Institute of Biochemistry Russian Acad. Sci., Moscow, Russia

Food Safety Symposium July 16, 2015 – University of Mautritius

IMMUNOTECHNIQUES IN FOOD SAFETY:

ADVANTAGES, LIMITATIONS AND SOLUTIONS

Zherdev Anatoly

Variety of analytical techniques for food control

MS-MS

LC/GC-MS

HPLC-F

HPLC-UV

ELISA

TEST STRIPS

MICROBIOLOGICAL

Two directions of analytical techniques

Stationery equipment for specialized laboratories

• More parameters

• Possibility of final decision

• High accuracy

• High cost

• Qualified personel

Portable devices for on site testing

• Low cost

• Simple protocols

• Possibility of wide screening

• Limited quantity of parameters

• Additional (confirming) assays may be needed

«Map of the battles» for immunoassays development

Dzantiev et al. Biotechnologia Acta, 2013, 6 (4), 94-104.

ELISA,

RIA,

FIA,

etc.

SPR-based

assays,

Flow- injection

assays,

etc.

Homo

+

heterogeneous

immunoassays:

magnetic separation,

polyelectrolyte

separation, etc.

Lateral flo w,

through- flow

immunoassays,

etc.

PFIA,

EMIT,

etc.

Kinetic

heterogeneous

immunoassays

IP etc.

Rapidity

Se

nsit

ivit

y

Fast

Low

LOD

Slow

High

LOD

New labels

FRET-basedimmunoassays

Bio-barcodeassay

Single molecular

detection

Cascadeamplification

Classic

heterogeneous

immunoassaysClassic

flow

immunoassays

Classic

membrane

immunoasssays

Classic

homogeneous

immunoassays

First immunoassays

New labelsand detectors

Main demands to screening tests

• Reduction of assay duration

(from hours to minutes)

• Lesser labor intensiveness

(from manual manipulations

to autonomous / automatic processes)

• Increased quantity of controlled parameters

(from mono-parametric to multi-parametric assays)

• More operate measurements

(from stationery equipment

to of out-of-laboratory analytical techniques)

The ideal POC diagnostic device quantitatively detects several analytes, within minutes, at femtomolar sensitivity from 1 µL of bodily fluid and reports the encrypted results to an electronic record. The device may use an increased volume of sample with very low concentrations of analytes. The chip is disposable and the

mass manufacturing device cost is less than $1. (Advanced Materials, © 2010 IBM Corporation)

Biosensor of dream (2010)

The future is here. It's just not evenly distributed yet. W. Gibson, American science fiction writer

Ideal analytical devices: 1. detect – several compounds 2. – quantitatively 3. – in a few minutes 4. – with femtomolar sensitivity 5. – in small (from 0.001 ml) samples 6. send annotated results 7. reach lower detection limits for larger samples 8. disposable 9. Cost less than $1 in mass production.

Biosensor of dream (2015)

Principle of immunochromatographic assay

Advantages of immunochromatography

• All reactants are applied onto membranes

• Contact of sample and test-strip initiates all further processes

• The assay can be carried out without any additional stages

• The assay results may be estimated visually

Immunochromatography – limitations and their overcoming

Dzantiev B.B. et al.

Trends in Anal. Chem.,

2014, 55, 81-93.

Factors influencing parameters of immunochromatography

Dzantiev B.B. et al.

Trends in Anal. Chem.,

2014, 55, 81-93.

The given factors allow to change cut-off levels of the tests at two orders (from 1000 to 10 ng/ml)

0,01 0,1 1 10 100 1000

0

10

20

30

40

50

Chloramphenicol-protein

molar ratios

[Chloramphenicol], ng/ml

Lin

es inte

nsity, arb

. units

1:40

1:15

1:5

0,01 0,1 1 10 100 1000

0

10

20

30

40

Antibody adsorbed on colloidal gold

from concentrations (ug/ml)

1

2.5

5

[Chloramphenicol], ng/ml

Lin

es inte

nsi

ty, arb

. units

10

Hapten : carrier ratio of immobilized conjugate

Antibody loading on colloidal gold

Composition of the conjugates influences on the assay cut-off

Zvereva E.A. et. al.

Analytical Methods,

2015 (accepted).

Nanoparticles for bioassays

Different

changing

parameters

A lot of perspective

labels and carriers

• Increased surface for immobilized molecules

• Possibility of pseudo-homogeneous reactions

• Different approaches for sensitive detection of labels

Factors determining advantages of nanoparticles

Gold nanoparticles

with different structure

and optical properties

Impact of gold conjugate size into the assay characteristics

• The variation of the conjugates composition leads to a change of the PVX

detection limit from 80 to 2 ng/ml

• The lowest limit of detection is reached for the conjugates with 33 nm diameter

6 nm

12 nm

23 nm 33 nm

52 nm

-20

0

20

40

60

80

1001.00E+08 1.00E+09 1.00E+10 1.00E+11 1.00E+12

Lim

it o

f d

ete

ctio

n,

ng

/ml

Equilibrium binding constant, M -1

10 8 10 9 10 10 10 11

IgG-Gold

Safenkova I. et al. Anal. & Bioanal. Chem.

2012. 403 (6): 1595-1605.

