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</o:shapelayout></xml><![endif]--></head><body lang=EN-US link="#0563C1" vlink="#954F72"><div class=WordSection1><p class=MsoNormal style='text-autospace:none'><span style='color:black'>Dear RASMB,<o:p></o:p></span></p><p class=MsoNormal style='text-autospace:none'><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal style='text-autospace:none'><span style='color:black'>This question, is in relation to a Biacore T200 instrument and an artifact we are seeing at high sample injection concentrations (20uM to 1mM). <o:p></o:p></span></p><p class=MsoNormal style='text-autospace:none'><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal style='text-autospace:none'><span style='color:black'>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. </span><span style='color:black'><o:p></o:p></span></p><p class=MsoNormal style='text-autospace:none'><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal style='text-autospace:none'><span style='color:black'>The observables are<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in;mso-list:l0 level1 lfo1;text-autospace:none'><![if !supportLists]><span style='color:black'><span style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span style='color:black'>Regardless of the duration of the injection, the signal response rises to a maximum halfway through the injection and then decreases <br><img width=544 height=158 id="Picture_x0020_3" src="cid:image007.jpg@01D37274.D3030680"><o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in;mso-list:l0 level1 lfo1;text-autospace:none'><![if !supportLists]><span style='color:black'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span style='color:black'>This behavior appears to be concentration dependent.<br><img width=346 height=268 id="Picture_x0020_4" src="cid:image008.jpg@01D37274.D3030680"><o:p></o:p></span></p><p class=MsoNormal>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. <o:p></o:p></p><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal>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.<o:p></o:p></p><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal>These types of concentrations regimes and exposure times are not the injection protocols I normally use, thus I have not seen this behavior previously. <o:p></o:p></p><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal><b>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?<o:p></o:p></b></p><p class=MsoNormal><o:p> </o:p></p><div><p class=MsoNormal><span style='color:black'>I would like to thank the list in advance for any comments or advice you may provide, and wish everyone a happy holiday season.<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>Best regards,<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><img width=119 height=48 id="Picture_x0020_2" src="cid:image009.jpg@01D37274.D3030680"><o:p></o:p></span></p><p class=MsoNormal><span style='color:black'>John Sumida<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'>Molecular Analysis Facility<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'>University of Washington<o:p></o:p></span></p></div><p class=MsoNormal><o:p> </o:p></p></div></body></html>