Identifying biological agents with surface-enhanced Raman scattering
The anthrax attacks which afflicted the United States in 2001 underscore the necessity of a quick response to biological terrorism agents. Optimal organization and capacity are critical to saving lives at minimal cost.1Important to this effort is rapid biological agent identification aided by a field-ready analytical protocol. Given its capacity for unique molecular identification, Raman spectroscopy is uniquely poised to contribute to this need.
Although Raman spectroscopy readily detects chemical species, direct identification of biological materials is often difficult due to spectral complexity and low signal intensity. Several direct detection methods are available, including extraction and detection of a specific molecular marker, as well as surface-enhanced Raman spectroscopy (SERS) spectral generation by adsorbing an intact biological species onto a roughened gold or silver surface.2–10Several indirect SERS detection methods are also available, such as labeling the analyte with a Raman-active dye and bringing it to the SERS substrate for readout.11,12 Nanometer-scale SERS-active tags conjugated with specific biological recognition moieties, thereby effecting direct binding to the analyte, is another possibility.13–19
We developed an indirect biological detection system compatible with the Raman-based StreetLab Mobile. It employs SERS tags as unique labels for each target of interest in a sandwich immunoassay format.20,21 Unique spectroscopic signatures are generated with SERS tags consisting of individual glass-encapsulated gold nanoparticles and surface-bound Raman active reporter molecules, as depicted in Figure 1. These SERS tags are bound to a specific antibody and provide a strong, spectroscopically-consistent label. Superparamagnetic particles conjugated to the antibodies capture and concentrate the SERS-labeled complex at the focal point of the Raman laser using a magnetic field. The simple SERS readout confirms the presence or absence of the analyte (see Figure 2).
SERS tags are typically resistant to photobleaching. Our silica-encapsulated tags are also immune to changes in the detection medium, such as pH or temperature, which renders our assay amenable to field use. Another important advantage is rapid interaction between the capture particles, targets, and SERS tags in solution, resulting in low-level target detection within five minutes. The pellet (formed by concentration) blocks most of the background SERS signal from the assay solution, thereby eliminating the need for a wash step. Furthermore, the characteristic Raman spectrum of the assay pellet is identical to the spectrum collected from the SERS tags alone. There is little to no spectral contribution from antibodies or cellular material in the assay pellet, thus there is no need for spectral deconvolution due to assay contaminants.
We originally developed our assay with Escherichia coli (E. coli) as a model organism. We collected proof-of-concept data with killed E. coli O157 H7, and subsequently characterized the full assay. We evaluated five different strains of live E. coli O157 H7 to determine the assay detection limit. By challenging the assay with 10 near-neighbor strains, we also investigated specificity. Since the assay is intended for field-use, we thoroughly analyzed reagent shelf-life and assay robustness to a variety of interferents.20
The ability to successfully detect very low levels of E. coli was fundamentally important for demonstrating assay capabilities. However, StreetLab Mobile is a rugged device intended for dealing with potential chemical or biological threat agents in real life. Our future work focuses on SERS-based sandwich immunoassays to detect potential biothreats, such as anthrax (Bacillus anthracis), ricin toxin from Ricinus communis, tularemia (Francisella tularensis), botulism (Clostridium botulinum toxin), plague (Yersinia pestis), and abrin toxin from Abrus precatorius.
Tracy L. Paxon received her PhD in analytical chemistry from Pennsylvania State University in 2005, focusing on detecting and quantitating biological samples such as RNA, DNA, and small-molecule neurotransmitters. Her current research ranges from energy (temperature sensing) to security (pathogen detection at the chemical sensing laboratory) applications. She possesses two patents, five patent applications, and 11 publications.
R. Scott Duthie received his MS in biology from the University of Wisconsin, Oshkosh, in 1983. He spent the following two years as a research assistant studying molecular biology at Marquette University. His career has included employment at Pharmacia Biotech, Inc. and Amersham Biosciences/GE Healthcare, and he currently focuses on molecular biology.
Casey L. Renko received her BS in chemistry from the State University of New York, Oneonta, in 2010. Her research currently focuses on analytical methods used in water analysis, as well as biological sandwich immunoassays.
Andrew Burns received his PhD in materials science and engineering from Cornell University in 2008. He developed fluorescent silica nanoparticles for targeted bioimaging and sensing. His research currently focuses on nanomaterial development for sensing and optics, polymeric materials for holography, and bio-interfacial materials for cell growth. He possesses seven patent applications and 15 publications.
Frank J. Mondello received his PhD in microbiology from the University of Tennessee, Knoxville, in 1982. His research originally focused on microbial degradation of chlorinated aromatic hydrocarbons. He currently focuses on biosensor development and co-invented the SERS-based immuno-magnetic bioassay for StreetLab Mobile. He possesses eight patents and 20 peer-reviewed publications.
Marie Lesaicherre received her MSc in chemical engineering from the Superior National School of Chemistry, France, and her PhD from the National University of Singapore. She is a chemical biologist and the biodetection program manager. Her experience includes over 10 years of research in biosensors, focusing on development and utilization of advanced detection technologies for DNA, RNA, and protein analysis for medical, water analysis, and security applications.
