REVIEWARTICLE

Recent developments in rapid multiplexed bioanalyticalmethods for foodborne pathogenic bacteria detection

Aldo Roda & Mara Mirasoli & Barbara Roda &

Francesca Bonvicini & Carolina Colliva &

Pierluigi Reschiglian

Received: 1 March 2012 /Accepted: 5 April 2012 /Published online: 22 May 2012# Springer-Verlag 2012

Abstract Foodborne illnesses caused by pathogenic bacte-ria represent a widespread and growing problem to publichealth, and there is an obvious need for rapid detection offood pathogens. Traditional culture-based techniques re-quire tedious sample workup and are time-consuming. It isexpected that new and more rapid methods can replacecurrent techniques. To enable large scale screening proce-dures, new multiplex analytical formats are being devel-oped, and these allow the detection and/or identification ofmore than one pathogen in a single analytical run, thuscutting assay times and costs. We review here recent

advancements in the field of rapid multiplex analyticalmethods for foodborne pathogenic bacteria. A variety ofstrategies, such as multiplex polymerase chain reactionassays, microarray- or multichannel-based immunoassays,biosensors, and fingerprint-based approaches (such as massspectrometry, electronic nose, or vibrational spectroscopicanalysis of whole bacterial cells), have been explored. Inaddition, various technological solutions have been adoptedto improve detectability and to eliminate interferences, al-though in most cases a brief pre-enrichment step is stillrequired. This review also covers the progress, limitationsand future challenges of these approaches and emphasizesthe advantages of new separative techniques to selectivelyfractionate bacteria, thus increasing multiplexing capabili-ties and simplifying sample preparation procedures.

Keywords Food safety . Pathogenic bacteria . Multiplexassays . Rapid assays . Biosensors

Introduction

Foodborne illnesses caused by microorganisms, such asbacteria, viruses, parasites, algae and fungi and related tox-ins, are a large, widespread and growing public healthproblem. Although food safety practices are being improveddue to severe regulatory actions, there remains a growingneed for enhanced rapid tools for food pathogen detection.The recent Escherichia coli Shiga toxin-producing O104:H42011 outbreaks in Europe are only one example, demon-strating the difficulties in promptly detecting contaminationsand avoiding their spread [1].

Traditional culture-based techniques are time-consuming(5–7 days) and labor-intensive, thus new rapid analyticalmethods are hot research topics [2–6]. To fulfill the

The work was presented at the workshop of Biosensors for food safetyand environmental monitoring, Ouarzazate, Morocco (October 06–08,2011).

A. Roda (*) :M. Mirasoli : C. CollivaAnalytical and Bioanalytical Chemistry Laboratory,Department of Pharmaceutical Sciences, University of Bologna,Via Belmeloro 6,40126 Bologna, Italye-mail: aldo.roda@unibo.itURL: http://www.anchem.unibo.it

A. Roda :M. MirasoliNational Institute of Biostructure and Biosystems, N.I.B.B,Interuniversity Consortium,Viale medaglie d’Oro 305,00136 Rome, Italy

B. Roda : P. ReschiglianDepartment of Chemistry “G. Ciamician”, University of Bologna,Via Selmi 2,40126 Bologna, Italy

F. BonviciniMicrobiology Section, Department of Haematology andOncological Sciences “L. e A. Seragnoli”, University of Bologna,Via Massarenti 9,40138 Bologna, Italy

Microchim Acta (2012) 178:7–28DOI 10.1007/s00604-012-0824-3

regulatory requirements of “absence of pathogen”, suchmethods should be characterized by high detectability andspecificity, providing the ability to exclude the presence ofeven one pathogenic microorganism in the sample, even inthe presence of a large number of other non-pathogenicbacteria.

The development of rapid, simple, cost effective, highlysensitive and robust methods for bacteria detection in foodmatrices still remains a challenge. Although rapid bioana-lytical methods shorten assay times from days to fewminutes or hours, in most cases they require a pre-analytical cultivation enrichment to reach necessary detect-ability and to remove interferents, thus increasing turn-around time. Cultivation enrichment can be substituted byor combined with biospecific purification and concentrationprocedures, such as immunomagnetic separation, to main-tain short analysis times [6, 7].

The necessity to ensure food safety along the entire foodchain requires the analysis of a large number of samples,searching for a wide range of analytes in each of them. Thedevelopment of rapid assays characterized by multiplexing(i.e., the ability to detect and/or identify more than oneanalyte in a single analytical run) would greatly reduceassay costs and times, thus enabling larger screeningprocedures.

In this review, the most recent advancements in rapidbioanalytical methods for pathogenic bacteria detection infood matrices will be reported and critically discussed(while bacterial toxins and non-bacterial pathogens willnot be treated here), with particular emphasis on the tech-nological solutions devised to reach multiplexing capabili-ties. Multiplexing can be usually realized by twoapproaches. The “signal encoding” approach is based onthe use of different labels, each targeted to one analyte.The number of analytes that can be detected with thisstrategy is limited by the number of labels that can beindependently measured in one assay. The “position encod-ing” approach, in which different analytes are captured inseparate positions and then detected by means of a commonlabel, can reach much higher multiplexing capabilities,mainly depending on the detector spatial resolution.

Along with the development of multiplex assays, suitablepre-analytical sample preparation techniques must be devel-oped, including mixed-culture enrichment, employment ofmixtures of antibody-coated paramagnetic particles, or otherseparation techniques [6, 7].

Rapid nucleic acid-based methods

In the last decade, foodborne pathogens detection has beensignificantly improved by the development of several mul-tiplex nucleic acid-based methodologies. Polymerase chain

reaction (PCR)-based assays and DNA microarray technol-ogies have been shown to combine rapid results and highspecificity of detection, with increased sensitivity compar-ing to culture-based methods. Moreover, the inability ofnucleic acid-based assays to distinguish between live anddead cells has been recently overcome by the use of ethi-dium monoazide and propidium monoazide compoundsduring sample preparation [8–10].

Many multiplex PCR-based assays have been recentlyapplied to simultaneously detect pathogens in food in lessthan 24–30 h, including a relatively short pre-enrichmentstep, with detectability in the order of 100 CFU g−1. Theycould serve as a rapid screening method for pathogen de-tection and they have the potential to be fully automated.Recent multiplex PCR-based applications are reported inTable 1 [11–16]. In PCR-based assays, a nucleic acid targetof definite length and sequence is amplified by repetitivecycles of strand denaturation, annealing, and extension ofoligonucleotide primers by a thermostable DNA polymer-ase. To amplify multiple bacterial targets in one singlereaction tube different approaches have been used exploitingbacterial genome characteristics. The genome contains bothhomologous DNA regions, common to all members of agenus, and sequences that are less conserved and are uniqueto a species, sub-species or isolate, thus suitable for specificbacterial identification. The ribosomal 16S rRNA gene hasbeen frequently targeted since it contains both these sequen-ces, it is ubiquitous and it provides a naturally amplifiedmolecular target within cells [17]. A Multiplex PCR-basedassay has been recently successfully performed targeting the16S rRNA gene of eight different pathogens (E. coli, Clos-tridium perfringens, Campylobacter jejuni, Salmonellaenterica, Listeria monocytogenes, Vibrio parahaemolyticus,Staphylococcus aureus, and Bacillus cereus) followed by aspecies-specific identification by separating PCR productsin capillary electrophoresis-based single-strand conforma-tion polymorphism (CE-SSCP) [15]. Multiplex PCR-basedassays have also been developed by using sets of primerstargeting different regions of foodborne pathogens (from 3to 6 different pathogens) with subsequent post-PCR analysis[16, 18–20]. A further improved multiplex methodology hasbeen developed to overcome amplification disparities due tothe use of several primers; this universal primer-multiplexPCR system (UP-M-PCR) has been successfully applied todetect E. coli, L. monocytogenes, and Salmonella spp. [14].This approach is based on the use in the amplificationreaction of four different primers, three selective primerscontaining both bacteria sequences and a 18-mer commonsequence at their 5′-end, which is also the sequence of thefourth universal primer. In the first PCR cycles, the selectiveprimers take action for amplification of the different patho-gens, while the universal primer does not have a targettemplate. In the former cycles when amplified products

8 A. Roda et al.

increase, universal primer starts to take the amplified prod-ucts as template, producing with the same efficiency specificfragments of 3 different lengths.

