Increase in KD (M) value

Determinant (Analyte) is nothing but the sample and its replicate.
I have run single sample 4 times to check the repeatability. 
What type of injection? can you simplify your question.

My question was about the sensorgram you show because we call that single cycle kinetics. this is more than one analyte injection followed by a longer dissociation and if necessary a regeneration. The multi cycle kinetics has one analyte injection plus dissociation and if necessary a regeneration step. The next analyte injection is separate from the first and the next. ( www.sprpages.nl/experiments/strategy )

You have checked the repeatability and I trust that was good.

So what is different between the first determinant and the second and the third? Obviously something changes. You have to look in sample purity, stability, stock concentration, regeneration etc. You know what you have done, look at all the steps and try to figure out.
I have no new insights for you now.

kind regards
Arnoud

Dear Arnoud,

Thank you for your continuous suggestions.
we have resolved the issue. It was related to startup cycles. So, we ran a dummy concentration in the startup to equilibrate the chip surface. 

Thanks and regards
SP

Dear Arnoud

Thank you for your continuous suggestions.
We have resolved the issue. 
it was related to startup cycle. it wasn't able to equilibrate the surface of chip Hence, we run a dummy concentration and resolved the asked issue.

Regards
SP