Post on 11-Jun-2018
Electrochemical Immunoassays
Mateusz Szymanski24.02.2009
Immunoassays
A biochemical test that measures the concentration of a substance in a biological liquid, typically serum or urine, using the reaction between antibody and its antigen.
Immunoassays
enzyme Immunoassay
radioImmunoassay
fluoroImmunoassay
metalloImmunoassays
Immunoassays
Lateral flow stripsELISA
The point of care device has to be:
Easy to useSmall and inexpensivePortableSpecificRobustFastWorking with low volume and turbid samples
The new electrochemical immunoassay has the potential to fulfil all the above requirements !!!
Electrochemical Immunoassayswith gold nanoparticle
HBr/Br2Au3+
Electrochemical Immunoassay Format
Electrochemical Immunoassay Format
Electrochemical Immunoassay Format
Electrochemical Immunoassay Format
Electrochemical Measurement Process:
Ag+ x 106 !
ASV
Magnetic Particle – Antibody Conjugate
Commercially available,Tosylactivated magnetic microbeads,
1 µm diameter
Amide bond
Silver Sol – Antibody Conjugate
Mechanism:
Proteins absorb to silver surfaces through:
1. Hydrophobic interaction (Tyr, Trp, His andPhe)
2. Electrostatic interaction (carboxylates of Asp, Glu and C-termini)
3. Dative bonds through sulphur atoms (Cysand Met) or nitrogen atoms (Trp and His)
Silver Sol – Antibody Conjugatepoor-quality conjugates:
Wrong orientation Aggregation
Denaturated/Inactive antibody
Silver Sol – Antibody Conjugateoptimisation:
1. Silver sol : antibody ratio2. Buffer type (PBS, TBS, Borate)3. Buffer molarity4. Buffer pH5. Blocking agent
Performance of 6 different silver conjugates
0
2
4
6
8
10
12
14
0 0.5 1 1.5 2
Log (Myoglobin [ng/mL])
Mic
roco
ulom
bs [µ
C]
0.2M H3BO3 pH 6,5
0.4M H3BO3 pH 6.5
0.1M H3BO3 pH 7.5
0.01M H3BO3 pH 7.5
0.1M H3BO3 pH 8.5
0.01M H3BO3 pH 8.5
Electrochemical Measurement Process:
Ag+ x 106
ASV
Silver Sol Dissolution
+++ +→+ 230 FeAgFeAg
oxidantAg+
Ag+
Ag+Ag+Ag+
Ag+
144 ])()([)()( +−−+−++ →++ nm
nm SCNNHAgSCNnNHmAg
61.268 61.518 61.768 62.018 62.268 62.518 62.768 63.018 63.268-4-0.484x10
-4-0.234x10
-40.016x10
-40.266x10
-40.516x10
-17.500x10-1
-15.000x10-1
-12.500x10-1
-10.000x10-1
-7.500x10 -1
-5.000x10 -1
-2.500x10 -1
0
2.500x10 -1
t / s
i / A
E / V
61.268 61.518 61.768 62.018 62.268 62.518 62.768 63.018 63.268-4-0.484x10
-4-0.234x10
-40.016x10
-40.266x10
-40.516x10
-17.500x10-1
-15.000x10-1
-12.500x10-1
-10.000x10-1
-7.500x10 -1
-5.000x10 -1
-2.500x10 -1
0
2.500x10 -1
t / s
i / A
E / V
ASV scan of Silver Colloid with and without Fe (III)
Silver Sol can be measured with no oxidant !
Silver Sol in the presence of thiocyanate forms aggregates with the SCN- layer surrounding it.
NH4SCN SCN-
SCN-
SCN-SCN-
SCN-SCN-
SCN-SCN-
SCN-
SCN-SCN-
Ø = 40 nmZeta Potential = -50 mV
Ø = 400 nmZeta Potential = -10 mV
UV-Vis spectrum of Silver Colloid
+ NH4SCN
SCN-
SCN-SCN-
SCN-SCN-SCN-
SCN-
SCN-
SCN-
SCN-
SCN-
SCN-
SCN-
SCN-
SCN-
+ + + + + + +- - - - - - - - - -+ + + + + + + +
e- e- e-
Ag+
Ag+ Ag+
Electrochemical Measurement Process:
Ag+ x 106
ASV
Carbon Screen Printed Electrodes
Working Electrode
Counter Electrode
Reference Electrode
Carbon Screen Printed Electrodes
The voltage value at WE during different steps of ASV
pre-treatment
Nucleation Step
Accumulation Step(Reduction)
Stripping step(Oxidation)
Pre-treatment: POTENTIALAg+ vs Silver Sol in 1M NH4SCN
The influence of pre-treatment potential (t=10s) on current signal. Signal from Ag+ is potential-independent; signal from silver sol is potential-dependent.
3
4
5
6
7
8
9
10
-1.6 -1.1 -0.6 -0.1 0.4 0.9 1.4potential [V]
Mic
roco
ulom
bs [µ
C] 2.5 ppm Ag+
80% Silver Sol
Pre-treatment: DURATIONAg+ vs Silver Sol in 1M NH4SCN
The influence of pre-treatment time (at U = +0.4V) on current signal. Signal from Ag+ is time-independent; signal from silver sol is time-dependent.
