Detection of pathogenic bacteria in large volume food samples using an enzyme-linked immunoelectrochemical biosensor
Introduction
The US Centers for Disease Control and Prevention estimates that 48 million people contract a foodborne illness in the United States every year. Over 125,000 will be hospitalized and 3000 will die from complications associated with food contracted illnesses. Despite the large number of different pathogens found in foods, it has been estimated that a mere 14 are responsible for 95% of illnesses (Hoffmann, Batz, & Morris, 2012) and, of these, only 7 pathogens are attributed to 90% of the foodborne illnesses. While there are a higher number of infections related to Salmonella and Campylobacter, pathogens such as Shiga-toxin producing Escherichia coli (STEC) and Listeria monocytogenes have higher hospitalization and fatality rates (Centers for Disease Control and Prevention, 2017).
Conventional screening assays for foodborne pathogens employ culture enrichment (Gill, 2017). These procedures are often implemented into protocols in order to increase the concentration of the microorganism to detectable levels (Francesca Losito, 2012). While effective, these protocols are not conducive to quantifying the initial bacterial load per unit mass of food products, and greatly extend the length of time needed to perform the assay.
The current sample collection protocols employed by industry and regulators alike use volumes of enrichment media that may be up to 5 orders of magnitude higher than the volumes of samples that can be used in screening assays. For example, the sample volume utilized for real-time PCR is typically ~2–10 μL whereas the enrichment volume is ~1000 mL (United States Department of Agriculture Food Safety and Inspection Service & Office of Public Health Science, 2019a; 2019b). Thus, there is a significant amount of uncertainty associated with the subsampling. To combat the issue with subsampling, filter-based techniques have been developed to separate and concentrate bacteria using size exclusion, density, and other physical properties (Li et al., 2013).
Graphite felt (GF) has shown utility as a porous 3-D electrode due to its reasonable electrical conductivity, mechanical flexibility, compressibility, and reasonable cost. GF can be used in electrochemical reactors in a flow-by, flow-through, or flow-across configuration (Nava, Oropeza, & Carreño, 2013). The flow-through reactor provides a more compact volume, diminished electrical resistance, and pressure drops, which consequently result in low pump requirements (Castañeda, Walsh, Nava, & Ponce de León, 2017). The high conversion can be attributed to the increase of volumetric area exposed to the electrolyte (Hussain, O'Mullane, & Silvester, 2018). It is worth mentioning that the high porosity of the GF creates turbulence, which enhances mass transport (Rivera, Ponce de Leon, Nava, & Walsh, 2015). The latter helps to capture targets, such as bacteria, while maintaining acceptable potential and current distributions. Combining these performance attributes enables the GF to serve as both a capture surface and transducer in a sensor format.
GF has been used in several electrochemical applications. Its principal use in electroanalysis is that of a sensor, identifying diverse metallic ions and chemical species owing to the high current intensity generated, which favors lower limits of detection. Although our prior manuscript (Capobianco, Lee, Armstrong, & Gehring, 2019) focused on Salmonella, the sensor is viewed as a platform technology. To evaluate the ability of the sensor to identify additional pathogens, the capture antibody and enzyme-linked antibody conjugate were substituted with those specific to E. coli O157:H7 and beef homogenate was included as a sample matrix. The protocol used for Salmonella was retained and applied to E. coli detection in 5, 60, and 1000 mL sample volumes.
Section snippets
Materials and methods
Reference Ag/AgCl electrodes, and electrode polishing suspension were sourced from Bioanalytical Systems, Inc., (West Lafayette, IN). A two-inch long, 0.5 mm diameter platinum wire counter electrode was sourced from VWR (Radnor, PA), the 0.25 in thick graphite felt electrode (GFE) from Electrosynthesis (Lancaster, NY). A second two -inch, 0.5 mm diameter platinum wire from VWR (Radnor, PA) was sourced to facilitate the electrical connection of the GFE to the electrochemical cell. Spherical
E. coli detection in buffer
In a previous proof-of-principle study, Capobianco et al. demonstrated the ability of an immunoelectrochemical sensor to detect heat-killed Salmonella in sample volumes up to 60 mL (Capobianco et al., 2019). The selectivity of this sensor was provided by an anti-Salmonella antibody, which was immobilized on the surface of the graphite felt electrode (GFE). The use of graphite felt as both a capture surface and a porous working electrode in this sensor was unique. However, this study had several
Conclusions
The enzyme-linked immunoelectrochemical biosensor displays the promising capability to detect bacteria in large sample volumes without the need for subsampling. In a flow-through configuration where the electrode serves as both a capture surface and transducer, the biosensor successfully detected the presence of 10,000 E. coli O157:H7 in 1 L of solution within 3 h. When applied to E. coli O157:H7 detection in a ground beef homogenate, with sufficient sample pretreatment the system displays a
Author contributions
J.A.C. and J.L. planned, conducted, and analyzed experiments, authored and edited the manuscript; C.M.A. and A.G.G. planned and analyzed experiments, authored and edited the manuscript.
Funding
This material is based upon work supported by the U.S. Department of Agriculture, Agricultural Research Service, under Project No. 8072-42000-084.
CRediT authorship contribution statement
Joseph A. Capobianco: Conceptualization, Methodology, Validation, Formal analysis, Writing - original draft, Writing - review & editing, Supervision. Cheryl M. Armstrong: Conceptualization, Methodology, Formal analysis, Validation, Writing - original draft, Writing - review & editing. Joe Lee: Conceptualization, Methodology, Investigation, Validation, Formal analysis, Data curation, Writing - original draft, Writing - review & editing. Andrew G. Gehring: Conceptualization, Validation, Writing -
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors thank Dr. George Paoli (USDA-ARS-ERRC) for providing the E. coli O157:H7-PC strain used in this study. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal opportunity employer.
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