[RASMB] SPR question

Wed Dec 13 12:11:04 PST 2017

Thank you all for your responses on and off list. I have attached a jpg file
in this email that illustrates the artifact we are observing.

I do have additional information that may be useful.

To answer Tom’s question, yes the sample prep in this current case included
an exhaustive dialysis against running buffer (10mM HEPES, 150mM NaCl, 3mM
MgCl2, 0.05%P20, pH=7.4)  – we’ve tried this experiment a number of times
with the same result and had been focused on incomplete dialysis of the
sample being the problem, so we were overly cautious in this regard for the
last experiment.  Additionally, samples were microfuged and pre-filtered
with a 0.22um syringe filter; additionally, there is no known propensity of
this protein to self-associate at these concentrations though this has not
been determined by this lab.

The term “flow reversal,” used in my initial post, is a term that was used
to describe the process to me, and it is inaccurate.  While there appear to
be examples of actual flow reversals in microfluidic channels in the
literature under specific conditions, this has not, to my knowledge been
reported, for the Biacore microfluidics system.  The use of the term refers
to the sequences of steps the instrument proceeds through during the
injection of sample where sample is initially aspirated into a sample loop
and then the flow is “reversed” and injected onto the sensor surface.
Similar deviations in surface responses are known to occur in microfluidic
systems, in general, where pulsed solution delivery is used and where the
direction of flow is reversed in order to inject sample into the channel (ie
this would include rigs that utilize sample loops for injection), and one
current working theory is that the artifact is associated with this
injection format.  Instruments, which do not use a sample loop, relying
instead on direct injection of the sample, do not, apparently, exhibit this
behavior. 

As this lab, and it appears now also other labs, have observed this effect
for proteins, I need to add that this behavior is usually associated with
small hydrophobic molecules. In our case, we are seeing these injection
phase deviations both for hydrophobic small molecule drugs as well as for a
small to medium sized protein at concentrations (12nM) well below the level
I expect to see noticeable changes in refractive index upon mixing. 

What is interesting to me, is why this happens for some molecules and not
others.  I suspect that one possible explanations would be a gradated change
in the refractive index of the solution as a result of reversing the
direction of flow, perhaps akin to what might happen in a synthetic boundary
experiment, but the deviations are quite reproducible and I would not have
expected that in the absence of a centrifugal field.

Thanks again for taking the time to consider this topic.

Best regards,

John Sumida

University of Washington.

Molecular Analysis Facility.

From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of Laue, Thomas
Sent: Wednesday, December 13, 2017 8:14 AM
To: Huber, Sylwia <sylwia.huber at roche.com>; John Sumida <jpsumida at uw.edu>
Cc: RASMB <rasmb at list.rasmb.org>
Subject: Re: [RASMB] SPR question

Hi-

I am not an SPR expert, so this may be a naive question on my part. Are you
dialyzing your samples against the solvent prior to running them? The idea
here is that you may be observing an refractive index artifact at the
midpoint injection time. Again, just a thought.

Best wishes,

Tom
University of New Hampshire

  _____  

From: RASMB <rasmb-bounces at list.rasmb.org
<mailto:rasmb-bounces at list.rasmb.org> > on behalf of Huber, Sylwia
<sylwia.huber at roche.com <mailto:sylwia.huber at roche.com> >
Sent: Wednesday, December 13, 2017 7:18 AM
To: John Sumida
Cc: RASMB
Subject: Re: [RASMB] SPR question 

Dear John, 

such deviation in the association phase we have observed in our laboratory
as well. We did comparison study with the same protein-sample system on the
Biacore 3000 and found that the deviation you observe are typical for the
T200 instrument. 

They occur only for some proteins, in our cases, very often for membrane
proteins, or systems where you have to work with detergents or exotic
buffers.

We found the same data as you. The deviation is independent on the injection
time, it occurs almost always around the middle of the association phase. We
found that this happens also for the buffer injection on the protein but not
on the reference (channel). We found also that it is dependent on the flow
rate. The highest flow rate the strongest the deviation.

I had a lot of discussions with Biacore Specialists from GE, but up to now
we did not find the reason for this observation. We speculated different
things, e.g. pressure lowering during the injection time, but up to now we
have still no answer on that. If anybody will find the solution of this
problem, please let us know. I am also very curious about this.

Kind regards,

Sylwia

Mit freundlichen Grüssen/With Kind Regards

Dr. Sylwia Huber
Principal Scientist, Biophysics Team Head

Roche Innovation Center Basel 

F. Hoffmann-La Roche Ltd., Chemical Biology

Building 65/203, Grenzacherstrasse 124

CH-4070 Basel, Switzerland
Phone: +41/61/6879507 

On Mon, Dec 11, 2017 at 8:40 PM, John Sumida <jpsumida at uw.edu
<mailto:jpsumida at uw.edu> > wrote:

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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