# Specific Detection of PE-Included Vesicles Using Cyclic Voltammetry
## 3 Results and Discussion
### CV Response Difference for Vesicle Composition  
The values of charge \(Q_{i}\) were calculated according to the equation \(Q_{i}=\int I\ \mathrm{d}t=\int I\ \mathrm{d}(E/\
u)=(1/\
u)\ \int I\ \mathrm{d}E=\int I\ \mathrm{d}t\), in which \(Q_{i}\) is integrated from the potential scan rate of \(-200\) mV to \(500\) mV, \(I\) is the current (\(\mu\)A), \(E\) is the potential (V), and \(v\) is the sweep rate \(0.05\) V/s [29]. The charge \(Q_{i}\) (\(\mu\)C) at each step is listed in Table 1.  
Ninety-five percent of charge transfer was blocked after the monolayer of the pure MUD or 90% MUD/10% MHA was formed (the charge transfer at the gold electrode was assumed as 100%). The compositions of the monolayer had little effect on the amount of the charge transferred to the electrode. The addition of the cinnamycin on the monolayer led to no reduction of permeability, either. For the injection of the vesicles, permeability was little changed except in the case where 90% DPPC/10% DPPE vesicles were injected  
Figure 3: CV responses before and after the 10% PE-vesicle addition on the monolayer prepared with 90% MUD/10% MHA.  
Figure 2: CV response after the monolayer formation on the gold electrode.  
to the monolayer of 90% MUD/10% MHA. This result indicates that the vesicles were bound to the monolayer at a specific composition. The response for 95% DPPC/5% DPPE vesicles was only sometimes different from that for pure-DPPC vesicles. This inconsistency indicates that the vesicle immobilization was less uniform on the surface.  
The consistency differences between 5% and 10% DPPE vesicles seemed to be caused by a steric hindrance effect. Since DPPC and DPPE are saturated phospholipids, no phase-separation occurs in the surface of the vesicles. Presumably, the distribution of the lipids is uniform on the surface. Considering the geometry of the cinnamycin, no additional specific binding seems located, on average, within 8 nm\({}^{2}\) around one binding site [28]. This surface density of the binding is corresponding to the composition between 5% and 10% DPPE, because the mean molecular area of DPPE is 0.6 nm\({}^{2}\)[30]. Therefore, as long as DPPE is 10% or more, the cinnamycin appears to bind to PE at any orientation. This interpretation of the surface density is also consistent in terms of the composition of the cinnamycin density. The amount of the MHA in the monolayer was adjusted from 10 to 0.5%, and the vesicles of 90% DPPC and 10% DPPE were added to the cinnamycin for each composition of the monolayer. The CV response varied little from 10 to 1% MHA but was different at 0.5% (Figure 4). Considering the projection area of the vesicle, at least one cinnamycin seemed to be placed on the monolayer surface from 10 to 1% MHA. The theoretical value required for little change in the CV response was estimated at 0.1% MHA. Therefore, the change at 0.5% MHA indicates that equidistance between the DPPE molecules was always not maintained, although the phase separation did not occur in the lipid layer.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multicolumn{2}{c}{**Charge (\(\mu\)C)**} & \multicolumn{2}{c}{**Charge Permeability (\%)**} \\ \cline{2-5}
**Step** & **MUD** & \begin{tabular}{c} **90\% MUD/10\%** \\ **MHA** \\ \end{tabular} & \begin{tabular}{c} **MUD** \\ **MHA** \\ \end{tabular} \\ \hline \begin{tabular}{c} Monolayer \\ \end{tabular} & 1.0 & 1.0 & 5 & 5 \\ \hline \begin{tabular}{c} Cinnamycin/Monolayer \\ \end{tabular} & 1.0 & 1.0 & 5 & 5 \\ \hline \
\begin{tabular}{c} Vesicles/Cinnamycin/ \\ \end{tabular} &
\begin{tabular}{c} DPPC \\ 90\% DPPC/ \\ 10\% DPPE \\ \end{tabular} & 1.0 & 0.6 & 5 & 3 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Charge permeability with respect to each case treated on the gold electrode.  
Figure 4: CV responses after the 10% PE-vesicle addition on the monolayer prepared respectively with 99.5% MUD/0.5% MHA and 99% MUD/1% MHA.