ARTICLE IN PRESS

FOODMICROBIOLOGY

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doi:10.1016/j.fm

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Food Microbiology 25 (2008) 1–12

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Review

Immunosensors for rapid detection of Escherichia coli O157:H7 —Perspectives for use in the meat processing industry

Oleksandr Tokarskyya, Douglas L. Marshallb,�

aDepartment of Food Science, Nutrition, and Health Promotion, Mississippi Agricultural and Forestry Experiment Station,

Mississippi State University, Box 9805, Mississippi State, MS 39762-9805, USAbCollege of Natural and Health Sciences, University of Northern Colorado, Gunter Hall 1000, Campus Box 134, Greeley, CO 80639, USA

Received 13 July 2007; accepted 22 July 2007

Available online 31 July 2007

Abstract

This review critically evaluates different types of immunosensors proposed for rapid identification of Escherichia coli O157:H7. The

methods are compared with approved USDA-FSIS standard procedures for determination of this pathogen in raw or ready-to-eat meat

products. Major advantages and disadvantages for each method are highlighted. Our analysis suggests that application of

immunosensors in the meat-processing industry may be limited to identification of uncontaminated samples after conventional selective

enrichment in broth. Use for detection appears limited at the present time.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Escherichia coli O157:H7; Immunosensors; Food safety

Contents

1. Enterohemorrhagic Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2. Biosensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

3. Immunosensors with direct measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3.1. Surface plasmon resonance immunosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3.2. Piezoelectric transducer immunosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3.3. Impedance-based immunosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

4. Immunosensors with indirect measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

4.1. Fluorescent label. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

4.2. Enzyme label . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

4.3. Luminescent label . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

4.4. Conductometric label . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

5. Application of immunosensors for examination of raw and ready-to-eat meats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

6. Comparison of immunosensors with other rapid test kits for E. coli O157:H7 detection. . . . . . . . . . . . . . . . . . . . . . . . . . . 10

7. Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

e front matter r 2007 Elsevier Ltd. All rights reserved.

.2007.07.005

ing author. Tel.: +1970 351 2877; fax: +1 970 351 2176.

ess: douglas.marshall@unco.edu (D.L. Marshall).

ARTICLE IN PRESSO. Tokarskyy, D.L. Marshall / Food Microbiology 25 (2008) 1–122

1. Enterohemorrhagic Escherichia coli

Enterohemorrhagic strains of Escherichia coli (EHEC) arewell-known enteric pathogens associated with life-threaten-ing symptoms of initial hemorrhagic colitis (Jay, 2000) andlater development of hemolytic uremic syndrome (Buchananand Doyle, 1997). The most challenging problem forenumeration, identification, and control of this pathogen isthe low infectious dose necessary to cause disease, which isbetween 2 to 2000 ingested cells (Buchanan and Doyle,1997). EHEC foodborne outbreaks have been caused byconsumption of sandwiches, undercooked ground beef, rawmilk, unpasteurized apple juice, water, and fecally con-taminated produce (Buchanan and Doyle, 1997).

Although the strains responsible for this gastrointestinaldisease are numerous worldwide, only one is predomi-nantly responsible for foodborne outbreaks in the US(Buchanan and Doyle, 1997). Based on serotyping, E. coli

O157:H7 is the predominant disease-causing strain. Thesomatic antigen is O157, while H7 refers to the flagellarantigen on the surface of the cells. Prevalence of thedisease-causing strain also depends on the geographicregion of foodborne outbreaks. For example, serotypeO111:H� and O157:H� outbreaks are predominant inAustralia, while O157:H� is important in central Europe(Buchanan and Doyle, 1997). Other serotypes of the EHECgroup are well known and listed elsewhere (Buchanan andDoyle, 1997; Jay, 2000).

In many cases, non-motile isolates of EHEC strains weremarked as H� serotypes of E. coli because flagellarantigens are usually responsible for bacterial motility.However, Feng et al. (1996) showed that these non-motileisolates could actually be H7 positive and even motile.Therefore, some strains of H� or non-motile clinicalisolates might be mis-typed. Feng et al. (1998) describedgenotypic and phenotypic changes in E. coli O157:H7 thatcould create challenges for future serological identificationof strains responsible for disease. Serotyping by itself doesnot provide sufficient information about pathogenicity ofstrains, and in most cases verification of toxin productionby EHEC strains serves as additional confirmation. It isknown that all EHEC strains produce Shiga-like toxinsverotoxin 1 and verotoxin 2 (Buchanan and Doyle, 1997).However, toxin alone is not responsible for pathogenicity,as some strains possessing genes for verotoxin productionare nonpathogenic. Another virulence factor, the proteinintimin, is required for bacterial attachment to theintestinal wall (Feng et al., 1996).

Biochemical markers for E. coli O157:H7 include itspartial or complete inability to ferment sorbitol and theabsence of b-glucuronidase (Okrend et al., 1990), which is aphenotype isolated on selective media. However, sorbitol-positive O157 isolates responsible for outbreaks ofhemolytic-uremic syndrome have been observed inGermany (Feng et al., 1998).

The current policy of the United States Department ofAgriculture—Food Safety and Inspection Service (USDA-

FSIS) towards this pathogen is defined as zero tolerance,which means absence of the pathogen using a well-definedsampling plan (USDA-FSIS, 2002). The current limit ofE. coli O157:H7/H� detection by the approved USDA-FSIS method is less than 1 cfu per 65 g sample of raw orready-to-eat meat product. Emphasis is not on enumera-tion, but rather on the presence and proper identification ofthe pathogen. The method is based on specimen enrich-ment in selective broth, rapid screening tests to discardnegatives, immunomagnetic separation, plating on highlyselective Rainbow agar, O157 serotype confirmation, andplating on Blood agar. Resultant colonies on Blood agarare confirmed as positive if biochemical and serologicaltests are positive, and Shiga-toxin presence is confirmed.To summarize, a potential positive sample can be identifiedby rapid screening tests after 20–24 h selective enrichment.Negative samples by this test are considered free of EHECand cleared for consumption. Presumptive positivesamples possess typical colonies on Rainbow agar, whichagglutinate with the O157 antibody. Confirmed positivesamples have typical colonies on Blood Agar plates andshould be additionally identified biochemically, serologi-cally, and by the presence of verotoxin(s) or responsiblevirulence gene(s).