Recently, a prototype of multichannel oscillatory-flowmultiplex PCR system for the detection of three foodbornebacterial pathogens has been developed, displaying greatpotential for high-throughput and fast DNA amplification[21, 22].

To further improve foodborne pathogenic bacteria detec-tion, Multiplex Real Time PCR assays have been designedboth exploiting SybrGreen-based and TaqMan-based tech-nologies. The combination of PCR chemistry with fluores-cent detection of amplified products allows achieving bothqualitative and quantitative analytical results sooner than byconventional PCR-based assays, with improved sensitivity(≤100 CFU mL−1) and reduced risk of false-positive sam-ples due to laboratory contaminations. Multiplexing isachieved employing different TaqMan probes labeled withspectrally distinct fluorophores. Selected recent examples[23–27], as well as commercially available PCR-based kitsare reported in Table 1.

Postollec and colleagues have recently provided an over-view on Real Time PCR assays in food microbiology andsome multiplex applications have been described [28 andreferences therein]. A further advanced strategy has beenproposed combining the SybrGreen-based universal ampli-fication of 25 different pathogens and the high-resolutionmelting analysis of products [29]. Moreover, a TaqMan-based Multicolor combinational probe coding (MCPC) RealTime PCR has been experimentally validated to detect8 foodborne pathogens in one tube (S. aureus, L. monocy-togenes, Salmonella typhi, Shigella spp., E. coli O157:H7,Vibrio cholerae, V. parahaemolyticus, and Streptococcuspyogenes) by using a variety of fluorophores and theircombinations [30].

Recently, DNA microarray-based methodologies havebeen proposed as promising powerful tools for the rapiddetection and identification of foodborne bacterial pathogens,combining PCR and probe hybridization technologies. Micro-array applications are reported in Table 1 [31, 32]. An im-proved Multiplex DNA microarray based on a 14-plex PCRamplification of target sequences followed by specific probehybridization reactions has been developed allowing the si-multaneously detection of E coli O157:H7, S. enterica, L.monocytogenes and Campylobacter jejuni [31]. Concerningthe identification of species, a DNA microarray platform ableto identify seventeen species of E. coli has been recentlydeveloped [32]. Finally, the DNA microarray potentiality infoodborne investigations has been further improved by thedevelopment of a bioinformatic tool (named ArrayTrack™)by FDA researchers [33]. The free software provides exten-sive functionalities to analyze and interpret data obtained bywhatever DNA microarray platforms [34].

Rapid immunoassays

Several immunological bioanalytical methods have beendeveloped for rapid foodborne bacteria detection exploitingpolyclonal or monoclonal antibodies [4]. The characteristicsof the used antibody, particularly its binding affinity con-stant and cross-reactivity, affect assay analytical perform-ances, such as assay sensitivity (low number of falsenegative samples) and specificity (low number of falsepositive samples), respectively. In addition, innovative bind-ing molecules with enhanced binding affinity, specificityand stability can be obtained by producing recombinantantibodies [4, 6, 35], phage-displayed antibody fragmentsor antibody-like binding molecules, such as aptamers [36].

Enzyme-linked immunosorbent assays (ELISA) in 96-wellmicrotiter plates are well-established screening tools in foodsafety assessment and many of them are commercially avail-able in the uniplex format. In most cases, the sandwich formatis employed, in which the target analyte (which might be awhole bacterial cell presenting a surface antigen or the antigenin solution within a bacterial lysate) is captured by an immo-bilized antibody and detected upon binding to a second la-belled antibody. The main limitations of ELISA are theirrelatively high Limits of Detection (LoD, normally 104–106 CFU mL−1) demanding for pre-analytical selective en-richment procedures, and long and labor-intensive analyticalprotocols. Many efforts are thus directed to the developmentof new immunoassay formats, offering assay rapidity, highsensitivity, multiplexing abilities and high sample throughput,possibly employing portable user-friendly devices, suitablefor point-of-care applications [4, 5].

Various multiplex immunoassays for foodborne pathogendetection were developed following the “signal encoding”approach, employing a combination of labels (e.g., quantumdots or time resolved fluorescence labels) that can be spec-trally distinguished when present in a mixture [6, 37–41].More frequently, the “position encoding” approach wasused, mainly exploiting electrochemical, fluorescence orchemiluminescence detection in microarray formats and/ormultichannel platforms. Suspension microarrays, based onthe use of diverse microsphere populations as a solid phasefor multiplex immunoassays, are briefly described in para-graph “Bacteria separation to assist bioassays”.

Microtiter plate-based planar microarrays have been de-veloped with the aim to facilitate their laboratory implemen-tation, being their format adaptable to automated 96-wellmicrotiter plate handling instrumentation (e.g., robotic dis-pensers, plate washers, incubators) [6, 42–44]. Recently, wedesigned a 96-well microtiter plate, in which each well isinternally divided into four subwells, thus allowing separateimmobilization of four distinct antibodies in the bottom ofeach well. Upon sample addition to the main well, eachanalyte was captured in one subwell and it was then detected

Developments in rapid multiplexed detection of foodborne bacteria 9

Table 1 Recent multiplex bioanalytical methods for foodborne pathogen bacteria detection

Microorganism Test characteristics Food matrix Notes Referencea

PCR-based methods

Vibrio spp. LoD: 1 CFU mL−1(18–24 henrichment)

Seafood Identification of species 11

Bacillus cereus group spp LoD: 2.9×103 CFU g−1 (24 henrichment)

Artificially contaminatedpowder milk

Detection of 117 strains 12

Salmonella spp, Listeria.monocytogenes, Escherichiacoli O157:H7

LoD: 5 CFU 25 g−1 (20 henrichment)

Meat, fish, and dairyproducts (fresh and afterfrozen storage)

Detection 13

Escherichia coli, Listeriamonocytogenes, Salmonellaspp.

Universal primer-multiplexPCR assay

Meat, fish, raw milk, eggs Detection 14

Escherichia coli, Clostridiumperfringens, Campylobacterjejuni, Salmonella enterica,Listeria monocytogenes,Vibrio parahaemolyticus,Staphylococcus aureus,Bacillus cereus

Capillary electrophoresis-basedsingle-strand conformationpolymorphism (CE-SSCP)coupled with multiplex PCR

Detection 15

Vibrio parahemolyticus,Salmonella spp., Escherichiacoli O157:H7, Shigella spp.

LoD: 5.1×101 - 2.9×102

CFU mL−1 depending on thetarget pathogen (12 henrichment)

Beef, vegetables, pork andshrimp

Detection 16

Salmonella spp., Listeriamonocytogenes, Escherichiacoli O157:H7

TaqMan-based Real Time PCRassay

Spiked pork samples Quantitative detection 23

LoD ≤2.0×102CFU mL

−1(20 h enrichment)

Salmonella spp., Listeriamonocytogenes, Escherichiacoli O157:H7

TaqMan-based Real Time PCRassay

Artificially contaminatedground beef

Quantitative detection 24

LoD ≤18 CFU 10 g−1 (20 henrichment)

Yersinia pestis TaqMan-based Real Time PCRassay

Milk, ground beef 25Yersinia pseudotuberculosis

LoD: 101–103 CFU mL−1 inmilk and 102–105 CFU g−1 inground beef (without any pre-enrichment step)

Campylobacter jejuni,Campylobacter coli,Campylobacter lari

TaqMan-based Real Time PCRassay

Chicken Quantitative detection 26

LoD: 1 CFU g−1 (24 henrichment)

Salmonella spp. TaqMan-based Real Time PCRassay

Inoculated Cheddar cheese,raw turkey, and cookedturkey

Identification of serovars 27

LoD: ≤1 CFU g−1 (enrichment)

Campylobacter jejuni,Campylobacter coli,Campylobacter lari

Bax® System Real Time PCRassay (DuPont Qualicon)

Validated on enrichedsamples of spiked turkeybreast and naturallycontaminated chickencarcass rinses

