how to get rid of a sticky compound in the injection system of my T200

Dear biosensor scientists,

After taking a look here and there to try to find a solution to my issue (and not finding it) I come to you hopping for some wisdom and advices.
I tried to post this this morning but i am not sure it worked properly so I give it an other try.

Here is the situation:
I'm running on a T200 a titration campaign for a few different small molecules on a CM5 functionalized with neutravidin by amine coupling on which I have captured 3 different protein (obviously one per flow channel)
I am using a Tris buffer with some NaCl, metals, 5%glycerol, BME. I have 0.005% tween20 to kill some potential aggregates without reaching the micelle formation concentration. And I have 2% DMSO in order to solubilize the analytes.
the top concentration on my titration scale variate from an analyte to the other from 1 µM to 100 µM in order to fit around the K_D value.

So far so good, but here come the troublemaker.

After a few compounds titrated my blank started to look like a binding event with a nice association and dissociation phase instead of the usual flat curve I use to have. It seems that one of those compound at 100µM (which seems soluble) is more sticky than expected in the system. after those compound get injected every single blank run by the machine look like a binding event. Therefore, even if I have quite good looking sensorgrams for the following compounds, because the sticky compound seems to be also injectent, I can’t decently use those sensorgrams to get a properly quantified K<sub>D[/sub.

Worst!!!
Even after a desorb (SDS, glycine pH9) a desorb and sanitize (SDS, glycine pH9, hypoclorite) and a shut down (70% ethanol) the first few startups injection on a freshly prepared chip seems to still show a binding event instead of the flat signal you could expect… (I haven’t run a super clean protocole yet, that will be my next step)

So that’s about the situation, now come the two question:

1 How can I get rid of this sticky compound? (without buying a brand new T200. my research grant doesn’t allow it)
Except from the super clean protocole, is there any other tips to be sure the machine is as clean as it could?

2 Once I’ve manage to get rid of those compounds stuck on the system, how can I titrate them again?

Of course the more reasonable thing to do would be to just discard those compound or use an orthogonal assay.

Fact is, those compounds belong to a series for Structure Kinetic Relationship study. They have really important structural features for out story. Also the other compounds of the series gave use good SPR kinetic behavior. Therefore we would like a full SKR story and for that we would need to mesure those compound as well, if possible with the same sort of assay.

The two obvious way would be to increase the tween concentration or the DMSO concentration. But somehow, for sticky compounds which resist a desorb and sanitize run, I doubt it would be enough.
Is there by any chance some of you encounter those sort of situation and discover some trick to solve it or am I asking too much to my poor T200?

Thank you very much for reading my complain and for the moral support.
May the Surface Plasmon Wave be with you.

Pierre</sub>

Hi,
If I understand correctly, some of your compounds seem to bind to the internal tubing of the T200 instrument. Even after cleaning there is a marked interaction on a native/deactivated dextran surface upon buffer injection.

Main issue is therefore cleaning of the injection system after compound injection. One option is to inject the compound that gives you issues and then clean the injection system by rinsing with a solution. In the T200 you can define an extra wash and define the wash solution position (pg 87 of the T200 software handbook). Then inject buffer to see if the compound is still present. It looks like the regeneration scouting procedure.
For instance, you can try different concentrations of DMSO, since your compounds are dissolved in this solution. Or any solution you seem to fit since the solution will not pass over the sensor surface.

If the non-specific interaction is to the matrix you can add 0.005% CM-dextran to the analyte to diminish this binding.

Some second thoughts about the persistence of the sticky compounds. It seems for me unlikely that when you clean the system and then inject standard flow buffer, the sticky compounds dissolves in the flow buffer and binds to the sensor matrix. What happens when you wash the system over weekend with flow buffer? Does this still happen on a fresh sensor chip?

You are welcome to relieve your heart.

Kind regards
Arnoud

Dear Arnoud,

Thank you very much for your reply.
My heart seems in good hand now.

I realized by reading your answer that I forgot to precise a few small information.

indeed some compounds seems to bind to the internal tubing. more precisely in the injection system.
If I do a manual run, as long as i keep the flow running nothing happen and i have normal flat line.
But, if I pipet some of the running buffer from the bottle, put it in the sample rack and ask the machine to inject it, then instead of still having a flat line as once could expect ( it is exactly the same buffer) then i have a nice increase of the ru until reaching a nice +10 RU plateau then when the injection stop the RU smoothly decrease to it's starting point this happen only on the Flow channel where my protein is. it doesn't have the same "kinetic" within the three different proteins and the RU from the reference flow channel stay perfectly flat.

In a nutshell it look like I am injecting a compound when I am actually injecting just the running buffer.
This look to me like I have some contamination in the injection tubing.

When I saw that, as I have said earlier, I have undocked the chip, run a desorb ans sanitize, wait overnight( as recommended in the manual) docked a new chip, immobilized my protein, (took a breath), run an other titration method.
AND already in the "primes" of the titration method, it seems that i still had some binding.

an other detail: in my titration protocole I already have an extra wash step with 50% DMSO.
it seems to be enough usually but not here.

It is still a pretty good advice and indeed I think I will try to probe some other wash condition.

As an update, I have run a thousand(more or less) prime, a few "desorb" and a "superclean" and it seems to be enough to finally wash out the sticky compound.
I will try later on to prob if an acidic or a basic wash can be a good solution.

I agree that it seems unlikely for a compound to resist a SDS wash but dissolve in a running buffer but it is what I observed.

Again thank you very much for the answer and suggestions.
I didn't thought of probing other wash conditions but it is actually quite obvious now.

I will keep you inform about how the situation evolve and if I finally manage to titrate this compound without wasting my T200 again.

With all my gratitude,
Best regards,
Pierre

Hello:

Couple of other things to look into. Do you have any additivies or compounds in your running buffer? How about detergent? Are your running buffer bottles cleaned/washed by a central dishwashing station? I've seen several instances of a core dishwashing group returning glass bottles to a lab wihtout being fully rinsed of detergent, which can cause all sorts of issues with data quality. I recommend that you wash/rinse all running buffer bottles by hand in your own lab. Often no dish washing detergent is needed, just a thorough rinse with DI water should suffice, assuming that the bottle didn't run dry and have buffer components crystalize in the bottle. If you are concerned with this, try washing your buffer bottles several times with NaOH, and then rinsing with water throroughly before preparing new running buffer in that bottle.

Since you are suing small molecules in your assay, and are already using an extra-wash step with 50% DMSO, you might want to try to do a "small molecule" desorb on your instrument. Instead of using Desorb 1 and Desorb 2, use 50% DMSO in place of Desorb 1 and 10% DMSO (in water) in place of Desorb 2. If there is ANY chance there could be some sort of small molecule contamination in the fluidics outside of the IFC/sample loop (i.e., your running buffer lines) then I would suggest your prepare a larger volume of 50% DMSO and 10% DMSO. place the buffer lines and water lines in 50% DMSO and run prime 1-2 times, then do the same thing with 10% DMSO, then prime with water. This might hopefully remove any contaminants elsewhere in the fluidics outside of the IFC. Just be sure to not walk away from the instrument and leave it sit in 50% DMSO for several hours as that could be detrimental as well.

If none of those things improve your response, you will probably need to contact service for a PM visit, or to consider replacing specific components, or the entirety of the fluidics system if some sort of large scale contamination has occured that can't be remedied by the "small molecule desorb" protocol, and/or superclean/desorb and sanitize procedures.

Hope this helps, and best of luck in your experiments!