2. Biosensors

One approach for rapid pathogen detection is the use ofbiosensors. One type of biosensor, also called an immuno-sensor, is based on specific antibody-antigen interactions(Ivnitski et al., 1999; Iqbal et al., 2000; Leonard et al.2003). Numerous immunosensors have been developed fordetection and enumeration of E. coli O157:H7 and werepromising candidates for rapid screening of foods. There isno strict definition of an immunosensor, and researchershave different perceptions of the concept. For example,Gizeli and Lowe (1996) emphasized that immunosensorsdetect antigen binding to antibodies by immobilization ofthe reaction to the surface of a device known as atransducer. The transducer converts surface change para-meters into a detectable electric signal. A similar definitionwas suggested by Pathak and Savelkoul (1997), whoemphasized the necessity for the presence of a biologicalrecognition mechanism together with a suitable transducer,which transforms recognition at its surface into measurablesignals. On the other hand, Marco and Barcelo (1996)stated that a biosensor should be a miniaturized device. Inevery case, immunosensors are based on principles of solid-phase immunoassays on the surfaces of transducers (Marcoand Barcelo, 1996). This does not include several ampero-metric immunosensors, in which immunological reactionsare carried out separately but not on the surface of thetransducer. In this case, the captured immunocomplextransforms a chemical compound known as a mediator,which is oxidized or reduced at the surface of an electrode.However, several researchers extended the definition ofbiosensors to include devices based on the later principle

ARTICLE IN PRESSO. Tokarskyy, D.L. Marshall / Food Microbiology 25 (2008) 1–12 3

(Marco and Barcelo, 1996; Leonard et al., 2003; Brewsterand Mazenko, 1998). For the purpose of this review, weincluded these ‘‘out-of-definition’’ immunosensors as well.

Ivnitski et al. (1999) divided biosensors into batch-typeand continuous-type functioning devices. However, Marcoand Barcelo (1996) reported that most immunosensorswere irreversible because of specific binding. Therefore,continuous functioning is difficult to achieve and transdu-cers are either disposable or regenerable. Thus, only batch-type sensing is currently possible, which either includes aregeneration step before the next measurement or replace-ment of the transducer surface (Marco and Barcelo, 1996).For regeneration, low-affinity antibodies, which mightcompromise sensitivity, or agents capable of disruptingantibody-antigen interaction, are needed (Marco andBarcelo, 1996). For example, Fratamico et al. (1998) usedguanidine-HCl to disrupt non-covalently bound E. coli

O157:H7 cells from immobilized antibodies at the surface.Immunological reactions that are slow due to diffusionlimitations of antigens to immobilized antibodies makereal-time measurement difficult, particularly for low levelsof contaminants. However, most biosensors give resultswithin 20–90min, which is close to real-time in comparisonwith conventional techniques and classical enzyme-linkedimmunosorbent assays (ELISAs). Another advantage ofbiosensor use is that results are read via digital signals andare not as dependant on personal factors such as bias,fatigue, level of training, or visual disorders. However, thisproperty is also shared by microtiter plate spectrophoto-metric immunoassays.

Velasco-Garcia and Mottram (2003) noted that a generalapproach to increase sensitivity of immunosensors is toconcentrate bacteria by capturing on immunomagneticbeads, membranes, or optic fiber tips. These capturematrices also can act as transducer surfaces. A pre-concentration step can help avoid diffusion limitationsand enable detection of pathogens at low concentrations.Pathak and Savelkoul (1997) mentioned one majordisadvantage of immunosorbent assays; namely, theimmobilization of antibodies in incorrect conformationfor interaction with antigens. This problem was solved bythe use of the biotin–streptavidin system (Yu and Bruno,1996; Paffard et al., 1997; Gehring et al., 1998) and proteinA and protein G as ligands (Fratamico, 1998) to improvesensitivity and specificity.

Given these observations, most sensors now operate asbatch devices, where the immunological reaction may becarried out not only on the surface of a transducer, but onother surfaces for concentration/capturing or even inhomogenous solution before ‘‘transferring’’ the signal tothe transducer. However, the signal is read from thetransducer itself, which may be separate from the capturingsurface. At this point, the difference between instrumentalmethods for immunological reaction measurement andbiosensors does not seem to be significant. In this review,we considered immunosensors to be portable, with rapiddetection and advanced technology for detection.

According to Gizeli and Lowe (1996), two large groupsof immunosensors exist, which use with direct (non-labeledantibody) and indirect (labeled antibody) measurements.Labeled substances are less desirable because of highercost, but they provide better sensitivity due to signalamplification (Gizeli and Lowe, 1996; Marco and Barcelo,1996). Another approach to amplify signal, improvesensitivity, and avoid non-specific binding is to usesandwich assays or even a complex of three differentantibodies known as a double sandwich assay.In the remainder of this review, only those immunosen-

sors that were used for E. coli O157:H7 (or occasionallyE. coli K12) detection are considered together with theirprinciples by which they function. Detection limits of theinstruments are expressed as ‘‘cells/ml’’ throughout thetext. This was done in order to add consistency andsimplicity, and because several studies used mortalizedbacteria instead of viable cells. For additional informationon other immunosensors, several references are available(Ivnitski et al., 1999; Harzanyi, 2000; Leonard et al., 2003).All spectrophotometric immnunoassays, where measure-ments were not done on the surface of a transducer, anddirect enumeration of bacteria by epifluorescence micro-scopy, were not considered for discussion. In some cases,immunoassays that were considered to be close tobiosensor functioning either by us or by the authors arediscussed (fluorescence-based and amperometric methods).Summary commentary on immunosensor use in the foodindustry is provided with emphasis on meat products.

3. Immunosensors with direct measurement

Few optical transducers that are based on surfaceplasmon resonance and piezoelectric transducers used inacoustic bulk-wave-based biosensors can utilize non-labeled antibodies. Recently, impedance-based immuno-sensors were proposed for direct measurements (Yanget al., 2004; Radke and Alocilja, 2005). Even if it looksadvantageous to use non-labeled antibodies because of lowprice and simplicity, the major shortcoming of these typesof sensors is low sensitivity, which becomes an importantlimiting factor for E. coli O157:H7 detection. Threeexamples of direct measurement immunosensors arediscussed in this section; surface plasmon resonance,piezoelectric transducer immunosensors, and impedance-based immunosensors.