Quantitative detection Commerciallyavailable

LoD: 104 CFU mL−1

PCR time: 1.5 h (after 24–48 henrichment)

Escherichia coli O157:H7 BAX® System Real-Time PCRAssay: STEC Panel ½(DuPont Qualicon)

Validated on raw groundbeef, beef trim, spinachand lettuce

Identification of serogroups Commerciallyavailable

LoD: 104 CFU mL−1

PCR time: 3.5 h (after 8–24 henrichment)

10 A. Roda et al.

Table 1 (continued)

Microorganism Test characteristics Food matrix Notes Referencea

Listeria species (except L. grayi ) BAX® System PCR Assay forGenus Listeria 24E (DuPontQualicon)

Validated on baggedspinach, processedcheese, frankfurters,cooked shrimp andstainless steel

Multiplex qualitative detection Commerciallyavailable

LoD: 104 CFU mL−1

PCR time: 3.5 h (after 20–24 henrichment)

Vibrio cholerae, Vibrioparahaemolyticus, Vibriovulnificus

Bax® System Real Time PCRassay (DuPont Qualicon)

Shrimp, tuna, oysters,scallops, crab

Commerciallyavailable

LoD: 104 CFU mL−1

PCR time: 1.5 h (after 18–20 henrichment)

Listeria spp. MicroSEQ® Listeria sppDetection Kit (AppliedBiosystems)

Meat and seafood products,milk

Commerciallyavailable

LoD: 1–3 CFU in 25 g−1 (27–31 h enrichment)

Salmonella spp. MicroSEQ® Salmonella spp.Detection Kit (AppliedBiosystems)

Meat and seafood products,poultry, fruits and juices,dairy products, chocolate/bakery products, peanutbutter, egg products

Commerciallyavailable

LoD: 1–3 CFU 25 g−1 (27–31 henrichment)

Oligonucleotide array-based methods

Escherichia coli O157:H7,Salmonella enterica, Listeriamonocytogenes,Campylobacter jejuni

Oligonucleotide microarray Detection 31LoD: 20 copies/reaction

Escherichia coli Oligonucleotide microarray Identification of species 32

Immunoassay-based methods

Salmonella typhimurium,Escherichia coli O157:H7,Listeria monocytogenes

Magnetic nanobeads-basedimmunoseparation followedby labeling with spectrallydistinguished quantum dotsand fluorescencemeasurement.

Artificially contaminatedwash solution fromchicken carcasses, groundbeef and fresh-cutvegetables.

38

LoD: 20–50 CFU mL−1

(without any pre-enrichmentstep). Assay time <2 h.

Salmonella typhimurium,Staphylococcus aureus,Legionella pneumophila,Escherichia coli O157:H7

Enzyme-linked immunosorbentassay (ELISA) performed onan immunochromatographicstrip. LoD: in the range 103–105 CFU mL−1 for the varioustarget pathogens.

49

Assay time: 20 min.

Escherichia coli, Salmonellaenteritidis

Immunomagnetic separationfollowed by labeling withspectrally distinguishedquantum dots andfluorescence measurement.

39

LoD: 102 CFU mL−1. Assaytime: 2 h.

Escherichia coli O157:H7,Salmonella spp.

Sandwich immunoassayperformed on an antibodyarray employingchemiluminescence detection.

Artificially contaminatedmilk samples (LoD inmilk samples is notreported).

50

Developments in rapid multiplexed detection of foodborne bacteria 11

Table 1 (continued)

Microorganism Test characteristics Food matrix Notes Referencea

LoD: 105–107 CFU mL−1.Assay time: 4 h

Salmonella typhimurium,Escherichia coli O157:H7,Shigella flexneri

Immunomagnetic separationfollowed by labeling withspectrally distinguishedquantum dots andfluorescence measurement.

Artificially contaminatedapple juice and milk.

37

LoD: 103 CFU mL−1. Assaytime: 2 h.

Salmonella typhimurium,Escherichia coli O157:H7,Legionella pneumophila

Multichannel flow−throughmicroarray chip exploitingCCD-based chemilumines-cence detection.

47

LoD: 1.8×104 CFU mL−1for E.coli O157:H7, 7.9×104 CFU mL−1for L.pneumophila, and 2.0×107 CFU mL−1for S.typhimurium. Assay time:18 min.

Campylobacter genus,Campylobacter jejuni

VIDAS Campylobacter Food products Detection CommerciallyavailableSingle-dose test composed of a

strip containing the ready-to-use reagents and a SPR (SolidPhase Receptacle) coated withspecific antibodies. Run:70 min (Total time 2 Days)

Listeria spp, Listeriamonocytogenes

VIDAS Listeria DUO (LDUO)Run: 120 min (Total time 2Days)

Food products Detection Commerciallyavailable

Biosensors

Escherichia coli O157:H7,Escherichia coli CECT 675(non pathogenic)

Covalently immobilizedaptamers on single-walledcarbon nanotubes as ion-to-electron transducers. Potentio-metric detection.

Milk, apple juice E.coli O157:H7 detected in thepresence of different bacteria(Salmonella enterica,Lactobacillus casei) and adifferent strain (nonpathogenic E. coli)

59

LoD: 6 CFU mL−1 in milk,26 CFU mL−1 in apple juice.Real time detection.

Filtration and washing pre-analytical step.

Enterococcus faecium,Staphylococcus aureus,Stenotrophomonasmaltophilia, and Vibriovulnificus

DNA multiple Au nanowireSERS sensors. (PCR as pre-analytical step).

LoD: 10 pM of DNA

Application to clinicalsamples, potentialities tofood samples analysis.

Self-assembly of Aunanoparticles on nanowire inthe presence of target DNAs.

58

Salmonella typhimurium,Salmonella dublin, Salmonellathompson, Staphylococcusaureus, Yersinia enterocolitica,Listeria monocytogenes,Shigella flexneri, Shigella bogdi,Campylobacter jejuni,Escherichia coli O157:H7,Vibrio parahaemolyticus, Vibriocholerae, Enterobacter sakazaki,Pseudomonas aeruginosa

DNA silicon-based optical thin-film biosensor. (PCR as pre-analytical step)

LoD: 8.5×101 CFU/mL. Assaytime: 30 min.

Meat Biotinylated PCR amplificon.Detection by color changesafter HRP-antibody binding tobiotinylated hybrid and a sub-strate addition.

60

Bacillus antracis, Yersiniapestis, Francisella tularensis

DNA array gold electrodes intoa silicon chip (methodincludes multiplexed PCR

Compact and fully automatedsystem; potentialities as POCTdevice.

61

12 A. Roda et al.

Table 1 (continued)

Microorganism Test characteristics Food matrix Notes Referencea

step). Enzyme-mediated elec-trochemical detection.

Assay time: 27 min (excludingPCR step)

Escherichia coli, Hafnia alvei,Listeria innocua, Bacillussubtilis, Bacillus atrophaeus

DNA/RNA electrochemicalbiochip and reporter enzyme.Amperometric detection

Endogenously infectedmeat, juice sample addedwith E. coli

Reporter enzyme: thermostableesterase from Alicyclobacillusacidocaldarius.

62

LoD: 550 CFU mL−1 for E.coli.

Pre-enrichment: 7 h.

Two bacteria mixtures:, -Cryptosporidium parvum,Giardia lamblia,Encephalitozoon intestinalis, -Escherichia coli O157,Salmonella typhimurium,Salmonella flexneri

Polydiacetylene (PDA)liposome-based biosensorcoated with antibodies. Fluo-rescence measurements.

Solid-phase PDA sensors,immobilization of PDAsupramolecules with specificoptical properties

63

LoD: 102 units mL−1for eachbacteria (106 units mL−1forbacteria mixture)

Salmonella enterica serovarTyphi, Staphylococcus aureus,Listeria monocytogenes, Vibrioparahaemolyticus, Shigellasonnei, Enterobacter sakazakii,Escherichia coli O157:H7,Campylobacter jejuni

Mesofluidic chip based on thecapture on fluorescently-labeled PCR products bymeans of oligonucleotide-coated glass microbeads.

LoD: 0.5–6×103 CFU mL−1

(8-h pre-enrichment).

