I am doing SPR experiment using a homemade setup. I have a bulk change problem during the exchange of solutions. This is what I did: PBS-water-2 propanol-water-PBS. According to my understanding, these solutions will not bind to the surface of the chip. So after the whole process, I should get back the baseline level. However, I observed a change in baseline level at the end. I can see a lot of published results with the commercial SPR machine are showing the same problem. May I know who can help me to explain what is happening? Thank you very much.
You are correct that the solutions should not bind. What you not say is what surface you use in the experiment. In case of a dextran matrix you can observe some baseline shift due to vertical shrinking or extension of the matrix. I expect that the 2 propanol has the largest effect.
Regeneration solution like high or low pH, and high salt also can change the base line. In addition, bound proteins can also change by this solutions.
I know that in all these cases there is no mass change on the sensor chip surface. However i think that somehow the density of the surface alters the refractive index of the system and therefore the baseline.
I am not familiar with this read out, so I cannot comment if it is high or low.
The 16-MHA has a free -COOH group. The NHS/EDC can activate the -COOH group to N-hydroxysuccinimide ester. This is slightly bigger so will give a positive signal. The ester will detoriate over time.