Approaches for signal amplification

in immunochromatographic assay

Signal amplification by the use of anti-species – colloidal gold conjugate

Antibody – anti-species antibody interactions as tool to improve sensitivity of immunochromatography

Decrease of the limit of detection in aflatoxin B1 assay

Magnetic separation and concentration: integration of homo- and heterogeneous assays advantages

Advantages: • High sorption capacity of dispersed nanoparticles • Reducing the time of diffusion processes • Easy separation of the reaction mixture

ELISA with magnetic nanocarriers

Traditional ELISA

ELISA of aflatoxin B1 (AFB) with the use of magnetic nanoparticles

Preconcentration allows 10-fold lowering

the limit of detection (up to 1 pg/ml)

0,01 0,1 1 10 100 1000

0

20

40

60

80

100

A,

%

Aflatoxin B1, pg/ml

ELISA using MP

ELISA using MP

with preconcentration

Two formats of aflatoxin B1 ELISA using magnetic nanoparticles

Assay time – 20 min

Property Result

Cores from elements

of II/VI and III/V

groups of the

Periodic Table

High storage stability

High photostability

Different polymeric

coatings

Biocompatible conjugates

Surface stabilization

High quantum yield

Variable core size

(1-10 nm)

Use of size-dependent

energy tranfers

Size-dependent

fluorescence

(emission peak at

500-1000 nm)

Possibility of multicolored

emission for multiassays

Absence of influence of

biomatrix fluorescence

Fluorescence

intensity in

accordance with light

excitation

«On-off» regulation of the

test-systems responce

Advantages of quantum dots as analytical labels

QD CG

Limit of detection, ng/ml 0.2 4.8

Working range, ng/ml 0.3-10 9-210

Portable fluorescent reader

Comparison of quantum dots (QD) and colloidal gold (CG)

y = 0.717-0.211 lg x R2 = 0.998

Application of quantum dots for immunochromatography of chloramphenicol

0 0.3 1 5 ng/ml

Calibration curve

Flu

ore

sce

nce

inte

nsi

ty, r

el.

un

its

Chloramphenicol, ng/ml

Berlina A.N. et al.

Anal. Bioanal. Chem.

2013, 405 (14), 4997-5000.

From mono- to multiparametric immunochromatography

F, test-strip with a two-dimensional array

of spots containing reagents of different

specificity

D, E, test-strips with successive binding

zones containing reagents with different

specificity

B, C, cassettes and devices to combine

single-parameter strips

A, single-parameter test-strip

Dzantiev B.B. et al.

Trends in Anal. Chem.,

2014, 55, 81-93.

Ofloxacin Chloramphenicol Streptomycin

Limit of detection, ng/ml 0.008 0.09 0.2

Range of measured concentrations, ng/ml

0.01-200 0.13-10 0.3-500

Assay duration, min 10

Fluorescent «traffic light» in immunochromatography

Zones:

OFL

CHL

STR

OFL –

CHL –

STR –

OFL +

CHL –

STR –

OFL –

CHL +

STR –

OFL –

CHL –

STR +

Re

lati

ve

in

ten

sit

y o

f fl

uo

res

ce

nce

, %

nm

Concept of multiplex immunochromatography

Immunochips Test strips

• 2-3 hours • specialized laboratory • up to 100 compounds • quantitative analysis

• 10-15 minutes • out-of-laboratory analysis • 1-4 compounds • qualitative (yes-no) analysis

dozens of compounds

10 minutes

out-of-laboratory analysis

quantitative results

Principle of «2-D immunochromatography» for multiplex assay

Construction

of test strip

Application

of reactants

by pins

Potato pathogens: PVX, PLRV, PVM, Cms,

PVYO, PVYN

Assay duration – 15 min

Control zone

PLRV

Сms

PVYO

PVM

PVX

PVYN

Simultaneous detection of six phytopatogens in plant samples

Co

ntr

ol

zon

e

Advantages of video digital registration

Traditional reflectometry:

• One data per sample • No storage of test image • Sequential work with series of samples

Video digital reflectometry:

• Multi-pixel massive of data from each sample • Storage of “as is” images of tests

• Simultaneous work with series of samples • Regulated processing of noise • Proper location of target zones

• Varying procedures to estimate coloration

Formal parameter of average intensity

Portable devices for recording the assay results

Camera-based solutions

Scanner-based solutions

• compact working place • non-contact measurements

• high resolution • only novel software for available equipment

Software for: • qualitative and quantitative analyses of assay results • creation, storage and processing measurements data

• Immuno-PCR, biobarcode enhancement

• Immunodetection of single molecules

• Direct detecton of antibody binding (Q-bodies)

• Microfuidics (lab-on-chip)

• Alternative receptors

• …

Directions that were NOT considered in the report

From first tests to integrated systems of data processing and transfer

Dzantiev B.B. et al.

Trends in Anal. Chem.,

2014, 55, 81-93.

Thank you for your attention!

Institute of Biochemistry Russian Acad. Sci. Immunobiochemistry Lab.

Leninsky prospect 33

119071 Moscow, Russia

www.inbi.ras.ru

E-mail: zherdev@inbi.ras.ru

Tel 7(495)954-28-04

Any questions?