Food Biochemistry and Food Processing (2 edition)

BLBS102-c45 BLBS102-Simpson March 21, 2012 14:38 Trim: 276mm X 219mm Printer Name: Yet to Come

45 Biosensors for Sensitive Detection of Agricultural Contaminants, Pathogens and Food-Borne Toxins 861

3

1

2

Resonance signal (RU)

4

Time (s)

1 2 3 4

Immobilised Association Dissociation Regeneration

surface

Figure 45.2.Schematic representation of a Biacore sensorgram,

which provides information relating to the biomolecular interaction

between the immobilised antibody and its cognate antigen

(relevant analyte).

(Markey 2000). However, when the light is above a particular

angle of incidence, no light is refracted across the interface and

TIR occurs. Even though the incident light is reflected back

from the interface, an electromagnetic field (called an evanes-

cent wave) penetrates a distance of the order of one wavelength

travelling into the less optically dense medium.

A key advantage of SPR over other optical methods of detec-

tion is that SPR measures the interaction between immobilised

molecules on a surface (e.g. an antigen, or an antibody im-

mobilised onto a sensor chip) and the corresponding ligand in

solution passing over this matrix. This means that the reaction

can be measured in a coloured solution or in a turbid complex

matrix, such as in fruit juice or vegetable preparations. Hence,

there is no requirement for food samples to be pre-treated to

remove contaminating coloured species prior to analysis.

The Biacore system has a lower limit of detection (LOD)

of approximately 10 RU (which is approximately 10 pg/mm^2 ),

which is suitable for the detection of trace amounts of analytes

of interest, such as pesticide or herbicide residues. There are

several platforms available for use. The Biacore Q instrument

has major potential for quality control analysis, whereas the

Biacore 3000 is particularly useful for high sensitivity antibody-

based analyte detection and kinetic analysis. The T100 and the

Table 45.3.The Ideal Characteristics of a Biosensor

1. The biorecognition element must be highly specific for
   the substrate or antigen.


2. Re-usable, with multiple readings permitted on a single
   device.


3. High sensitivity.


4. Cost-effective.


5. Permit rapid or ‘real-time’ analysis of biomolecular
   interactions.


6. Use of ‘on-line’ or ‘in-situ’ measurement.


7. Good signal to noise ratio.


8. The device should be robust.


9. The ability to measure samples in a high throughput
   fashion, if required.


10. Fast turnaround time on analysis.


11. Ease of use.

more recently developed A100, permit high-throughput analy-

sis of protein–protein and protein–ligand interactions that may

be useful in the judicious selection of antibodies for specific

diagnostic applications.

Sensor Surfaces

When selecting a biosensor-based platform, such as Biacore, for

the evaluation of quality of food produce, several key consid-

erations should be made. The first of these relates to the nature

of the analytical surface that will be used in the assay. One of

the primary reasons why Biacore is commonly selected for food

analysis (aside from the excellent sensitivity and flexibility for

designing a suitable assay) relates to the fact that many different

surface chemistries are available for conjugating molecules onto

the sensor chip surface to perform the assay. Biacore uses sensor

chips, such as those mentioned in Table 45.4, and the choice of

chip chemistry is dependent on a number of different factors,

including the desired application of the assay, the physical and

chemical properties of the binding partners and the nature of the

biomolecular interaction.

The CM5 chip is the most versatile Biacore chip currently

available and is the most frequently used for food analysis. Its

matrix consists of a completely modified carboxy-methylated

dextran covalently attached to a gold surface, and is ideally

suited for the analysis of a variety of different binding events,

ranging from those involving small organic molecules to multi-

domain proteins. Analytes of interest can readily be coupled to

the sensor chip surface through the application of a number of

different cross-linking chemistries (e.g. amine, thiol or aldehyde

coupling), and a stable surface can be produced that permits ac-

curate and repeated analysis on a single surface (Dillon et al.

2005) as non-covalently bound entities can readily be removed

from the surface by a process termed regeneration. Typically,

this is achieved with low concentrations of acid (HCl) or base

(NaOH). As an example, for the detection of a bacterial strain in

a complex sample matrix (e.g. coleslaw), a pathogen-specific

antibody may be immobilised on a CM5 surface by amine