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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
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