[RASMB] SPR question
John Sumida
jpsumida at uw.edu
Fri Dec 15 09:51:41 PST 2017
Dear RASMB,
The sample loop is constructed from poly ether ether ketone (PEEK), and should have low adsorptive properties. Having said that however, the idea that adsorption of sample the sample loop results in a concentration gradient that persists through the injection phase is worth looking at. My limited understanding of microfluidic systems is that diffusion across the flow field is limited and thus any gradient that is set up in the sample plug would persist. I am not completely sure if this is conceptually accurate.
Currently the plan is to replace the tubing with a new injection module, (in these instruments the sample loop and injection block are molded as one unit). Once we have a pristine sample loop in place we may be able to take the old injection block, cut the sample and rejoin it to the injection block in a reversed orientation. Perhaps we can then distinguish between a possible sample fouling of the sample loop versus a fundamental issue with instrument format.
Thanks again to you all for your insights.
Best regards
John Sumida
Molecular Analysis Facility
University of Washington.
Sent from my Verizon 4G LTE Droid
On Dec 14, 2017 8:26 AM, Andrew Leech <andrew.leech at york.ac.uk> wrote:
>
> Hello all,
>
> I have been wondering whether this effect is related to laminar flow
> in the injection system. As far as I know the Biacore system includes
> air "breaks" in the injections to avoid mixing effects with contiguous
> buffer in the tubing. However it strikes me that if the sample is prone
> to stick to the tubing surface, a decrease in concentration might then
> be converted by the flow into a concentration variation over the
> injection period. However, the details of the injection system and
> flow path are rather limited in the machine manuals so I can't say
> whether this is plausible or not. I suspect that testing this out might
> need more detailed control over the flow system than is allowed by the
> standard control software.
>
> Kindest regards,
>
> Andrew
>
> On 13/12/2017 20:11, John Sumida wrote:
> > 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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> --
> Dr Andrew Leech * Laboratory Head
> Technology Facility * Molecular Interac
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