EXAMENSARBETE INOM MILJÖTEKNIK,AVANCERAD NIVÅ, 30 HPSTOCKHOLM, SVERIGE 2017

Advanced Technologies for Detection of Cryptosporidum parvum in Drinking water: capture and detection using Microfluidic devices and Imaging Flow Cytometer

SAFA KARIMI MOLAN

KTHSKOLAN FÖR ARKITEKTUR OCH SAMHÄLLSBYGGNAD

Advanced Technologies for Detection of Cryptosporidum

parvum in Drinking water: capture and detection using

Microfluidic devices and Imaging Flow Cytometer

Safa Karimi Molan

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TRITA LWR Degree ProjectISSN 1651-064XLWR-EX-2017:06

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ھمسرمتقدیم به مادرم، پدرم و

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Summary Protecting drinking water supplies from pathogens such as Cryptos-poridium parvum is a major concern for water treatment plants worldwide. The sensitivity and specificity of current detection meth-ods are largely determined by the effectiveness of the concentration and separation methods used. In this study, disposable microfluidic micromixers were fabricated to effectively isolate Cryptosporidium parvum Oocysts from water samples, while allowing direct observa-tion of Oocysts captured in the device using high quality immunofluo-rescence microscopy. In parallel, quantitative analysis of the capture yield was carried out by analyzing the waste from the microfluidics outlet with an Imaging Flow Cytometer. At the optimal flow rate, cap-ture efficiencies higher than 95% were achieved in spiked samples, suggesting that scaled microfluidic isolation and detection of Cryptos-poridium parvum will provide a faster and more efficient detection method for Cryptosporidium compared to other available laboratory-scale technologies. Key Words: Drinking water treatment, pathogen detection, Cryp-tosporidium, microfluidic devices, Imaging flow Cytometry, Capture efficiency.

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Acknowledgements I would like to express my gratitude for the support of my supervisor and advisor Professor Prosun Bhattacharya. I would also like to sincerely thank my co-supervisor at University of South Australia, Ian Wark Research Inistitute Associate Professor Benjamin Thierry for his continual support and advice during my all time at the Ian Wark Research Institute.

I acknowledge Dr Lorena Dieguez for her assistance at the begin-ning of my experiments with Microfluidic devices. I would also like to express my sincere appreciation to Marnie Winter for the training of Image Stream X Cytometre. The financial support of IAN WARK RESEARCH INSTITUTE is grateful-ly acknowledged. Finally, I would like to thank SA WATER for provid-ing Cryptosphoridium solution samples for my research.

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Table of Content Summary v Acknowledgements vii Table of Content ix List of figures xi abbreviations xiii 1. Introduction 1 2. AIMS, OBJECTIVES AND HYPOTHESIS 3

2.1. Aims and objectives 3 2.2. Hypothesis 3

3. Materials and Method 3 3.1. Materials 3 3.2. Experimental Methods 3

3.2.1. Fabrication of microfluidic devices 4 3.2.2. Functionalization of the microfluidic devices 7 3.2.3. Capture of Cryptosporidium parvum Oocysts 9 3.2.4. Fluorescence microscopy studies 10 3.2.5. Imaging Flow Cytometry studies using Image Stream X Cytometer 10

4. Results and discussion 12 4.1. Microscope Imaging 12

4.1.1. Evaluating the effect of device pattern on capturing Cryptosporidium Oocysts 12 4.1.2. Evaluating the effect of flow rate on capturing Cryptosporidium Oocysts 13 4.1.3. Distribution of Cryptosporidium Oocysts into the microfluidic devices 15 4.1.4. Flow Cytometry image analysis 16 4.1.5. Capture efficiency by Imaging Flow Cytometry 21

5. Conclusion 23 6. References 25

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List of figures Fig. 1. SU-8 master moulds on 4 inch silicon wafers. ............................................................ 6

Fig. 2. Master covered with PDMS and placed under a vacuum desiccator to remove any bubbles from the PDMS. ..................................................................................... 6

Fig. 3. A digital photo of oxygen plasma cleaner (PDC-002, Harrick Plasma, USA). .......... 7

Fig. 4. A schematic representation of surface chemistry for the functionalisation of a microfluidic device. ............................................................................................................... 8

Fig. 5. A digital photo of microfluidic experimental set up after Glutaraldehyde functionalisation in a fume hood. Note the yellow colour within the withdrawing waste syringes which indicates a reaction between ATPES and the Glutaraldehyde. ......... 9

