Antibody Immobilization on Waveguides Using aFlow–Through System Shows Improved Listeria monocytogenesDetection in an Automated Fiber Optic Biosensor: RAPTORTM

Antibody Immobilization on Waveguides Using aFlow–Through System Shows Improved Listeria monocytogenesDetection in an Automated Fiber Optic Biosensor: RAPTORTM

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  Sensors   2006  ,   6  , 808-822  sensors ISSN 1424-8220 © 2006 by MDPI Full Research Paper  Antibody Immobilization on Waveguides Using aFlow–Through System Shows Improved  Listeria monocytogenes Detection in an Automated Fiber Optic Biosensor: RAPTOR ™ Viswaprakash Nanduri 1 , Giyoung Kim 2 , Mark T. Morgan 1,* , Daniel Ess 3 , Byoung-KwonHahm 1 , Aparna Kothapalli 1 , Angela Valadez 1 ,   Tao Geng 1  and Arun K. Bhunia 1,* 1 Department of Food Science, Purdue University, 745 Agriculture Mall Dr., West Lafayette,IN,47907, USA 2 National Institute of Agricultural Engineering, RDA, 249 Seodun-dong, Suwon, 441-100, Republic of Korea 3 Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN,47907, USA* Authors to whom correspondence should be addressed; E-mails: (Arun K.Bhunia ) or (Mark T. Morgan)  Received: 6 February 2006 / Accepted 9 June 2006 / Published: 19 August 2006  Abstract : Recent outbreaks of food borne illnesses continue to support the need for rapidand sensitive methods for detection of foodborne pathogens. A method for detecting  Listeriamonocytogenes  in food samples was developed using an automated fiber-optic-basedimmunosensor, RAPTOR ™ . Detection of  L. monocytogenes  in phosphate buffered saline(PBS) was performed to evaluate both static and flow through antibody immobilizationmethods for capture antibodies in a sandwich assay. Subsequent detection in frankfurtersamples was conducted using a flow through immobilization system. A two stage blockingusing biotinylated bovine serum albumin (b-BSA) and BSA was effectively employed toreduce the non-specific binding. The sandwich assay using static or flow through mode of antibody immobilization could detect 1×10 3  cfu/ml in PBS. However, the effectivedisassociation constant K d  and the binding valences for static modes of antibodyimmobilization in spiked PBS samples was 4×10 5 cfu/ml and 4.9 as compared to 7×10 4 cfu/ml and 3.9 for flow through method of antibody immobilization. Thus the sensitiveflow-through immobilization method was used to test food samples, which could detect5×10 5 cfu/ml of  L. monocytogenes  in frankfurter sample. The responses at the lowestdetectable cell numbers in the frankfurter samples was 92.5 ± 14.6 pA for  L. monocytogenes to comparative responses of 27.9 ± 12.2 and 31 ± 14.04 pA obtained from  Enterococcus  Sensors 2006 , 6    809  faecalis  and  Lactobacillus rhamnosus  (control species), respectively. The effective K d  andbinding valency from spiked frankfurter samples was 4.8×10 5 cfu/ml and 3.1, thus showinghighly sensitive detection can be achieved using the RAPTOR ™  biosensor even in thepresence of other bacterial species in the matrix. Keywords: Biosensor,  Listeria monocytogenes , fiber optic sensor, immunosensor,RAPTOR Introduction  Listeria monocytogenes  is one of the major foodborne pathogens and current U.S. regulatory policymaintains a “zero tolerance” in ready-to-eat (RTE) foods. It is a gram-positive, rod-shapedintracellular pathogen that causes listeriosis in elderly, those with weakened immune systems, andpregnant women. Recent  L. monocytogenes -related outbreaks from various food sources [1] haveincreased public awareness of this pathogen. The greatest threat of listeriosis is from RTE productsthat do not require further cooking at home. A recent risk assessment study estimated the risks of serious illness and death associated with consumption of RTE foods possibly contaminated with  L.monocytogenes . The results included a list of 23 food categories of seafood, produce, dairy and meatwhich were classified as very high risk (> 100 cases per year), high risk, moderate risk and low risk (<1 case per year). The very high and high risk categories included: deli meats, pasteurized fluid milk,other dairy products, and frankfurters (not reheated). The Healthy People 2010 goals for nationalhealth promotion and disease prevention called on federal food safety agencies to reduce foodbornelisteriosis by 50% by the end of the year 2005. A recent risk assessment study conducted by Food andAgriculture Organization (FAO) and the World Health Organization (WHO) indicated that the ready-to-eat products are of highest risk for  L. monocytogenes  and the risk increases with increase dose at thetime of consumption [2].Conventional methods for  Listeria  detection and identification involve prolonged, multipleenrichment steps. Even though some rapid immunological and nucleic acid-based assays are available,these assays still require enrichment steps and give results in 24-48 h [3]. Other methods for thedetection of  Listeria  species include reverse transcription polymerase chain reaction [RT-PCR]; realtime quantitative PCR; nucleic acid sequence-based amplification (NASBA); DNA microarrays; PCR-based microarrays and oligonucleotide-based microarrays [3] Fiber-optic biosensors have proven tobe a promising new technology for rapid detection of food borne pathogens [4]. Fiber-optic biosensorsuse light transmittable tapered fibers to send excitation laser light and receive emitted fluorescence,usually from a fluorophore-labeled antibody. The fluorescent light excited by an evanescent wavegenerated by the laser is quantitatively related to the number of labeled biomolecules in closeproximity to the fiber surface [5]. A fiber-optic biosensor (Analyte 2000, Research International, Monroe, WA) has been used todetect various microorganisms including: Vaccinia  virus [6],  Escherichia coli  O157:H7 [7],  Bacillusglobigii  [8], Salmonella  Enteritidis [9], and  L. monocytogenes  [10, 11]. Improvements in theportability and automation of the fiber-optic biosensor (RAPTOR™, Research International, Monroe,  Sensors 2006 , 6    810 WA) have increased the usefulness of this detection device. The RAPTOR™ system has been used todetect  Bacillus anthracis  and Francisella tularensis  [12]. Salmonella  Typhimurium [13] andstaphylococcal enterotoxin B [14].