Helen Bridle

Detection of Cryptosporidium in miniaturised fluidic devices

Contamination of drinking water with the protozoan pathogen, Cryptosporidium, represents a serious risk to human health due to the low infectious dose and the resistance of this parasite to chlorine disinfection. Therefore, several countries have legislated for the frequent monitoring of drinking water for Cryptosporidium presence. Existing approved monitoring protocols are however time-consuming and do not provide essential information on the species, virulence or viability of detected oocysts. Rapid, more information-rich and automatable systems for Cryptosporidium detection are highly sought-after, and numerous miniaturised devices have been developed to address this need. This review article aims to summarise the state-of-the-art and compare the performance of these systems in terms of detection limit, ability to determine species, viability and performance in the presence of interferents. Finally, conclusions are drawn with regard to the most promising methods and directions of future research.

Application of microfluidics in waterborne pathogen monitoring: A review

A review of the recent advances in microfluidics based systems for the monitoring of waterborne pathogens is provided in this article. Emphasis has been made on existing, commercial and state-of-the-art systems and research activities in laboratories worldwide. The review separates sample processing systems and monitoring systems, highlighting the slow progress made in automated sample processing for monitoring of pathogens in waterworks and in the field. Future potential directions of research are also highlighted in the conclusions.

Targeting Cryptosporidium parvum capture

Polymer microarrays offer a high-throughput approach to the screening and assessment of a large number of polymeric materials. Here, we report the first study of protozoan-polymer interactions using a microarray approach. Specifically, from screening hundreds of synthetic polymers, we identified materials that either trap the waterborne protozoan parasite, Cryptosporidium parvum, or prevent its adhesion, both of which have major practical applications. Comparison of array results revealed differences in the adhesion characteristics of viable and non-viable C. parvum oocysts. Material properties, including polymer composition, wettability and surface chemistry, allowed correlation of binding and identification of structure function relationships. Understanding C. parvum binding interactions could assist in improved water treatment processes and the identified polymers could find applications in sensor and filter materials.

On-chip fabrication to add temperature control to a microfluidic solution exchange system

We present a concept for the post production modification of commercially available microfluidic devices to incorporate local temperature control, thus allowing for the exact alignment of heating structures with the existing features, e.g. wells, channels or valves, of a system. Specifically, we demonstrate the application of programmable local heating, controlled by computerized PI regulation, to a rapid solution exchanger. Characterisation of the system to show that both uniform temperature distributions and temperature gradients can be established, and to confirm that the solution exchange properties are undisturbed by heating, was achieved using in situ thermometry and amperometry.

Exploitation of Nanotechnology for the Monitoring of Waterborne Pathogens: State-of-the-Art and Future Research Priorities

Contaminated drinking water is one of the most important environmental contributors to the human disease burden. Monitoring of water for the presence of pathogens is an essential part of ensuring drinking water safety. In order to assess water quality it is essential to have methods available to sample and detect the type, level and viability of pathogens in water which are effective, cheap, quick, sensitive, and where possible high throughput. Nanotechnology has the potential to drastically improve the monitoring of waterborne pathogens when compared to conventional approaches. To date, there have been no reviews that outline the applications of nanotechnology in this area despite increasing exploitation of nanotechnology for this purpose. This review is therefore the first overview of the state-of-the-art in the application of nanotechnology to waterborne pathogen sampling and detection schemes. Research in this field has been centered on the use of engineered nanomaterials. The effectiveness and limitations of nanomaterial-based approaches is outlined. A future outlook of the advances that are likely to emerge in this area, as well as recommendations for areas of further research are provided.

Analysis of Parasitic Protozoa at the Single-cell Level using Microfluidic Impedance Cytometry.

At present, there are few technologies which enable the detection, identification and viability analysis of protozoan pathogens including Cryptosporidium and/or Giardia at the single (oo)cyst level. We report the use of Microfluidic Impedance Cytometry (MIC) to characterise the AC electrical (impedance) properties of single parasites and demonstrate rapid discrimination based on viability and species. Specifically, MIC was used to identify live and inactive C. parvum oocysts with over 90% certainty, whilst also detecting damaged and/or excysted oocysts. Furthermore, discrimination of Cryptosporidium parvum, Cryptosporidium muris and Giardia lamblia, with over 92% certainty was achieved. Enumeration and identification of (oo)cysts can be achieved in a few minutes, which offers a reduction in identification time and labour demands when compared to existing detection methods.