3.1. Surface plasmon resonance immunosensors

Surface plasmon resonance (SPR) is based on the factthat electrons of a metal layer become excited and absorblight at a particular incident angle of appropriatewavelength. This causes reflection of light to become lessintense. The incident angle depends on the dielectricproperties of the medium adjacent to the metal surface,which is affected by analytes (antibody–antigen complex)bound to that surface (Ivnitski et al., 1999; Leonard et al.,

ARTICLE IN PRESSO. Tokarskyy, D.L. Marshall / Food Microbiology 25 (2008) 1–124

2003; Velasco-Garcia and Mottram, 2003). The firstcommercial SPR-based biosensor was marketed by Phar-macia BiosensorAB, now called BIAcoreAB. AffinitySensors commercialized IASys plusTM and the fullyautomated IASys Auto +AdvantageTM systems. AnotherSPR-based device was recently marketed by XanTechBioanalytics GBR. Other commercial devices are availablefrom Texas Instruments USA and Biotul, which marketsthe Kinomics PlasmoonTM instrument. The differentinstruments provide varying limits of detection, which aredependent upon signal to noise ratio of the instruments(Leonard et al., 2003).

Fratamico et al. (1998) utilized the BIAcore instrumentto detect E. coli O157:H7. Antibodies, either monoclonalor polyclonal against E. coli O157:H7, were bound to thesensor surface either directly or via protein A or protein Gligands. In order to amplify a signal, a sandwichimmunoassay was performed. The sensor was found tobe specific for E. coli O157:H7 (4 strains) and no cross-reactivity was observed with Salmonella enterica serovarTyphimurium or Yersinia enterocolitica. E. coli O103:H2did not generate a notable signal. The pre-coating of thesurface with ligands in order to bind the antibody in theproper direction did not enhance detection in comparisonwith results of antibody binding to the surface in theabsence of ligands. The detection limit of E. coli O157:H7was 5� 107 cells/ml for a total injection time of 10min. Asnoted by Fratamico et al. (1998), usage of the SPRimmunosensor was excellent to study interactions betweenE. coli O157:H7 and different antibodies, and if appro-priate antibodies were used, it may be possible to use thebiosensor for detection of target microorganisms inenrichment cultures of foods.

3.2. Piezoelectric transducer immunosensors

Piezoelectric transducers are used in acoustic bulk-wave-based biosensors. Measurable signal is produced by a shiftin resonant frequency of a whole piezoelectric crystal dueto an increase in mass. This is the result of antigen–anti-body complex formation on the crystal surface (Marco andBarcelo, 1996; Ivnitski et al., 1999; Leonard et al., 2003).Resonant frequency is a result of shear deformation of thecrystal that is provoked through the application ofalternating current between two electrodes on both sidesof the crystal. Disadvantages of acoustic-wave immuno-sensors are the need for long incubation time withcontaminated samples, numerous washing and dryingsteps, and problems with crystal regeneration (Ivnitskiet al., 1999; Leonard et al., 2003).

He et al. (1994) proposed the use of piezoelectric crystalas an immunosensor for generic E. coli. This methodinvolved separation of one of the electrodes from thesensor, and the change of resonant frequency of the crystalwas only due to the change of chemical composition of themedium between the separated electrode and the crystal.The frequency detection time (100–500min) was inversely

dependent with initial concentration (10 to 106 cells/ml) ofE. coli. However, this approach was neither specific forE. coli strain, nor even for general E. coli. Generallyspeaking, the method was based on a change in con-ductivity and impedance of the medium, which thencontributed to the shift of resonant frequency.Plomer et al. (1992) utilized a piezoelectric device for

E. coli K12 detection, with an immobilized antibody on thecrystal sensor against the enterobacterial common antigen(ECA). Antibody binding to the crystal surface wasachieved by three different methods — via polyethylenei-mine, via silianization, and via protein A-gold. Referencecrystal was used to measure non-specific binding forfurther subtraction. The sensor was capable of detecting106 to 109 cells/ml in 40min. Reusability of the crystalwithout damaging antibody layer was impossible; there-fore, for each new determination, a new coat of antibodywas applied.Su and Li (2004) proposed a monolayer-based piezo-

electric immunosensor for E. coli O157 detection. Anti-bodies were attached to the surface of quartz crystal viaeither protein-A or NHS ester (derived from 16-mercapto-hexadecanoic acid). Detection limit for heat-killed patho-gen was 103 cells/ml using phosphate buffered saline as adiluent. Time to detection ranged from 30 to 50min.

3.3. Impedance-based immunosensors

These immunosensors consist of an electrode withattached antibodies. After binding of bacterial cells to thesurface of the electrode while being immersed in the testedsample, detectable changes in the electrochemical proper-ties of the electrode occur due to bacterial monolayerpresent on its surface. Radke and Alocilja (2005) developeda microelectrode array immunosensor for E. coli O157using polyclonal antibodies attached to the surface of anelectrode. The biosensor was sensitive to viable E. coli

O157 in peptone water at 104 cells/ml. The same immuno-sensor had detection limit of only 107 cells/ml in romainelettuce wash fluid. Yang et al. (2004) proposed anelectrochemical impedance microelectrode-immunosensorusing a redox chemical probe added to the test solution.Detection limit for heat-treated E. coli O157 diluted inphosphate-buffered saline was 106 cells/ml.

4. Immunosensors with indirect measurement

Labeled antibodies are more often used for immunosen-sor development as a biological recognition mechanism.Four types of antibody labels have been used for E. coli

O157:H7 detection — fluorophore, enzyme, luminescentcompound, and conductive polymer. Measurements weredone using fluorescence, chemiluminescence (with inter-mediate chemiluminescent compounds), or electrochemicalproperties (with intermediate electroactive compounds).

ARTICLE IN PRESSO. Tokarskyy, D.L. Marshall / Food Microbiology 25 (2008) 1–12 5

4.1. Fluorescent label

Czajka and Batt (1996) utilized a simple solid-phasefluorescent capillary immunoassay to detect E. coli

O157:H7. The principle used was antigen down competi-tion immunoassay. The proposed method had a limit ofdetection of 1 cell per 10 g of ground beef after selectiveenrichment for 7 h at 371C in m-EC broth. The minimaldetectable level in apple cider samples was around 0.5 cells/ml. In this food sample, E. coli cells were first captured withimmunomagnetic beads before incubation in selectivebroth.

Yu et al. (2002) coupled immunomagnetic separationwith time-resolved fluorometry for the detection of E. coli

O157:H7. Immunomagnetic separation, using immuno-magnetic beads (Dynal) specific to O157, is a highlyinnovative and effective technique for bacterial pre-concentration (Okrend et al., 1992) and is currentlyapproved (USDA-FSIS, 2002) as a pre-concentrationmethod for E. coli O157:H7 from enrichment broth offood samples. The procedure developed by Yu et al. (2002)included trapping bacterial cells on immunobeads andseparation of immunobeads with the addition of a secondantibody labeled with a fluorescent compound. Thesensitivity of the assay was 103 cells/ml directly. Whenapple cider was spiked with E. coli O157:H7 and enrichedin selective broth for 4 h, the detection limit was 10 cells/mlof initial contamination. Under the same conditions, butwith additional initial contamination with 106 cells/ml ofE. coli K12, the detection limit of E. coli O157:H7increased to 102 cells/ml of initial contamination. Thiswas explained by the reduction of pathogen recoveryduring enrichment in the presence of E. coli K12.