Fig. 6. A digital photo of experimental set up under biocabinet; Device connected to the syringe pump. A biocabinet was used to help maintain a sterile environment for the duration of the experiment. ......................................................................................... 9

Fig. 7. A digital photo showing configuration of the Imaging Flow Cytometer Image Stream X (ISX, AMNIS, Seattle, WA, USA). ........................................................................ 10

Fig. 8. A digital photo of high speed centrifuge for eppendorf tubes (left) and a centrifuge for larger tubes 10 and 50ml Falcon tubes ;(right). ............................................ 11

Fig. 9. Photomicrograph of the microfluidic “Post” device after running the control experiment at Cryptosporidium concentration of 1500 000 per ml and flow rate of 2 µl/min. The posts are 50 µm in size, the image was taken at100x magnification. ......... 12

Fig. 10. Microscopic images of Micromixer microfluidic devices at 10x magnification with captured Cryptosporidium (red circles), stained in blue (DAPI). The devices were run at the concentration of 1500 000 Cryptosporidium per ml. .......... 13

Fig. 11. Microscopic images of microfluidic serpentine devices with Cryptosporidium run at the flow rate of 2 µl/min. These images show the mixing process of Oocysts from different microfluidic channels within the microfluidic device (Images are extracted from a video record). ............................................................. 14

Fig. 12. Microscopic images at 40x (a, b, d) and 60x (c) of the Cryptosporidium Oocysts stained in blue with DAPI (a,b,c), captured inside the functionalized microfluidic devices at the optimal flow rate of 2 µl/min. , stained with both DAPI and FITC (d) ........................................................................................................................... 15

Fig. 13. The distribution of Cryptosporidium Oocysts in the microfluidic device decreases from inlet to outlet (left to right). This decrease in Oocysts towards to end of the microfluidic device demonstrates the strong specific binding of the Oocysts to the functionalised device. .................................................................................... 16

Fig. 14. Average number of Cryptosporidium Oocysts captured in a micromixer with a flow rate of 2 µl/min at different penetration lengths from inlet to outlet. The dark grey line shows a functionalized experiment device and the light grey is the non-functionalised control device. ................................................................................. 16

Fig. 15. Example of the overall snapshot from the IDEAS software for image analysis. .................................................................................................................................. 17

Fig. 16. Cryptosporidium images from the different channels in the Imaging Flow Cytometer (Brightfield (BF), Darkfield) and the combined image with (a) only DAPI staining, and (b) DAPI and secondary FITC antibody (binding to the Cry104 primary antibody). Images were taken at 40× magnification. ........................................... 18

Fig. 17. (a) Images of Cryptosporidium clusters from different channels in the Imaging Flow Cytometer (b) Composite image -combination of three channels, Brightfield, DAPI and FITC- for a cluster of Cryptosporidium Otocysts. Images were taken at 40× magnification. ......................................................................................... 18

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Fig. 18. Images of (a) beads, and (b) debris from different channels (composite, DAPI, FITC, Darkfield and Brightfield) in the Imaging Flow Cytometer. Images were taken at 40× magnification. ......................................................................................... 19

Fig. 19. A graph of the normalised frequency against the area of brightfield demonstrating the area for classified population of both single oocytes and cluster of Oocysts. .............................................................................................................................. 19

Fig. 20. The circularity graph of single Cryptosporidium particles based on the brightfield image of each particle. The particles can be seen to be fairly homogenous with a small number presenting as less circular, potentially due to their orientation or due to their shape..................................................................................20

Fig. 21. Single oocytes with (a) circular and (b) non circular shapes at different channels of Flow Cytometry. (b) demonstrates the change when Oocysts present in a different orientation and therefore are seen as less circular. Images were taken at 40× magnification..................................................................................................................20

Fig. 22. Three stages of categorizing and differentiating the Cryptosporidium oocyte population from the other particles, the frequency of the particles at different brightfield gradients (a) the intensity of darkfield versus DAPI (b) and the intensity of FITC versus DAPI (c). .................................................................................. 21

Fig. 23. Capture efficiency of Cryptosporidium Oocysts in the microfluidic devices at different flow rates calculated with Imaging Flow Cytometry. Lined bars are the functionalised experimental results and the checked bars are the non-functionalised control devices. .............................................................................................. 22

Fig. 24. Dependence of the capture efficiency of Cryptosporidium Oocysts in microfluidic micromixers at 2 µl/min at concentrations 15,000 and 1,500,000 Cryptosprodium/ml, using Imaging Flow Cytometry. Lined bars are the functionalised experimental results and the checked bars are the non-functionalised control devices. .............................................................................................. 23

