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Diffusivity Measurements of Bacteriophages by Gradient Diffusion and Dynamic Light Scattering

Appala Raju Badireddy, Department of Civil and Environmental Engineering, University of Houston, 4800 Calhoun Rd., Houston, TX 77204-4003 and Shankar Chellam, Department of Civil and Environmental Engineering, Department of Chemical Engineering, University of Houston, 4800 Calhoun Rd., Houston, 77204-4003.

Virus diffusion coefficients and their hydrodynamic radii are crucial in quantitatively understanding their behavior in natural (e.g. subsurface transport and riverbank filtration) as well as in technological systems (e.g.coagulation/flocculation, media filtration, and membrane filtration). To date, investigators have exclusively employed dimensions obtained from crystallography or microscopy where viruses had been dehydrated prior to observations and calculated their diffusivity using the Stokes-Einstein relationship.The applicability of such approximations even for near-spherical viruses has not yet been rigorously tested. Additionally, several viruses of public health and environmental interest are non-spherical for which simplistic models relating virus diameter and diffusivity are not available.

The principal objective of this work is to experimentally measure the aqueous bulk diffusivities of several near-spherical and non-spherical bacterial viruses.A specially constructed cell was employed to measure the net diffusive flux of various near-spherical (Qβ, MS2, ΦΧ174, and PRD1), and non-spherical (Mu1, T4, P1, and P22) bacteriophages of varying dimensions in the absence of a pressure gradient. A track-etched polycarbonate membrane having near-cylindrical pores of large (8 micron) diameter was employed to avoid steric interactions between the phage and the pore walls. Experiments were conducted using phosphate buffered saline solutions to reduce electrostatic interactions and maintain virus viability during the course of the measurements. First, cell constants were obtained by performing separate experiments using salts, organics, and colloids of known diffusivities. Next, virus diffusion coefficients were obtained by using it as a fitting parameter to quantitatively model virus concentration profiles using a simple model of diffusive mass transfer arising from Fick's first law and the previously obtained cell constants.

Diffusion coefficients measurements based on macroscopic observations in the diffusion cell were compared with those obtained from local concentration fluctuations at the microscopic level using Rayleigh scattering. Virus self-diffusivities were measured using dynamic light scattering or photon correlation spectroscopy (Zetasizer Nano ZS, Malvern Instrument Ltd., Worcestershire, UK). For our measurements, a He-Ne laser (633 nm) was employed, the sample cell temperature was maintained at 22 C, and the decay of the scattered light intensity arising from Brownian motion was monitored at a fixed 90 angle.

Gradient diffusivity values (in 10-12 m2s-1) from the diffusion cell for various bacteriophages employed; Qβ, MS2, ΦΧ174, PRD1, Mu1, T4, P1, and P22 were 16.6 0.9, 16.3 0.9, 14.8 0.7, 6.1 0.1, 4.9 0.1, 4.9 0.1, 4.1 0.1, and 3.8 0.2, respectively. These were in excellent agreement from dynamic light scattering measurements. Stokes-Einstein hydrodynamic diameters for the bacteriophages were 29.1 1.6, 29.8 1.7, 32.8 1.5, 79.3 9, 98.1 13.9, 98.8 14.1, 118.5 20.5, and 127.7 23.9 for Qβ, MS2, ΦΧ174, PRD1, Mu1, T4, P1, and P22 respectively. These measurements are in close agreement with dimensions obtained by electron microscopy for the near-spherical viruses (Qβ, MS2, ΦΧ174, and PRD1). The values reported for the non-spherical viruses correspond to effective values that can quantitatively capture their diffusive transport in aqueous systems. These effective diameters can be employed in previously derived mass transfer models to better predict their fate and transport in natural and technological systems.