why analyte (SSDNA) binding to the activated referance channel-CM chip

we are doing aptamer protien binding interaction. recently we got some weird results. normally i don't inject Ethanolamine after the protein immobilization because we didn't get any results after EA injection. my first question what is the reason for that?
recently we got some weird results mean we got some binding KD value in pMol range. but this date is too good to be true. so accidently we found that this aptamer can bind to the activated reference channel we are using CM chip. i don't normally activate the reference channel . this signal same as SPR normal signal. it is not like non specific binding. it is also hard to belive how this aptamer can bind to the activated channel because there are no free amine in ssDNA (aptamer) my second question is that one?

so we think CM chip is contaminated. do you any way to clean or remove the contaminates of the CM chip. can we send some literature to find this

in the attachment channel 3 is the reference. i activated that. then i injected the analyte.when we used
EA we coulnt see any binding for same analyte

1) The ethanolamine is generally 1 M and pH 8.0–8.5. The purpose of the high concentration and high pH is to deactivate the activated sites and to wash away non-specific bound protein from the sensor surface. The high concentration and higher pH value can induce some unfolding of the protein which can be permanent and result in an inactive protein (no binding of aptamer). Deactivation can be done with other solutions which contain primary amines such as 50 mM Tris pH 7.4. Longer contact times may be necessary. Just waiting (>20' maybe longer) (Lahiri et al., 1999) will also revert unoccupied activated sites to –COOH groups which are more charged than groups deactivated with ethanolamine.

2) I am not aware of publications investigating possible contaminations on sensor chips.
I can suggest washing the sensor surface until a stable baseline is seen. Injections with low pH (high salt) and NaOH can remove possible contaminations as long as they are non-covalently bound. Wash again until a stable baseline.

Lahiri, J., Isaacs, L., Tien, J., & Whitesides, G. M. (1999). A Strategy for the Generation of Surfaces Presenting Ligands for Studies of Binding Based on an Active Ester as a Common Reactive Intermediate: A Surface Plasmon Resonance Study. Analytical Chemistry, 71(4), 777–790. doi.org/10.1021/ac980959t

kind regards
Arnoud

Thank you very much for reply. its very much helpful for me i have another question do you think ssDNA can bind to the active channel with out immobilization of protein. it is not like normal non specific binding. you can check in my SPR graph.
do we have to activate the reference channel always?

Finding the best reference surface is always difficult. Ideally the reference surface mimics the active surface as close as possible in origin and density. However, generally we don't have such a surface available and look for a different solution. Activating/deactivating removes the (charged) carboxyl groups and replaces them with less charged –OH groups. You can let the reference surface as is.
The fact that you find binding to the sensor surface indicates that the aptamer has some affinity to the sensor surface. Maybe it is possible to prevent this with a slight change in buffer such as more NaCl (150-250 mM) or a different pH (6.8-8.0).

As an alternative switch to a sensor surface with a short dextran matrix or one without. Although we use the carboxylated dextran matrix a lot wil not say it is the best surface for your experiments. See www.sprpages.nl/sensor-chips-intro/xantec-sensor-chips for more surface possibilities.

thank you . i will try that. do you have any idea why aptamer bind to the activated reference channel. because aptamer doesnt have free amines. i don't have no idea how it bind

From what I read in literature aptamers can recognise small moleculaes such as antibiotics. I can imagine that some aptamers can recognise the dextran structure of the sensor surface with or without the carboxyl group or the deativated surface with the etanolamine.


Try surfaces with a different matrix such as PEG or alginate.