Egg, pork, chicken,shellfish, fish, ice cream,and milk powder.

64

Staphylococcus aureus,Escherichia coli O157:H7,Salmonella typhimurium

Integrated microdevice formultiplex bio-barcode assay.Assay time 30 min. Targetbacteria detection at thesingle-cell level, without PCRamplification.

Multiplex pathogenidentification. The devicehas a sample-in-answer-outcapability, with point-of-careapplicability.

84

Electronic nose

Escherichia coli, Listeria spp. E-nose MOS sensor-AlphaMOS

Bacteria colony takendirectly from the surfaceof agar medium andresuspended in buffer

100

Uncorrelated linear discriminantanalysis.

Classification accuracies of92.4 %.

Spectroscopic methods

Salmonella enterica serovarTyphimurium, Staphilococcusaureus

SERS. Multiplexing is obtainedby employing antibody-labelled nanoparticles labelledwith different Raman reporters

Spinach solution Detection 82

Staphilococcus aureus,Lactobacillus lactis spp.Cremoris (non pathogenic)

FT-IR coupled withchemometric spectral analysis.

Milk Detection, enumeration andinvestigation of growthinteraction

105

Bacteria enumeration isobtained in cocultures withoutthe need for their separation.

Escherichia coli, Salmonellathyphimurium

SERS. Multiplexing is obtainedby employing antibody (oraptamer)-nanoparticleslabelled with different Ramanreporters.

Detection 83

LoD 102–103 CFU mL−1

a Only papers published on 2009 or later (or currently commercially available kits) have been included in the table.

LoD Limit of Detection

Developments in rapid multiplexed detection of foodborne bacteria 13

by sandwich-type immunoassays, employing a mixture ofenzyme-labelled detection antibodies. Simultaneous analytedetection was performed by chemiluminescence, employinga charge-coupled device (CCD) imaging system, or a conven-tional luminometer suitable for 384-well microtiter plates.With this format a chemiluminescent four-plex immunoassaywas developed, able to detect down to 1 CFU g−1 for eachtarget bacteria in ground meat, upon 9-h enrichment (Fig. 1)[42]. Employing the same outline, other multiplexed bindingassays can be developed, by customizing the plate layout todivide each main well in the number of subwells required forthe analysis [43].

With a similar approach, significantly higher multiplexingabilities can be reached (e.g., 813 spots array per well) bydepositing an antibody microarray at the bottom of each wellof a 96-well microtiter plate employing a microarray printingsystem [6]. Employing this system, a fluorescence multiplexsandwich immunoassay was developed for simultaneous de-tection of E. coli O157:H7 and S. typhimurium in culture-enriched ground beef filtrate down to 106–107 cells mL−1 and2.5 h total assay time [44]. The technology developed byMeso Scale Diagnostics, LLC (Gaithersburg, MD, http://www.mesoscale.com) also allows the development of

microtiter plate-based multiplexed immunoassays employingelectrochemiluminescence (ECL) detection. Nevertheless,applications in food safety assays are very few [45] and theneed for dedicated instrumentation might hamper its wide-spread acceptance in this field.

The necessity to perform the analysis directly where thesample is obtained (point-of-use testing) prompted the devel-opment of portable hand-held analytical devices for multiplexfoodborne pathogens detection, exploiting the lab-on-a-chiptechnology, as recently reviewed [5]. Many of such devices,displaying full integration of the transduction system with thebiospecific recognition element can be categorized as biosen-sors and they will be treated in paragraph “Biosensors”.

Lab-on-a-chip and multichannel devices for multipleximmunoassays were developed exploiting high detectabilityoffered by fluorescence and chemiluminescence optical de-tection techniques [46, 47]. However, integration of opticaldetection systems in fully portable analytical devices stillrequires further studies. Exploiting a recently describedportable device based on contact CCD-based imaging ap-proach [48], we have developed a multiplex sandwich-typeimmunoassay with chemiluminescence detection, able toperform simultaneous detection of four pathogenic bacteria(i.e., E. coli O157:H7, Yersinia. enterocolitica, S. typhimu-rium, and L. monocytogenes) by immobilizing four specificcapture antibodies on a microfluidic platform. With thissystem, LoD values of 104–105 CFU mL−1 were achievedfor each of the four bacteria (unpublished results). Prelimi-nary results are shown in Fig. 2.

The most recently reported multiplex immunoassays forfoodborne pathogens are reported in Table 1 [37–39, 47, 49,50].

Biosensors

Biosensors, in which the biospecific recognition element(often an antibody or a nucleic acid probe) is integrated withthe transduction system, have been proposed in a variety offormats for foodborne pathogens detection, as described inrecent reviews [3, 6] and references therein, [51–54]. Inmost cases electrochemical or optical transduction systemsare employed. Despite usually providing better sensitivitythan electrochemical techniques, optical methods are gener-ally less suitable for routine use owing to their higher costand complexity; although some improvements have beenreported exploiting the development of technologies foroptical signal’s transmission and systems readout, as it willbe discussed later in this paragraph. On the other hand, theperformance of electrochemical techniques in terms of sen-sitivity and multiplexed capabilities are not still satisfactory.Electrochemical biosensors have attracted much attention,since they are generally characterized by high rapidity, low

Fig. 1 Chemiluminescence multiplex immunoassay for Escherichiacoli O157:H7, Yersinia enterocolitica, Salmonella typhimurium, andListeria monocytogenes pathogenic bacteria employing a custom-designed microtiter plate format and CCD-based imaging detection.Reprinted with permission from [42]. Copyright 2007 AmericanChemical Society

14 A. Roda et al.

cost and amenability to miniaturization, so that they are inmost cases proposed in a disposable format. Today, the greatchallenge in the field for the simultaneous detection ofmultiple target analytes is constituted by the developmentof automated electrochemical microarray platforms. Fara-bullini et al. developed a DNA array of four gold electrodeson which four foodborne pathogens (L. monocytogenes, E.coli O157:H7, Salmonella spp., and S. aureus) weredetected by enzyme-mediated differential pulse voltamme-try. Mixtures of DNA sequences from the four bacteria weredetected at the nanomolar level with high selectivity inabout 1 h [55]. Recent examples of these biosensors towardsmultiplexed analysis are reported in Table 1.

Most biosensors proposed for multiplex bacteria detectionexploited optical transduction, particularly surface plasmonresonance (SPR), Raman spectroscopy and surface enhancedRaman spectroscopy (SERS), Fourier transform infrared (FT-IR) and optical fiber devices employing luminescence techni-ques. Most of them can be classified as label-free biosensorsbased on the direct measurement of a signal that is generatedby the biospecific recognition event (i.e. changes in mass orrefractive index upon antigen-antibody binding).

SPR biosensors measure the changes of the refractive indexin close proximity to the sensing metal surface as a conse-quence of the biorecognition event. Many commercial SPRsystems are now available (e.g. BIAcore™, GE Healthcare,UK, http://www.biacore.com) even in a portable formatSPREETA (Texas Instruments Inc. Dallas, TX, http://www.ti.com). An interesting example reports the detection of E. coliO157, S. choleraesuis typhimurium, L. monocytogenes and C.jejuni by means of a sandwich immunoassay label-free SPRbiosensor based on wavelength division multiplexing wherethe SPR wavelength for each of the eight flow channels is

monitored by four spectrophotometers. LoD values rangingfrom 3.4×103 to 1.2×105 CFU mL−1 in apple juice wereobtained [56]. Multiplexing capabilities are also offered by anapproach combining SPR imaging and fluorescence. A hybridmicrofluidic biochip consisting of an array of gold spots, eachfunctionalized with a capture biomolecule targeting a specificpathogen, was enclosed in a flow chamber delivering a mag-netically concentrated sample. The sample was detected bySPR on the bottom of the biochip, while fluorescencemeasuredfrom the top was used to verify the pathogen state [57]. Aschematic view of the system is reported in Fig. 3.