Deformability Assessment of Waterborne Protozoa Using a Microfluidic-Enabled Force Microscopy Probe

Many modern filtration technologies are incapable of the complete removal of Cryptosporidium oocysts from drinking-water. Consequently, Cryptosporidium-contaminated drinking-water supplies can severely implicate both water utilities and consumers. Existing methods for the detection of Cryptosporidium in drinking-water do not discern between non-pathogenic and pathogenic species, nor between viable and non-viable oocysts. Using FluidFM, a novel force spectroscopy method employing microchannelled cantilevers for single-cell level manipulation, we assessed the size and deformability properties of two species of Cryptosporidium that pose varying levels of risk to human health. A comparison of such characteristics demonstrated the ability of FluidFM to discern between Cryptosporidium muris and Cryptosporidium parvum with 86% efficiency, whilst using a measurement throughput which exceeded 50 discrete oocysts per hour. In addition, we measured the deformability properties for untreated and temperature-inactivated oocysts of the highly infective, human pathogenic C. parvum to assess whether deformability may be a marker of viability. Our results indicate that untreated and temperature-inactivated C. parvum oocysts had overlapping but significantly different deformability distributions.

Silver Nanoparticles Decrease the Viability of Cryptosporidium parvum Oocysts

ABSTRACT Oocysts of the waterborne protozoan parasite Cryptosporidium parvum are highly resistant to chlorine disinfection. We show here that both silver nanoparticles (AgNPs) and silver ions significantly decrease oocyst viability, in a dose-dependent manner, between concentrations of 0.005 and 500 μg/ml, as assessed by an excystation assay and the shell/sporozoite ratio. For percent excystation, the results are statistically significant for 500 μg/ml of AgNPs, with reductions from 83% for the control to 33% with AgNPs. For Ag ions, the results were statistically significant at 500 and 5,000 μg/ml, but the percent excystation values were reduced only to 66 and 62%, respectively, from 86% for the control. The sporozoite/shell ratio was affected to a greater extent following AgNP exposure, presumably because sporozoites are destroyed by interaction with NPs. We also demonstrated via hyperspectral imaging that there is a dual mode of interaction, with Ag ions entering the oocyst and destroying the sporozoites while AgNPs interact with the cell wall and, at high concentrations, are able to fully break the oocyst wall.

Cascading and Parallelising Curvilinear Inertial Focusing Systems for High Volume, Wide Size Distribution, Separation and Concentration of Particles

Inertial focusing is a microfluidic based separation and concentration technology that has expanded rapidly in the last few years. Throughput is high compared to other microfluidic approaches although sample volumes have typically remained in the millilitre range. Here we present a strategy for achieving rapid high volume processing with stacked and cascaded inertial focusing systems, allowing for separation and concentration of particles with a large size range, demonstrated here from 30 μm–300 μm. The system is based on curved channels, in a novel toroidal configuration and a stack of 20 devices has been shown to operate at 1 L/min. Recirculation allows for efficient removal of large particles whereas a cascading strategy enables sequential removal of particles down to a final stage where the target particle size can be concentrated. The demonstration of curved stacked channels operating in a cascaded manner allows for high throughput applications, potentially replacing filtration in applications such as environmental monitoring, industrial cleaning processes, biomedical and bioprocessing and many more.

Analysis of Giardia lamblia Interactions with Polymer Surfaces Using a Microarray Approach

The interaction of the waterborne protozoan parasite, Giardia lamblia, with polymeric materials was investigated by microarray screening of 652 polymers. Polymers were identified which either bound G. lamblia cysts or prevented their binding. Correlation of material properties such as wettability and surface roughness with cyst attachment revealed no influence of these factors upon Giardia adhesion. However, the study of polymer composition allowed the correlation of binding and generation of polymer structure function relationships; glycol and aromatic functionalities appeared to prevent adhesion, whereas secondary amine groups promoted adhesion, in agreement with previous literature. A significant reduction in attachment was observed following both cyst treatments with proteinase K and performing experiments at extremes of pH (2 and 12). It is suggested that proteinase K removes the proteins needed for specific surface interactions, whereas extremes of pH influence either protonation of the polymer or the surface charge of the cysts. The mechanism by which the protozoa attach to polymeric surfaces is proposed to be through ion-pair interactions. Improved understanding of G. lamblia surface interactions could assist in predicting transport and fate behavior in the environment and contribute to better design of water treatment processes, while the polymers identified in this work could find use in sensor applications and membrane filtration.