Elsevier

Biosensors and Bioelectronics

Volume 21, Issue 7, 15 January 2006, Pages 1283-1290
Biosensors and Bioelectronics

A novel FRET-based optical fiber biosensor for rapid detection of Salmonella typhimurium

https://doi.org/10.1016/j.bios.2005.05.017Get rights and content

Abstract

A biosensor that is portable and permits on-site analysis of samples would significantly reduce the large economical burden of food products recalls. A fiber optic portable biosensor utilizing the principle of fluorescence resonance energy transfer (FRET) was developed for fast detection of Salmonella typhimurium (S. typhimurium) in ground pork samples. Labeled antibody–protein G complexes were formed via the incubation of anti-Salmonella antibodies labeled with FRET donor fluorophores (Alexa Fluor 546) and protein G (PG) labeled with FRET acceptor fluorophores (Alexa Fluor 594). Utilizing silanization, the labeled antibodies–PG complexes were then immobilized on decladded, tapered silica fiber cores to form the evanescent wave-sensing region.

The biosensors were tested in two different solutions: (1) PBS doped with S. typhimurium and (2) homogenized pork sample with S. typhimurium. The fiber probes tested in a S. typhimurium doped phosphate buffered solution demonstrated the feasibility of the biosensor for detecting S. typhimurium as well as determined the optimal packing density of the labeled antibody–PG complexes on the surface of fibers. The results showed that a packing density of 0.033 mg/ml produced the lowest limit of detection of 103 cells/ml with 8.2% change in fluorescence. The fiber probes placed in homogenized pork samples inoculated with S. typhimurium showed a limit of detection of 105 CFU/g with a 6.67% in fluorescence within a 5-min response time. These results showed that the FRET-based fiber optic biosensor can become a useful analytical tool for detection of S. typhimurium in real food samples.

Introduction

Foodborne pathogens have remarkably caused food contamination at every stage of food production, processing, and distribution. Since 1973, about 44–61% of foodborne disease outbreaks have been caused by contaminated muscle food (Bryan, 1988a, Bean and Griffin, 1990). Salmonella is one of the main organisms causing the outbreaks of foodborne illnesses, and pork is one of the major vehicles of foodborne salmonellosis throughout the world (Bryan, 1988b). A recent study of pork in U.S. retail stores indicated that 9.6% of the samples obtained were contaminated by Salmonella (Duffy et al., 2000), resulting in the large economical burden for the food industry due to product recalls.

Various monitoring techniques have been developed to detect foodborne pathogens. Conventional detection methods are cumbersome and time-consuming, requiring several days for presumptive results and confirmation (Jofre et al., 2005). Although enzyme-linked immunosorbent assays (ELISAs) are the most frequently used methods to detect pathogens, most require time-consuming pre-enrichment steps and 2–3 h for assaying (Hayes et al., 1991, Beckers et al., 1998). The polymerase chain reaction (PCR) technique has proved to be a powerful tool for the detection of pathogens in foods (Luk, 1994, Simon et al., 1996, Karpiskova et al., 2000), but this technique suffers from false positives by amplifying dead cells and making data interpretation complex (Deisingh and Thompson, 2004). Also, this technique requires multi-step processing and well-trained personnel, which adds considerable time and expense to the overall detection (Oberst et al., 1998).

Fiber optic biosensors have been investigated for the detection of pathogens, but many still require comparably long reaction time and multi-step processing, such as washing, filtration, enrichment step, sandwich assay, and immunomagnetic separation (Starodub et al., 1994, Zhou et al., 1997, Che et al., 2000, Liu et al., 2003, Geng et al., 2004, Kramer and Lim, 2004). Thus, the need remains for more accurate, inexpensive, simple, and rapid monitoring methods capable of detecting Salmonella contamination with a minimum of manipulations in food processing facilities.

We have been developing a fiber optic biosensor based on fluorescence resonance energy transfer (FRET). The phenomenon of FRET involves non-radiative energy transfer from a fluorescent donor molecule to an acceptor molecule due to dipole–dipole interactions when in close proximity (Lakowicz, 1999). The rate of energy transfer is inversely proportional to r6, where r is the distance between the donor and acceptor molecules. This results in a sensing mechanism that is extraordinarily sensitive to donor–acceptor separation distances.

For the development of our FRET-based fiber optic biosensors, antibodies and protein G were labeled with donor and acceptor fluorophores, respectively. Protein G binds specifically with the Fc portion of the IgG antibodies, enhancing proper orientation of antibodies immobilized onto the fiber while forming tertiary structures that will not interfere with the antibody's ability to bind to the antigen (Boyle and Reis, 1987). Utilizing evanescence sensing, the evanescent wave penetrates out of the fiber core and excites the labeled complexes, so only the interaction between the analyte and the immobilized molecular complexes can be sensed as shown in Fig. 1a, while Fig. 1b details the antibody–pathogen binding. In the absence of the pathogen, the fluorescence will be at the emission wavelength of the donor (λ1), with little or no fluorescence from the acceptor (λ2). However, conformational change in the 3D structure of an antibody occurs as the antibody binds with the target pathogen, leading to a decrease in distance between the two fluorophores (Roitt and Delves, 2001). This results in a non-radiative transfer of energy from the excited donor fluorophores to the acceptor fluorophores and consequently an emission from the acceptor (λ2). Ratiometric detection is then possible and the ratio of donor to acceptor emission provides a measure of the binding state between the antibody and targeted pathogen on the surface of the fiber.

The feasibility of the FRET immunosensor technique to detect Salmonella antigen via “in-solution test” was previously demonstrated by our group (Ko and Grant, 2003). In this paper, we now report on the rapid detection of S. typhimurium in ground pork with minimal sample preparation using a fiber optic FRET biosensor coupled to a custom-built benchtop fluorometer. The advantages of this biosensor are that it integrates the basic principles of FRET with the inherent conformational change of the antibody as it binds to antigens on the surface of the fiber. Thus, the biosensor is capable of rapidly detecting analytes while drastically reducing false positives since conformational changes are measured. Multi-step analysis, which is common for the competitive and non-competitive sandwich assays, is not necessary.

Section snippets

Materials

Affinity purified antibodies (goat immunoglobin, IgG) against Salmonella common structure antigens-1, recognizing all Salmonella serotypes available from ATCC, were obtained from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD). Alexa Fluor 546 (AF546) and Alexa Fluor 594 (AF594) (Molecular Probes, Eugene, OR) were utilized as the donor and acceptor fluorophores, respectively, in this study since they have high spectral overlap and energy transfer (Panchuk-Voloshina et al., 1999).

Immobilization of the labeled antibodies–PG complexes

The immobilization method is based on the covalent interaction between protein G and the silica fiber core by means of the bifunctional crosslinkers bound to the silane film on the fiber surface. To investigate if the labeled antibody–PG complexes were effectively immobilized on the fiber tip, the intensities of the donor and acceptor fluorophores were compared to that of a bare tapered fiber. As shown in Fig. 3a and b, the donor intensity increased from approximately 0.4 to 0.62 V while the

Conclusion

A novel FRET-based fiber optic biosensor for the detection of S. typhimurium in pork samples was developed. SalAb-AF546/PG-AF594 complexes were successfully immobilized onto the surfaces of silica optical fibers using the silanization method. The immobilized labeled complexes would change the intensity of their fluorescence based upon binding of S. typhimurium. The biosensors were tested in two different solutions: (1) PBS doped with S. typhimurium and (2) homogenized pork sample with S.

Acknowledgements

This project was funded, in part, by the National Pork Board and the Missouri Food for the 21st Century.

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