Technological advances and new tools for food safety control

Katrina Campbell Lecturer in Bioanalytical Systems

(katrina.campbell@qub.ac.uk)

2nd International Symposium on Innovation in marine products and food industry Vigo, 15th September 2014

Global Food Safety System Food Safety has always been essential for existence

Supply versus

demand

Impact: Biological

Economical Social

Political

Farm to Fork Under the current EU Food Hygiene legislation Producing safe food is

the responsibility of Food Business Operators (FBOs)

Hazard Analysis and Critical Control Points (HACCPs) checks

• Source of raw materials • Production site • Processing sites and distribution • End product testing

FOOD CONTAMINANT TESTING IS MAINLY ONLY PERFORMED IF

LEGISLATIVELY REQUIRED AND IF SUITABLE METHODS ARE AVAILABLE

What is there in food worth measuring? Basic components (protein, carbohydrate, fibre fats etc)

Additives (vitamins, colourants, stabilizers, nanoparticles)

Contaminants

Chemical Microbiological Process Natural Man made Bacterial / Viral

Heat Contact

RASFF Contaminants

Why food safety analysis?

Measuring Food Contaminants Verify the presence or absence of contaminant in a sample:

Qualitative Test

Quantify the amount of contaminant in a sample: Quantitative Test

An analytical method which will give a strong indication that there is some form of chemical contaminant present

Screenings tests

Confirmatory tests An analytical method which gives unequivocal proof that there is some form of chemical contaminant present

1. Functional Assays a. Animal assays b. Cell based c. Receptor based d. Enzyme based e. Fluorescence based

2. Biochemical Assays a. ELISA b. Lateral flow devices c. Biosensor

Level of contaminant measured is relative to the biological effect of the sample

May detect new toxic analogues Contaminant identification is not unequivocal Technology transfer of methods is difficult

Binder assays Toxicity does not always correlate with binder

cross-reactivity Sample preparation and data analysis is fast Screening tools for HACCP management and

rapid response

Methods applied to food analysis

4. Analytical methods a. HPLC b. LC-MS c. GC-MS d. ICP-MS

Contaminants can only be identified and quantified for available analytical standards

Toxicity equivalent factors must be applied Sample clean-up is extensive with oxidation

steps being required in cases Data analysis is laborious LC-MS is unequivocal for identification

3. Spectroscopic methods a. Near IR b. Mid IR c. RAMAN d. SERS

Fingerprinting techniques Non-destructive methods little to no sample prep Require chemometric models of known samples Sensitivity is questionable

Methods applied to food analysis

ANALYTICAL METHODS TRADITIONAL CONFIRMATORY METHODS

Screening analysis by animal assays, functional methods,

immunochemical

Selected samples taken from farms (blood) and slaughterhouses (meat

and urine) and feedmills (feed), production sites (various foods)

Monitoring plan for food analysis

Compliant result No further action -VE

Confirmatory analysis and quantification by

physiochemical methods usually LC-MS

+VE

BELOW

Non compliant result Action required

ABOVE

Screening Tests for Food Analysis Animal Based Cell Based Receptor Based Antibody Based

Antibiotics residues in milk

Dioxins in feed & food

Chemical contaminants in foods

Toxins in food Botulism

Marine toxins

Screening tests that require special facilities for use

Low molecular

weight Contaminant

Antibody Production

Protein Conjugation

Protein carrier

Immunisation Programme Polyclonal

antibody production

Titre Specificity

Major Problems:

Toxicity

Source of contaminant

Specificity

Antibodies specific for a desired antigen can be conjugated with a radiolabel, fluorescent label, or colour-forming enzyme & are used as a "probe" for detection.

Immunological methods for food analysis

1960: Radioimmunoassay was first described in a scientific paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960 1971: Peter Perlmann and Eva Engvall at Stockholm University invented ELISA 1975: Generation of the 1st monoclonal antibodies (George Kohler & Cesar Milstein)

Lateral Flow Tests Rapid & simple

Antibody Coated Competitive ELISA

Antibody coated

No toxin in sample Antibody captures labelled toxin

High response

Toxin in sample Antibody captures toxin

Wash step removes labelled toxin Low response

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Antigen Coated Competitive ELISA

Toxin protein conjugate (TPC)

No toxin in sample Antibody binds to TPC

Labelled antibody binds to antibody High response

Toxin in sample Antibody binds to toxin

Wash step removes antibody Low response

Screening Tests for Food Analysis Rapid Reliable Low cost Low false positives No false negatives Safe

Speed – higher throughput Cost Portability Multiplexing Remote or in situ sensing Requirements of regulators or industry End product testing for release systems