DeMarco et al. (1999) utilized a fiber-optic evanescentbiosensor with a sandwich immunoassay. In their method,signal was detected by measuring fluorescence generated bya second labeled antibody. This immunosensor detected3–3� 101 cells/ml of E. coli O157:H7 in spiked ground beefwithin 20min without enrichment. The influence ofnaturally occurring microflora and intentional bacterialcontaminants were negligible. Later investigations by thesame researchers (DeMarco and Lim, 2002) revealed thatin real-life bulk measurements with specific signal break-point stringency and dilution scheme to avoid interferencewith food matrix, sensitivity with 100% correct identifica-tion decreased to 9� 103 cells/g for 25-g ground beefsample and to 5.2� 102 cells/g for 10-g ground beefsample.

Ho et al. (2004) proposed an immunosensor based onflow injection liposome immunoanalysis. This systeminvolved a sandwich assay on the surface of a fused-silica microcapillary with second antibody being taggedwith liposomes encapsulating a fluorescent dye. Uponlysis of liposomes in the formed sandwich, the dye wasreleased and fluorescence measured. As claimed by theauthors, the detection limit for heat-killed E. coli O157 was3.6� 102 cells/ml.

4.2. Enzyme label

Enzyme labeled antibodies are widely used amplificationtools for E. coli O157:H7 identification. In most cases, thislabel works indirectly by changing its substrate to product(fluorescent, chemiluminescent, electroactive compound),prior to detection by a sensor.Paffard et al. (1997) developed an enhanced immunoassay

for E. coli K12 detection. Bacteria were entrapped on amembrane by filtration through a 0.45mm nitrocellulosemembrane followed by bovine serum albumin masking ofthe unreacted membrane surface. This assay involvedspecific rabbit anti-E. coli antibody binding to entrappedbacterial cells. Amplification was achieved by biotinylatedgoat anti-rabbit IgG with final attachment of avidinconjugate with horseradish peroxidase. At this point, achemiluminescent test based on peroxidase activity wasperformed. E. coli K12 cells were cultivated alive and wereresuspended in phosphate-buffered saline before entrapping.The lowest detectable level was 6� 101 cells/ml within 1 h.A chemiluminescence multichannel immunosensor for

E. coli O157:H7 was developed by Yacoub-George et al.(2002). This device used capillary sandwich ELISAcombined with a miniaturized fluidics system. The secondantibody was conjugated with horseradish peroxidase. Thelowest detectable limit for E. coli O157:H7 was 105 cells/mlafter an analysis time of 24min.Gehring et al. (1998) developed a light-addressable

potentiometric sensor for detection of E. coli O157:H7.The sensor consists of a silicon sensor and a pH-sensitiveinsulating layer that is placed in contact with an aqueoussolution in which the immunological reaction takes place.Changes in pH associated with enzyme reactions aretransformed into potential change. Thus, photocurrentcan be measured on the discrete zones where the sensor isilluminated. In this method, a double-sandwiched immu-nocomplex of bacterial cells was filtered through a 0.45-mmmembrane with biotinylated bovine serum albumin forfixation on the membrane. After washing, the membranewas touched by a pH-sensitive insulator with urea insolution. Enzyme catalyzed changes in pH were measuredand correlated with bacterial concentration. Affinitypurified anti-E. coli O157:H7 antibodies raised againstthe heat-killed pathogen were used for the assay. Interest-ingly, the sensor was sensitive towards both heat-killed andlive E. coli O157:H7, with higher specificity for heat-killedcells. The limit of detection was 7.1� 102 cells/ml for heat-killed pathogen and 2.5� 104 cells/ml for live E. coli

O157:H7, with an assay time of 45 and 30min, respectively.Three possibilities may explain the detection limit differ-ence: (1) shorter exposure time for binding of live E. coli toantibodies; (2) enhanced binding of heat-killed E. coli dueto release of highly immunogenic fragments; and (3) higherreactivity towards heat-killed E. coli due to the fact thatantibodies were raised against heat-killed cells.Enzyme labels also are used in amperometric detection.

This method measures current that is generated by oxidation

ARTICLE IN PRESSO. Tokarskyy, D.L. Marshall / Food Microbiology 25 (2008) 1–126

or reduction of electrochemically active compounds on thesurface of an electrode. Antibody labels are usuallyoxireductases or hydrolytic enzymes, which yield electro-active compounds as products of reaction with mediators(Marco and Barcelo, 1996). Amperometric sensors withoxireductase as a label may have low selectivity, especiallyfor oxygen/hydrogen peroxide detection. Low selectivity isdue to interference with electroactive contaminants; there-fore, application of indirect mediators is highly desirable.Filtration/immunofiltration capture or immunomagneticbead separation can increase the selectivity of amperometricbiosensors. In addition, the flow injection techniqueincreases the potential for assay automation (Leonardet al., 2003).

Perez et al. (1998) combined immunomagnetic separa-tion with amperometric flow injection analysis for detec-tion of viable E. coli O157. Immunobeads with trappedE. coli were incubated with mediators (potassium hex-acyanoferrate (III) and 2, 6-dichlorophenolindophenol) fordetermination of E. coli respiration response. Next,immunobeads were separated and supernatant was injectedinto an electrochemical cell with working and referenceelectrodes. The heights of amperometric current peaks wereplotted against initial E. coli concentration. Possibleinterferences were not studied. Dilutions of bacteria weremade in phosphate buffered-saline with KCl. The detectionlimit was 105 cells/ml for a two-hour assay, but additionalpre-enrichment in broth is required for food samples,which may affect the detection limit.

Abdel-Hamid et al. (1999) developed a flow-throughimmunofiltration assay system for the detection of E. coli

O157:H7. A peroxidase-labeled antibody in a sandwichassay that was combined with an amperometric sensor wasutilized in this study. Iodide ions served as mediators,which reacted with hydrogen peroxide to form iodine. Thereaction was catalyzed by captured peroxidase in animmunocomplex on the filter. Iodine served as theelectroactive compound. The lowest detectable limit was102 cells/ml with an assay time of 30min, which was 1000times more sensitive than a classical ELISA test with thesame immunochemicals. However, dead cells were utilizedin this study for safety reasons.