The increasing availability of new nanostructures has ledto the development of application of SERS biosensors forbacteria analysis. As an example, multiplex DNA detectionwas obtained employing multiple Au particle-on-wire sys-tems as a SERS sensing platform [58]. Selected recentapplications of biosensors to multiplexed bacteria analysisin food samples are reported in Table 1 [58–64]. A chemi-luminescence antibody microarray was developed for therapid and simultaneous detection of Escherichia coli O157:H7, Salmonella typhimurium, and Legionella pneumophilain water samples using a sensitive CCD camera for signaldetection. The detection limits were comprised between 3×103 and 3×106 cells mL−1 for the different target bacteria,with a 13-min assay time [46]. Mutagenesis of enhancedgreen fluorescent protein was performed to develop afluorescence-based biosensor for gram-negative bacteria,like E. coli and Pseudomonas aeruginosa [65]. However,multiplexed capabilities are still to be demonstrated.

Biosensors based on optical measurement in combinationwith the use of fiber optic were also developed for the rapid,high sensitive and multiplexed detection of foodborne patho-gens. Biosensors exploiting the fluorescence resonance energy

Fig. 2 Chemiluminescence multiplex immunoassay for Escherichiacoli O157:H7, Yersinia enterocolitica, Salmonella typhimurium, andListeria monocytogenes pathogenic bacteria employing an array ofspecific capture antibodies immobilized on a glass support and contactCCD-based imaging detection. a Calibration curve obtained employingthis system (open symbols) is shown in comparison with the calibra-tion curve obtained employing the microtiter plate-based multiplexsystem previously described (black symbols) [42]. b Chemiluminescence

image of four spots obtained by performing the multiplex sandwich-typeimmunoassay upon depositing specific antibodies for E. coli, Y. enter-ocolitica, S. typhimurium, and L. monocytogenes in four separate posi-tions on the glass slide. The image was acquired employing a lenslessCCD imaging device previously described [48]. Bar corresponds to1 mm. c Three-dimensional plot showing the spatial distribution of thechemiluminescence signal

Developments in rapid multiplexed detection of foodborne bacteria 15

transfer and evanescent-wave principles in a portable formatwere applied to the detection of Salmonella typhimurium andE. coli O157:H7 from meat samples. Moreover, the rapiddetection of Escherichia coli in the presence of other bacteriasuch as S. typhimurium, C. jejuni, and L. monocytogenes wasobtained with a chemiluminescence measurement based on theuse of fiber optic with an immunomagnetic separation. A LoDof 1.8×102 CFU mL−1 was obtained within an assay time of1.5 h. This biosensor shows potentialities toward the detectionof bacteria in various real samples with lower LoDwith respectto other rapid analytical methods [66].

Approaches for multiplexed bacteria analysis employingbiosensors arrays based on optical phenomena have alsobeen reviewed by Raz et al [5] and recent applications arereported in Table 1. An example is the Naval ResearchLaboratory (NRL) biosensor based on planar waveguideand evanescent excitation light for fluorescent measurementof signal from immobilized target analytes. The commercialversion is based on the use of a portable system employing aCCD camera. Immunoassays based on the NRL biosensorwere used for Escherichia coli O157:H7 and Staphylococ-cus aureus with LoD of 5×103 and 106 CFU mL−1 withpotentialities toward multiplexed analysis. Multiplexed ca-pabilities were also explored using alternative capture mol-ecule such as cellular receptors or antimicrobial peptides.The NRL biosensor was also used in FDA blind trials for thedetection of multiple foodborne pathogens in food samplessuch as water, juice and milk [67]. Among very recentdevelopments, nanostructured optical transducers wereimplemented in biosensors format for multiplexed bacteriaanalysis. A representative example shows target bacteriacaptured onto the surface of the optical label-free porousSi-based biosensors, inducing changes in the thin-film

optical interference spectrum. Low bacterial concentrationsfor E. coli, in the range of 103–105 cells mL−1, could dedetected in few minutes without the need of any pretreat-ment cell lysis [68, 69].

Among different transducing systems used for the analy-sis of foodborne pathogens, quartz crystal microbalance(QCM) and surface acoustic wave (SAW) technologies arewidely employed in label-free biosensors to detect changesin mass due to the biospecific binding event (e.g., antigen-antibody complex formation) occurring on the surface of aquartz crystal [70]. An example based on lectins immobi-lised on a gold-plated quartz crystal of a QCM biosensorwas described able to give different response profiles for E.coli, S. aureus and Mycobacterium phlei [71]. However,only few applications to multiplexed analysis are described.

In conclusion, biosensors offer an interesting alternativeto the conventional methods for bacteria identification sincethey potentially allow multiplex analyses with very shortassay times. Their routine use could be enabled by theimprovements on transducer sensitivity, miniaturization,and immobilization process of biospecific probe. However,much effort has to be addressed to the direct application tobacteria analysis in complex matrices, since at the momentvery few examples were developed for biosensors directlyused for bacteria detection without the need of an enrich-ment, separation or purification step.

Nanotechnologies

Thanks to the continuous advances in the field of nanotech-nology, new classes of materials with peculiar chemical andphysical properties (e.g., nanoparticles, nanotubes, nanowires,

Fig. 3 Multicomponentschematic of the overallpathogen detection systemshowing light paths anddetection system. Reprintedwith permission from reference[57]. Copyrignt 2009 JohnWiley and Sons

16 A. Roda et al.

nanofunctionalized surfaces) have become available. Theirimplementation in bioanalytical assays enhanced assay per-formance, opened new possibilities for devices miniaturiza-tion and allowed the development of entirely new assayformats. In particular, nanomaterials have generated greatexpectations with respect to increasing assay detectabilityand selectivity and reducing assay times [72–76] and to en-hance analytical performance in a variety of analytical appli-cations, such as microfluidic platforms [77], electrochemical[78], optical [79], and microgravimetry [80] biosensors, thuspotentially providing significant advancements for multi-pathogen detection.

Various authors have also exploited the peculiar charac-teristics of nanometer-sized materials to reach multiplexcapabilities. Different populations of nanosized labels, eachtargeted to a pathogen of interest, have been used for multi-plexed assays based on optical detection, such as quantumdots [37–39], dye-doped nanoparticles [41], compositenanoparticles [40, 81] or nanoparticles doped with differentRaman reporters [82, 83]. The recently developed bio-barcode assay offers great potentialities for high detectabil-ity (nucleic acids can be detected without PCR amplifica-tion) and multiplexing. In this assay, the target analyte (aprotein or a nucleic acid) is captured by a biospecific bind-ing element immobilized on a magnetic microparticle and isthere sandwiched with a second binding element carrying agold nanoparticle (Au-NP) loaded with hundreds of thio-lated oligonucleotide barcodes. A magnetic field is appliedto collect the sandwich structures and the DNA barcode isthen released from Au-NP and detected. Jung JH et al haverecently reported an integrated microdevice able to sequen-tially perform all the assay steps within 30 min for multiplexpathogens detection at the single-cell level [84].

The application of nanomaterials in multiplex foodbornepathogens detection is still a relatively new research area,which is rapidly growing and holds considerable promise tolead to a new generation of rapid, portable and multitargetanalytical platforms. Nevertheless, only in few cases thedeveloped assays have been applied to real food samplesanalysis, which is crucial for their future widespread accep-tance and commercialization.

Bacteria fingerprint-based approaches

The bioanalytical methods described above are based onthe use of one or more binding molecules, such as anti-bodies or nucleic acids, directed towards a biomarkerspecific of a given bacterial species and are thus restrictedto the availability of such probes. As an alternative,reagentless approaches based on the acquisition of awhole-cell biochemical fingerprint have been proposedas rapid, simple and cost-effective foodborne pathogen

detection tools, exploiting mass spectrometry (MS), vibra-tional spectroscopy (Raman and infrared), electronicnoses and light scattering analyses. Such methods arebased on the acquisition of a complex fingerprint-likepattern of instrumental signals that is distinctive of themicroorganism of interest, which can be identified bycomparison with data libraries. In most cases, mathemat-ical deconvolution and chemometric analysis tools arenecessary to simplify complex raw signals and to extractthe useful qualitative and quantitative analytical informa-tion from the minimum number of variables. One of themain issues of such approach is reproducibility of thefingerprint, especially when real food matrices are ana-lyzed and when public, commercial, or between-laboratory shared libraries are employed.

