**4. Estimation of zeta potential of prepared blood sample by zeta meter system 4.0**

The zeta potential of the RBCs was is measured using zeta meter system 4.0. Zeta potential is purely an electro kinetic property of the electrical double layer surrounding the system but not the surface of the system itself. The value of zeta potential gives an indication about the stability of the system under study. This quantity is measured by determining the mobility/velocity of the particle under an applied electric field. The value of zeta potential can be obtained from the equation given by Helmholtz-Smoluchowski.

$$\mathcal{L}\_{\rm d} = \left(\mathsf{4}\,\mathsf{π}\mathfrak{η}/\mathsf{e}\right)\,\mathsf{V} \tag{2}$$

where ζd = electro kinetic potential/zeta potential, η = viscosity of dispersion medium, ϵ = dielectric constant of the dispersion medium, V = v/E (mobility of the particle), v = velocity of the particle in cm/s, E = potential gradient in V/cm [20].

A special capillary cell called electrophoretic cell is used for the measurement of zeta potential. The capillary is embedded inside a chamber having electrodes at either of the two ends. Sample is placed from any one end of the electrophoretic cell and electrodes are connected to the cell and electric field at specific voltage is applied (200 V). Charged particles move towards oppositely charged electrode and their velocity is measured and expressed in terms of electro kinetic potential/ zeta potential which indicates the mobility of particle under applied electric field. Recently this method is widely used for determining the membrane potential of biological membranes.

In this experiment, fresh capillary blood samples were obtained from volunteer by puncturing the skin with a lancet and blood suspension was prepared as described in above procedure. Prior to zeta potential measurement temperature of the RBC suspensions were measured and detection parameters for ZP measurements such as light intensity, focal plane and tracking duration were optimized for stable data collection. The RBC suspensions were then added to the previously cleaned and calibrated (using min-u-sil) zeta-meter cell placed under the zeta-meter stage and the mobility of individual RBCs was tracked by equipped

Zeta meter-ZM4DAQ software using microscopically-acquired video images, and data were recorded 10 times for each sample and average zeta-potential in mv was determined using a standard Helmholtz-Smoluchowski formula.