Another assay format proposed by Abdel-Hamid et al.(1998) used a novel amperometric immunoelectrode, whichacted as both a working electrode and a surface for theimmunological reaction. A sandwich immunoassay wasfixed on the surface of a transducer and the secondantibody was labeled with peroxidase. The mediator for theenzymatic reaction was 5-aminosalicylic acid. Achievedworking concentration range was 2� 102–7� 103 cells/mlfor a total assay time of 40min. The electrode was partiallyimmersed in solution to accelerate the rate of immunolo-gical, enzymatic, and electrochemical reactions. Non-viableE. coli O157:H7 were used for safety reasons.

Brewster and Mazenko (1998) used filtration capturecombined with electrochemical E. coli O157:H7 detection.Cells were prepared by binding to phosphatase-labeled

antibodies and captured on a membrane. The capturedenzyme catalyzed the degradation of the substrate to anelectroactive product, which produced an electric current atthe measuring electrode. Bacterial cells were killed byirradiation and dilutions were prepared in phosphate-buffered saline. Assay time was 25min with a detectionlimit of 5� 103 cells/ml.Ruan et al. (2002) developed an immunosensor for

E. coli O157:H7 detection based on a sandwich assay onindium tin oxide electrode chips. The second antibody waslabeled with alkaline phosphatase. During enzymaticallycatalyzed reactions, precipitation of product occurred. Theprecipitate led to a change in electrode resistance that wasmeasured and correlated with concentration of presentantigen. Detection limit of heat-killed E. coli O157:H7 was6� 103 cells/ml.

4.3. Luminescent label

Yu and Bruno (1996) proposed an immunomagnetic-electrochemiluminiscent method for detection of E. coli

O157:H7. This assay was based on immunomagneticseparation of heat-killed bacteria on immunobeads thatwere pre-coated with goat polyclonal anti-E. coli O157antibody. Upon capture of heat-killed bacteria from serialdilutions or artificially contaminated food samples, theimmunobeads were washed, resuspended in phosphate-buffered saline, and the second polyclonal anti-E. coli O157antibody labeled with Ru(bpy)3

2+ was sandwiched. Afterwashing unbound labeled antibodies, the suspension ofbeads was captured on a magnetized anode where anelectrochemical reaction took place with generation of asubstance capable of reacting with tripropylamine resultingin light emission. Detection limit was 102 cells/ml in pristinebuffer and 103–2� 103 cells/ml in artificially contaminatedfood products with a total assay time of 1 h. The assay wasaccomplished in milk, juices, supernatant fluids fromground beef, finely minced chicken, and fish suspensions.Cross-reactivity of the anti-E. coli O157:H7 antiserum waslow when tested against several different E. coli strains.

4.4. Conductometric label

Muhammad-Tahir and Alocilja (2003) developed aconductometric immunosensor for the detection of E. coli

O157:H7. Their system involves an electrochemical sand-wich assay, such that the first antibody is trapped in thespace between two electrodes. An antigen with a secondantibody conjugated to a conducting polymer is trappedthrough its flow through the space between the electrodes.A conductive bridge is then monitored as an increase inconductance. The detection limit was approximately7.9� 101 cells/ml in pure culture with a ten-minute assay.Live E. coli O157:H7 were serially diluted in 0.1% peptonesolution before application to the sensor. The responses ofthe biosensor to the pathogen were significantly differentbetween control and any level of contamination above the

ARTICLE IN PRESSO. Tokarskyy, D.L. Marshall / Food Microbiology 25 (2008) 1–12 7

lowest detection limit; however, there was no statisticaldifference from one level to another. Analysis of a mixtureof five microorganisms (two strains of E. coli O157:H7,non-pathogenic E. coli, Salmonella enterica serovarThompson, and Salmonella enterica serovar Typhimurium)showed that this immunosensor was specific towardsredundant E. coli O157:H7. The researchers used a testvolume of 0.1ml for analysis, which implied detection of aslow as eight cells in the sample. Such low detection limit, inour viewpoint, is questionable.

The same research group also utilized an immunosensorbased on the same technology to analyze artificiallycontaminated produce (Muhammad-Tahir and Alocija,2004). However, they pre-concentrated samples by filtra-tion of peptone water used to release cells from thecontaminated produce. The filter was later vortexed in asmall volume of peptone water. Standard plate count wasused as an alternative procedure. The investigators claimeda detection limit as low as 8.1� 101 cells/ml.

5. Application of immunosensors for examination of raw and

ready-to-eat meats

A summary of the reviewed E. coli O157:H7 immuno-sensors/immunoassays is presented in Table 1. Sensitivity,assay time, methods employed in immunosensor function-ing, compliance with immunosensor definition, and severalother characteristics are included for each immunosensordescription. These characteristics include number of anti-bodies involved, type of label for indirect measurements,pre-concentration step (if any), assay diluents, and state ofpathogen. If applicable, cross-reactivity with other bacter-ial species and direct measurements of contaminated foodproducts also are given. Hereafter, we discuss the potentialof these immunosensors as potential tools to help the meatprocessing industry deal with E. coli O157:H7.

As was shown in the USDA-FSIS approved method(USDA-FSIS, 2002), initial enrichment is necessary torecover the pathogen and increase its population size.However, the predominant selling point of immunosensorsis direct measurement without enrichment. Enrichment inselective broth is an essential step, since sensitivity of thecurrent USDA-FSIS approved method for raw meat andcooked ready-to-eat meat products is much greater thanthe detection limits of immunosensors (Table 1). DeMarcoet al. (1999) noted that analysis of a 25-g ground beefsample after homogenization with 225ml of buffer couldcreate problems in the detection of low numbers ofbacteria. In their study, 1ml of liquid sample was usedfor analysis. Thus, the probability of capturing the fewbacteria present in a 225ml sample in a 1ml subsample islow. Methods that included simple filtration capture orimmunofiltration/active filtration (Paffard et al., 1997;Brewster and Mazenko, 1998; Gehring et al., 1998;Abdel-Hamid et al., 1999) also utilized small sample sizesfor analysis, ranging from 0.1 to 1ml. Therefore, combina-

tion of enrichment broths with biosensors might reduce theincidence of false-negative results.In most cases, positive results obtained with a biosensor

are only presumptive, because polyclonal antibodies aregenerally effective in binding both live and dead bacteriaand may cross-react with other bacterial species (DeMarcoand Lim, 2002; Liu and Li, 2002). This is supported by thefact that commercially available inexpensive antibodies areraised against an inactivated pathogen through injectioninto an animal. Therefore, epitopes of mortalized pathogensare determinants for antibody specificity. In one study(Gehring et al., 1998), which used both live and deadbacteria, a higher affinity of polyclonal antibody was foundto dead bacteria. DeMarco and Lim (2002) suggested thatE. coli O157:H7 polyclonal antiserum could easily cross-react with non-O157 microorganisms, including Escherichia

hermanii, Brucella melitensis, and Brucella abortis. There-fore, false-positive results may occur by polyclonal antibodybinding to non-target microorganisms or non-viable targetmicroorganisms. For example, three false-positive resultswere found during analysis of fifteen non-contaminated 25-gbeef samples. (DeMarco and Lim, 2002).According to standard procedure to detect E. coli

