, allowing for rapid visible readouts with high sensitivity and high throughput. For the samples that generate a positive optical signal, the identity of the antigens in solution will be obtained by labeling bacteriophage with specific DNA oligonucleotides and nanoparticle reporters that will be detected with high sensitivities by surface enhanced Raman spectroscopy (SERS). Because the SERS analyses need only be performed after a positive optical signal is obtained, the need for expensive spectrophotometric sample testing will be greatly reduced. This sensor design is made possible by the dual, orthogonal biomolecular recognition of the bacteriophage to both the antigen and the nanomaterial developer. The proposed research will be divided into the following four phases: (1) discovery and isolation of antigen-specific binding bacteriophage with pH-sensitive affinities, (2) study of nanoparticle aggregation by modified bacteriophage to create easily observed visible changes in solution color, (3) implementation of these viruses into enzyme-free amplifiable sensors for rapid detection and maximum sensitivity, and (4) engineering of these viruses to couple qualitative detection with highly sensitive, amplified modes for antigen identification.
BROADER IMPACTS: The proposed biosensing scheme has easily recognized advantages over so-called "sandwich" assays that have been used to sense the presence of, and identify, specific proteins/antigens. These methods involve relatively unstable components, e.g., enzymes that catalyze reactions needed to amplify the signal. Such methods are therefore not practicable in environments where refrigeration and specialized detection apparatus are not available. An important feature of this proposal lies in the promise of providing selective and highly sensitive sensors sufficiently robust to be used under sub-optimal conditions in the field to identify antigenic agents and other disease markers. The PI is a member of the new Nanoengineering Department at UCSD. She will contribute to building the undergraduate and graduate curricula of this department through courses in Intermolecular and Surface Forces and Advanced Nanofabrication that she is teaching. In efforts to broaden participation in science and engineering, she has established relationships with the UCSD Society of Women Engineers, the National Society of Black Engineers, and the Society of Hispanic Professional Engineers. These organizations provide direct access to underrepresented undergraduate students and also a bridge to the local K-12 community. As a specific K-12 outreach activity, the PI will work with the UCSD BioBridge program to integrate the proposed sensing diagnostics into existing middle and high school chemistry and biology classes. She is also a participant in the annual San Diego Science Festival that hosts high school students for science demonstrations in working laboratories. A particularly important feature of her plans for integration of research and education involves her prior experience as an industrial scientist and her desire to convey to her students the challenges associated with implementing nanoscience and nanoengineering into real-world, commercially viable technologies.