Their main advantage is their potentiality for largescreening procedures. Indeed, as data libraries are beingcontinuously updated with an increasing number of finger-printed bacterial strains, this approach offers great potenti-alities for the identification of a pathogen bacterium within alarge cohort of candidates, with a single and rapid analysis.However, the analysis of samples in which two or morebacterial strains are present is hampered by the inherentcomplexity of the raw signals, which might become over-whelming in the analysis of mixed populations. The differ-ent strategies devised to overcome this limitation arediscussed in the following.

Mass spectrometry

For the identification of both known and unknown bacteria,rapid methods based on MS were developed, exploiting softimpact ionization technologies suitable for proteins analysis.This approach offers high rapidity and amenability to auto-mation, although requirement of expensive instrumentationmight hamper its wide applicability. In most cases, matrix-assisted laser desorption/ionization time-of-flight MS(MALDI/TOF-MS) is employed, which has demonstratedto be particularly suited for a fingerprint-like approach,thanks to its characteristic of generating mostly singlycharged ions and to its high sensitivity for intact proteinsin the Mr range 4,000–15,000, which mainly constitute thebacterial proteome population. The ability to detect down to104 microorganisms also provides a rather high detectabilityfor foodborne bacteria [85].

In whole-cell MALDI-TOF MS, characteristic “finger-print” spectra are obtained from whole cells, without bio-marker prefractionation, digestion, separation, or cleanup[86–88]. The observed protein biomarkers are typicallyhighly expressed and highly conserved proteins in bacteria,possessing housekeeping functions, such as ribosomal ornucleic acid-binding proteins. The method is therefore char-acterized by universal applicability [89]. Target bacteria

Developments in rapid multiplexed detection of foodborne bacteria 17

identification can be accomplished either employing a pro-teomic approach, based on the detection of specific proteinbiomarkers that are identified by means of proteome data-base searching [85], or a chemometric approach in whichbacterial species are identified by comparing the wholespectrum of the unknown bacteria with a library of massspectra of reference strains [90].

Use of MS for foodborne bacteria detection requires astrict and standardized protocol to obtain reproducible andcomparable spectra, especially when real samples are ana-lyzed, in which non-target bacterial populations or foodsample matrix components might act as interferents [87,88, 91, 92]. With this respect, several technologicalapproaches have been proposed, such as accurate protocolsstandardization, bio-inspired bacteria purification systems,coupling with separative techniques, high-resolution MSmethods, and bioinformatic tools [92–95]. Employing thepathogenic bacteria E. coli O157:H7 and Yersinia enter-ocolitica and the non-pathogenic E. coli MC1061 strainas model samples, we recently proposed the use of chemo-metric tools to extract relevant information, useful for com-paring the fingerprint MALDI-TOF spectra of unknownbacteria with a spectral library, to accomplish robust bacteriaidentification and classification [96].

When a mixture of two or more bacteria need to beanalyzed by MALDI-TOF MS fingerprint techniques, spec-tra complexity and signal suppression phenomena posesevere limitations for their identification. Therefore, com-plementary separative techniques have been proposed toprepare the sample and provide a fraction enriched in thebacteria of interest for MALDI-TOF analysis. With thisrespect, field-flow fractionation (FFF) techniques haveproved a useful non-invasive sample preparation tool [95],as better described in the paragraph “Bacteria separation toassist bioassays”.

Electronic nose

Whole-cell biochemical fingerprint can also be obtained byexploring the bacterial metabolome employing electronicnose technology, providing, in perspective, rapid, simpleand sensitive methods for routine and screening purposes.Volatile organic compounds (VOCs) are the most common-ly species produced and released from bacteria sources.Electronic noses comprise an array of chemical sensorsbased on different detection principles, able to give a distinctresponse to different VOCs resulting in a “smell fingerprint”useful for sample identification. Pattern recognition techni-ques can be used to analyse the raw response and to dis-criminate the data signals. Conventional techniques basedon statistical/mathematical analysis, such as principal com-ponent analysis (PCA) and linear discriminant analysis(LDA); or data processing systems that do not employ

conventional mathematical models, e.g. artificial neural net-works and fuzzy-inference systems, are used [97]. Electron-ic noses have been applied to bacteria analysis in food,employing various types of sensors including conductingpolymers sensors (CP, exploiting the changes in polymersconductivity), metal oxides semi-conducting sensors (MOS,sensible to resistance changes), amperometric gas sensors(AGS) and field effect transistors (MOSFET), as well asQCM and SAW mass sensors (already mentioned in para-graph “Nanotechnologies”).

Commercially available electronic noses based on CP(e.g. Cyranose-320™, Cyrano Sciences Inc, Pasadena CA,http://www.smithsdetection.com), MOS (i.e., EOS system,SACMI Imola, Italy http://www.sacmi.it; AlphaMOS, Tou-louse, France http://www.alpha-mos.com; PEN 3, AirsenseAnalytical GmbH, Schwerin, Deutschland, http://www.airsense.com) were applied to food and water analysis, asrecently reviewed [98] and references therein]. In recentyears combined technologies based on electronic noses cou-pled to detection systems such as mass spectrometry andultra fast gas chromatography were also developed: SAWsensors and GC (zNoseTM, Electronic Sensor Technology,Newbury Park, CA, http://www.estcal.com); or MOS sen-sors and MS (Kronos AlphaMOS Toulouse, France, http://www.alpha-mos.com) are two examples [99]. An exampleof the most recent multiplexed applications is reported inTable 1 [100].

New generation electronic noses are hybrid systems in-cluding more than one type of sensor principle. A culture of5×10 8 cells mL−1 of Enterobacter aerogenes was readilydiscriminated from an E. coli strain using an array of eightquartz microbalance (QMB), eight metal oxide semiconduc-tor (MOS), and four electrochemical gas sensors, after aPCA analysis [101].

Despite the improvement of technologies employingarrays of sensors using hybrid formats the identification ofbacteria in real complex samples still requires a separative/enrichment step.

Spectroscopic methods

Besides being explored as transduction principles in biosen-sors, as described in Section 5, vibrational spectroscopytechniques coupled with multivariate statistical analysis,have been proposed as metabolic fingerprinting techniquesfor pathogenic bacteria detection and identification in foodmatrices. Automated instrumentation, such as Fourier trans-form infrared (FT-IR) platforms from Thermo Fisher Scien-tific (Madison, WI, http://www.thermofisher.com) and

Fig. 4 HF FlFFF MALDI/TOFMS of a 1:1 mixture of lyophilized E.coli and B. subtilis cells: fractionation profile (a), fractions spectra (b)and (c). Adapted with permission from reference [127]. Copyright(2004) American Chemical Society

b

18 A. Roda et al.

Developments in rapid multiplexed detection of foodborne bacteria 19

Bruker (Ettlingen, Germany, http://www.bruker.com), makethese techniques suitable for large scale screening proce-dures, while the development of portable devices, such as aportable SERS system [102] opened the way for their point-of-use application.

Fourier transform infrared spectroscopy provides infor-mation about the biochemical composition of cells and ithas demonstrated to possess a high discriminatory power,being able to detect, identify, and classify different food-borne bacteria at the species, subspecies, and strain levels[103], also discriminating between live and dead cells[104].

One main limitation of such techniques is the necessity ofa pre-analytical enrichment step, which is necessary both toobtain sufficient biomass for the analysis and to bring bac-terial cells in a “clean” environment, eliminating possibleinterferents from the food matrix.

With this respect, signal amplification offered by surface-enhanced Raman scattering microscopy (SERS) [82, 83], ora combination of Raman spectroscopy with other opticaldetection techniques have been proposed to eliminate theneed for enrichment culturing.

Recently, the use of chemometric data analysis tools hasbeen employed to allow bacterial identification in mixturesemploying FT-IR analysis [105].

Bacteria separation to assist bioassays

For most of the methods described in the previous para-graphs the main critical aspect is the possibility to directlyanalyze highly complex samples, such as food samples. Apre-analytical separation/enrichment step is thus oftensought.