0
2
4
6
8
10
0 5 10 15 20 25 30 35 40time [s]
Mic
roco
ulom
bs [µ
C]
2.5 ppm Ag+80% Silver Sol
Pre-treatment: THIOCYANATE MOLARITYAg+ vs Silver Sol in 1M NH4SCN
The influence of NH4SCN molarity on current signal. Signal from Ag+ is basically constant; signal from silver sol is thiocyanate-dependent. (Pre-treatment time t=10s at 0.4V).
0
2
4
6
8
10
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2[NH4SCN] [mol/L]
Mic
roco
ulom
bs [µ
C]
2.5ppm Ag+80% Silver sol
Electrochemical reactions at the WE:
−+ +↔ eAgAg 0
144 ])()([)()( +−−+−++ →++ nm
nm SCNNHAgSCNnNHmAg
1. Silver sol dissolution and formation of electroactive chelate:amonium thiocyanatosilver(I) at +0.4V
nmnm
nmnm SCNNHAgeSCNNHAg −−+−+−−+ →+ ])()([])()([ 4
14
2. Accumulation step of ASV (reduction) at –1.2V
−+−−+−−+ +→ eSCNNHAgSCNNHAg nmnm
nmnm
144 ])()([])()([
3. Stripping step of ASV (oxidation) -1.2V +0.1V
+ + + + + + + +
SCN-
SCN- SCN-
SCN-
SCN- SCN-SCN-
SCN- SCN-SCN- SCN-
SCN-
SCN-SCN-
SCN- SCN-
SCN-
SCN-
SCN-
e- e- e-
Ag+ Ag+
Ag+
Anodic Stripping Voltammetry
0.4 V/15s
+ + + + + + + + 0.4 V
Ag+
Ag+Ag+
Ag+
Ag+
Ag+
Ag+
Ag+Ag+
Ag+
Ag+
Ag+ Ag+
Ag+
Ag+
Ag+
Ag+
Ag+
Ag+
Ag+
Ag+Ag+
Ag+
Ag+
Anodic Stripping Voltammetry
- - - - - - - - -1.2 V
Ag+
Ag+Ag+
Ag+
Ag+
Ag+
Ag+
Ag+Ag+
Ag+
Ag+
Ag+ Ag+
Ag+
Ag+
Ag+
Ag+
Ag+
Ag+
Ag+
Ag+Ag+
Ag+
Ag+
Anodic Stripping Voltammetry
- - - - - - - - -1.2 V, t=55s
Ag+ Ag+Ag+
Ag+Ag+Ag+
Ag+Ag+
Ag+Ag+
Ag+Ag+Ag+ Ag+Ag+Ag+
Ag+Ag+
Anodic Stripping Voltammetry
Ag+ Ag+Ag+
Ag+Ag+Ag+
Ag+Ag+
Ag+Ag+
Ag+Ag+Ag+ Ag+Ag+Ag+
Ag+Ag+
Anodic Stripping Voltammetry
+ + + + + + + + + +
Ag+ Ag+Ag+
Ag+Ag+Ag+
Ag+Ag+
Ag+Ag+
Ag+Ag+Ag+ Ag+Ag+Ag+
Ag+Ag+
Anodic Stripping Voltammetry
+ + + + + + + + + +
Ag+ Ag+Ag+Ag+Ag+
Ag+Ag+
Ag+Ag+
Ag+Ag+Ag+Ag+Ag+
Ag+
Analytical Signal
Electrochemical signal is related to analyte concentration
Q = I x t [Ag+]
[Analyte]
Assay for Myoglobin
0
2
4
6
8
10
0.0 2.0 2.8 3.2 4.0
Log Myo [pM]
Mic
roco
ulom
bs [µ
C]
0
0.4
0.8
1.2
1.6
Abs
orba
nce
ArgentoELISA
0
2
4
6
8
10
0.0 2.0 2.8 3.2 4.0
Log Myo [pM]
Mic
roco
ulom
bs [µ
C]
0
0.4
0.8
1.2
1.6
Abs
orba
nce
ArgentoELISA
Multiplexing
Au Ag Cu
58.801 59.301 59.801 60.301 60.801 61.301 61.801-3-0.019x10
-30.031x10
-30.081x10
-30.131x10
-30.181x10
-30.231x10
-30.281x10
-30.331x10
-17.500x10-1
-12.500x10-1
-7.500x10 -1
-2.500x10 -1
2.500x10 -1
7.500x10 -1
12.500x10 -1
t / s
i / A
E / V
ASV scan of 40ppm Ag+ and 40ppm Au3+ in 0.1M HCl
Multiplexing
Ag+
Au3+
Summary
1. A new sensitive platform technology. Myoglobin down to 0.2 ng/mL.
2. The problematic oxidative dissolution step eliminated.
3. The assay can be used in Point of Care devices.
Acknowledgements:
Dr Robert PorterDr Eleonora CerasoliLIRANSBritish Biocell InternationalHyTest
Thanks to the UK Government for funding the project through the Chemistry and Biology NMS Programme.