Polyelectrolyte Multilayers As Antiadhesive Sacrificial Membrane Films: A Novel Approach to Virus Concentration for Detection

Prevention of disease outbreaks relies on the efficient detection of pathogens in drinking and recreational water. Because waterborne pathogens are typically present in low concentrations, a fast and reliable sample concentration process is a crucial step in the overall detection procedure. An emerging method of sample concentration for the detection of waterborne pathogens is crossflow membrane filtration, which offers several important advantages over traditional methods of pathogen concentration. First, membranes of an appropriate pore size can simultaneously concentrate a wide range of microbial pathogens from protozoa to bacteria to viruses without concentrating molecular toxins and low molecular weight qPCR inhibitors. Second, crossflow decreases membrane fouling and results in higher rates of concentration. Third, in the presence of crossflow a larger fraction of viruses remains suspended in the membrane retentate throughout the concentration process facilitating virus recovery for subsequent detection. To prevent virus passage through and adsorption to the membrane and to further improve recovery, the membranes are usually “blocked” with a proteinaceous solution (e.g. calf serum) and sometimes are pretreated with a chemical dispersant. Despite these measures, however, the recovery of certain pathogens such as adenoviruses remains low and difficult to reproduce.

To overcome this limitation, we propose a conceptually different approach based on anti-adhesive sacrificial membrane films (Fig. 1). These membranes are prepared by coating an ultrafiltration support with a polyelectrolyte multilayer (PEM) film consisting of alternating layers of polycations and polyanions. The procedure can be regarded as a highly controlled approach of membrane blocking by two oppositely charged polymers selected to design a film with desirable properties.  This is in contrast to blocking with calf serum wherein pores of the supporting ultrafiltration membrane are plugged by components of a complex mixture of macromolecules of various molecular weights. Main advantages of the proposed approach are 1) fast and reproducible preparation of membranes, 2) versatility in designing the coating to be antiadhesive, 3) possibility of sacrificing the coating to recover viruses that did attach to the membrane surface.

The objectives of this study were:

1)    To design anti-adhesive sacrificial membrane coatings using the PEM approach and assess their efficiency in virus recovery.

2)    To comparatively evaluate the effects of filtration conditions on the rate of concentration, rejection, and the recovery of viruses using traditional pre-concentration approach and novel PEM-coated membranes.

A polyethersulfone membrane with a MWCO of 50 kDa served as the support for PEM films.  Chitosan and heparin were coated on the support using a layer-by-layer technique [1]. In the crossflow filtration experiments, a peristaltic pump was used to draw the DI water spiked with P22 bacteriophage (10⁷ PFU/ml) from the feed tank to the filtration cell. A pressurized air tank was used to apply a transmembrane pressure of 7 psi to 40 psi. An in line flowmeter was used to monitor the crossflow. Experiments were performed at crossflow rate over initial permeate flow rate ratio, Jcf/Jp, in the (1,500 to 13,000) range. Crossflow rate during filtration was between 0.11 m/s and 0.42 m/s (Reynolds number between ca. 400 and ca. 1600). Following filtration the membrane was eluted at the same crossflow rate than the filtration with a solution containing 0.01% Tween 80 and 0.01% NaPP. Grab samples of the feed, retentate, permeate and eluate were taken and analyzed by qPCR. For qPCR analysis, P22 DNA was extracted using MagNA Pure Compact System.

We demonstrate that coating a membrane with a PEM film, a 1-hour process, results in removal and post-elution recovery values that are at least as high as those obtained with conventional membranes (95% on average), and significantly better pre-elution recovery for all types of water tested (Fig. 2). Pre-elution P22 recovery is significantly higher at medium or high values of crossflow-to-permeate flux ratio.

The high pre-elution recovery values observed for PEM-coated membranes show potential for eliminating the need for an elution step, thus reducing both the time required for pathogen concentration and the volume of the final sample. Moreover, the possibility of removing the coating [2] should increase recovery of highly adhesive and difficult to concentrate viruses such as adenoviruses.

References

1. G. Decher, Fuzzy nanoassemblies: toward layered polymeric multicomposites, Science 277 (1997) 1232.
2. Shan, W., P. Bacchin, P. Aimar, M. L. Bruening, V. V. Tarabara. Polyelectrolyte multilayer films as backflushable nanofiltration membranes with tunable hydrophilicity and surface charge. J. Membr. Sci. 349 (2010) 268.