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Abbreviations

DAPI 4′,6-Diamidino-2-phenylindole dihydrochloride PDMS Polydimethylsiloxane IMS immuno-magnetic separation IFA Immunofluorescence microscopy GPTMS 3-Glycidyloxypropyl)trimethoxysilane APTMS (3-aminopropyl)trimethoxysilane GA glutaraldehyde PBS phosphate buffered saline UV Ultra Violet EVA Ethylene-vinyl acetate APTES 3-Aminopropyl triethoxysilane MQ water Milli-Q Water BSA Bovine Serum Albumin RCF relative centrifugal force ISX Image Steam X FITC Fluorescein isothiocyanate

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1. Introduction Cryptosporidium is a highly resistant protozoan commonly encoun-tered in surface waters (Kotloff et al. 2013).Cryptosporidium contam-inates drinking water and may have severe health consequences for human being. Among 20 species of Cryptosporidium only Cryptos-poridium parvum is recognized by such severe health effects. This kind of Cryptosporidium is a major cause of acute diarrhoea. Three main reasons has been identified for such serious risks which are (1) the resistance of Cryptosporidium to chlorine disinfection, (2) its long term survival in water supplies and (3) its low infectious dose. Apart from human health problems, Cryptosporidium parvum Oocysts are environmental contaminants because of their strong shells, their widespread presence at source water supplies and their continual ex-isting in the environment. The Oocysts shells have two different inner and outer layers, mostly composed of proteins, which are responsible to protect them from environmental harsh conditions. There is an in-verse relationship between temperature and Oocyst survival. At higher temperatures, Oocyst wall tends to loose its integrity (Butkus, Bays & Labare 2003; Carey, Lee & Trevors 2004; Kuznar & Elimelech 2006). Although Cryptosporidium infections are self-limiting in healthy indi-viduals (Kotloff et al. 2013), the consequences can be far more serious for three vulnerable groups of people causing moderate to severe diar-rhea. The first group is infants and young children in developing world. The second affected group is elderly people all over the world. Cryptosporidium is also fatal for immuno-compromised individuals, people with deficiency in their body’s immune system, which might be more problematic in developed countries. In fact, contamination of drinking water supplies with Cryptosporidium is a serious threat for large scale outbreaks of infections both in developing and developed countries. Some recent outbreaks of this kind of infectious disease, Cryptosporidiosis, have been recorded in UK, Australia and Sweden (Bridle et al. 2012). In order to protect water supplies from Cryptos-poridium, various barriers at different stages should be considered; Surface and ground water protection, optimization of the water treat-ment processes, and maintaining distribution systems all contribute to the safe water provision. Cryptosporidium outbreaks are often linked to treatment failures or treatment deficiencies at water treatment plants, allowing contamination of drinking water. Risk management of source waters requires cost effective, rapid and efficient monitoring of Cryptosporidium (Betancourt & Rose 2004). Cryptosporidium Oocysts are resistant to inactivation by common dis-infections such as chlorine and monochloramine. Ozone is more effi-cient in inactivating Oocysts since the free radicals of the ozone attacks the oocyst wall, but the efficiency decreases at lower water tempera-tures and Oocysts are still resistant to ozone concentrations which are commonly used in water treatment processes. Among advanced water treatment processes, Micro filtration and Ultra filtration as well as Re-verse osmosis filters are capable of complete removal of Oocysts. Moreover, UV light, as an alternative disinfection method, also inacti-vates Oocysts effectively. However, these advanced treatment process-es are not commonly used all over the world due to their cost (Betancourt & Rose 2004; Carey, Lee & Trevors 2004; Chalmers & Katzer 2013). There is a huge concern about transmission of Cryptosporidium in drinking water supplies. The risk for such a global problem is consid-ered to be high when the number of Oocysts is increased to 10-30 in 100 L of treated water. The presence of Cryptosporidium has to be monitored regularly for the water supplies as part of guidelines for drinking water quality. Procedures for Cryptosporidium detection in-cludes collection of a large volume of water sample (10 -1000 L), fol-

