Transcript Protocol for immobilisation of antibodies on to Gold electrodes

Capability of Immobilisation
techniques and detection of Ab-Ag
interactions
by
Eyad Hamad
November 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 11mercaptoundecanoal (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 -20⁰C.]
•
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.
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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
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Preliminary results (Maskless IDEs)
10000
9000
8000
7000
C1= 1 μg/ml
ΔZ [Ω]
6000
C2= 10 μg/ml
5000
C3= 100 μg/ml
4000
3000
2000
1795
1000
175
0
-1000 0
50
128
100
gap size [ μm]
•
•
ΔZ =ZAb-Ag-Zab
ΔZ is distinguishable for S< 50 µm
93
150
200
Sensitivity of detection
•
Sensitivity curve in the range of 0-500
µg/mL of Ag
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Sensitivity increased dramatically by
reducing the gap size less than 50 µm
Limit of Detection
-2e7
Z'' (ohm)
7.5 µm gap-size with Ab only
7.5 µm gap-size with Ab with 5 µL of 1 ng/mL of Ag
7.5 µm gap-size with Ab with 5 µL of 10 ng/mL of Ag
-1e7
0
0
1e7
2e7
3e7
Z' (ohm)
Reducing the gap size, increased the limit of detection ( 1 ng/mL)
Reproducibility
•
Replacing pads soldering with electrode holder initiates reproducibility of
the experiments (especially at high frequency range)
108
E7 in Ferrocouple 10 mV.z
E7 in Ferrocouple 10 mV with new holder.z
E7 in Ferrocouple 10 mV with new holder next day.z
|Z|
107
106
105
104
103
10-1
100
101
102
103
104
105
106
104
105
106
Frequency (Hz)
-100
-75
theta
-7.5e7
bare_IDE8_1.z
bare_IDE8_2.z
bare_IDE8_4.z
-50
-25
108
|Z|
Z[Ohm]
107
-2.5e7
0
10-1
bare_IDE7_1.z
bare_IDE7_2.z
bare_IDE7_3.z
bare_IDE7_4.z
bare_IDE7_5.z
-5.0e7
106
0
0
2.5e7
5.0e7
Re(Z) [Ohm]
7.5e7
100
101
102
103
Frequency (Hz)
104
105
101
102
103
Frequency (Hz)
105
104
10-1
100
106
Sensitivity versus gap size
S=10m
S=7.5m
S=5m
S=2.5m
1500
Change of Impedance ()
1250
Slope =4.04
2
R =0.99
Slope =3.09
1000
Slope =2.85
750
Slope =2.44
500
2
R =0.99
250
0
50
100
150
200
250
300
350
Myoglobin Antigen (ng/mL)
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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
Selectivity
Myoglobin Ab-Myoglobin Ag
Myoglobin Ab- Rabbit IgG Ag
Myoglobin Ab only
-40000000
Im (Z) ()
-30000000
-20000000
-10000000
0
0
10000000
20000000
30000000
40000000
50000000
Re (Z) ()
•
Selectivity test was conducting by the used of different types of antigen
(Rabbit IgG), which showed no response in the signal obtained.