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. • 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) 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 • 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=10m S=7.5m S=5m S=2.5m 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) • 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.