1. C. parvum polyclonal antibodies (Abcam Inc., Cambridge, MA) are adsorbed to the surface of the gold nanoparticles. To create stable conjugates, the surface of the nanoparticle must be completely saturated with the antibodies. A flocculation assay was performed to determine the minimum number of antibodies necessary to saturate the nanoparticle surface. In a flocculation assay, the gold nanoparticle solution is exposed to varying concentrations of antibodies. Upon addition of salt, aggregation will occur if the nanoparticle surface is not saturated. This results in a blue shift of the 530 nm gold sol absorption peak. Approximately 30 antibody molecules are required to coat one 41 nm diameter gold nanoparticle surface (Figure 5). of Cryptosporidiosis remain common in the United States. Cryptosporidium parvum is resistant to common disinfectants and has a very low infectious oocyst dose. Current Cryptosporidium detection methods are expensive, slow, and plagued by variable recovery rates. The Development of an Immunoassay for the Detection of Cryptosporidium parvum in Drinking Water Krista L. Rule and Peter J. Vikesland The Charles E. Via, Jr. Department of Civil and Environmental Engineering Cryptosporidium parvum is a waterborne pathogenic protozoan that causes the gastrointestinal illness Cryptosporidiosis. Although typically self-limiting in healthy adults, Cryptosporidiosis can be chronic and deadly to the immunocompromised. Outbreaks 2) Cryptosporidium parvum oocysts are captured by the antibody-functionalized membrane as water containing the oocysts is filtered through the membrane. 1) Capture antibodies (monoclonal anti- Cryptosporidium parvum IgG) are immobilized on a polycarbonate membrane filter. 3) The captured oocysts are labeled by gold nanoparticle conjugates that have anti- Cryptosporidium parvum antibodies and fluorescent labels bound to the gold surface. Figure 1: AFM image of C. parvum oocyst trapped in filter membrane pore. Preparation of Immunogold SERS Labels The SERS immunogold labels are prepared by conjugating gold nanoparticles with C. parvum antibodies and with fluorescent dye molecules (Figure 3). To obtain reproducible SERS results, it is imperative that the gold sol demonstrate little particle size heterogeneity. For this reason, gold nanoparticles of diameter ~41.3 nm are synthesized by seeding preformed 13 nm gold nanoparticles with HAuCl 4 and citric acid (Figure 4). The size distribution of synthesized gold sol is determined by transmission electron microscopy (TEM). TEM images of the gold nanoparticles are analyzed for size heterogeneity using Scion Image software. Functionalization of the Polycarbonate Membrane Funding for this project is provided by a National Science Foundation Grant and a National Science Foundation Graduate Research Fellowship. Introduction Proposed Method for the Detection of Cryptosporidium 4) The membrane is analyzed for the presence of the fluorescent label using Surface Enhanced Raman Spectroscopy (SERS). Our proposed assay involves the following steps (see Figure 2): Figure 2. Schematic of the proposed immunoassay protocol for the detection of Cryptosporidium parvum in drinking water (see text above for an explanation of the steps). Millipore Isopore polycarbonate membrane filters with 5 μm pores were characterized by Atomic Force Microscopy before and after anti-Cryptosporidium parvum antibody functionalization (Figure 9). As seen in the figures below, globular entities of approximately 20- 30 nm (in the range of the expected size of an IgG antibody) are visible in the AFM height images after antibody functionalization. Figure 8. Schematic of antibody functionalization of a polycarbonate membrane via a poly-(L)-lysine spacer molecule. Figure 9. AFM height image of a polycarbonate membrane before and after anti-Cryptosporidium parvum antibody functionalization. AFM imaging was performed in a water- filled fluid cell operating in TappingMode. Figure 4. TEM images of the gold seeds (left) with average particle diameters ~15.0 (± 0.98) nm and the seeded larger particles (right) with average particle diameters ~ 41.3 (± 2.6) nm. Figure 7. Raman spectra of RBITC immunogold conjugates at varying concentrations. Inset: Relationship between the intensity of the Raman peak at 12700 cm -1 and the immunogold particle concentration. Abstract This research involves the development of a sandwich immunoassay for the detection and quantification of Cryptosporidium parvum in drinking water. The protocol couples an immunofilter for the capture of Cryptosporidium oocysts with Surface Enhanced Raman Spectroscopy (SERS) for the detection and quantification of the oocysts. Acknowledgments Future Work Testing of immunoassay with waters spiked with viable Cryptosporidium parvum oocysts Expansion to multi-pathogen detection immunoassay A monolayer of anti-Cryptosporidium parvum antibodies is immobilized on the polycarbonate membrane surface using poly-(L)-lysine as a spacer molecule (Figure 8). In this process, an amino group of poly-(L)-lysine is bound by activated carbonate groups on the polycarbonate membrane. A second amino group of poly-(L)-lysine is then activated with glutaraldehyde. When the activated layer is exposed to anti-Cryptosporidium parvum antibodies, a uniform layer of antibodies is formed. Figure 3. Schematic of immunogold synthesis. Figure 5. Flocculation assay results for the 41 nm gold nanoparticle/C. parvum antibody conjugates. 30× indicates that the conjugates consist of thirty antibodies per gold nanoparticle. A shift in the gold sols 530 nm absorbance peak indicates aggregation has occured five minutes after the addition of NaCl to a concentration of 0.1 M. SERS is capable of detecting extremely low levels of rhodamine B isothiocyanate present in the immunogold conjugates and the concentration of the immunogold conjugates is relative to the Raman peak intensities (Figure 7). This method therefore has the potential of providing quantitative data in addition to providing positive and negative results. A Raman label must also be bound to the gold nanoparticle surface. We have chosen rhodamine B isothiocyanate (RBITC) for our nanoparticle conjugates as it can covalently bind to the gold surface via the dyes isothiocyanate functional group (Figure 6). Figure 6. Structure of rhodamine B isothiocyanate. The red circle illustrates the functional group responsible for covalent bonding with the gold nanoparticle.