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ENH fix the snb decay curve method and results to not overreach

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Nathan Dwarshuis 2021-07-28 12:32:22 -04:00
parent 0e52b87b04
commit 395efa6657
1 changed files with 32 additions and 18 deletions
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@ -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 were removed from the retronectin plates using vigorous pipetting and
transferred to another 96 well plate wherein expansion continued. transferred to another 96 well plate wherein expansion continued.
% METHOD snb decay curve generation and analysis (including the equation used to \subsection{sulfo-NHS-biotin hydrolysis quantification}
% fit the data)
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 % 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 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 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, increase the number of deprotonated amines on the surface of the microcarrier,
it also increases the number of OH\textsuperscript{-} groups which can it also increases the number of \ce{OH-} groups which can spontaneously
spontaneously hydrolyze the \gls{snb} in solution. hydrolyze the \gls{snb} in solution.
Furthermore, we observed that washing the microcarriers after autoclaving Furthermore, we observed that washing the microcarriers after autoclaving
increases the biotin binding rate (\cref{fig:dms_qc_washes}). While we did not 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 lightly-suspended peptides/protein fragments are also measured and therefore
inflate the readout. inflate the readout.
% TABLE decay curve half lives
Lastly, we asked what the effect on reaction pH had on spontaneous degradation 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 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 minutes of preparation, then it is important to carefully control the timing
between \gls{snb} solution preparation and addition to the microcarriers. When between \gls{snb} solution preparation and addition to the microcarriers. We
buffering \gls{pbs} to different pH's, analyzing the decay curves using UV plate found that in the presence of \gls{di} water, \gls{snb} is extremely stable
reader, and fitting an exponential decay equation to the data, we observed that (\cref{fig:dms_snb_decay_curves}) where it decays rapidly in the presence of
the half-life of \gls{snb} in solution decreases \gls{pbs} buffered to pH of 7.1. In fact, the \gls{di} water curve actually
(\cref{fig:dms_snb_decay_curves}). However, these half-lives are large enough decreases slightly, possibly due to \gls{snb} absorbing to the plate surface.
(on the order of several hours) not to be of concern assuming that the \gls{snb} \gls{snb} is known to hydrolyze in the presence of \ce{OH-}, but the lack of
solution is added within a few minutes of preparation (which it was in all our hydrolysis in \gls{di} water can be explained by the fact that biotin itself is
cases). Furthermore, we dissolved our \gls{snb} in \gls{di} water and not acidic, and thus the reaction is self-inhibitory in an unbuffered and neutral pH
\gls{pbs} which means the pH is even lower and thus the half life is even system. Because we dissolve our \gls{snb} in \gls{di} water prior to adding it
higher, further showing that the decay of \gls{snb} is not a concern. 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 % TODO use the water vs pbs curve here
% concern
\begin{figure*}[ht!] \begin{figure*}[ht!]
\begingroup \begingroup