M.Jimenez, B. Miller and H. Bridle

       Access to safe drinking is “a human right that is essential for the full enjoyment of life and all human rights" as recognized by the United Nations General Assembly resolution (A/RES/64/292-2010). However, despite this, several outbreaks are reported each month across Europe. For instance, 47,617 episodes of illness have been reported between 2000 and 2007 in Europe by the European Environment and Health Information Systems¹ while the Drinking Water Inspectorate² reported around 60 significant events caused by waterborne pathogens in England and Wales in 2012. Cryptosporidium is one well-known and highly resistant protozoa encountered in water systems³. Its presence does not correlate well with indicators, and it has been detected in water despite the absence of the four microbiological parameters (E.Coli (or faecal/thermotolerant coliforms), total coliforms, enterococci (faecal streptococci) and Clostridium perfringens) designated by the European Union for monitoring the water quality⁴.

A specific standardized procedure is thus required for detecting its potential presence relying on (i) a filtration allowing large volume of water to be treated while retaining all the particles of the same size or bigger than Cryptosporidium, (ii) an elution to remove Cryptosporidium from the filter while (iii) centrifugation and immuno-magnetic-separation are used for concentrating captured pathogens for detection. Experienced staff then performs the detection by (iv) fluorescent labelling and microscopy³. This detection is long (several days) and non-automated. The task becomes even more challenging if other pathogens have to be separated for detection after filtration. The development of new tools for easing the separation of pathogens after filtration is thus required for approaching a more automated/rapid process. This is particularly important with the growing interest in the use of molecular methods, as optimal lysis methodologies vary between different pathogen kingdoms.

Microfluidics represents an interesting approach for this purpose since the scale is appropriate for working with small particles (size of Cryptosporidium around 5 um). Some teams have proposed a direct miniaturisation of the current process for microfluidic filtration and immuno-magnetic-separation although clogging issues and the specificity to one single pathogen are still limiting their practical use. Dielectrophoresis is another technique developed for concentrating and separating Cryptosporidium but the working flowrates are usually small, while hundreds of millimetres can potentially need to be analysed after filtration⁵.

Inertial focusing is an interesting microfluidic approach for separating particles without any external forces at high flowrates. In a straight channel, particles experience a lift force due to the Poiseuille velocity profile, which leads to a displacement of particles towards the wall, perpendicularly to the fluid motion. When particles approach the wall, another lift force emerges in the opposite direction.  The equilibrium of these two forces implies a net lift force allowing particles to align along specific positions in the channel that can be controlled for efficiently separation of particles⁶. In a spiral channel, secondary rotating flows appear, namely Dean flows, and particles in such devices experience a supplementary force, the Dean drag. The equilibrium of the net lift force and this Dean drag allows particles to be focused along a single position in the channel, near the inner wall. This spiral inertial focusing is function of the size of the particles. Large particles will tend to focus on the equilibrium position while smaller ones will follow the Dean flows. Since the focusing position is predictable, an appropriate design of the outlets allows a size-based separation by separating large particles from a sample. Along with the size, other parameters such as the flowrate, channel aspect ratio, curvature, particle deformability, density and symmetry are of prime importance for controlling efficiently this separation⁷. Despite being characterized by high flowrate (dozen of mL/min reported in the literature), spiral inertial focusing has already been proposed for the separation of non-spherical biological matters such as circulating tumor cells in blood⁸. It represents thus an interesting approach for separating Cryptosporidium from other particles after filtration in drinking water. Moreover, pathogens, depending on their kingdom (virus, bacteria, protozoa), present characteristic sizes offering an interesting sorting parameter. However, a strong comprehension of the focusing effect on non-spherical biological particles is still lacking in the literature, which hinders any prediction of the behaviours of these pathogens in such a device.

       The purpose of this paper is to investigate spiral-inertial focusing for the separation of Cryptosporidium and potentially other harmful pathogens after filtration in drinking water at high flowrate to propose an alternative to the current procedure of separation.

       Harmful pathogens can be listed in three kingdoms: (i) viruses presenting a spherical shape (average size around 50 nanometers), (ii) bacteria usually characterised by a rod-shape (average size around 1 um) and protozoa such as Cryptosporidium that present an ellipsoidal shape with an average equivalent diameter around 5 micrometres. It can be noted that some protozoa present a different shape than Cryptosporidium. Giardia, for instance, presents a much more flattened shape. A wide range of size and shape thus characterizes pathogens that can be encountered in drinking water. The task being already complex enough, experiments focus first on Cryptosporidium, which can be appropriately modelled as a first approach by polystyrene beads. A spiral channel has been designed and manufactured for focusing spherical rigid beads around 4 um. This device presents a single inlet for the sample, and two different outlets: one for collecting particles being focused and one for the waste (non-focused particles). The device is 165 um wide and 30 um high with 6 loops for generating the secondary Dean flows. A first set of experiments with polystyrene beads of different sizes down to 4 um has confirmed the single equilibrium position of particles near the inner wall to be then successfully separated in the relevant outlet. The focusing effect of 4 um beads, presumably representing the behaviour of Cryptosporidium, has been observed with a minimum flow rate of 300 uL/min and up to 1 mL/min.

Based on these successful results, experiments have then been carried out with pathogens. For the first time in the literature, focusing effects of isolated Cryptosporidium parvum have also been observed in the device (minimum of 400 uL/min). Interestingly, pathogens seem less sensitive to the focusing effect when forming clusters. As mentioned previously, a lot of different parameters interact with the focusing efficiency and the high flowrates used make the tracking of single pathogens in the device complicated. Experiments with other pathogens such as Giardia emphasises the impact of the particle shape and symmetry on the focusing effects. The impact on the focusing position as a function of the pathogen viability has also been investigated. The last experiment carried out here relies on the separation of a sample with protozoa and polystyrene beads (1 um and 50 nm) to characterize the behaviour of bacteria and virus on the separation efficiency. Recovery rates for experiments involving pathogens are proposed by analysing the inlet and both outlets with a flow cytometer while fluorescence intensities are used for the separation of polystyrene beads.

       For the first time in the literature, a spiral inertial focusing device is designed for gaining an in-depth understanding of the focusing effects on harmful pathogens in drinking water. The promising results represent an interesting first step for developing an automated separation device for waterborne pathogen monitoring.

References

1- European Environment and Health Information System-Outbreaks of waterborne diseases- Fact Sheet 1.1, 2009

2- Drinking Water Inspectorate (2012). Drinking Water 2012.

3- H. Bridle, M. Kersaudy-Kerhoas, B. Miller, D. Gavriilidou, F. Katzer, EA. Innes, et al. Detection of Cryptosporidium in miniaturised fluidic devices. Water Research, 2012.

4-WHO. Report on regulations and standards for drinking water quality. June 2014.

5- H. Bridle, B. Miller, and M. P. Desmulliez. Application of microfluidics in waterborne pathogen monitoring: A review. Water Research, 2014.

6- J. Martel, and M. Toner. Inertial Focusing in Microfluidics. Annual Review of Biomedical Engineering,  2014.

7- H. Amini, W. Lee, and D. Di Carlo. Inertial Microfluidic Physics.Lab on a Chip, 2014.

8- J. Sun et al. Double spiral microchannel for label-free tumor cell separation and enrichment. Lab on a chip, 2012.