From 395efa6657b6cd443d007f57404f6ec69a681597 Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Wed, 28 Jul 2021 12:32:22 -0400 Subject: [PATCH] ENH fix the snb decay curve method and results to not overreach --- tex/thesis.tex | 50 ++++++++++++++++++++++++++++++++------------------ 1 file changed, 32 insertions(+), 18 deletions(-) diff --git a/tex/thesis.tex b/tex/thesis.tex index 37e3204..5acdb9e 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -957,8 +957,23 @@ plates and incubated for another \SI{24}{\hour}. Cells and beads/\glspl{dms} were removed from the retronectin plates using vigorous pipetting and transferred to another 96 well plate wherein expansion continued. -% METHOD snb decay curve generation and analysis (including the equation used to -% fit the data) +\subsection{sulfo-NHS-biotin hydrolysis quantification} + +The equation for hydrolysis of \gls{snb} was assumed to follow +\cref{chem:snb_hydrolysis}. + +% TODO make this look prettier +\begin{equation} + \label{chem:snb_hydrolysis} + \ce{NHS-CO-Biotin + OH- -> NHS- + Biotin-COOH} +\end{equation} + +The hydrolysis of \gls{snb} was performed spectroscopically. \gls{snb} was added +to either \gls{di} water or \gls{pbs} in a UV-transparent 96 well plate. Kinetic +analysis using a Biotech Plate Reader began immediately after prep, and readings +at \SI{260}{\nm} were taken every minute for \SI{2}{\hour}. + +\subsection{reaction kinetics quantification} % METHOD add reaction kinetics diffusion mathy stuff @@ -1103,8 +1118,8 @@ other variables, which is not surprisingly given the behavior observed in We also observed that the reaction pH does not influence the amount of biotin attached (\cref{fig:dms_qc_ph}), which indicates that while higher pH might increase the number of deprotonated amines on the surface of the microcarrier, -it also increases the number of OH\textsuperscript{-} groups which can -spontaneously hydrolyze the \gls{snb} in solution. +it also increases the number of \ce{OH-} groups which can spontaneously +hydrolyze the \gls{snb} in solution. Furthermore, we observed that washing the microcarriers after autoclaving increases the biotin binding rate (\cref{fig:dms_qc_washes}). While we did not @@ -1115,24 +1130,23 @@ and when measuring the supernatent using the \gls{haba} assay, these soluble or lightly-suspended peptides/protein fragments are also measured and therefore inflate the readout. -% TABLE decay curve half lives - Lastly, we asked what the effect on reaction pH had on spontaneous degradation of \gls{snb} while in solution. If the \gls{snb} significantly degrades within minutes of preparation, then it is important to carefully control the timing -between \gls{snb} solution preparation and addition to the microcarriers. When -buffering \gls{pbs} to different pH's, analyzing the decay curves using UV plate -reader, and fitting an exponential decay equation to the data, we observed that -the half-life of \gls{snb} in solution decreases -(\cref{fig:dms_snb_decay_curves}). However, these half-lives are large enough -(on the order of several hours) not to be of concern assuming that the \gls{snb} -solution is added within a few minutes of preparation (which it was in all our -cases). Furthermore, we dissolved our \gls{snb} in \gls{di} water and not -\gls{pbs} which means the pH is even lower and thus the half life is even -higher, further showing that the decay of \gls{snb} is not a concern. +between \gls{snb} solution preparation and addition to the microcarriers. We +found that in the presence of \gls{di} water, \gls{snb} is extremely stable +(\cref{fig:dms_snb_decay_curves}) where it decays rapidly in the presence of +\gls{pbs} buffered to pH of 7.1. In fact, the \gls{di} water curve actually +decreases slightly, possibly due to \gls{snb} absorbing to the plate surface. +\gls{snb} is known to hydrolyze in the presence of \ce{OH-}, but the lack of +hydrolysis in \gls{di} water can be explained by the fact that biotin itself is +acidic, and thus the reaction is self-inhibitory in an unbuffered and neutral pH +system. Because we dissolve our \gls{snb} in \gls{di} water prior to adding it +to the microcarrier suspension (which itself is in \gls{pbs}) this result +indicated that hydrolysis is not of concern when adding \gls{snb} within +minutes. -% TODO add the water curve to the figure just to make it clear this is not a -% concern +% TODO use the water vs pbs curve here \begin{figure*}[ht!] \begingroup