O157:H7 (USDA-FSIS, 2002), commercial rapid test kitsare used after enrichment of food samples for 20–24 h inmodified EC broth with novobiocin (mEC+n broth).Positive results are presented as presumptive positives, butnegative samples are considered to be negative anddiscarded after rapid screening. In food analysis, rapidmethods for direct bacterial detection in foods can beinaccurate and enrichment in specific media is necessary.Moreover, only viable cells of E. coli O157:H7 canproliferate in m-EC+n broth (Okrend et al., 1990), whilethe growth of other bacteria is partially suppressed. Inorder to prove viability of bacteria and therefore to confirmpresence of E. coli O157:H7, USDA-FSIS requirespresence of visible colonies on very specific media (Rain-bow agar, Blood agar). In addition, final serological (O157and H7 antigens), biochemical, and Shiga toxin confirma-tion of the colonies is necessary.Immunosensor results also depend on the type of antibody

used. Most immunomagnetic separation techniques usebeads coated with an anti O157 antibody. Possession of thisantigen is not the only requirement for pathogenicity ofenterohemorrhagic strains of E. coli. Monoclonal antibodiesare specific, but only to one antigen that may not be a crucialdisease-causing virulence factor. Vernozy-Rozand et al.(1998) compared the efficiency of an automated enzyme-linked fluorescence immunoassay (ELFA VIDAS method)with immunomagnetic separation followed by conventionalplating. They found that the ELFA VIDAS method detectedseventeen positive samples from 496 retail food samples.Among these positives, the VIDAS immunoconcentrationsystem (VIDAS ICE) confirmed nine samples. However,eight of them were sorbitol-positive, O157-positive, H7-negative, motile, nonverotoxin-producing E. coli and onesorbitol-negative nonverotoxin-producing E. coli. In this

ARTICLE IN PRESS

Table 1

Summary of immunosensors for E. coli O157:H7 detection

Method Group Detection

limit,

cells/ml

Assay

time,

min

Label Number

of

antibodies

involved

Pre-

concentration

State of

pathogen

Dilution

diluent

Interferences with other

bacterial species

Direct application

foods, detection limit

SPR IS 5–7� 107 10 NA 2 NA V HBS NCR: S. typhimurium, S.

enterocolitica

NT

PS,

E.coli

K12

IS 106 - NA 1 NA V PS NT NT

PS IS 103 30–50 NA 1 NA D PBS NCR: S. typhimurium NT

EI IS 106 - NA 1 N/A D PS NT NT

EI IS 104 10 NA 1 NA V PW NCR: S. infantis 107 cfu/ml romaine

lettuce wash

SPFCI IA - 10 +

enr

FC 1 m-EC broth;

+ IMS for

cider

V PS NT 1 cfu/10 g beef; 0.5 cfu/

ml apple cider

FIA-

SPFCI

IA-IS 3.6� 102 45 FC 2 N/A D - NT NT

TRF IA 103 120

+

enr

FC 2 IMS,+ m-

EC broth for

cider

V AS NCR: C. freundii, E. coli

O9:K99, S. choleraesuis, S.

typhimurium,Y.

enterocolitica

10–102 cfu/ml apple

cider

EWFB IA-IS – 20 FC 2 NA V PBS NT 3–30 cfu/g beef; 9.0 �

103cfu/g in 25 g beef;

5.2 � 102 cfu/g in 10 g

beef

CL,

E.coli

K2

IA �6x101 �60 EZ 3 F V PBS NT NT

CL IA-IS 105 24 EZ 2 NA - - NT NT

LAPS IS 2.5� 104

7.1� 10230 EZ 3 IMF VD TBS

with

BSA

NCR: non-pathogenic

E.coli, S. typhimurium

NT

A, FIA A 105 120 NA 1 IMS V PBS

with

KCl

NT NT

A, FIA A 102 30 EZ 2 IMF D PBS NT NT

A IS 2� 102 40 EZ 2 N/A D AB with

BSA

NT NT

ECL IA 102 o60 CLC 2 IMS D PB NCR: non-pathogenic E.coli 103 – 2� 103 cells/ml

A A 5 � 103 25 EZ 1 F D TBS NT NT

EI IS 6 � 103 - EZ 2 NA D TBS NT NT

C IS 7.9� 101 10 CP 2 NA D 0.1 %

PW

NCR: non-pathogenic

E.coli, S. enterica

NT

C IS - 6 CP 2 F V 0.1 %

PW

NCR: non-pathogenic E.

coli

8.1� 101 cfu/ml lettuce,

alfalfa strawberry wash

Method: SPR, – surface plasmon resonance; PS,-piezoelectric sensor; CL, – chemiluminescence; ECL,-electrochemiluminescence; SPFCI,-solid-phase

fluorescent capillary immunoassay; TRF, – time-resolved fluorometry; EWFB, – evanescent-wave fluorescence biosensor; LAPS, – light addressable

potentiometric sensor; C, – conductometry; A, – amperometric; FIA, – flow-injection analysis; EI,-electrochemical impedance.

Group: IS, – ‘‘strict’’ immunosensor. Immunological reaction is carried out on the surface of the transducer, which transforms recognition at its surface

into measurable signals; A, – amperometric sensor with antibody-based technique involved; IA, – immunoassay; IA-IS, – immunoassay called

immunosensor by authors.

Labels: FC, – fluorescent compound; EZ, – enzyme; CP, – conducting polymer; CLC, – chemiluminescent compound.

State of pathogen: V, – viable; D,-dead.

Pre-concentration: IMS, – immunomagnetic separation; IMF, – immunofiltration or other active filtration; F, – filtration capture.

Diluent: PS,-physiological saline; PBS, – phosphate-buffered saline; BSA,-bovine serum albumin; KCl, – potassium chloride; PW, – peptone water; PB, –

pristine buffer; AB, – acetate buffer; TBS, – Tris-buffered saline; HBF, – HEPES-buffered saline; AS, – assay buffer containing Tris-HCl, NaCl, NaN3,

BSA, bovine gamma globulins, Tween 40 and diethylenetriamine-penta-acetic acid.

