A Bifunctional Nanocomposites Based Electrochemical Biosensor for in-field Detection of Pathogenic Bacteria in Food (Biomedical Project)

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ABSTRACT:

This research focused on the application of electrochemical biosensors for the rapid detection of pathogenic bacteria, Escherichia coliO157:H7 and Salmonella Typhimurium, in foods. The possible presence of pathogenic bacteria in foods has always been a great threat to the wellbeing of people and the revenue of food companies.

Therefore, the demand for rapid and sensitive methods to detect foodborne pathogens is growing. In this research, an impedimetric immunosensor was first developed for the rapid detection of E. coliO157:H7 and S. Typhimurium in foods. It was based on the techniques of immuno magnetic separation, enzyme labelling, and electrochemical impedance spectroscopy (EIS).

This impedimetric immunosensor was capable of specifically detecting E. coliO157:H7 and S. Typhimurium within the range of 10 2to 10 6 colony forming unit (cfu)/ml in the pure culture. The limits of detection (LODs) of E. coliO157:H7 in ground beef and S.Typhimurium in chicken carcass rinse water were 2.05x103cfu/g and 1.04x103cfu/ml, respectively.

The second electrochemical biosensor was designed for rapid detection of E. coli O157:H7. This biosensor integrated magnetic GOx-polydopamine (PDA) based polymeric nanocomposites (PMNCs) which served dual functions as both the carrier and the label, and Prussian blue (PB) modified SP-IDMEs for measurement.

The core-shell Abs/GOxext/gold nanoparticles (AuNPs)/magnetic beads (MBs)-GOx@PDA PMNCs acted efficiently to get a high load of enzyme onto the surface of bacterial cells.Afiltration step separated the free PMNCs from the bonded ones and reduce the background noise to achieve better sensitivity.

The constructed biosensor had been proved to be able to detect E. coli O157:H7 with the LOD of 52cfu/mlin the pure culture. The third ectrochemical aptasensor was developed to detect S. Typhimurium based on the concept of the bifunctional nanocomposites. The ssDNA aptamers were used as the biorecognition element. The achieved LOD in the pure culture was 96 cfu/ml.

The biosensors developed in this research exhibited good specificity, reproducibility, and easy-to-operate, and are expected to find broad applications in the detection, especially in-field detection, offoodborne pathogens.

OBJECTIVES

The overall goal of this research is to develop electrochemical biosensors for rapid detection of foodborne pathogens. E. coliO157:H7 and S.Typhimurium are chosen to be the model targets. The purpose of this research essentially is to use the screen-printed interdigitated electrodes and to improve the performance of the electrochemical biosensors to achieve lower limit of detection, shorter detection time, and more suitable for in-field detection of foodborne pathogens.

  • To develop an electrochemical biosensor based on electrochemical impedance spectroscopy using screen-printed interdigitated microelectrodes for the detection of E. coliO157:H7 and S. Typhimurium.
  • To develop an amperometric biosensor based on the use of bifunctional magnetic GOx-PDA PMNCs and PB-modified SP-IDMEs for the detection of E. coliO157:H7.
  • To expend the concept of using bifunctional magnetic PMNCs for detection of
    S.Typhimurium with aptamer-based electrochemical biosensor.

 LITERATURE REVIEW

Foodborne Pathogens and Illnesses:

Foodborne illnesses caused by pathogenic bacteria have always been a serious threat to the health of people and to the economics of nations. The Centers for Disease Control and Prevention (CDC) has estimated that roughly 48 million people get sick, 128,000 are hospitalized, and 3,000 are dead every year in the United States due to foodborne illness (Scallan et al., 2011).

Among all the cases of foodborne illnesses, about 9.4 milllion cases (20%) are caused by 31 types of known pathogens, including norovirus, Salmonella, Clostridium, Campylobacter spp. Staphylococcus, E. coli, etc. There are also 38.4 million cases (80%) are caused by a broad spectrum of unknown agents (Scallan et al., 2011).

Conventional Methods for the Detection of E. coli O157:H7 and S. Typhimurium:

Even though there have been many legislations, regulations, and methodical programs like good manufacturing practices (GMP) and hazard analysis and critical control point (HACCP) which can greatly reduce the occurrence of foodborne pathogens in food, the methods for sensitive, reliable, and rapid detection of these pathogens are still critical to identify and prevent the problems from pathogenic bacteria.

Biosensors for the Detection of Foodborne Pathogens:

The acknowledged world’s first biosensor was fabricated by Clark and Lyons(1962) for the detection of glucose using immobilization of glucose oxidase on the electrode and the electrochemical detection of hydrogen peroxide generated in the reduction-oxidation of glucose. Since then, incredible innovations have been integrated into the development of biosensors using novel approaches to improve their sensing performance.

The schematic frame of a typical biosensor

The Schematic Frame of a Typical Biosensor.

Electrochemical Biosensors for the Detection of E. coliO157:H7 and S.Typhimurium:

Electrochemical biosensors are intensively studied and well developed for the detection of foodborne pathogens. Some advantages of electrochemical biosensors over others include comparable sensitivity, fast response, possibility to operate in turbid solution, and possibility to be miniaturized.

Furthermore, due the current tate-of-the-art techniques used in the fabrication of electronics, the electrochemical biosensors can be integrated into one simple, automated, and low cost device (Law et al., 2015; Palchetti & Mascini, 2008; Sharma & Mutharasan, 2013; Velusamy et al., 2009; Wang & Salazar, 2016). On the other hand, to apply electrochemical biosensors to the detection of foodborne pathogens, there are still some drawbacks need to be overcome.