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 nmdiodes to excite each of four, 4.5 cm long fiber-optic probes. The fibers are assembled in a couponwhich has fluidic channels for automated operation. Fluorescent molecules bound on the surface of thesensing region are excited by an evanescent wave generated by the laser. Photodiodes collect emissionlight at wavelengths over 670 nm. The emission signal is recorded in pico amperes (pA) and related toconcentration of analyte [4].The purpose of this study was to develop an automated assay method for detecting  L.monocytogenes  using the RAPTOR™ system. The packing and orientation of antibodies on the sensorsurface play a crucial role in determining the sensitivity and detection limit in a biosensor. In an effortto increase the detection limit, both static and flow through methods for immobilization of antibodieson the fiber optic waveguide were investigated. Ideal blocking steps were also developed in an effortto reduce non-specific binding. Sandwich assays were tested for detection of  L. monocytogenes in theboth phosphate buffered saline (PBS) buffer and samples from frankfurter previously spiked with lownumbers of  L. monocytogenes and incubated for 20 h. Results and Discussion  Instrument set up and fiber preparation for antibody immobilization The RAPTOR system uses a disposable coupon that holds four optical fibers (waveguides) whichare immobilized with capture antibody. Flow through set up (Fig 1) employed in this study forantibody immobilization on waveguides and subsequent detection for pathogen using a detailedprocedure outlined in Fig 2.Effect of two-stage blocking employing biotinylated bovine serum albumen (b-BSA) and BSAalone was examined. Fig 3 shows the comparative binding responses (pA) for  L. monocytogenes  fromboth antibody (PAb – P66) immobilized sensor surfaces and control (without antibody) sensorsurfaces. As it can be seen, the deployment of dual blocking clearly reduced the background noise thatmay be generated by the non-specific binding of  L. monocytogenes  occurring at the sensor surface. Optimization of sandwich assay for detection of Listeria monocytogenes Initially two different capture antibodies (PAb-P66 and LM PAb) were attached using the staticimmobilization method onto the fibers at low concentrations and the responses of biosensors toincreasing concentrations of  L. monocytogenes  spiked into PBS were tested. PAb-P66 produced highersignal than LM PAb for both 10 µg/ml and 20 µg/ml concentrations (Fig. 4). Using higherconcentration of capture antibody at the fiber preparation step generally increased responses. For LMPAb, the signal increase from using higher concentration of capture antibody, continued up to 10 6 cfu/ml. At lower concentrations of antibody and using static immobilization on the waveguides, thedifferent lowest detectable cell numbers and their corresponding responses are observed (Table 1).  Sensors 2006 , 6    811Figure 1. The flow through setup with the waveguide coupon. Figure 2. Preparation of the waveguides.  Sensors 2006 , 6    812Table 1 . Detection limits for  L. monocytogenes  in PBS using staticmode of capture antibody immobilization.Preliminary tests established that PAb-P66 antibody proved more efficient as a capture antibodythan LM PAb. Further detection trials were conducted using PAb-P66 at higher concentrations (200µg/ml) and comparing static and flow through modes of antibody immobilization on the wave guides.The high affinity of the antibody P66 to  L. monocytogenes  was also confirmed in an independent study[17].Figure 5 shows the dose responses from PBS containing different concentrations of  L.monocytogenes  using static and flow through modes of capture antibody immobilization for PAb-P66.The two upper lines representing bacterial binding are sigmoid fits to the mean values obtained fromsix experimental data, each one derived from individual coupons tested with the same sample ( ■ - χ 2 =6.02, R 2 =0.92; ▲ - χ 2 = 4.6 × 10 -1 , R 2 =0.99) and repeated under similar conditions. The two lowerCaptureantibodyConcentration(µg/ml)Lowest detectablecell numbers(cfu/ml)Response at thelowest detectablecell numbers (pA)10~1×10 8 85.6LM PAb20~1×10 4 18.310~1×10 3 26.4PAb-P6620~1×10 3 71.1   Figure 3. Comparative binding response of  L. monocytogenes  to sensor surface with orwithout dual blocking agents. The upper line ( ■ ) represents the signal obtained from anantibody (PAb-P66) immobilized sensor surface that had not been blocked with the dualblockers as compared to the signal ( ●  ) that had been obtained by similar sensor surfacewhich had been blocked using the dual blockers. The lower lines represent the signalsobtained from sensors surface devoid of antibody [control] ( ▲ - without dual blocking) and asimilar antibody devoid surface ( ▼ ) after undergoing dual blocking.
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