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lowed by concentration using various techniques including filtration and centrifugation to obtain a concentrated sample. The standardized procedure EPA 1623 describes a complete isolation/detection protocol based on filtration, elution from the filter and centrifugation to obtain a concentrated sample. The recovered sample then undergoes im-munomagnetic separation to isolate Oocysts from debris and the iso-lated Oocysts are stained with specific fluorescent antibodies and DAPI for detection by immunofluorescence and microscopy (Carey, Lee & Trevors 2004). Despite wide application, the EPA 1623 detec-tion method has several limitations. Purification of the samples in or-der to separate Oocysts from other particles by Immuno Magnetic Separation (IMS) may not be sufficient for some particle removal, for example Algal cells. Besides, IMS and staining protocols is a slow pro-cess. Moreover, Immunofluorescence microscopy (IFA) for Cryptos-poridium detection requires skilled technicians and are time and re-source-consuming. While immunofluorescence microscopy is the gold standard other antigen based detection systems including ELISA and immunochromatographic assays are also commercially available (Bri-dle et al. 2012; Carey, Lee & Trevors 2004; Checkley et al. 2015; Cheng et al. 2012; Edzwald et al. 2001; Kar et al. 2011; Smith & Nichols 2010). A number of alternative approaches have also been developed (Bridle et al. 2012), including those based on Surface Plasmon Resonance (Kang et al. 2006) and nucleic acid detection (Crannell et al. 2014) as well as microfabricated (Taguchi et al. 2007; Warkiani et al. 2011) or microfluidic filters (Kim et al. 2014), micromeshm (Taguchi et al. 2007), and microwells (Taguchi, Takeyama & Matsunaga 2005). More recently, microfluidics has been advanced with some success to enrich Cryptosporidium (Jimenez, Miller & Bridle 2015) and Giardia (Ganz et al. 2015) from water and food samples. However, microfluidic ap-proaches lack detection specificity. The use of biofunctionalized mi-crofluidic devices have been very successful in the isolation of circulat-ing tumor cells (CTC) from the blood of cancer patients (Nagrath et al. 2007). The application of a biofunctionalized microfluidic device for the immunospecific capture of Cryptosporidium as presented in this study has not been investigated. (Diéguez et al. 2015). Resistance to conventional water treatment processes and the absence of adequate methods for quick detection of the Oocysts necessitates the development of efficient detection methods for Cryptosporidium parvum Oocysts. Applying particularly automated methods may pre-vent outbreaks of Cryptosporidium to the distributing system by sav-ing time and providing more precise results. Recent developments in microfluidic systems led us to focus on evaluating the efficiency of this method for capturing Cryptosporidium parvum Oocysts (Bange, Hal-sall & Heineman 2005), (Bridle et al. 2012), (Ramirez & Sreevatsan 2006), (Smith & Nichols 2010), (Straub & Chandler 2003). Recent re-search studies have also shown that flow cytometry is potentially an effective tool to quantify Cryptosporidium Oocysts from water sam-ples compared to fluorescence microscopy. Moreover, problems that conventional flow cytometry faces including the presence of auto-fluorescent debris and clumps of Cryptosporidium, inhibiting proper identification, are potentially eliminated with Image Stream X flow cy-tometry (Hsu et al. 2005),(Keserue, Füchslin & Egli 2011), (Lepesteur, Blasdall & Ashbolt 2003).

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2. Aims, objectives and hypothesis 2.1. Aims and objectives

The aim of this project was the capture and detection of Cryptosporid-ium Oocysts spiked in water applying novel microfluidic devices and Imaging Flow Cytometry. Currently, the most common monitoring protocol for detecting Cryp-tosporidium Oocysts is EPA method. However, the whole process is time consuming. This project aimed at examining the efficiency of mi-crofluidic device capture and Flow cytometry studies in producing re-liable results to detect and quantify Cryptosporidium and the ability to analyses a sufficient volume of sample. The aim was achieved by investigating the following objectives: • Evaluating the effect of geometrically diverse microfluidic devices • Evaluating the effect of different flow rates on Oocysts capture and

optimising flow rate for best capture in microfluidic devices • Evaluating the effect of Cryptosporidium concentrations by spiking

different numbers of Cryptosporidium in to water samples

2.2. Hypothesis • Functionalised microfluidic devices can be used for the specific cap-

ture of Cryptosporidium parvum Oocysts allowing the isolation, identification and enumeration of oocyts in a water sample.

• Imaging flow cytometry can be used to detect and enumerate Cryp-tosporidium parvum Oocysts in a water sample and can also be used to enumerate capture efficiency of microfluidic devices.

