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.