Rapid detection of ricin in liquid foods using surface-enhanced Raman spectroscopy

Abstract

The potential for bioterror agents to be distributed using existing supply chains for food with the purpose of causing mass casualty, terror, and/or economic damage is a hazard to homeland security. Therefore, it is necessary that bioterror agent detection procedures be developed which are appropriate and can serve as tools for food companies and other security personnel to monitor the U.S. food supply. The present research develops an appropriate assay which could be used to detect the toxin ricin in fresh liquid foods, specifically milk and orange juice. Ricin toxin is a category B bioterror agent as defined by the Centers for Disease Control and Prevention due to its potent toxicity and relative ease of access. For this application, an appropriate detection assay was rapid, inexpensive, easy to conduct, and sensitive enough to detect ricin at concentrations below toxic doses if consumed. These criteria were important since food surveillance requires frequent monitoring by personnel who do not necessarily have backgrounds in chemistry nor laboratory techniques. Additionally, tested foods should not proceed through the processing/distribution chain until a negative result was received; therefore the assay must be very rapid. Research was conducted and procedure developed using an immuno-based separation technique to capture dilute concentrations of ricin, (or a surrogate), from foods spiked with the toxin. Two separation techniques were utilized and evaluated for speed, ease, and final sensitivity. The first separation technique utilized a commercially available polyclonal ricin antibody conjugated to magnetic beads in order to concentrate and separate ricin from a sample. The second separation technique used a single-stranded DNA aptamer to perform the capture step. Following either separation technique, surface-enhanced Raman spectroscopy (SERS) was used to directly detect the target. Raman is a mode of vibrational spectroscopy where a sample is exposed to a monochromatic laser and a spectrum of Raman-active vibration modes is obtained. These spectra contain a large amount of information about the sample's molecular structure, and therefore act as `fingerprints' for a sample. Surface-enhanced Raman exploits cutting edge nanotechnology to significantly increase the sensitivity of Raman analyses by placing a sample on or near noble metal nanostructures. Immunomagnetic separation (IMS), the first of the separation techniques, produced limits of detection of 4 micrograms/mL in milk, in less than 20 minutes. According to current toxicological data, these limits of detection are sufficient for providing proper protection in the case of a ricin-related attack. Additionally, the procedure was conducted with a portable, hand-held Raman instrument to validate its compatibility with technology that could be used in a harsh environment. The second separation technique used an aptamer which had been covalently bound to the nano-enhancing substrate to capture ricin from food. Aptamers are oligomers of single-stranded DNA which can be used like antibodies to selectively concentrate antigens from complex solutions. This separation protocol delivered a final detection limit of 25 ng/mL, 100 ng/mL, and 100 ng/mL, in buffer, milk, and orange juice, respectively. This assay could also be completed in less than 20 minutes. This project has developed novel procedures for the rapid detection of ricin in liquid foods. These procedures were designed to be easily integrated into existing food quality monitoring programs within food plants or at other sensitive locations, such as national borders or international shipping ports. Additionally, these methods serve as a proof-of-concept since their flexibility allows for easy adaptation to provide detection of other targets as well by replacing the capture molecule.