3. Materials and Method

3.1. Materials 3” silicon wafers were purchased from Micro Materials & Research Cons. Pty Ltd (Australia), SU-8 50 photoresist was purchased from MicroChem (USA) and Polydimethylsiloxane (PDMS) elastomer SYLGARD 184was purchased from Dow Corning (USA).(3-Glycidyloxypropyl)trimethoxysilane (GPTMS), trichloro 1,1,2,2-perfluorooctyl-silane, (3-aminopropyl)trimethoxysilane (APTMS), glutaraldehyde (GA), phosphate buffered saline (PBS), formaldehyde, Triton™ X-100, 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) and goat anti-mouse IgG FITC secondary were purchased from Sigma Aldrich (USA). Irradiated Cryptosporidium parvum were kind-ly donated from SA Water (Australia). Specific monoclonal antibody cry104 was obtained from (BTF Biomerieux). Chemicals were pur-chased from Sigma-Aldrich (USA) unless stated otherwise. All other chemicals were analytical grade. The syringe pumps (model KDS-212-CE, KDS-210) used were obtained from KD Scientific. A FITC goat anti mouse IgG secondary antibody was purchased from Sigma Aldrich (USA). Polydimethylsiloxane (PDMS) elastomer SYLGARD 184 was obtained from Dow Corning (USA) and SU-8 10 photoresist was pur-chased from MicroChem (USA). All other chemicals were analytical grade. Silicon wafers with a 3” diameter were obtained from Micro Materials & Research Cons. Pty Ltd (Australia), and the syringe pumps KDS-212-CE and KDS-210 used in this study were purchased from KD Scientific.

3.2. Experimental Methods Standard soft lithography was used to produce microfluidic devices. Disposable PDMS (Poly-di-methyl siloxane) microfluidic devices were fabricated and sealed by means of oxygen plasma. Then, the devices were functionalized with an antibody specific against antigens ex-

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pressed on the oocyst surface. Using a silane-based functionalisation strategy, antibodies were immobilized on the channel surface. Known numbers of Cryptosporidium were introduced through the microfluid-ic devices at different flow rates. The Oocysts captured in the device were washed, fixed, permeabilized and stained with DAPI prior to mi-croscope examination. The waste solution recovered from the devices was also analysed with a state-of-the-art Imaging Flow Cytometer Im-age Stream X (ISX, AMNIS, Seattle, WA, USA). The following sections explain in detail all the experimental procedures (Bridle et al. 2012).

3.2.1. Fabrication of microfluidic devices Soft photolithography technique is generally applied in different areas for creating patterns in materials such as polymers. Due to the intro-duction of soft lithography using moulding polymers in the late 90’s, a significant decrease in the cost of fabricating microfluidic devices oc-curred. Poly-di-methyl siloxane (PDMS) is currently the most popular and widely used elastomer for fabrication of microfluidic devices for cell biological applications. PDMS has appropriate mechanical proper-ties (mouldable) as well as being permeable to gas, biocompatible and transparent; all of these properties make the material as a perfect choice for microfluidic device fabrication. Applying Soft photolithography technique, Masters for PDMS mould-ing were fabricated. A silicon master mould, designed with a specific pattern and fabricated from a photoresist material (most commonly SU-8). The process of fabricating microfluidic devices based on this method is composed of several steps. Before starting the fabrication process, the pattern of the device should be designed and sent to the manufacturer to produce the mask. The produced pattern contains chambers, posts and or channels etc. and can be utilized to fabricate dozens of PDMS replicas. The type of material used for the master production is Chrome. The reason for that is it is possible to illuminate selected areas of a sub-strate through the mask by UV exposure. When ordering the mask, it is required to specify whether it is Brightfield or Darkfield. Brightfield means that the produced mask is exactly the same as the designed lay-out, while darkfield means the opposite; in which the shape of the pat-tern is excluded from the master. It is also important to know which type of resist is being applied in the process, positive or negative. In a positive resist, areas exposed to UV light will be removed in the devel-opment stage, but in a negative resist the exposed areas remains. For the current experimental study darkfield master with a negative SU-8 was applied. The procedure for master mould fabrication requires the use of a spin coater and UV source. It is recommended that the master fabrication procedure is carried out in a clean room to ensure the masks features are developed to the desired precision and accuracy. The following separate processes have been conducted for device fabrication. Coating a substrate with SU-8 photoresist The substrate is a silicon wafer which needs an epoxy structures on top. The epoxy used in this instance is SU-8. SU-8 photoresist is man-ufactured in different grades, determined by the amount of solids with respect to the solvent. A number followed by the ‘SU-8’ indicates the grade of the specific SU-8 type, those numbers can range from 2 to 500 indicating the lowest and highest viscosity of the SU-8 respective-ly. Since the viscosity differs, the spin speed has to be adjusted ac-cordingly. For appropriate adhesion between the Si wafer and the SU-8 resist the wafer needs to be clean. Silicon wafers were cleaned by subsequent

