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