Food Biochemistry and Food Processing (2 edition)

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870 Part 8: Food Safety and Food Allergens

associated risk of fatality (Hearty et al. 2006). While the natural

ecosystem of this bacterial strain includes soil, water, plant ma-

terial and decaying plant detritus (Suihko et al. 2002), numerous

foods, such as raw vegetables, fruits, and horticultural samples,

are also prone to infection. Furthermore, the psychrophilic nature

of this strain, conferring the ability to grow at refrigeration tem-

peratures, and the ability of this bacterium to withstand high salt

concentrations and tolerate a wide pH range suggest that contam-

ination of foodstuffs such as coleslaw is a frequent occurrence.

Leonard et al. (2005) devised a rapid SPR-based immunosensor

assay for the detection ofL. monocytogenes, which incorpo-

rated a polyclonal antibody specific for internalins. Internalin

B (InlB), a protein on the surface ofL. monocytogenes, partic-

ipates with Internalin A (InlA) in the invasion of mammalian

cells. In this assay format, the antibody was immobilised on a

CM5 sensor chip and varying concentrations of cells were then

injected over the surface. A detection limit level of less than 2×

105 cells/mL of sample was reported. Hearty et al. (2006) gen-

erated a monoclonal antibody that specifically interacted with

the InlA surface protein, and demonstrated the efficacy of this

antibody by developing a Biacore-based assay capable of detect-

ing 1× 107 cells/mL. Finally, Tully et al. (2006) described the

use of quantum dot-labelled antibodies, specific for InlA, for the

immunostaining ofL. monocytogenescells, and demonstrated

that these have major potential for use with fluorescence-based

sensor formats. These three examples demonstrate the efficacy

of detecting bacterial pathogens through the identification of cell

surface epitopes and subsequent generation of antibodies. Bac-

terial cells, in particular, present an array of different antigenic

determinants, including capsular, flagellar and surface antigens.

While carbohydrate elements may also, in theory, be targeted,

these typically have lower immunogenic potential than their

proteinaceous counterparts, and therefore are rarely used for

biorecognition.

Many of the more recent examples of biosensor-based bacte-

rial pathogen detection have focused on the use of electrochemi-

cal platforms, including impedimetric assays. Tully et al. (2008)

described an impedance-based assay for the analysis of theL.

monocytogenesInB protein, which used a leporine host-derived

polyclonal antibody as a bioligand. The assay format developed,

which used planar screen-printed carbon electrodes modified

with PANI for antibody capture, had an excellent LOD for InlB

(4.1 pg/mL). Additional electrochemical platforms have also

been developed for the detection ofS. typhimurium, a pathogen

typically transmitted to human hosts through the ingestion of

contaminated animal produce, such as meat, eggs or milk. Nan-

dakumar et al. (2008) devised an antibody-based impedimetric

assay capable of detecting 500 colony forming unit(CFU)/mL

ofS. typhimuriumin less than 10 minutes. Interestingly, the au-

thors suggested that the developed methodology would be of

particular use in portable pathogen detectors, due to the rela-

tively low complexity of the sensor format and the necessity

for a small number of data samples (30) to provide accurate

pathogen detection.

Biosensors are particularly useful for the rapid detection of

analytes of interest in complex sample matrices, and this also

applies to the detection of bacterial pathogens where additional

methodologies may be used in conjunction with immunosensing

for pathogen retrieval. Immunomagnetic separation (IMS) may

be applied to initially pre-concentrate bacterial cells from a food

sample prior to biosensor-based analysis (Byrne et al. 2009)

and, as with all immunodetection-based strategies, the efficacy

of this approach is dependent on the quality of the antibody that

is selected for biorecognition. Li ́ebana et al. (2009) used a com-

bination of IMS and magnetic electrode-based electrochemical

biosensing for detectingS. typhimuriumcells in milk, with the

antibody capable of selectively differentiating between target

andE. colicells in this matrix. Detection was verified through

the introduction of a second HRP-labelled anti-Salmonellaan-

tibody, thereby creating a sandwich assay format on the sensor

surface that could be completed in less than one hour. Interest-

ingly, the sensitivity of this assay was influenced by the nature

of the samples selected for analysis. WhenS. typhimuriumcells

were propagated in Luria Bertani (LB) medium, the LOD was

5 × 103 CFU/mL. In spiked milk samples diluted tenfold in LB

medium, the sensitivity was 7.5× 103 CFU/mL. However, when

the milk was pre-enriched for six and eight hours, the limits of

detection could be significantly reduced to 1.4 CFU/mL and

0.1 CFU/mL, respectively, suggesting that sample preparation

has a significant impact on assay sensitivity.

Salam and Tothill (2009) also developed an electrochemical

immunosensor forS. typhimuriumdetection that was applied for

the analysis of chicken breast samples. The assay format con-

sisted of a murine monoclonal antibody captured on a screen-

printed gold electrode via amine coupling. Similarly to the ex-

ample described above by Li ́ebana et al. (2009), a secondary

HRP-labelled polyclonal antibody was selected for enhancing

specificity. The assay format permitted rapid cross-reactivity

analysis to be performed with additional bacterial strains, in-

cludingStaphylococcus aureus,Pseudomonasspp. andKleb-

siella pneumonia(all at a concentration of 1× 109 CFU/mL).

This is an important consideration for pathogen detection, and

the ability to develop a species-specific immunoassay and sub-

sequently, screen for non-specific binding to additional bacterial

strains/pathogens is essential.

Liao and Ho (2009) describe a novel electrochemical sen-

sor for the detection ofE. coliO157:H7. Instead of selecting

an epitope on the bacterial cell for immunorecognition, a gene

encoding an enzyme that participates in O-antigen assembly

(rfbE) was targeted by a single-stranded DNA probe, and the re-

sultant assay had excellent sensitivity (0.75 amol) for the target

gene. In another excellent example, Koo et al. (2009) devel-

oped an ‘antibody-free’ biosensor platform for the detection of

L. monocytogenes. Here, the biomolecular interaction between

theListeriaadhesion protein (LAP, also referred to as alcohol ac-

etaldehyde dehydrogenase) and biotin-tagged heat shock protein

60 (HSP60) was used to facilitate the detection of this oppor-

tunistic pathogen in a matrix containing additional pathogenic

bacterial strains, includingBacillus cereusandProteus vulgaris.

Of particular interest in this study was the observation that the

efficiency of detection observed through the use of the HSP

biorecognition element was over 80-fold higher than for an anti-

L. monocytogenesmonoclonal antibody previously developed

in the same laboratory.