Detection of pathogenic bacteria in large volume food samples using an enzyme-linked immunoelectrochemical biosensor

Highlights

• Rapid, field portable biosensor for detecting microbial pathogen in foods.

• Flow-through system enables screening of large sample volumes without subsampling.

• The working electrode serves as selective capture surface and as signal transducer.

Abstract

Increased speed and sensitivity of testing are always desired for the detection of pathogens in foods. Presented a test sample with low microbial analyte concentration, it is an advantage to analyze as much volume as possible of the sample to attain the best limit of detection (LOD). Therefore, a rapid screening method using a novel flow-through immunoelectrochemical biosensor was developed for the detection of pathogenic bacteria (E. coli O157:H7 and Salmonella) in food (ground beef). As the working electrode employed was comprised of a porous, antibody-coated graphite felt electrode that served as both a biorecognition-element coated solid support for capture of targeted pathogens as well as a signal transducer, high volumes of aqueous sample could be rapidly exposed to the solid support via gravity flow. Flow rates as high as 16.7 mL/min and 12.3 mL/min could be achieved for bacterial samples in buffer and 1:4 ground meat (beef) homogenate, respectively, with no significant effect on LOD. Fastest flow rates for beef homogenate, without clogging of the porous electrode as well as reduction in apparent electrochemical interference, was realized with a tandem combination of sample pretreatment strategies that included filtration with glass wool and graphite felt as well as continuous flow centrifugation. The LOD for 10,000 E. coli O157 cells in 5, 60, and 1000 mL of buffer was 2000, 170, and 10 cells/mL, respectively in a total assay time of 3 h whereas the LOD for E. coli O157 was 400 cells/mL in 1:4 beef homogenate.

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.