Immunochemical Methods and Immunochemical Methods and Biosensors for pollutants Biosensors for pollutants

determinationdetermination

(General principles and application)(General principles and application)  

Danila MosconeDanila MosconeDepartment of Chemical Science and TechnologyDepartment of Chemical Science and Technology

University "Tor Vergata"University "Tor Vergata"Rome, ItalyRome, Italy

danila.moscone@uniroma2.itdanila.moscone@uniroma2.it

These methods already play an important role, These methods already play an important role, especially in clinical chemistry, being used for especially in clinical chemistry, being used for

the fast and safe detection of proteins, the fast and safe detection of proteins, hormones, and pharmaceutical agents. hormones, and pharmaceutical agents.

Immunoassays (IAs)Immunoassays (IAs) are techniques based on are techniques based on the formation of a thermodynamically stablethe formation of a thermodynamically stable

antigen – antibody complex.antigen – antibody complex.

Immunoassays become important whenImmunoassays become important when::

Fast measurement and evaluation are required Fast measurement and evaluation are required  Highest possible detection strength is required Highest possible detection strength is required  Large numbers of samples are to be expected Large numbers of samples are to be expected  Only complex and expensive analytical methods are Only complex and expensive analytical methods are

otherwise available.otherwise available.

The greatest potential for the use of immunoassays in The greatest potential for the use of immunoassays in environmental analytical chemistryenvironmental analytical chemistry is in is in

SCREENINGSCREENING i.e., for the selection of contaminated i.e., for the selection of contaminated and and uncontaminated samples for further validation uncontaminated samples for further validation analysis.analysis.

TerminologyTerminology

Immunogen: Substance able to generate an immune response

Antigen: Original - Substance able to generate antibody.More general - Substance that can be recognized by antibody or T cells

Hapten: Non-immunogenic substance. Usually low molecular weight. Induces antibody formation when coupled to a larger “carrier” molecule. Can bind antibody

Immunize with Antibodies formedDNP NoneBSA Anti-BSA

DNP-BSA Anti-DNP Anti-BSA Anti-DNP-BSA

Protein Carrier -Bovine Serum Albumin

Hapten - DNP

Antibody structureAntibody structure

Antigen binding sites

Light Chain

Heavy Chain

.ANTIBODY (immunoglobulin)

A biological molecule (protein) that specifically recognizes a foreign substance (antigen) as a means of natural defence

Antibodies: production and labellingAntibodies: production and labelling

LABELLINGLABELLING

Radio-isotopes, Enzymes, Fluorescent, probes (Quantum dots), Chemi-luminescent probes, Metal tags

PRODUCTIONPRODUCTION

•Animals have a large number of antibody producing cells, all producing a different antibody. When an animal (rabbit) is injected with antigen, proliferation of the corresponding antibody producing cell is promoted. Blood from the rabbit contains antibodies, originating from different cells with slight variations.

Antibodies Antibodies

PolyclonalPolyclonal MonoclonalMonoclonal

Antibodies that are collected from sera of exposed animal

recognize multiple antigenic sites of injected biochemical.

Individual B lymphocyte hybridoma is cloned and cultured. Secreted antibodies are collected from culture media

recognize ONE antigenic site

of injected biochemical

Antigen-antibody InteractionsAntigen-antibody Interactions

Features of the Antigen-Antibody Interaction

•ReversibilityReversibilityNon-covalent Interactions

•AffinityAffinityMeasure of the strength of the bindingEase of association or dissociation

•AvidityAvidityIncrease in affinity due to multivalent

bindingThe summation of multiple affinities

Non-covalent Non-covalent bindingbinding

Affinity and AvidityAffinity and Avidity

Antibody-based Antibody-based assaysassays

EEnzyme-nzyme-LLinked inked IImmunosorbent mmunosorbent AAssayssay

ELISAELISA

Specific Ab

Ag E antigen- enzyme conjugate

immobilisation surface

Affinity reaction

EE

EE

S

P

Enzym. reaction

Productmeasurement

EE

Incubation

EEEE

Coating

I. No analyte - high detection signal

E E EE E E

II. Analyte present - detection signal reduced

E E

The enzymatic product concentration is inversely proportional to the analyte

(standard or sample) amount

ANTIGEN COATING

ENZYMATIC REACTION

S

P

Indirect competitive ELISA formatIndirect competitive ELISA format

BLOCKING

FREE Ag and SPECIFIC Ab ADDITION

SECONDARY

LABELLED Ab

ELISA SANDWICH FORMATELISA SANDWICH FORMAT

Y Y Y

Y Y Y

Y

YY

Y Y Y

Y Y Y

2nd antibody with enzyme

Antibody/Antigen

Antibody

Y Y YY Y Y

enzyme produces colour

signal/concentration curvesignal/concentration curveS

ign

al (

enzy

me

acti

vity

)S

ign

al (

enzy

me

acti

vity

)

Antigen concentrationAntigen concentration

Functional concentration range

ELISA PLATE WASHER

ELISA PLATES

SPECTROPHOTOMETER ADAPTED FOR ELISA PLATES

Lateral Flow StripsLateral Flow Strips(Dipsticks)(Dipsticks)

• Immunochromatography (Lateral Flow)

• Biochemical components are separated across an absorbent membrane into discrete distinct regions.

Apply sample solution, upon application of sample biochemicals dissolve

Immobilised Antibody area

Control area

Positive: no antigen

Negative: antigen present

analyte

Test line

Ab-colloidal gold

Predator

support

Sample pad

QUALITATIVE TESTQUALITATIVE TEST

Test line

Sample pad

QUALITATIVE TEST:QUALITATIVE TEST: Analyte absent in the sampleAnalyte absent in the sample

Analyte Ab-colloidal gold

If the analyte is ABSENT in theIf the analyte is ABSENT in the sample the line will be coloredsample the line will be colored

Test line

Sample pad

Analyte Ab-colloidal gold

Test line

Sample pad

Analyte PRESENT in the sampleAnalyte PRESENT in the sample

Analyte Ab-colloidal gold

Test line

Sample pad

Analyte Ab-colloidal gold

Test line

Sample pad

Analyte Ab-colloidal gold

Sample pad

Test line

If the analyte is present in the If the analyte is present in the samplesample the line will be not coloredthe line will be not colored

Analyte Ab-colloidal gold

We can use these immunochemical We can use these immunochemical elements to assemble a special kind of elements to assemble a special kind of biosensors calledbiosensors called

ImmunosensorsImmunosensors

AnalyteAnalyte Biological component

Signal transducer

Recorder

What do they have in common?

Analyte / bioreceptor / transducer / processor

BiosensorBiosensor

Small molecules / olfactory membrane / nerve cells / brain

Visible light / rods and cones / nerve cells / brain

NoseNose

EyeEye

Staphylococcus aureusStaphylococcus aureus

Enterotoxins: A, B, C, D, E (thermostable);Enterotoxins: A, B, C, D, E (thermostable); Coagulase;Coagulase; Thermonuclease.Thermonuclease.

100-200 ng of enterotoxins are sufficient to 100-200 ng of enterotoxins are sufficient to cause toxinfection in immunocause toxinfection in immuno--compromised subjects.compromised subjects.

gram-positive, non spore-forming bacterium

able to synthetise:

gram-positive, non spore-forming bacterium

able to synthetise:

Conventional ELISA Proteina AConventional ELISA Proteina A

Conventional ELISA S. aureusConventional ELISA S. aureus

ELISA/AMPLI S. aureusELISA/AMPLI S. aureus

ELIMC S. aureusELIMC S. aureus

ELIME S. aureusELIME S. aureus

DEVELOPED TEST:DEVELOPED TEST:

Spectrophotometric ELISA

Protein A/S.aureus

Protein A/S.aureus

Human IgG Human IgG

Specific antibody (MAb o PAb)

Specific antibody (MAb o PAb)

Secondary antibody-AP

Secondary antibody-AP

AP

p-NPPp-NITROPHENOL

protein A (ng/mL)

0,1 1,0 10,0 100,0

AB

S @

405

nm

0,00

0,30

0,60

0,90

protein A (ng/mL)

0,01 0,10 1,00 10,00 100,00

AB

S @

405

nm

0,00

0,80

1,60

2,40

 

MAb

IgG 10 mg/mL

MAb 1:10000

Ab2-AP 1:1000

PAb

IgG 10 mg/mL

PAb 1:10000

Ab2-AP 1:1000

LODLOD

SensitivitySensitivity

0.6 ng/mL

7.6 ng/mL

LODLOD

SensitivitySensitivity

0.07 ng/mL

0.6 ng/mL

y =(a – d)

x

c+1

b+ d

y = <x0> + 3s

ELISA Protein AELISA Protein A

Sensitivity was calculated as tha amount of protein A needed to produce a 25% increase in the signal

[S.aureus] (cell/mL)

1,e+3 1,e+4 1,e+5 1,e+6 1,e+7 1,e+8

AB

S @

40

5 nm

0,00

0,50

1,00

1,50

2,00

2,50

[S.aureus] (cell/mL)

1,e+6 1,e+7 1,e+8

AB

S @

405

nm

0,00

0,30

0,60

0,90

1,20

MAb

IgG 10 mg/mL

MAb 1:10000

Ab2-AP 1:1000

PAb

IgG 10 mg/mL

PAb 1:10000

Ab2-AP 1:1000

LODLOD

SensitivitySensitivity

2 106 cell/mL

9 106 cell/mL

LODLOD

SensitivitySensitivity

2 104 cell/mL

2 105 cell/mL

ELISA S.aureusELISA S.aureus

No cross-reactivity

Acetaldehyde

NADPH

NADH

NAD+

INT

FORMAZANEthanol

Alcohol deydrogenase Diaphorase

Alkaline phosphatase

Pi

N N+

NN

NO2

I

Br

NH2 N CH N NH

DAKO, Handbook for AmpliQ, 1997

AMPLI QAMPLI Q

[S.aureus] cell/mL

1,e+3 1,e+4 1,e+5 1,e+6 1,e+7 1,e+8

AB

S @

490

nm

1,00

1,50

2,00

2,50

3,00

[S.aureus] (cell/mL)

1,e+3 1,e+4 1,e+5 1,e+6 1,e+7 1,e+8

AB

S @

490

nm

0

1

2

3

4

ELISA S.aureus AMPLIQ

MAb

IgG 10 mg/mL

MAb 1:10000

Ab2-AP 1:1000

PAb

IgG 10 mg/mL

PAb 1:10000

Ab2-AP 1:1000

LODLOD

SensitivitySensitivity

6 104 cell/mL

2 105 cell/mL

LODLOD

SensitivitySensitivity

7 102 cell/mL

6 103 cell/mL

Good results in immunological field

Ø 1-5 µm

Measurements on real samples

Magnetic Beads Magnetic Beads

Magnetic particles are particles constituted from a dispersion of magnetic material (Fe2O3 and Fe3O4) and then covered with a thin shell of polymer which contains the magnetic material and also serves to define a surface area for the absorption or coupling of a large variety of other molecules.

Magnetic particles are particles constituted from a dispersion of magnetic material (Fe2O3 and Fe3O4) and then covered with a thin shell of polymer which contains the magnetic material and also serves to define a surface area for the absorption or coupling of a large variety of other molecules.

ELIMC (Enzyme Linked ImmunoMagnetic Colorimetry)

O

P ONaOH

O

NO2

All reactions were carried out in eppendorf tubes No intermediate washings

AP

p-NPP

p-NITROPHENOL

O H

N O 2

+ N a H 2 P O 3

MicrotitreELISA

ELIME (ELIME (Enzyme Linked ImmunoMagnetic ElectrochemistryEnzyme Linked ImmunoMagnetic Electrochemistry))

OH

a-naphthyl phosphate

a-naphthol

AP

+

+ NaH2PO3

•Selectivity Ag-Ab;•Sensibility of electrochemical detection;•Possibility of concentrating magnetic particles on the electrode surface.

