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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
[email protected]@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