**2.5 Zeta potential measurement by zeta meter system 4.0**

Zeta meter System 4.0 instrument was used to measure the zeta potential of the Erythrocytes [12]. Zeta potential of the system is measured by applying the electric field to the samples using electrodes and determining the mobility/velocity of the particle under the applied field. Zeta potential was calculated according to the simplified Helmholtz-Smoluchowski equation as follows: Mean velocity of the 10 readings was used to calculate the zeta potential Eq. (1).

$$\textbf{Zeta} \cdot \textbf{Potential}, \,\,\xi = \frac{\textbf{113000} \times \nu \times EM}{D} \tag{1}$$

where ν = viscosity of sample in poise at temperature "t"; *EM* = electrophoretic mobility; *D* = dielectric constant.

The electrophoretic cell consists of capillary which is embedded inside a chamber having electrodes at both ends having cavity for sample connection with electrodes. From the cavity of any one end of the electrophoresis cell the Sample is introduced into the capillary to fill it completely. Electrodes are connected to the cell with the applied electric field at specific voltage of 200 V. Due to the applied electric field charged particles move towards oppositely charged electrode. Their velocity under the applied electric field is measured and expressed in terms of electro kinetic potential/zeta potential. Nowadays, this system is preferably used for determining the zeta potential of various types of biological membranes.

In this method, fresh blood samples were collected from volunteer and blood suspension was prepared as described in preparation of sample. Prior measurement of zeta potential temperature of and other parameters for ZP measurements were adjusted such as light intensity; focal plane and tracking duration were optimized for stable data collection with minimal error. The collected and prepared samples were then added to the previously cleaned zetameter cell, which were then connected to electrodes and placed under the lens over stage. The Erythrocytes were tracked by using remote by microscopicallyacquired video images, values were recorded 10 times for each sample and average zeta-potential in mv was obtained standard deviation from software Zeta meter-ZM4DAQ software.

## **2.6 Statistical analysis**

The experimental results for ZP are expressed as mean ± standard (SD). Investigated groups were compared by the statistical one way analysis of variance (ANOVA) and evaluated by Tukey's multiple comparison tests using Graph Pad Prism version 5.00 for Windows. Experimental Results were considered significant at p < 0.05 for the different groups which indicates that the control and other patient groups differ significantly from one another in all situations.
