Real-time pathogen monitoring during enrichment: a novel nanotechnology-based approach to food safety testing Kristin Weidemaier a, ⁎, Erin Carruthers a , Adam Curry a , Melody Kuroda a , Eric Fallows a , Joseph Thomas a , Douglas Sherman a , Mark Muldoon b a BD Technologies; 21 Davis Drive; P.O. Box 12016; Research Triangle Park, NC 27709, USA b Romer Labs Technology, Inc; 130 Sandy Drive; Newark, DE 19713, USA abstract article info Article history: Received 28 August 2014 Received in revised form 11 December 2014 Accepted 14 December 2014 Available online 24 December 2014 Keywords: Surface Enhanced Raman Scattering SERS Nanotechnology Food pathogen detection We describe a new approach for the real-time detection and identification of pathogens in food and environmen- tal samples undergoing culture. Surface Enhanced Raman Scattering (SERS) nanoparticles are combined with a novel homogeneous immunoassay to allow sensitive detection of pathogens in complex samples such as stomached food without the need for wash steps or extensive sample preparation. SERS-labeled immunoassay reagents are present in the cultural enrichment vessel, and the signal is monitored real-time through the wall of the vessel while culture is ongoing. This continuous monitoring of pathogen load throughout the enrichment process enables rapid, hands-free detection of food pathogens. Furthermore, the integration of the food pathogen immunoassay directly into the enrichment vessel enables fully biocontained food safety testing, thereby significantly reducing the risk of contaminating the surrounding environment with enriched pathogens. Here, we present experimental results showing the detection of E. coli, Salmonella, or Listeria in several matrices (raw ground beef, raw ground poultry, chocolate milk, tuna salad, spinach, brie cheese, hot dogs, deli turkey, orange juice, cola, and swabs and sponges used to sample a stainless steel surface) using the SERS system and demonstrate the accuracy of the approach compared to plating results. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Food-borne illnesses significantly impact society, not only with respect to health, but also health-care costs. The CDC has estimated that each year about 1 in 6 Americans (or 48 million people) gets sick, 128,000 are hospitalized, and 3,000 die of food-borne disease (Centers for Disease Control, 2012). It has also been estimated that food-borne illnesses contribute to $152 billion in health-related expenses each year in the U.S., particularly for bacterial infections caused by Salmonella, Listeria monocytogenes, E.coli and Campylobacter spp. (Scharff, 2010, 2012). The current level of food safety found in the U.S. is the result of government regulations combined with industry self-monitoring influ- enced by market incentives, such as legal liability, brand value, reputa- tion, and sales volume. Highly publicized outbreaks of food-borne illness in samples ranging from spinach to cantaloupe to peppers has prompted increased public concern and culminated in the 2011 passage of the Food Safety Modernization Act (Neuman, 2010; Sutton, 2009). Food pathogen testing may occur on food samples ranging from raw materials to end products or on environmental samples acquired from surfaces, floors, drains, and processing equipment as part of a site's overall Hazard Analysis and Critical Control Point (HACCP) plan (U.S. Food and Drug Administration, 1997). For many samples, a “zero tolerance” requirement is imposed on key human pathogens such as Listeria monocytogenes and E. coli O157:H7 (Fratamico et al., 2005). The requirement to detect even a single, potentially highly damaged, viable pathogen in a food sample has driven the selection and develop- ment of today's state of the art pathogen tests. In particular, all food pathogen testing today requires a culture step to enrich the potentially low levels of pathogens contained in a sample and ensure that the limit of detection of the analytical test is reached. This workflow inherently limits time-to-results, since it is impossible to know a priori the starting pathogen load of any given sample. Therefore, the enrichment protocol lengths must be set for the “worst case” scenario (i.e. the need to recover one damaged pathogen). As a consequence, samples with higher pathogen loads are cultured longer than may be strictly necessary, leading to a delay in time-to-results. Following the culture step, a portion of the sample is removed and tested by conventional plate- based technologies or, more commonly, by rapid tests, including immu- noassays and PCR-based tests (Velusamy et al., 2010). While culturing food and environmental samples prior to the analyt- ical pathogen test provides the necessary sensitivity, these methods suffer from well-known disadvantages. First and foremost, time-to- results can be long. Methods to reduce these times are currently under development by a number of groups and rely primarily on improving the analytical test by further lowering the test's limit of detection or International Journal of Food Microbiology 198 (2015) 19–27 ⁎ Corresponding author. Tel.: +1 919 597 6383; fax: +1 919 597 6402. E-mail address: Kristin_Weidemaier@bd.com (K. Weidemaier). http://dx.doi.org/10.1016/j.ijfoodmicro.2014.12.018 0168-1605/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro