Highly Sensitive and Rapid Detection of Salmonella typhimurium Using a Colorimetric Paper Based Analytical Device Coupled with Immunomagnetic Separation

Salmonella causes over a million foodborne illnesses per year in the United States resulting in more hospitalizations and deaths than any other foodborne bacteria pathogen. To help prevent outbreaks a rapid, portable, sensitive, and reliable method for onsite detection of bacteria that can be used in different sample matrices would be beneficial. Herein, we present a colorimetric paper-based analytical device (PAD) combined with immunomagnetic separation (IMS) for detecting Salmonella typhimurium. IMS anti-Salmonella coated magnetic beads was applied to capture and separate bacteria from the sample matrix and preconcentrated it into small volumes before testing on paper. To directly detect S. typhimurium after IMS, a sandwich immunoassay was implemented into the procedure with β-galactosidase (β-gal) as the detection enzyme. Using the antibody/enzyme complex, we performed a colorimetric assay with chlorophenol red-β-D-galactopyranoside (CPRG) for bacteria quantification. The method was confirmed to be highly specific to S. typhimurium without interference from other pathogenic bacteria like Escherichia coli. Using this system, the limit of detection of S. typhimurium was found to be 102 CFU mL-1 in culturing solution without any pre-enrichment. In addition, distance-based detection where the concentration is read as the length of colored band formed on reaction was also demonstrated. This assay had a detection limit of 102 CFU mL-1 for S. typhimurium, providing instrument-free quantitative analysis alternative to spot tests, which require image analysis. Finally, the proposed platform was applied for detection of S. typhimurium in inoculated Starling bird fecal samples and whole milk with detection limits of 105 CFU g-1 and 103 CFU mL-1 respectively, describing the first published paper-based detection method for S. typhimurium in bird feces and whole milk. Salmonella is widely known as one of the most prevalent pathogens causing foodborne illness outbreaks.1 Per the United States Center for Disease Control and Prevention (CDC), Salmonella causes an estimated one million illnesses in the United States resulting in 19,000 hospitalizations and 380 deaths, more than any other pathogenic bacteria.2 Infected individuals encounter symptoms, such as fever, abdominal cramps, diarrhea, and even death within 12 to 72 h of exposure.3 Salmonella contaminates food products, like eggs, fruits, vegetables, meat, poultry, and milk4 typically through animal fecal contamination.5 This is because Salmonella lives and replicates in the intestinal tracts of humans and animals, and therefore is present in their feces.6 Studies have shown a strong correlation between skin and meat contamination of Salmonella, and prevalence in the animal’s feces, making it an important sample matrix to detect the pathogen’s presence.7 Traditional methods to detect Salmonella are based on bacterial culture, which is time-consuming and laborious, often taking at least a few days to complete detection.8,9 In addition, this conventional method is not practical for on-site detection of bacteria because it requires a central laboratory. The need for expensive equipment and trained lab personnel increases testing costs, making large scale studies of Salmonella epidemiology difficult.10 Using polymerase chain reaction (PCR) as a method for detecting food borne pathogens has attracted considerable attention because of its high selectivity and accuracy. The overall analysis time of PCR methods was reported to take approximately 24 h which is much improved compared to the traditional culture method.11 However, PCR methods encounter a problem associated with sample matrix which could contain PCR inhibitors normally found in biological samples (i.e. urine, feces and blood), food, and humic substances.12 In addition, results from PCR could be unreliable when there is a low number of pathogens within a relatively large amount of sample, thus concentrating samples is unavoidably required.13 Accordingly, a rapid, userfriendly, cost-effective, reliable, and specific approach for Salmonella detection is required to overcome the drawbacks of conventional methods. Page 1 of 13 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 Over the past decade, paper-based analytical devices (PADs) have become attractive analytical tools. The advantages of PADs include small sample and reagent consumption, rapid analysis, simple operation, disposability, and portability.14,15 PADs hold great promise for use as analytical tools in remote areas or areas where minimal instrumentation is available due to their natural fluid wicking properties and ability to store reagents. These properties make PADs attractive and simple platforms for analysis in fields such as environmental monitoring, medical diagnostics, point-of-care testing, and food safety control.14,16 However, there have been only a few reports on using PADs for rapid detection of bacteria, including Pseudomonas aeruginosa,17 Staphylococcus aureus,17,18 Escherichia coli,19-21 Salmonella typhimurium,20,22 Enterococcus spp.21 and Listeria monocytogenes.20 Our previous work demonstrated the application of PADs for detecting Salmonella spp. , L. monocytogenes, E. coli, and antimicrobial resistant bacteria using both colorimetric detection 20,21,23 and electrochemical measurements.21 However, an enrichment step was required to detect lower bacteria concentrations and the detection motif relied solely on enzyme production by the bacteria. Immunomagnetic separation (IMS) is a powerful analytical method because of its ability to separate targets of interest from complex sample matrices. It can also be used as an alternative to culture enumeration as a preenrichment step for the target of interest. IMS is a procedure where antibodies specific to an analyte or cell are covalently attached to a magnetic particle.Antibodymodified magnetic particles are added to the sample matrix to adhere to the target and are separated from the matrix with a magnet and re-suspended in buffer. After separation from the sample matrix, many detection methods have been used including microscopy, broth enrichment, immunoassays, and PCR.24 Because IMS does not require bulky and expensive equipment to complete the procedure, this makes it ideal for in-field measurements. IMS is also a faster technique with total enrichment time of less than an hour compared to 4-16 h for culture enrichment depending on initial bacteria concentration. Unlike culture enrichment, IMS has been demonstrated for efficiently separating target analytes and cells from complex mixtures such as blood,25 milk,26 meat,27 cheese and yogurt,28 and even bovine feces.29 With IMS, the antibodies attached to the beads can be specific to any analyte or cell of interest. Because of this, IMS has been demonstrated for detecting many biomarkers,25,30 along with various bacteria27-29 and viruses.31-33 Combining IMS with paper-based devices has been previously described for the detection of E. coli in contaminated water.19 In this work, the authors describe the use of IMS to pre-concentrate samples from contaminated water before lysing the bacteria and detecting bacterial enzymes β-galactosidase and β-glucuronidase. To the best of our knowledge, there has not been a paper-based device that is coupled with IMS for the detection of bacteria in more complicated sample matrices, such as animal feces and whole milk. Furthermore, despite the prevalence of Salmonella in bird feces, to the best of our knowledge, there has not been a proposed alternative detection method to traditional culture en-