I have been working to develop a microfludic protein analysis system and the current prototype system utilizes eppendorf-type vials to contain samples and protein reagents. The vials are connected to the analysis device via peek tubing, so that the fluid flows from the vials to the device. The peek tubing is re-usable and thus needs to be cleaned or flushed between assay runs.
We typically block the tubing and device prior to the assay, but after the assay we see a significant amount of residual protein analyte that affects the next assay run. The assay is a protein binding sandwich assay.
What have we tried?
-wash buffers containing Tween20
-high pH buffers with 100mM or more NaOH
-low pH buffers with 100mM or more HCl
-isopropanol, methanol, ethanol.
-combinations of the above with and without soaking times.
Results?
-the alcohols seem to do the most, but still not a one-step solution. We would like a method that works every time, a single washing reagent would be nice. We start the system dry, so even a gas based cleaning procedure might work. I may not necessarily need to remove the protein, but rather effectively denature the proteins from the previous assay run.
Few Questions:
What temperature are you running the assay (4C or room temp)?
Why do you need to block the peek tubing as these tubings are highly inert and should not bind to any protein.
If the proteins are highly sticky then you can try using 1N NaOH to clean the peek tubing. Other alternatives are 2% Contrad (detergent), 0.5% SDS or 70% Isopropanol.
All the Best
Nitin
The assay temperature is around room temp or slightly above at 26C. Sonication is not an option since the tubing is embedded in the system and can't be easily removed between assay runs. We block the tubing as a matter or course in the process of blocking the assay device (the blocking solution flows through the tubing before hitting the assay device).
I see several options listed that we have not tried, so thanks for the answers.
Another idea I had yesterday is to try using an enzymatic cleaning solution (like those used to clean contact lenses).
Thanks,
Jim
Erwin Metzmann;If you start always dry you may think about heat-denaturing (microwave).
I would try pepsin / HCl. This mixture can be obtained commercially (e.g. from VWR) for cleaning purposes or it can be prepared in the lab. Similar preparations based on other proteases are used for the removal of proteins from contact lenses (enzymatic cleaners) as mentioned by Jim. http://www.ncbi.nlm.nih.gov/pubmed/2186082
Hi James, when we work with proteins - we usually used 0,1M citric acid for regeneration of biofunctionalized magnetic particles and also columns after elution during immunoaffinity chromatography. But we wash by citric acid several times to be sure that all proteins are gone out and it works well.
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