The need exists to improve sensitivity of detection of toxic pollutants and pathogenic microorganisms, ensuring food and water safety. Developing methods that would increase antibody binding surface area and/or improve the sampling process by specifically concentrating the analyte of interest from the diluted extracted food sample would increase the chances of finding and detecting food pathogens and their toxins. Our approach to improve sensitivity was to generate high surface nanofibrous membranes with covalently attached molecular recognition elements (MREs, e.g. antibodies and peptides) for the selective capture of target analytes through the use of electrospinning. Electrospinning is a process by which high static voltages are used to produce an interconnected membrane-like web of small fibers with diameters ranging from 50-1000 nanometers. These nanofibrous membranes can have surface areas approximately one to two orders of magnitude higher than those found in continuous films. The association of MREs with electrospun fibers presents the opportunity for developing both biosensor detection platforms with increased surface area and membrane concentrators. It is expected that the available surface area demonstrated by this technique will provide increased sensitivity, capture efficiency and fast response time in sensing applications. Antibodies and peptide-based receptors were selectively immobilized onto these nanoporous membranes for bioaffinity capture. Initial results involving fluorescent and chemiluminescent imaging for quantifying attachment and activity in association with the electrospinning process will be discussed.
Developments in rapid detection technologies have made countless improvements over the years. However, because of the limited sample that these technologies can process in a single run, the chance of capturing and identifying a small amount of pathogens is difficult. The problem is further magnified by the natural random distribution of pathogens in foods. Methods to simplify pathogenic detection through the identification of bacteria specific VOC were studied. E. coli O157:H7 and Salmonella typhimurium were grown on selected agar medium to model protein, and carbohydrate based foods. Pathogenic and common spoilage bacteria (Pseudomonas and Morexella) were screened for unique VOC production. Bacteria were grown on agar slants in closed vials. Headspace sampling was performed at intervals up to 24 hours using Solid Phase Micro-Extraction (SPME) techniques followed by GC/MS analysis. Development of unique volatiles was followed to establish sensitivity of detection. E. coli produced VOC not found in either Trypticase Soy Yeast (TSY) agar blanks or spoilage organism samples were - indole, 1-decanol, and 2-nonanone. Salmonella specific VOC grown on TSY were 3-methyl-1-butanol, dimethyl sulfide, 2-undecanol, 2-pentadecanol and 1-octanol. Trials on potato dextrose agar (PDA) slants indicated VOC specific for E. coli and Salmonella when compared to PDA blanks and Pseudomonas samples. However, these VOC peaks were similar for both pathogens. Morexella did not grow on PDA slants. Work will continue with model growth mediums at various temperatures, and mixed flora inoculums. As well as, VOC production based on the dynamics of bacterial growth.
Using a chemiluminescent fiber optic biosensor and magnetic particles, a simple, sensitive and rapid method to determine Staphylococcus aureus enterotoxin A (SEA) in military ration components was developed. Anti-staphylococcal enterotoxin A (Anti-SEA) was immobilized on magnetic particles and incubated with SEA. The beads were then collected and rinsed on a membrane filter (0.45um). The captured toxin was then selectively labeled with a monoclonal-horseradish peroxidase (POD) conjugate. SEA concentration was detected with a luminometer and a chemiluminescent enhancing reagent. Total assay time was 1.25 hours. Chemiluminescent signal due to nonspecific binding was tested with various blocking agents. Phosphate buffered saline with casein had the lowest background signal. Primary antibody concentration, secondary labeled antibody concentration and chemiluminescent substrate type were also evaluated to optimize signal intensity. The chemiluminescent fiber optic biosensor assay was compared to the Analyte 2000, a commercial fluorescent fiber optic biosensor. This assay consisted of immobilizing Anti-SEA on polystyrene waveguides, and incubating the waveguides with the toxin. The waveguide was incubated with a selectively labeled monoclonal-CY5 Dye conjugate. The sensitivity of chemiluminescent and fluorescent immunoassays were 1 ng, significantly lower than the levels needed to cause illness.
A simple, sensitive and rapid chemiluminescent fiber optic biosensor utilizing monoclonal antibodies to S. aureus was developed to detect the pathogen in food. The S. aureus cells were selectively labeled with a monoclonal-horseradish peroxidase (POD) conjugate, collected by membrane filtration, and detected with a luminometer and an enhanced chemiluminescent luminol reagent. Two different diameter membranes, 25 mm and 13 mm, were first tested in a luminometer tube format assay. A hand operated syringe filtration unit was used to capture cells and the membrane was then transferred to a luminometer tube for the chemiluminescent reaction. An improved system utilized a simple but efficient microwell plate vacuum filtration unit with an 8 mm membrane sealed at the bottom of the sample well. The sample was concentrated on the membrane and positioned directly in front of a fiber optic light guide to effectively collect and transmit the signal to the luminometer. Labeling S. aureus in solution proved to be much more effective than on the membrane surface. Using the microwell plate filtration system resulted in less sample handling, better reproducibility, and dramatically reduced assay time. The variability for 25 mm and 13 mm assays were 24.7% and 13.3%, while the microwell plate assay reduced this to 4.0%. The ability of the fiber optic probe to effectively collect the signal meant the sensitivity of the assay was not compromised with smaller membrane and sample size. The sensitivity of the biosensor was 3.8 X 104 CFU/ml, adequate to detect the organism at concentrations lower than the level that could result in food poisoning. The performance of the biosensor was not effected by the food materials and by the presence of other bacteria.
Proceedings Volume Editor (1)
This will count as one of your downloads.
You will have access to both the presentation and article (if available).
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.