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***This subsection needs to be checked (+Eylea)***
Ranibizumab was purchased from Novartis (Frimley, UK) (Lucentis
10mg/mL) 10~mg/ml) and bevacizumab from Roche (Welwyn Garden City, UK) (Avastin
25mg/mL). 25~mg/ml). All other reagents were of the highest available purity and were either from Sigma (Poole, UK), VWR (Lutterworth, UK), or Merck (Hoddesdon, UK) unless otherwise specified.
\subsection{Sample preparation}
***This subsection needs to be checked (+Eylea).***
Ranibizumab and bevacizumab solutions were twice dialysed against phosphate buffered saline pH 7.4 (PBS) using D-Tube Midi Dialyzer units from Novagen
(6-8kD (6-8~kD cut-off). Subsequently, the purity of the dialyzed proteins was checked by standard SDS-PAGE. The proteins were then conjugated to the fluorophore Bis(2,2′-bipyridine)-4′-methyl-4-carboxybipyridine-ruthenium bis(hexafluorophosphate) (synonym, N-succinimidyl ester-Ru(bpy)2(mcbpy-O-Su-ester)(PF6)2)(Sigma-Aldrich) by using a succinimidyl ester-modified fluorophore with a short linker (Invitrogen, F6130). Conjugation reactions were performed in PBS adjusted with bicine buffer to pH 8.6 at
2mg/mL 2~mg/ml protein concentration with the activated fluorophore ester being used in excess. The reaction was stopped after two hours and conjugated proteins were separated from remaining free dye by size exclusion chromatography (7kD cut-off; Thermo Fisher). Using this method we generated ranibizumab conjugated to dye with a dye:protein ratio of 1.1:1 and bevacizumab with a dye:protein ratio of
1.7:1. 1.3:1. Azide
(2.0mM) (2.0~mM) was added to all fluorophore-conjugated drugs to protect them from microbial deterioration.
\subsection{Tr-FAIM measurements}
The Ru-labelled drug molecules in buffer were mixed with glycerol
and PBS in different proportions to produce solutions with different viscosities. A drop of each mixture was placed in a multiwell plate with \#1.5 coverslip glass bottom. The refractive index of each solution was measured with a refractometer (Bellingham+Stanley, U.K.) before and after the fluorescence measurement and converted to viscosity using a function fitted to a conversion chart.\cite{Glycerine1963}
A simplified diagram of the data acquisition setup is shown in Fig~\ref{fig:setup}a. The anisotropy experiments were performed with Leica SP2, a standard confocal inverted microscope. A pulsed diode laser (PLP-10 470, Hamamatsu, Japan; optical pulse width 90~ps) was used as the excitation source at 200~kHz repetition rate. The beam was
focussed focused in the middle of the well containing the sample solution with a 20$\times$ NA0.5 air objective (Leica HC PL Fluotar). The emission was collected with the same
objective, objective through a
green 550~nm long-pass emission
filter (550LP, details?) filter. A polariser was inserted in the emission path and
completely open pinhole. Parallel parallel and perpendicular
polarization polarisation components of the fluorescence emission were
separated with a polarising beam splitter (details?) and recorded
simultaneously sequentially with
two a photomultiplier
tubes tube (PCM-100, Hamamatsu, Japan) connected to a time-correlated single photon counting (TCSPC) acquisition card (SPC 830, Becker\&Hickl GmbH, Berlin, Germany). The measurement time window was 5~$\mu$s, with
total data acquisition time of
$\sim$60~min 30-60~min per data set.
\subsection{Calculation of hydrodynamic radii}
...
\end{equation}
where $A_1$ and $A_2$ are the amplitudes and $\phi_1$ and $\phi_2$ the rotational correlation times of the two different components.
The longer rotational correlation time for each protein obtained from the fitting was plotted against the viscosity. For each protein this yields a straight line, from which the hydrodynamic radius $R_h$ of the rotating unit
can be was calculated by combining the Stokes-Einstein-Debye equation (eq~\ref{eq:SED}) with the equation for the volume of a sphere:
\begin{equation}
R_h=\sqrt[3]{\frac{3kT}{4\pi}\frac{\phi}{\eta}} \label{eq:R_h}
\end{equation}
