Rapid and Sensitive Detection of Foodborne Pathogens Using Bio-Nanocomposites Functionalized Electrochemical Immunosensor with Dielectrophoretic Attraction.

Lee, Inae

Rapid and Sensitive Detection of Foodborne Pathogens Using Bio-Nanocomposites Functionalized Electrochemical Immunosensor with Dielectrophoretic Attraction.

Files

2017-12-phd-lee.pdf (3.87 MB)

Date

2017-12

Authors

Lee, Inae

Department

Molecular Biosciences & Bioeng

Abstract

The rapid detection and identification of potentially harmful microorganisms in food are essential to prevent foodborne outbreaks and ensure our safety. The faster response time and relatively high sensitivity and selectivity of biosensor-based detection methods, as compared to conventional methods, have increased the attention towards alternative approaches for the early inspection of foodborne pathogens in a variety of food products. The recent advances in micro- and nanotechnologies are attributed to the improvement of the sensor’s performance. The methods of using single-walled carbon nanotubes (SWCNTs) to enhance the sensing signal response, as well as dielectrophoresis (DEP) and fluidics techniques to improve bioaffinity reactions, create unique bio-nano sensing devices for fast and reliable microbial analysis. The goal of this study was to develop SWCNT functionalized electrochemical immunosensors for the rapid and sensitive detection of foodborne pathogens assisted with dielectrophoretic and fluidic technologies. The biosensor design, which involved electrode configuration, electrode surface modification, and detection mode, were gradually transformed to achieve the sensitive, selective, specific, or simultaneous detection of bacteria and viruses. The functionalized microwire-based electrochemical immunosensor (MEI sensor) was designed and fabricated for the selective detection of target bacteria from non-target bacteria. The Escherichia coli specific MEI sensor was prepared to test the functionalization process and the Staphylococcus aureus specific MEI sensor was used to validate the proposed sensor concept for other bacteria. The combination of double-layered SWCNTs and 5% bovine serum albumin coating contributed to signal enhancement and cell binding specificity. The selective capture of E. coli or S. aureus cells was achieved when the electric field was generated at a frequency of 3 MHz and 20Vpp. A linear trend in the change of electron transfer resistance (ΔRet) was observed as E. coli concentrations increased from 5.32 × 102 to 1.30 × 108 CFU/mL (R2 = 0.976) and S. aureus concentrations from 8.90 × 102 to 3.45 × 107 CFU/mL (R2 = 0.983). Both MEI sensors could detect target bacteria cells without interfering with the other bacteria in the mixed suspensions. The detection time was 10 min including cell concentration and signal measurement. The developed MEI sensor was evaluated for its detection of target bacteria from non-target materials in food. The E. coli specific sensor and the Salmonella specific sensor were fabricated to individually detect E. coli K12 and S. Typhimurium contaminated baby spinach. The estimated concentrations of E. coli in the spinach extracts corresponded well with the concentrations determined by the plate counting method, with an R2 value of 0.972 and a detection range from 8.33 × 102 to 7.97 × 105 CFU/g for the surface contamination method. A linear relationship was observed between ΔRet and S. Typhimurium concentrations from 1.43 × 103 to 1.67 × 107 CFU/g with an R2 value of 0.942. Both E. coli and Salmonella MEI sensors are specific towards the target bacteria in the sample despite the interference of spinach debris and non-target bacteria. The continuous flow multi-junction biosensor was fabricated and characterized for the simultaneous detection of E. coli and S. aureus. The developed continuous flow junction sensor showed an increase of sensing sensitivity by a factor of 10 in the detection of E. coli K12, as compared to the stationary sensor. A linear regression was observed for both the E. coli and S. aureus functionalized multi-junction array sensors with a detection range of 102 to 105 CFU/mL. Multiplexed detection of bacteria at sensing levels as low as 102 CFU/mL for E. coli K12 and S. aureus were accomplished within 2 min. Lastly, the flow-based dielectrophoretic biosensor was designed and tested for the detection of bacteriophage MS2 as a norovirus surrogate. The cyclic voltammogram showed that the current for the PEI-SWCNTs electrode was higher than the current of the PEI film surface, and was followed by a decrease in the current after antibody immobilization and MS2 attachment. Antibody immobilization on the detector with electric field applied to the fluidic channel at 10 Vpp and 1 MHz showed higher current changes by antibody-MS2 complexes than the assay without antibody immobilization and DEP. The changes in current signal displayed a dependence on the concentration of MS2 in the sample solution. The total assay was completed within 15 min. The developed MEI sensor with DEP-assisted cell trapping has the potential for fast, simple, and selective detection of low levels of target bacteria in the presence of mixed bacteria communities and food matrices. The CNTs functionalization and continuous flow assay could offer advances in sensitivity and detection time. The proposed sensing technology and the device can have a beneficial influence on the food industry by offering the rapid detection of multiple pathogens in food. It will also result in the development of new approaches to monitor and control biological hazards, which can be incorporated into food production and processing facilities to improve the safety of our food products.

Keywords

biosensor, carbon nanotubes, dielectrophoresis, impedance, microfabrication, virus

URI

http://hdl.handle.net/10125/62561

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Ph.D. - Molecular Biosciences and Bioengineering

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