Food is produced on an ever-increasing scale

To enhance and ensure GLOBAL FOOD SECURITY

new biosensor technologies and applications are needed

Antibody Based

Chemical contaminants in foods

Biosensors

http://packagingtech.net/34-biosensor-technology-for-food-packaging-applications.html

“A biosensor is an analytical device incorporating a

biological or biologically derived sensing element either intimately associated with or

integrated within a physicochemical transducer. The usual aim is to produce a digital

electronic signal which is proportional to the

concentration of a specific analyte or group of analytes”

Turner, A.P.F., Karube, I. and Wilson, G.S. (1987). Biosensors: Fundamentals and Applications. Oxford University Press, Oxford.

Biacore’s methods: Vitamin analysis for fortified foods Antibiotics in honey and screening for veterinary drugs Mycotoxin analysis in cereals and feeds Marine toxins: Domoic, okadiac and DTXs, PSPs, TTX, BTX Freshwater toxins: Microcystins and cylindrospermospin

SPR biosensor technology

Benefits: Simplicity of operation Minimal sample preparation Interchangeable chips for multiple targets Single channel operation

SPR Biosensor Invented by

Liedberg, Nylander,

Lunström (1983)

Drawbacks: Single target analysis, primarily laboratory based and relatively expensive

http://www.google.co.uk/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=GXHWCFK6xnNasM&tbnid=X55vlhQZdCNRMM:&ved=0CAUQjRw&url=http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/GELifeSciences/14110011&ei=nT9BUog5w9XiBL2OgDA&bvm=bv.52434380,d.bGE&psig=AFQjCNGxLLLwgkj3YAmhqh1p3cSfASktfw&ust=1380094233067556�

The use of multiplexing surface plasmon resonance

platforms is a major research focus at IGFS.

• 3 channel, fully automated SPR system • Control and data analysis software • Temperature controlled reagent racks

Nomadics SensiQ Platform

SPR biosensor technology

Individual SPR assays for DON & NIV and T2 &HT2 combined as a proof of concept

Meneely, J. P., Fodey, T., Armstrong, L., Sulyok, M., Krska, R., Elliott, C. T., 2010, Rapid surface plasmon resonance immunoassay for the determination of deoxynivalenol in wheat, wheat products, and maize-based baby food, Journal of Ag and Food Chem, 58, 8936-8941. Meneely, J. P., Sulyok, M., Baumgartner, S., Krska, R., Elliott, C. T., 2010, A rapid optical immunoassay for the screening of T-2 and HT-2 toxin in cereals and maize-based baby food, Talanta, 81, 630-636.

•Thermally insulated flow-cell and fluidics • Multiple sample injection options • Process over 300 samples in 24 hours

Multichannel Multiplexing

Sample compartment

Chip dockingcompartment

IFC

Running buffers

and waste

Peristaltic pumps

Injection needles

A 16 channel SPR biosensor

developed as part EU Biocop project

Campbell, K., McGrath, T., Sjölander, S., Hanson, T., Tidare, M., Jansson, O., Moberg, A., Mooney, M., Elliott, C., Buijs, J. (2011) Use of a novel micro-fluidic device to create arrays for multiplex analysis of large and small molecular weight compounds by surface plasmon resonance. Biosensors and Bioelectronics, 26: 3029-3036. McNamee, SE, Elliott, CT, Delahaut, P & Campbell, K 'Multiplex biotoxin surface plasmon resonance method for marine biotoxins in algal and seawater samples 2013 Environmental science and pollution research international, vol 20, no. 10, pp. 6794-807.

Toxin Antibody Titre IC50 (ng/ml) IC20 – IC80 (ng/ml)

Domoic Acid DA-Ab 1/200 2.6 1.0 – 6.4

Okadaic Acid OA-Ab 1/4000 4.9 1.7 – 14.4

Neosaxitoxin NEO-Ab 1/25 2.6 1.1 – 6.0

Saxitoxin STX-Ab 1/1000 1.9 1.0 – 3.7

Assay principle: indirect competitive assay with chemiluminescence detection

Munich MCR3 - Chemiluminescence

MCR3 Instrumentation

Toxin (ng/ml)

Domoic Acid Okadaic Acid Saxitoxin

Toxin IC50

(ng/ml) Saxitoxin 1.3

Domoic Acid 6.5 Okadaic Acid 2.9

Why nanosensors? Smaller and faster Require less power to run Greater sensitivity Better specificity Cost-effective Remote use Simple to use

SPR Biosensor

Bio to nanosensors

High Tech MS analysis

Multi mycotoxin methods Multi pesticide methods Untargeted analysis Fingerprint profiling

