Capability of Immobilisation techniques and detection of Ab-Ag interactions

by

Eyad HamadNovember 2010

Protocol for immobilisation of antibodies on to Gold electrodes

Cleaning of Gold surfaces• The gold surface should be absolutely clean, and this can be done

using “Piranha Solution” so preparation of piranha solution is needed which; it is a 3:1 mixture of concentrated Sulphuric acid (H2SO4) and 30% hydrogen peroxide (H2O2).

Note: Applying the sulfuric acid first, followed by the peroxide.

Do not store piranha.  Mix fresh solution for each use.  Excess solutions should be disposed via the drain, followed by flushing with copious amounts of water.

• Rinse in DI water in 3 stages, followed by N2 dry

Protocol for immobilisation of antibodies on to Gold electrodes

Preparation of alkanethiol self-assembling monolayer (SAM)• SAM comprises a mixture of 11 mercapto-1-undecanoic acid (MUA) and 11-

mercaptoundecanoal (MU). 4mM of MUA and 1 mM of MU are prepared in ethanol respectively and then can be stored at room temperature.Note: Then equal volumes of solution are mixed together and then added immediately to the gold surface which is exposed to the solution for about 12 hours (overnight).

• After adsorption electrode with alkanethiol SAM , electrode should be washed with ethanol to remove unbound thiols.

Protocol for immobilisation of antibodies on to Gold electrodes

Preparation of EDC/NHS• The next step is preparation of EDC and NHS. ( Peptide conjugates ):• 100 mM of NHS (N-hydroxysuccinimide) in freshly Millipore water, 400 mM

EDC (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) in freshly Millipore water separately. [Should be aliquot and stored separately at -20 C.]⁰

Activate SAM :• Can be done by mixing equal volume of 100 mM NHS and 400 mM EDC

together (V/V).Note: mix immediately prior to use and don’t re-use once mixed.

• The Monolayer coated gold surface is exposed to EDC/NHS mixture for 10 minutes.

• The excess of EDC/NHS solution on the electrode is removed with a pipette or rinsed once with phosphate buffered saline (PBS) buffer.

Protocol for immobilisation of antibodies on to Gold electrodes

Preparation of Myoglobin antibodies (MyAbs)• Apply MyAbs to the functionalised gold surface for 30 minutes• After 30 minutes the antibody solution is gently rinsed from the surface with

running PBS buffer. • A 1M Ethanolamine hydrochloride, pH 8.5 (stored in the fridge at 40 C),

should be exposed for 7 minutes to the gold electrode, in order to terminate the un-reacted sites.

• The ethanol amine hydrochloride solution should be added for no more than 10 minutes. The electrode is further rinsed by washing with running PBS buffer.

• The antibody should now be successfully coupled and ready for assay.

Preparation of Myoglobin antigens (MyAg)• MyAg are diluted in PBS to the desired concentatrion.

To avoid the possibility of non specific binding, a detergent such as Tween20 is added to the PBS solution while preparing the Myoglobin antigens MyAg. Typically 0.5ml of Tween20 in 1 litre of PBS is sufficient

Protocol for immobilisation of antibodies on to Gold electrodes: Summary

Step I: SAM formation

Step II: SAM activation

Step III: Antibodies applying

Step IV: Antigen Binding

Antigen Detection

• Preliminary results (Maskless IDEs)

• ΔZ =ZAb-Ag-Zab

• ΔZ is distinguishable for S< 50 µm

1795

175 128 93

-1000

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

0 50 100 150 200

gap size [ μm]

ΔZ

[Ω]

C1= 1 μg/ml

C2= 10 μg/ml

C3= 100 μg/ml

Sensitivity of detection

• Sensitivity increased dramatically by reducing the gap size less than 50 µm

• Sensitivity curve in the range of 0-500 µg/mL of Ag

Limit of Detection

Reducing the gap size, increased the limit of detection ( 1 ng/mL)

0 1e7 2e7 3e7

-2e7

-1e7

0

Z' (ohm)

Z'' (

ohm

)

7.5 µm gap-size with Ab only7.5 µm gap-size with Ab with 5 µL of 1 ng/mL of Ag7.5 µm gap-size with Ab with 5 µL of 10 ng/mL of Ag

Reproducibility

• Replacing pads soldering with electrode holder initiates reproducibility of the experiments (especially at high frequency range)

10-1 100 101 102 103 104 105 106103

104

105

106

107

108

Frequency (Hz)

|Z|

E7 in Ferrocouple 10 mV.zE7 in Ferrocouple 10 mV with new holder.zE7 in Ferrocouple 10 mV with new holder next day.z

10-1 100 101 102 103 104 105 106

-100

-75

-50

-25

0

Frequency (Hz)

thet

a

0 2.5e7 5.0e7 7.5e7

-7.5e7

-5.0e7

-2.5e7

0

Re(Z) [Ohm]

Z[O

hm

]

bare_IDE8_1.zbare_IDE8_2.zbare_IDE8_4.z

10-1 100 101 102 103 104 105 106104

105

106

107

108

Frequency (Hz)

|Z|

bare_IDE7_1.zbare_IDE7_2.zbare_IDE7_3.zbare_IDE7_4.zbare_IDE7_5.z

10-1 100 101 102 103 104 105 106

-100

-75

-50

-25

0

Frequency (Hz)

thet

a

Sensitivity versus gap size

• Sensitivity of IDE sensors is increased by reducing the gap size of IDE• Detection limit of the reduced gap size was optimised by a factor of 1000

0 50 100 150 200 250 300 350

250

500

750

1000

1250

1500 Slope =4.04

Slope =3.09

Slope =2.85

R2=0.99

R2=0.99

S=10m S=7.5m S=5m S=2.5m

Cha

nge

of I

mpe

danc

e (

)

Myoglobin Antigen (ng/mL)

Slope =2.44

Selectivity

• Selectivity test was conducting by the used of different types of antigen (Rabbit IgG), which showed no response in the signal obtained.

0 10000000 20000000 30000000 40000000 500000000

-10000000

-20000000

-30000000

-40000000

Im (

Z)

()

Re (Z) ()

Myoglobin Ab-Myoglobin Ag Myoglobin Ab- Rabbit IgG Ag Myoglobin Ab only