The use of electrophoresis for the separation of bacteriawas studied from many authors and a miniaturized systemfor bacteria separation in microchip format has been recent-ly described [106]. The main obstacle in its developmentwas related to cells aggregation and adhesion to separativedevices during the process and detection issues. A papershows an interesting solution based on the in-line connec-tion of fluorescence stereomicroscope to a capillary electro-phoresis system for the observation of the behaviour ofbacterial cells during capillary zone electrophoresis. Authorsunderline the interesting perspectives on the use of theproposed coupling to collect separated microorganism forfurther analysis [107]. Electrophoretic techniques, such asdielectrophoresis, despite displaying sufficient sensitivity,selectivity and precision for the selective trapping and sep-aration of bacteria [108], are characterized by high variabil-ity due to the dependence of electrophoretic mobilities oncell dispersions preparation [109, 110]. Despite the evolu-tion of dielectrophoresis as powerful, robust and flexible

method for cellular characterization, manipulation, separa-tion, its development as automated, rapid, simple and bio-compatibile method for bacteria analysis is still at an earlystage [111 and references therein].

Fluorescence-activated cell sorting (FACS) techniques areused to define different types of bacterial cells as positive ornegative for the given marker, and to consequently isolate themarked cells. Applications of FACS for bacteria separation arereported, in particular for clinical samples [112]. FACS meas-urements require high cost instrumentations and high-specialized personnel, thus limiting their use as separativetechniques for the rapid and simple routine analysis ofbacteria.

Flow cytometers have also been exploited to realizesuspension microarray multiplex immunoassays, based onthe use of fluorescent microspheres as a solid phase. Suchsystems are commercially available (e.g., xMAP Technolo-gy, Luminex Corporation, Austin, TX http://www.luminex-corp.com; Bio-Plex 200 System, Bio-Rad, Hercules, CA,http:/www.bio-rad.com) and their applications in pathogenicbacteria detection have been reviewed by Dunbar et al. [113,114]. The availability of a high number of spectrally distin-guished microsphere populations, which can be singularlyinterrogated by flow cytometry instrumentation, providesextremely high multiplexing capabilities. New miniaturizedflow cytometers based on microfluidic systems open newexciting perspectives for the point-of-use applicability ofsuch bioanalytical platform [115–117].

Field-flow fractionation

Fast, simple and portable systems for bacterial cells separa-tion/enrichment are still missing. With this respect, Field-flow fractionation (FFF) techniques show interesting per-spectives towards the development of new pre-analyticaland/or analytical formats for whole cells and bacteria.

FFF are a family of separation techniques able to separateanalytes on a wide dimensional range, from relatively smallbiomolecules to cells, from complex samples. The FFFseparation device is basically a capillary empty channel inwhich a laminar flow of mobile phase and a field appliedorthogonally make the analytes be driven into differentlaminar flows due to their differences in size, density, andsurface properties, resulting in different elution times. Dif-ferent types of field can be used, realizing different FFFvariants [118, 119].

Several FFF-based methods were developed to separatebacteria according to their differences in shape and mor-phology from environmental and clinical samples, withshort analysis times and high sensitivity [120–124]

Being FFF characterized by a “soft” separation mecha-nism, bacteria are separated without modifications of theirnative properties (e.g., enzymatic activity, cell vitality,

20 A. Roda et al.

quaternary proteins structure) and they can thus be collectedand re-cultivated or subjected to further analysis [125, 126].

Such a unique peculiarity of FFF, along with the possi-bility to use biocompatible devices and the possible integra-tion with other analytical methodologies soon showedappealing perspectives. Analytical information on a com-plex sample can be considerably increased through thehyphenation of FFF with high-sensitivity, orthogonal meth-ods giving rise to the development of new fast and multiplexanalytical formats. Examples of new approaches are de-scribed below.

FFF and MALDI/TOF MS

It has been proven that the use of the hollow fiber flow-fieldflow fractionation (HF FlFFF, HF5), the microcolumn variantof FFF employing a hydrodynamic field to structure the sepa-ration, is able to pre-purify or fractionate different species ofwhole bacteria using a MS-compatible mobile phase, allowingdirect MALDI/TOFMS analysis of fractionated samples. Frac-tions purified from non-cellular species (e.g. bacteria metabo-lites) obtained by HF5 were analyzed by MALDI/TOF MSwith a significant improvement of spectra quality. Moreover,after the fractionation of a mixture of two bacteria (Bacillussubtilis and E. coli), enriched fractions were obtained andanalyzed by MALDI/TOF MS showing characteristic ion sig-nals of a species without the presence of characteristic signalsof the other species, as reported in Fig. 4 [127].

FFF and immunological reactions

Gravitational FFF (GrFFF) is the FFF variant that employsEarth’s gravity as the applied field. A new format of flow-assisted non-competitive immunoassay based on the use ofGrFFF and chemiluminescence detection was recently pre-sented for the detection of intact pathogenic microorganisms.A horseradish peroxidase (HRP)-labelled monoclonal antibodyspecific for Y. enterocoliticawas added to the sample containingthe target bacteria, and the mixture was injected into the GrFFF

channel where, upon 30-min incubation, the free and bacterium-bound antibody fractions were separated and on-line detectedand quantified by addition of a chemiluminescent substrate forHRP and photons detection employing a flow-through chem-iluminometer. Figure 5 reports a scheme of the GrFFF-immunoassay. The assay showed analytical performance simi-lar to amicrotiter plate format (LoD 2×106 CFUml−1 and linearcalibration curves in the range between 106 and 108 CFU ml−1)with a reduction in analysis costs and time. Such approachshows interesting perspectives towards the multiplexed analy-sis, since immunocomplex of different bacteria strains could beseparated trough the GrFFF systems due the morphologicaldifferences among cells [128, 129].

The possibility to immunomodulate the fractionation pro-cess in FFF was explored by implementing the biospecificantigen-antibody recognition in a GrFFF channel. Suchhybrid system combining two orthogonal separative princi-ples (antigen-antibody binding and FFF separation) wasemployed to separate mixtures of E. coli O157:H7 and Y.enterocolitica. An anti-Y. enterocolitica antibody wasimmobilized in the injection area of the channel, so that Y.enterocolitica cells were specifically captured, while non-bound analytes were eluted and fractionated under theGrFFF principle. Upon addition of a mobile phase gradientwith a solvent mixture able to break the antibody-antigenbonds, the captured bacterial cells could be eluted from thechannel and subjected to further analysis. The hybrid immu-nomodulated GrFFF system represents a high versatile toolthat could be integrated as a module of pre-analytical clean-up in a point-of-care testing device [126].

FFF and olfactory systems

As discussed in the paragraph “Electronic nose”, electronicnoses are not selective enough to identify bacteria when presentin a complex sample and a preliminary separation is thereforerequired. Recently, some of the authors have developed amethod based on the use of the GrFFF system coupled with aMOS olfactory sensor (EOS 835, SACMI, Imola, Italy, http://

Fig. 5 (a) GrFFF-chemiluminescence immuno-metric method for pathogenicbacteria determination; (b) out-put of the method: representa-tive fractograms. A 0 peak areaof the retained immuno-complex used for the calibrationcurve. Reproduced by permis-sion of The Royal Society ofChemistry from reference [129]

Developments in rapid multiplexed detection of foodborne bacteria 21

www.sacmi.it) for the analysis of volatile metabolites producedby pathogenic bacteria in complex samples, employing E. coliO157:H7 and Y. enterocolitica as model samples. GrFFF sep-aration was accomplished on a 2-mL plastic GrFFF channel, inwhich the mobile phase was delivered by a peristaltic pump(Miniplus Gilson Plus, http://www.gilson.com). Upon sampleinjection through the channel an inlet port, eluting fractionswere detected by a flow-through UV–vis detector (UV6000,ThermoQuest, Austin, TX, http://www.thermo.com) connectedto the channel outlet. Two fractions, corresponding to theelution time characteristic for each strain, were on-line collectedin a fixed volume. They were then subjected to further analysiswith the EOS system, comprising an array of six thin-filmMOSsensors. Data was subjected to principal component analysis(PCA) and linear discriminant analysis (LDA), using V-PARVUS software (http://www.parvus.unige.it). A schematicview of the GrFFF-EOS system is reported in Fig. 6, whileFig. 7 shows typical fractographic profiles obtained when

single strains of E. coli and Y. enterocolitica or bacteria mix-tures were separated through GrFFF. Upon training the EOSsystem employing fractions collected from the injection of asingle strain, the ability of the system to correctly identifybacteria present in a complex sample was tested on fractionscollected after the GrFFF of a bacteria mixture. Figure 8 showsthe score plot obtained by PCA of data obtained from EOSanalysis, which shows satisfactory discrimination between thetwo species. Subsequent LDA analysis yielded a correct clas-sification and prediction ability of respectively 100 and 90 %for Y. enterocolitica and E. coli in the mixture. The combinedGrFFF-EOS system proved to be able to distinguish betweentwo different pathogenic bacteria species present in a samplebased on a simple and versatile approach, which shows poten-tialities towards the rapid analysis of complex samples. Inperspective, the use of GrFFF as a versatile enrichment stepcould be addressed to the selective detection of cells withmetabolic and respiratory activity while excluding inactive,dead cells from the detection. By GrFFF, dead cells could beseparated from live cells by FFF based on different morpho-logical properties.