Difficulties comes from complex food samples are the most concerned. The bacteria are highly unlikely distributed in/on foods in the pattern of uniformity. Therefore, it makes the direct use of electrochemical biosensors for the detection of pathogenic bacteria very hard without appropriate sample collections and preparations.

Recent Trends in the Development of Electrochemical Biosensors for E. coliO157:H7and S. Typhimurium:

Significant efforts have been focused on the detection of E. coliO157:H7 and S.Typhimurium in research because their important role as the sanitary indicator and the zero-tolerance policy regarding its presence in foods (FDA, 2013; USDA-FSIS, 2015).

The development of biosensors for whole bacterial cells is challenging due to the much larger size of the analyte than regular biochemical molecules, and also the surface of the bacterial cells are comprised of various epitopes that allow nonspecific interactions with the impurities or the sensor surface.

 Rapid Detection of Escherichia Colio157:h7 and Salmonella Typhimurium :

Materials and Methods:

Biochemical Materials

Stock phosphate buffered saline (PBS, 0.1 M, pH 7.4), glucose, glucose oxidase, and bovine serum albumin (BSA) were bought from Sigma-Aldrich (St. Louis, MI). Stock PBS solution was diluted at a ratio of 1:10 to prepare 1×PBS (10 mM, pH 7.4), and used throughout all tests. 1% BSA solution (wt/vol) was prepared in PBS as a blocking buffer. The ultrapure deionized water (18.2 MΩ∙cm) was obtained from Millipore Milli -Q, Bedford, MA). 10 mMglucose solution was made by dissolving glucose into deionized water. Streptavidin-coated magnetic beads (MBs) with a diameter of 130 nm were manufactured by Kisker Biotech GmbH&Co. KG (Steinfurt, Germany).

AN ELECTROCHEMICAL BIOSENSOR FOR RAPID DETECTION OF E. COLIO157:H7

Experimental:

Materials and apparatus

Phosphate buffered saline 1 (PBS1, 0.1 M, pH 7.4), dopamine (DA), gold (III) chloride hydrate, potassium ferricyanide (III), glucose, and glucose oxidase (128200 U/g solid) were bought from Sigma-Aldrich (St. Louis, MI). Ferric chloride hexahydrate was purchased from MP Biomedicals, LLC. (Solon, OH). PBS2 (10 mM, pH 7.4).

(A) Overall Structure of the Magnetic Beads From Ocean Nanotech, and (B) Tem Images of the Magnetic Beads at Different Sizes.

(A) Overall Structure of the Magnetic Beads From Ocean Nanotech, and (B) Tem Images of the Magnetic Beads at Different Sizes.

Preparation of Bifunctional PMNCs:

The schematic description of the preparation of the PMNCs. First, 20 μl of streptavidin-coated MBs were washed with 200 μl of PBS2 to remove the preservative content in 1.5 ml protein low binding tubes (SARSTEDT AG & Co., Germany) and magnetically separated with the magnetic separator for three min.

The Schematic Illustration of the Abs/GOxext/AuNPs/MBs- GOx@PDA PMNCs Synthesis.

The Schematic Illustration of the Abs/GOxext/AuNPs/MBs- GOx@PDA PMNCs Synthesis.

 AN ELECTROCHEMICAL APTASENSOR FOR RAPID DETECTION OF SALMONELLA TYPHIMURIUM

Methods for Culture Preparation and Media Plating Enumeration:

The test cultures of S. Typhimurium were prepared by growing the stock cultures in brain heart infusion (BHI) broth (Remel Microbiology Products, Lenexa, KS) at 37 oC for 16-18h. A series of 1:10 dilutions for each bacterial culture were made with PBS2. To determine the viable cell numbers of tested bacteria, 100 μl of each dilution were plated onto the surface of either non-selective Trypsin Soy Agar (TSA) or selective Xylose-Lysine-Tergitol 4 (XLT4) agars. The number of bacterial colonies formed on the media after incubation at 37 oC for 18 to 24 h was counted to determine the concentration of viable bacteria cells in the terms of colony forming units per milliliter (cfu/ml). All the cultures were prepared on the test days.

CONCLUSIONS

In this dissertation three major parts of research were conducted to develop electrochemical biosensors for the detection of foodborne pathogens. E. coliO157:H7 and S.Typhimurium were chosen as the model bacteria targets because their significance regarding to the food safety.

The first developed immunosensor was based on a sandwich-like immunoassay and bare screen-printed interdigitated microelectrodes for the detection of E. coliO157:H7 and S.Typhimurium. In this study, the 130 nm MBs were conjugated with antibodies specifically targeting the bacteria of interest, and had achieved high capture efficiencies. Then the antibody-conjugated GOx was used to label the bacterial cells and converted the antibody-antigen interaction into the electrochemical signal.

The electrochemical impedance analysis was used and revealed a simple linear relationship of the impedance changes versus the log concentration of bacteria in a broad range from 10 2to 106cfu/ml for both pathogens. The developed impedance immunosensor demonstrated that it can detect both of the target bacteria at 103cfu/ml with the detection time of 2 h without any pre-enrichment procedures.

This immunosensor gave up the strategy of using complicated immobilization procedures thatattach the bioreceptors on the surface of electrodes, reducing the cost to fabricate the biosensor.The concept can also be easily adapted to detect other pathogens if 129 other antibodies/aptamers with corresponding specificity are available. By coupling with inexpensive, mass-producible bare or PB-modified SP-IDMEs, the developed lectrochemical biosensors have great potentialfor in-field application.

Source: University of Arkasas
Author: Meng Xu

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