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On site or end product testing

Production of nanosensors

Localised SPR analysis - miniaturisation

Multiplexing ELISA

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Multiplex approaches for emerging concerns

ergotamine

ergocristine

CTRL

Ergot Alkaloid

scopolamine

atropine

CTRL

Tropane Alkaloid

CTRL

Pyrrolizidine Alkaloid

Analysis time = 45mins Detection of 15 PA compounds

Semi-portable multiplexing technology Luminex

Technology Toximet

Technology

Mycotoxin analysis - Aflatoxins

Portable SPR

Sensing Unit: high resolution camera Operation via touch screen Graphics processing unit Analysis Tools: Smart applications Large memories Data transmission via mobile networks

Seattle Sensors Multiple target detection for

remote analysis 4 x 3 channel

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

140.00%

0.01 0.1 1 10

[OA] (ng/ml)

Perc

ent R

ate

of B

indi

ng

Okadaic Acid Detection

Electrochemical Sensors

Environmental or chemical sensors for toxin analysis

Calibration curve for a biosensor prototype with 40 um gold microdisc array

Calibration curve for a biosensor prototype with 10 um gold microband array

Rapid Multiplex Portable Diagnostics

Aim to produce a lower cost platform offering • Low cost analysis • Simplicity in use • Highly specific single target analysis • Multiplexing – multiple target analysis • Bespoke sensitivity • Robust – high performance • Field deployable

Suitable for source to product testing

Up to 100 feature microarray

Planar waveguide with custom surface

chemistry

Multiplex Mbio Diagnostics

Scientific know how is based in the cartridge Different approaches in how this can be designed

• Molecular level – DNA / RNA for pathogen and speciation testing • Protein Level – Allergen testing eg milk, nuts, eggs, seafood • Residual level – Low molecular weight toxins / antibiotics / contaminants

Nanotechnology in Microarrays

Competitive inhibition assay

• Toxins are immobilised on the cartridge surface through nanospotting.

• Sample/antibody are mixed and added to the cartridge.

• Incubation with a secondary labelled antibody .

• Light directed to the bottom of the cartridge allows excitation of the fluorophores of the labelled antibody.

• The sensitive detection of binding events on the cartridge surface is observed.

• When toxin is present there is reduced antibody binding to surface.

Important to implement simple testing regimes to allow FBOs to perform testing

Offer a simple device requiring minimal sample preparation through either simple fluid application (blood, milk, juice) or dissolution of solid foods in buffering reagents

Simplicity in Use

Depending on user Qualitative – YES/NO Answer Quantitative – Concentration

Add Sample to Assay Buffer

Dye-labeled Ab

Transfer to Cartridge

Read at 5-10 min

Biotoxin Assay n IC50 (ng/ml)

Dynamic Range IC20 – IC80 (ng/ml)

Domoic acid Single 2 0.13 0.02 – 1.13

Multi 4 1.86 0.52 – 5.71

Okadaic acid Single 2 0.35 0.12 – 0.68

Multi 4 0.42 0.20 – 0.88

Saxitoxin Single 2 0.04 0.02 – 0.06

Multi 4 0.06 0.02 – 0.16

Microcystin Single 2 1.48 0.52 – 3.71

Multi 4 1.40 0.48 – 3.64

Cylindrospermopsin Single 2 0.23 0.07 – 0.55

Multi 4 0.19 0.08 – 0.52

Aquatic Toxins

Marine and fresh water toxin assay

Antibiotics in milk

Multiplex Test for detecting

antibiotics at MRL values

Synthetic Binders

Recombinant antibodies No use of animals

Phage display technology Synthetic binders from libraries Tolerant solvent and pH

Aptamers for food contaminants

single stranded DNA or RNA oligonucleotides that range in size

from 20-80 bases

Spectroscopic techniques “fingerprinting” technique giving unique spectra Little or no sample preparation Ideal technique for use with adulteration of food eg fats and oils Multivariate techniques can be used to extrapolate the desired chemical information

Image Detector Processor Data Answer

Animal Dog

Alsatian German Shepherd

Speed – higher throughput Simplicity in use Minimal sample preparation Relatively low cost Multiplexing Portability Remote sensing Requirements of regulators or industry End product testing for release systems

Summary for Rapid methods

EU Project Collab4Safety

http://web.spi.pt/collab4safety/collab4safety

Technological advances and new tools for food safety control

Katrina Campbell Lecturer in Bioanalytical Systems

(katrina.campbell@qub.ac.uk)

2nd International Symposium on Innovation in marine products and food industry Vigo, 15th September 2014