Quick question, if I'm just using a bare gold chip and not a CM or dextran chip, how would the amine coupling work using injection and not incubation? SO far I just incubated the chip under different solutions to do an amine coupling.
Should I make a SAM on the chip and then insert into machine for EDC-NHS, ligand and deactivation injections? If yes, what would be your SAM recommendation?
PS. I'm using Biosensing instruments 2500 and my ligand is cytochrome c oxidase on gold chip.
I would highly appreciate the help, since I had no training and after a year and a half I still can't get descent results. Thank you
On a bare gold surface proteins only adsorb. There is no covalent interaction between the gold and protein. To immobilize via the amine coupling both sensor surface and ligand should have appropriate chemical groups. In the case of amine coupling a carboxyl group (-COOH) and an amine (-NH2).
Thus the gold surface must be modified with a compound that will have either of these groups. In the case of protein immobilization it is easier to make a sensor surface with the carboxyl groups since proteins contain primary amine in the form of lysines.
Modifying the gold sensor surface with a SAM is the easiest. There are many protocols and since I used none of them I cannot recommend any.
1. Arya, S. K., Solanki, P. R., Datta, M., et al.; Recent advances in self-assembled monolayers based biomolecular electronic devices. Biosensors and Bioelectronics (24) 9: 2810-2817; 2009.
2. Ko, S., Park, T. J., Kim, H.-S., et al.; Directed self-assembly of gold binding polypeptide-protein A fusion proteins for development of gold nanoparticle-based SPR immunosensors. Biosensors and Bioelectronics (24) 8: 2592-2597; 2009.
3. Masson, J. F., Battaglia, T. M., Davidson, M. J., et al.; Biocompatible polymers for antibody support on gold surfaces. Talanta (67) 5: 918-925; 2005.
Please let us know what you did and what you recommend.
The following user(s) said Thank You: Maedehmozneb
Well, As I mentioned I have done the SAM on a gold chip and been pretty successful with the results, but the problem and my main question was that can I inject my ligand to be imooblized instead of just putting the chip over night in a solution of ligands? And your website helped great in figuring out my concentrations and that I'm getting an unexpected high result since Im conjoining the chip and proteins with gold nanoparticles.
I used a 20 mM solution of L-cystein and incubated the chips for 12 hours in them for SAM formation. another good SAM is thiols but they have a harder protocol and also longer. Cysteamine is also good for bare gold chips.
I just want to know if I can try injecting instead of incubating for immobilization.
I will try that this week ad update the forum with any results I got.
I would choose for immobilization during flow (in the machine). Then you can monitoring the activation, immobilization level and deactivation. This gives you a better idea of how much ligand is immobilized.