General: NCR,-no cross-reactivity; NT, – not tested; NA, – not applied;

O. Tokarskyy, D.L. Marshall / Food Microbiology 25 (2008) 1–128

study, ELFA VIDAS method was applied after 6-h selectiveenrichment in broth and 100% positive samples wereobtained with an initial artificial contamination of 8 cellsper 25 g of ground beef.

Given these observations, immunosensors, which arebased on the same principles as rapid ELISA test kits, mayhave limited use for confirming the absence of bacteria infood samples after selective enrichment.

ARTICLE IN PRESSO. Tokarskyy, D.L. Marshall / Food Microbiology 25 (2008) 1–12 9

Most of the immunosensors are not tested with real foodsamples. Use of real samples may significantly decreasesensitivity. In this case, direct detection of bacteria in foodbecomes even more questionable. Dilution during enrich-ment in broth may decrease concentration of interferingsubstances that are present in food matrices. In addition,use of further dilutions, such as physiological saline,phosphate-buffered saline, and tris-buffered saline, hasbeen reported (Table 1). In the absence of food biomole-cules in bacterial stock cultures and dilutions, possibleinterferences of macromolecules is rarely analyzed. Onecan imagine the complexity of a food matrix in terms ofchemical composition and the presence of interferingbacteria. In techniques utilizing filtration with membranepore size ranging from 0.2 to 3.0 mm, applying large samplesize becomes difficult because of noise signal increase dueto non-specific binding and presence of interfering sub-stances. To illustrate this concern, Brewster and Mazenko(1998) showed a detection limit of 500 cells when a 0.1mlsample size was used. With a 0.01ml sample size, less than50 cells could be detected. In this case, greater backgroundimmunoelectrochemical response was seen with the largersample size. Yu and Bruno (1996) tested their immuno-sensor using food samples and found the detection limit ofE. coli in milk was greater than in other foods. This wasrelated to inactivation of immunobeads in milk. They alsofound that interfering background luminescence of fishsamples gave false-positive results. Their detection limitwas 102 cells/ml in pristine buffer versus 103 to 2� 103 cells/mlin artificially contaminated food. It stands to reason thatdilution of food products or enrichment broth can decreasethe concentration of interfering substances. If the immu-nosensor is sensitive enough, a ten-fold decrease inconcentration may still result in an acceptable detectionlimit while diminishing the impact of food matrix or broth.DeMarco and Lim (2002) showed that a change from 25-gbeef sample:25ml buffer dilution to 10-g beef sample:40mlbuffer dilution decreased both the number of false-positiveand false-negative results.

Another point noted by DeMarco and Lim (2002) wasthat during artificial contamination of ground beef,bacteria are not firmly attached to the meat surface andare easily removed during mixing with diluent. In the caseof natural contamination, bacteria are trapped within thefood matrix and are firmly attached to food particles,making more vigorous actions such as homogenization orstomaching required for their release. These vigorousactions may result in homogenates with greater concentra-tions of interfering substances (DeMarco and Lim, 2002).

Immunosensors based on amperometric detection mighthave low sensitivity in food samples with regard to directmeasurements. For example, amperometric methods withperoxidase enzyme label may be influenced by the enzymescatalase and peroxidase, which are present in viablerespiring bacteria and some food tissues. Interferingelectroactive substances that are naturally present in foodscan compete with mediators. However, intensive washing

of transducers after sample application may decrease thisbackground level. It is possible to argue that enrichment inbroth as a tool for dilution of food matrix and bacterialnumber increase might be more effective. Amperometricflow-injection analysis that was proposed by Perez et al.(1998) for detection of viable E. coli O157:H7 with initialimmunomagnetic separation might be insufficient forbacterial mixtures. In this study, the mediator, whichmeasures respiratory activity of E. coli O157:H7, is generaland can measure the respiratory activity of all otherbacteria as well. Immunomagnetic separation depends onthe quality of the antibody that is attached to the beads.Okrend et al. (1992) demonstrated that immunobeads arecapable of adsorbing a high number of non-specificmicroorganisms that are respiring as much as the targetbacterium, E. coli. Therefore, background bacteria fromfoods that are in enrichment broth may make thedetermination of E. coli O157 difficult.Cross-reactivity with other bacterial species was not

tested for many immunosensors (Table 1). Therefore, moreinformation is needed pertaining to cross-reactions thatoccur both individually with interfering species and duringanalysis of mixed bacterial cultures. This informationwould take into consideration the natural contaminationof raw food products including meats (Jackson et al., 2001;Marshall and Bal’a, 2001). The problem of cross-reactionsmay be reduced through use of more specific antibodies,such as monoclonal or recombinant antibodies (Fitzpatricket al., 2000).There is a need for immunosensors that are more

sensitive than commercial rapid test kits and offer shorterenrichment time for determination of uncontaminatedsamples. If such devices are available, time and moneysavings for industry may be realized. Some investigators(Abdel-Hamid et al., 1998) have demonstrated that theirimmunosensor was 1000 times more sensitive than classicalELISA when the same reagents were utilized. Anotherpoint to consider is the fact that detection time usingimmunosensors can be less than that of ELISA assays,which require long assay times and more manipulations(Liu and Li, 2002). However, new immunoprecipitationtechniques are characterized by both short time (within10min) and simplicity combined with acceptable detectionlimit (Giese, 2003). For the reviewed immunosensors, someassumptions about the time needed for enrichment beforedetection are shown in Table 1 (column for immunosensordetection limit). Okrend et al. (1990) showed increasednumbers of E. coli O157:H7 from 1.6� 101 cells/ml to2.8� 108 cells/ml at 351C within 18 h in mEC+n broth, orfrom 0.5 cell/ml to 1.9� 102 cells/ml within 6 h at 371C. Itis helpful to keep in mind that the zero tolerance definitionfor E. coli O157:H7 has a detection limit of 1 cfu per 65 g ofsample (USDA-FSIS, 2002).Problems with immunosensors calibration with known

bacterial populations generally are not of concern. In thecase of E. coli O157:H7, one needs to know if pathogen ispresent or absent from the sample. Thus, only breakpoint

ARTICLE IN PRESSO. Tokarskyy, D.L. Marshall / Food Microbiology 25 (2008) 1–1210

of detection is important. Moreover, with low initialbacterial contamination levels after selective enrichmentin broth, it is difficult to establish initial concentration.Variability of responses among immunosensors that aremanufactured under the same conditions also can be aproblem (DeMarco et al., 1999). Therefore, positive andnegative controls are still needed for breakpoint verifica-tion.

