Detection of Listeria Monocytogenes Using an Automated Fiber-optic Biosensor: RAPTOR

Fiber-optic biosensor uses light transmittable tapered fiber to send excitation laser light and receive emitted fluorescent light. The fluorescent light excited by an evanescent wave generated by the laser is quantitatively related to biomolecules immobilized on the fiber surface [1]. An automated fiber-optic biosensor based detection method for Listeria monocytogenes was developed in this research. Detections of Listeria monocytogenes in hotdog sample were performed to evaluate the method. By using the detection method with automated fiber-optic biosensor, 5.4×10 7 cfu/ml of Listeria monocytogenes was able to detect. Introduction Conventional methods for Listeria detection and identification involve prolonged multiple enrichment steps. Even though some immunological rapid assays are available, these assays still require enrichment steps and give results in 18 to 48 hours. Fiber-optic biosensors have shown great potential for rapid detection of foodborne pathogens. Portable types of fiber-optic biosensors (Analyte 2000, Research International, Monroe, WA, USA) have been used to detect various microorganisms including Escherichia coli O157:H7 [2], Salmonella typhimurium [3], and L. monocytogenes [4]. Improvements in the portability and automation of a fiber-optic biosensor (RAPTORTM, Research International, Monroe, WA, USA) increased the usefulness of this detection device. The RAPTOR can perform four assays on the same sample allowing replicate measurements of the same analyte or simultaneous detection of four different targets. The RAPTOR uses four 635 nm diodes to excite each of four, 4.5 cm long fiber-optic probes. The fibers are assembled in a coupon which has fluidic channels for automated operation. Fluorescent molecules bound on the surface of the sensing region are excited by an evenescent wave generated by the laser. Photodiodes collect emission light at wavelengths over 670 nm. The emission signal is recorded in picoamperes (pA) and related to concentration of analyte. The purpose of this study was to develop an automated assay method for detecting L. monocytogenes using the RAPTOR system. A sandwich assay was devised and performed to evaluate the method. Materials and Methods Bacteria and regeants Listeria monocytogenes strain V7, from our collection was used for in the experiments. The bacteria were maintained on brain heart infusion (BHI) agar (1.5%) slants (Difco Laboratories) at 25°C for the duration of this study. Fresh cultures of Listeria monocytogenes were prepared by incubating the slant Key Engineering Materials Vols. 321-323 (2006) pp. 1168-1171 online at http://www.scientific.net © (2006) Trans Tech Publications, Switzerland All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of the publisher: Trans Tech Publications Ltd, Switzerland, www.ttp.net. (ID: 152.99.32.30-23/02/07,01:09:17) cultures in BHI broth at 37°C for 16 hours. In some cases, bacteria were adjusted to approximately the same concentration by using a spectrophotometer (Beckman-Coulter, Fullerton, CA, USA). For sample preparation, buffered Listeria enrichment broth (BLEB) and Oxford Listeria agar base were purchased from Acumedia (Baltimore, MD, USA). Modified oxford antimicrobic supplement was purchased from Becton, Dickinson (Sparks, MD, USA). Package of hotdogs was purchased from a local grocery store. Purified anti-Listeria monoclonal antibody (C11E9) [5] and polyclonal antibody (LM Pab and P66) were provided by Dr. Arun Bhunia (Purdue University, IN, USA). Casein, bovine serum albumin (BSA), and phosphate buffer were purchased from Sigma (St. Louis, MO, USA). SuperBlock was purchased from Piece (Rockford, IL, USA). Antibody labeling and biotinylation was prepared according to the method described by Tims et. al [4]. Hotdog sample preparation 10 g of each hotdog sample was spiked by dropping 100 μl of 1 × 10 9 CFU/ml of the Listeria monocytogenes cell suspension onto the sample surface. The spiked hotdog samples were placed in a sterilized enrichment container filled with 30 ml of buffered Listeria enrichment broth (BLEB) media at 37°C for 20 hours for enrichment. The enriched cell suspensions were collected after filtration to remove any remaining food particles and used for the immunoassay. Enumeration of the enriched L. monocytogenes was performed using the modified oxford (MOX) plate method. After 20 hours incubation, the numbers of enriched L. monocytogenes cells were counted using MOX plate. The numbers of L. monocytogenes after 20 hr enrichment were 5.4±0.6 × 10 cfu/ml and 6.2±0.6 × 10 cfu/ml for 10 cfu/g and 100 cfu/g inoculation, respectively. Fiber preparation and instrument setup Fibers were prepared to have active binding sites for L. monocytogenes detection as shown in Fig. 1. A fiber was inserted into a 100 μl pipette tip and incubated overnight (18 to 22 hours) at 4 °C with 90 μl of 100 μg/ml streptavidin. Fibers were rinsed with PBS-Triton (0.02 M phosphate buffered saline (PBS) containing 0.05 % Triton X-100) and incubated with 90 μl of 100 μg/ml biotinylated capture antibody (PAb or P66) in PBS at room temperature for 1 hour. Finally, the fibers were incubated with 90 μl of SuperBlock followed by 90 μl of 1 mg/ml biotinylated bovine serum albumen (bBSA) at room temperature for 1 hour each. Prepared fibers were mounted into the disposable coupon. And then the assembled coupon was inserted into the RAPTOR and measurements were done automatically by running preprogrammed a baseline recipe for each baseline reading and an assay recipe for each sample reading (Fig. 2). Fiber-optic Streptavidin