Fig. 7 GrFFF fractionation of Escherichia coli O157:H7 (light grayline), Yersinia enterocolitica (gray line) and a mixture of E. coli and Y.enterocolitica (black line). F1 (4.5 to 7 min), F2 (13 to 16.5 min):collected fractions

Fig. 8 Score plot of the first two principal components obtained byperforming PCA on data obtained by subjecting to EOS system anal-ysis the fractions collected from the injection of Escherichia coli O157:H7 (black circles) and Yersinia enterocolitica (open circles) in theGrFFF device

Fig. 6 GrFFF separation system-EOS. Aliquots of 50 μL of Escher-ichia coli O157:H7 and Yersinia enterocolitica bacteria (single strainsand mixture) at a concentration of 2.4×109 CFU mL−1, were injectedin the GrFFF system and fractionated using phosphate buffered saline

(PBS) as mobile phase at 1.5 mL min−1 flow rate. Fractionated sampleswere placed in the sensor chamber and incubated at 45 °C for 15 min,and then the headspace was sampled. Five repeated measurement wereperformed for each samples

22 A. Roda et al.

Table 2 Characteristics of bioanalytical methods for foodborne pathogen bacteria detection.

Advantages Drawbacks

Biospecific binding assaysa - Generally offer good specificity in complexmatrices

- Need to obtain biospecific binding molecules

- Skilled manpower required

PCR-based nucleic acids detection - High sensitivity - Laborious and time consuming, especially concerningnucleic acids extraction from the sample

- Real-time PCR provides quantitativeinformation

- Sample matrix contaminants may inhibit polymeraseenzyme activity

- Amenability to miniaturization and automation - Viable and non-viable cells are not distinguished (ethi-dium and propidium monoazides have been proposed tosolve this issue)

- Limited multiplexing capacity

- Generally not applicable to on-site measurements

DNA microarrays - High multiplexing capacity - Long assay times (analyte binding is usually driven bypassive diffusion)

- Laborious data elaboration

- Viable and non-viable cells are not distinguished

- Not applicable to on-site measurements

Immunoassays - High specificity, even in complex matrices - Low sensitivity, usually pre-analytical enrichment isrequired

- Recombinant antibodies and other bindingmolecules (e.g., aptamers) are available toovercome antibodies limitations

- Specificity and sensitivity are largely dependent on thequality of antibodies

- Some assay formats are applicable to on-sitemeasurements (e.g., lateral flowimmunoassays)

- Difficulties in obtaining high performance antibodies forcertain strains

- The antibody binding ability might be compromised incase of changes of expression or denaturation of thetarget antigen

- Antibodies might be unstable in complex samplematrices

- Viable and non-viable cells are not distinguished

Biosensorsa - Rapidity - The applicability on real food samples is still to bedemonstrated in most cases

- Low sample and reagents volumes

Integrated miniaturized analyticaldevices (e.g., lab-on-chip; suspen-sion microarrays)

- Automated portable devices, suitable for point-of-care applications

- Further technological advances required to integrate allthe analytical process steps in the device

- Potential high multiplexing abilities

Optical biosensors - High sensitivity - Limitations in miniaturization possibilities

- Multiplexing abilities -High-cost and complex instrumentation, especially in thecase of label-free systems

Electrochemical biosensors - Amenability to miniaturization - Limited sensitivity

- Low-cost; possibility to develop disposableformats

- Multiplexing can be hampered by technical difficultiesin obtaining an output form multi-electrode arrays

Nanotechnologies (e.g.,nanoparticles, nanotubes,nanowires, nanofunctionalizedsurfaces)

- Suitable for device miniaturization - Few applications to real sample analysis

- High assay detectability and selectivity - Need of characterization and standardization

- Reduced assay times

- Potential high multiplexing abilities

- Able to improve performances of several assayformats

Fingerprint-based assaysa - No need for biospecific binding molecules(e.g., antibodies, nucleic acid probes)

- Strict protocol standardization is required to obtainreasonable reproducibility

- Rapidity - Predictive performance depends on the quality oflibraries

Developments in rapid multiplexed detection of foodborne bacteria 23

Conclusions and future perspectives

In the last ten years, despite relevant improvements in foodsafety procedures adopted in developed countries, the mi-crobial contamination deriving from foodborne pathogenicbacteria still represents an unsolved problem and strategiesfor prevention and early detection are the main tasks. Un-fortunately conventional pathogen detection methods, al-though sensitive enough, are often too slow with analysistimes on the scale of hours to days and therefore not appli-cable for large-scale surveillance efforts and on-field use.

In recent years, several bioanalytical methodologies andbiosensors have been developed for foodborne bacteria detec-tion, achieving in some cases very low LoD and multiplexcapabilities. Portable and user-friendly analytical formats havebeen also proposed, allowing screening procedures to beperformed directly where the sample is obtained. Neverthe-less, combining all such features on a single analytical plat-form still represents a challenge for academic researchers anda stimulating commercial opportunity for R&D companies.

The development of multiplex assays is currently a hot topicof research, since they would allow larger screening proceduresfor a variety of pathogens in one assay, cutting assay time andcosts, thus providing powerful tools to increase food safety. Inaddition, multiplex abilities could also increase assays reliabil-ity, especially when highly specific capture probes are notavailable, by requiring a response from multiple less-specificprobes for a positive result on the target pathogen. Table 2

reports the main advantages and drawbacks of the principalbioanalytical methods described in the present review.

New analytical formats and the hyphenation of identifi-cation techniques with separation step such as field-flowfractionation open interesting perspectives in the field ofrapid and multiplexed foodborne pathogens analysis.

Despite large number of technological solutions de-scribed to increase analytical performance, only very fewmethods have been validated and thoroughly characterizedfor their applicability to real food samples. Thus, furtherwork is required before new analytical platforms are accept-ed for their application in screening procedures.

It is presumed that in the near future multiplex bioanalyticalassays will be commonly applied for on-site screening proce-dures, followed by confirmation analyses employing referenceanalytical methods.

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Table 2 (continued)

Advantages Drawbacks

- Possibility to create and share betweenlaboratories libraries of spectra/fingerprint

- Difficulties in analyzing samples in which two or moremicroorganisms are simultaneously present (couplingwith separative techniques is a possible solution)

- The target bacteria can be identified within alarge cohort of candidates, based on theavailable libraries

- Applicability to large screening procedures

Vibrational spectroscopy - Non destructive - Pre-analytical enrichment is necessary to eliminate ma-trix interferents and/or to obtain sufficient biomass formeasurement

- High discriminatory power (at the species,subspecies and strain level)

- Ability to distinguish between viable and non-viable cells

MALDI/TOF mass spectrometry - Large libraries available - High cost instrumentation

- Possibility to exploit the continuous growingproteomic data libraries

- Non applicable in point-of-care settings

- Sample matrix interferents may lead to ionizationsuppression phenomena

Electronic nose - Non destructive - Difficulties in sharing libraries between laboratories

- Relatively simple instrumentation that in somecases can be operated on site

- Pre-analytical enrichment is necessary for real sampleanalysis

a The reported characteristics also apply to the subsequent sub-categories.

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