In order to increase sensitivity and selectivity ofimmunosensors, several well-known tips are helpful:(1) Use a sandwich format with more than one antibodyto increase selectivity and avoid non-specific binding. Theorder of antibody binding with different specificity alsomay influence results (Fratamico et al., 1998). (2) Uselabeled antibodies to amplify signal. As shown in Table 1,direct methods are less sensitive. (3) Use the most specificantibody available. In the case of a labeled antibody, theantigen determinants present on the surface of bacteriashould be in high abundance so that the label can providemaximum amplification of the signal. (4) Optimizeconcentrations of antibodies. (5) Pre-concentrate sampleby applying immunomagnetic separation or immunofiltra-tion. (6) Use a sensitive type of transducer. For example,spectrophotometric assays are generally less sensitive thanfluorescent assays.

6. Comparison of immunosensors with other rapid test kits

for E. coli O157:H7 detection

There are three major types of rapid methods, which arebased on biochemical patterns, DNA sequence, or anti-body interactions (Feng, 2001). For the purpose of thisreview, we compare immunosensor performance with otherrapid methods for E. coli O157 immunological detection.According to FDA (Feng, 2001), immunological methodsconstitute the largest group of rapid test kits commerciallyavailable for food testing. Feng (1997) grouped antibody-based methods into five major divisions: latex agglutina-tion/reverse passive latex agglutination, immunodiffusiontest format, ELISA, immunomagnetic separation, andimmunoprecipitation. According to Boer and Beumer(1999), detection limit for immunological methods isapproximately 105 cfu/ml. The variety of commerciallyavailable immunological rapid test kits are well documen-ted (Feng, 2001, Fung, 2002), but the most promising rapidchecking of enrichment cultures are ELISA, immunopre-cipitation, and visual immunoassays. Given earlier ob-servations about possible application of immunosensorsonly after selective enrichment, their comparison with thesetechniques might be of interest.

As proposed by Boer and Beumer (1999), a ‘‘rule ofthumb’’ says that detection limit of classical ELISAmethod lies in the range of 103–105 cfu/ml. Immunopreci-pitation, or Lateral Flow Assay, is also based on the‘‘sandwich’’ procedure (Giese, 2003). According to theauthor, these assays are extremely easy, with result beingread visually within 10min without washing or other

manipulations. RapidChecks Lateral Flow Test Kit isdesigned to detect viable E. coli O157 in raw beef afterselective enrichment for 8–18 h with sensitivity of 1 cfu/25 gprior enrichment (Performance Tested AOAC, LicenseNumber 070201). As stated by the manufacturer, detectionlimit in enrichment broth is 104 cfu/ml. According toSinglepaths E. coli O157 rapid Lateral Flow Assaymanufacturer (Performance Tested AOAC, License Number010407), detection limit of their method is 104–106 cfu/mldepending on serological characteristics of the pathogen.Their procedure involves 18–24 h enrichment with 20minactual assay time.Boer and Beumer (1999) noted 10 factors influencing

advantages of rapid method use, and all of them can befound in currently proposed antibody-based rapid test kits.Accuracy, which is judged by sensitivity and specificity, is acurrent control for performance check of rapid methods tobe approved for standard USDA-FSIS E. coli O157method (USDA-FSIS, 2002). Accuracy of most commer-cially available rapid immunological test kits is validatedagainst standard methods and evaluated by collaborativestudies (AOAC performance method). As of this writing,we are not aware that any of the reviewed immunosensorshave had accuracy performance validated. As a majorfactor for rapid method use, time to detection is excellentfor immunoprecipitation methods. In most cases, immu-nosensor detection times are longer, with some proceduresthat include washing/binding steps. As a general rule(Su and Li, 2004), immunosensors can have decreasedassay times compared to corresponding ELISA tests;however, lateral flow assays may be even faster and easier.Automation and computerization of testing are added

benefits of some test formats. Microtiter plate basedmethods, such as TECRA VIATM, allow for simultaneousdetermination of 46 or 94 samples. Results are availableafter 1.5 h following an 18-h enrichment. The VIDASsystem (bioMerieux) is a fully automated ELIFA (similarto ELISA but uses fluorescence) with no need for manualprocedures. It gives result within 45min to 2 h. Simplicityand reagent availability are also pluses for commercialrapid assay kits. Most immunosensors require complexequipment, laborious preparation of transducer by im-mobilization of antibodies, and long-lasting regenerationof the transducer surface. Immunosensors generally requireinvestment in specialized equipment, while immunoassaysmight not require any at all (visual immunoassay, VIA)and can be performed in minimally equipped laboratories.As for detection limits, immunosensors remain unim-

pressive (Table 1). Taking into account the fact thatenrichment is a prerequisite step we believe immunosensorsremain outside the field of potential application comparedto regular immunoassays. Moreover, the literature containsabundant misuse of the term ‘‘immunosensor’’, whereimmunoassays are being called immunosensors, presum-ably to add panache to the paper. In future work, it wouldbe of value to directly compare biosensor performance tothat of commercially available assays.

ARTICLE IN PRESSO. Tokarskyy, D.L. Marshall / Food Microbiology 25 (2008) 1–12 11

7. Recommendations

The field of immunosensors for detection of E. coli

O157:H7 is still evolving. Presently, the advantages ofimmunosensors are not compelling other than for identi-fication of uncontaminated food samples after enrichmentin selective broth. In this case, they might serve as asubstitute for rapid antibody-based screening tests asproposed by USDA-FSIS. If immunosensor sensitivity isbetter than that of the currently available immunologicalmethods, it may be a valuable tool to decrease bothenrichment procedure and detection times, therefore,saving time and money. Major advantages of immunosen-sor use may be high sensitivity, short assay time,automation, and absence of manual procedures. Resultsare straightforward and might eliminate human biases.However, these features are currently present in rapidimmunological test kits. The major shortcoming of thistechnology is the cost of expensive equipment, which todate does not significantly contribute to decreased detec-tion limits. The current state of knowledge on immuno-sensors, which does not contain sufficient information onthe influence of food matrix and the influence of cross-reactivity with other bacterial species, is not sufficient forus to recommend immediate use of immunosensors.Methods for improvement of immunosensor sensitivityand selectivity are described and well known, so imple-mentation of these current trends may further facilitatefuture use of immunosensors for E. coli O157:H7 detectionby the food industry.

Acknowledgements

Approved for publication as Journal Article No. J-10640of the Mississippi Agricultural and Forestry ExperimentStation. This work was supported in part by a USDA-ARSFood Safety Cooperative Agreement (58-6202-5-083) andby the Mississippi Agricultural and Forestry ExperimentStation under project MIS-371071.

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