[RASMB] SPR question

[John Sumida]

Dear RASMB,

 

This question, is in relation to a Biacore T200 instrument and an artifact
we are seeing at high sample injection concentrations (20uM to 1mM).  

 

In discussing these observations, it has been suggested that these
observations are a function of the T200 microfluidics which exhibit a
reversal of sample/analyte flow at high  sample concentrations. 

 

The observables are

1.       Regardless of the duration of the injection, the signal response
rises to a maximum halfway through the injection and then decreases 


2.       This behavior appears to be concentration dependent.


It's been suggested that this is a sample dependent phenomenon, where a
reversal of flow occurs in the microfluidic channel above the sensor if the
sample concentration exceeds some value that is a function of the molecule
size and its diffusivity.  

 

The effect I am seeing appears to be concentration dependent, and the
response above the sensor surface is unusual in that above a certain
concentration, the response reaches a maximum value almost exactly halfway
through the injection time/volume (63% in both examples above) regardless of
the injection time.  It makes sense that at long injection times there would
be a decrease in the response during the end of the injection time due to
dispersion of the sample plume in the running buffer, but in this particular
case, while this response is observed at 240 seconds, a relatively long time
to use on the Biacore instrument, it is also observed at 120sec which I
would not consider a long injection time.

 

These types of concentrations regimes and exposure times are not the
injection protocols I normally use, thus I have not seen this behavior
previously.  

 

In your estimation, is it possible for flow to be reversed within a
microfluidic channel at high sample concentration and for this to result in
the observed behavior?

 

I would like to thank the list in advance for any comments or advice you may
provide, and wish everyone a happy holiday season.

 

Best regards,



John Sumida

Molecular Analysis Facility

University of Washington

 

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