O

PO O

ONa

DPV

Potential range 0-600 mV

Scan speed 100 mV/s

Pulse width 50 ms

Modulation time 60 ms

Interval time 0.16 s

Magnetic tube

Addition ofEnzymatic substrate

for

Electrochemicalmeasurement

ELIMC S.aureusELIMC S.aureus ELIME S.aureusELIME S.aureus

[S. aureus] cells/mL

1,e+3 1,e+4 1,e+5 1,e+6 1,e+7

Cur

rent

(µA)

0,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0

103 104

0,0

1,0

2,0

3,0

4,0

MAb

IgG 1.2 mg/mL

MAb 1:1000

Ab2-AP 1:100

LODLOD

SensitivitySensitivity

1 103 cells/mL

2 104 cells/mL

MAb

IgG 0.5 mg/mL

Mab 1:50000

Ab2-AP 1:300

LODLOD

SensitivitySensitivity

1 104 cells/mL

2 105 cells/mL

[S. aureus] cells/mL

1,e+4 1,e+5 1,e+6 1,e+7

ABS @

405

nm

0,0

1,0

2,0

3,0

4,0

1,e+4 1,e+5

0,0

0,2

0,4

0,6

0,8

Mab ELIME

Mab ELIMC

Pab ELISA AmpliQ

Mab ELISA AmpliQ

Pab ELISA S.a

Mab ELISA S.a

Pab ELISA prot A

Mab ELISA prot. A

SensitivityLOD

0.6 ng/mL

0.07 ng/mL

2 106 cell/mL

2 104 cell/mL

6 104 cell/mL

7 102 cell/mL

1 103 cell/mL

1 104 cell/mL

9 106 cell/mL

2 105 cell/mL

2 105 cell/mL

6 103 cell/mL

2 104 cell/mL

2 105 cell/mL

0.6 ng/mL

7.6 ng/mL 22 h

22 h

22 h

22 h

22 h

22 h

4 h

4 h

Analysis Time

Air samplesAir samples

Sample 1Sample 1 Sample 2Sample 2

660 cell/m660 cell/m33 ± 15%± 15% 11700 cell/m11700 cell/m33 ± 11%± 11%

Two air samples from hospital rooms Two air samples from hospital rooms

Sampling carried out by a SAS air-Sampling carried out by a SAS air-

sampler.sampler.

Flow rate 35 litri/min, for 30 minuts, collin Flow rate 35 litri/min, for 30 minuts, collin

30 ml of buffer30 ml of buffer

Immunoassay test products Immunoassay test products validated by OSWvalidated by OSW

Anticholinesterase activity Anticholinesterase activity

measurement by an enzyme biosensor:measurement by an enzyme biosensor:

application in water analysisapplication in water analysis

This method is a fast, cheap, and good analytical choice to measure the total anti-ChE charge in the sample, an important toxicological index defined as the amount of compounds which causes a % of ChE inhibition equivalent to that produced by a known amount of a pesticide (e.g. Paraoxon) taken as reference compound.

Acetilcholine Choline + Acetic ac. Acetilcholinesterase

Inhibited byInhibited bypesticidespesticides

Choline + O2 + H2O Betaine + H2O2 Choline oxidase

Not Inibited

H2O2 O2 + 2H+ + 2e-Electrode

Trasmission of the nervous impulse

Acetilcholinesterase

Time (min)

I%=[( EI%=[( E00-E-Eii)/E)/E00]]•100•100

Non inhibited enzyme (E0)

inhibited enzyme (E1)

Inhibition measurementsInhibition measurements

0 1 2 3 4TIME (minutes)

I (n

A)

0

2

4

6

Inhibition of AChE with ParaoxonInhibition of AChE with Paraoxon

Blank

2 ppb

6 ppb

10 ppb

HEAVY METALS DETERMINATION HEAVY METALS DETERMINATION BASEDBASED

ON THE USE OF INVERTASE ENZYMEON THE USE OF INVERTASE ENZYME

REACTIONS

Sucrose

E1

D-GlucoseD-Glucose + D-Fructose

D-GlucoseD-Glucose

E2 + O2

Gluconic acid + HH22OO22

HH22OO22 Electrode

O2 + 2H++ 2e2e--

E1= Invertase

E2 = Glucose Oxidase+ H2O

INV

INV + Inhibitor

Sucrose

Sucrose

I1

I2

Reaction TimeA

B

Time

Cu

rren

tINHIBITION MEASUREMENTS

0 20 40 60 80

[Hg2+] (ppb)

Inib

itio

n %

0

20

40

60

100

FIA Calibration with sucrose 10mM

80

Biosensors applied to the Biosensors applied to the determination of pollutants in real determination of pollutants in real

samplessamples

From: S. Rodriguez-Mozaza, M. J. L´opez de Aldaa, M.-P. Marcob, D. Barcel´oa,, Talanta 65 (2005) 291–297