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sonication steps in acetone and isopropanol for 5 minutes each, dried with nitrogen gas and activated in an oxygen plasma for 5 minutes. Upon activation the wafers were immersed in 5% GPTMS in ethanol for 1h, rinsed in ethanol and cured at 80ºC for 1 hour. The master mould was fabricated by spin coating (Karl Suss Delta 80 spin coater, Germany) the negative photoresist SU8-50 on to a clean silicon wafer. SU8-50 was dispensed on the wafer, which was then ac-celerated at 250 rpm to 500 rpm for 5 seconds and further to 1600 rpm for 30 seconds to produce a SU-8 layer of desired thickness of about 25µm. Pre-exposure bake/soft bake The wafer with the SU-8 film needs to be baked prior to the UV expo-sure. This is done to remove any remaining solvent and stabilize the film. The pre-exposure bake also makes the surface non-stick, which prevents it from leaving remains on the Cr mask. The pre-exposure bake is done on a hot plate in 2 steps, first at 65ºC for 10 minutes and second at 95ºC for 30 minutes. After the bake the resist needs to cool off before UV exposure. Ultra Violet Exposure SU-8 is a negative resist, meaning exposed areas remains after devel-opment. The exposure time/dose depends on resist thickness. The wa-fer was exposed through the designed darkfield mask with a UV lamp with the exposure intensity of 250 mJ/cm2. Post-exposure bake The post-exposure bake is done on a hot plate for 3 minutes at 65oC and 10 minutes at 95oC in order for the pattern to stand out. Silanisation The process of Silanisation is required prior to master replication, oth-erwise; PDMS sticks to the SU-8. The process must be completed un-der fume hood since it is highly toxic. The wafer was hydrophobized with a vapour-phase treatment in trichloro 1,1,2,2-perfluorooctyl-silane for 1h in a desiccator and cured for 1h at 80ºC. Master mould replication and device sealing Once the surface of the master is silanised, it can be used to produce PDMS replicas up to 20 times. Figure 1 shows a SU-8 master mould on a silicon wafer which is used for producing PDMS replicas. A new master should be fabricated as the master gets older since the PDMS attaches to the master surface and makes it difficult to peel off the rep-licas. Polydimethylsiloxane (PDMS) was prepared at a 10:1 (w/w) ra-tio, degassed and was cured against the master. The replicate was then cut out, removed, the holes for the inlet and outlets were punched, the devicecleaned and treated by Oxygen plasma and finally sealed to the clean glass slide. All the steps for PDMS device fabrication are ex-plained in detail as follows:

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PDMS preparation Polydimethylsiloxane (PDMS) or Sylgard 184 is used to generate the pattern. PDMS is transparent and has low viscosity as uncured, both favourable features for soft lithography. To mould PDMS devices from the master, a curing agent is mixed with liquid PDMS at the ratio of 1:10 for a few minutes which incorporates a huge amount of air in the solution. Then the air bubbles were removed by degassing which can be conducted in a vacuum desiccator. Figure 2 shows a master with PDMS on top under a vacuum desiccator. The sample is removed when it becomes clear and transparent. After PDMS is poured over the master and degassed it is cured at 80ºC for at least 60 minutes. After curing, the PDMS was unmoulded and the inlet and outlet were punched to create 1.5 mm diameter holes for tube insertion. Then to clean the replicas from residual PDMS, they were rinsed with Isopro-panol, dried by N2 gas and left to evaporate in the oven at 80oC for at least 15 minutes.

Figure 2. Master covered with PDMS and placed under a vacuum desicca-tor to remove any bubbles from the PDMS.

Figure 1. SU-8 master moulds on 4 inch silicon wafers.

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PDMS-Glass Binding Oxygen plasma (PDC-002, Harrick Plasma, USA) (Figure 3) was uti-lised to further clean the surface of any organic residue and activate the surface for binding to seal the device with a clean glass slide. The plasma preparation will incorporate oxygen atoms in the PDMS sur-face, leaving it hydrophilic. This can be used to bond PDMS to glass, or even to another PDMS stamp. The surface of clean glass slides and PDMS replicas were activated with oxygen plasma at low power for 15 seconds. Then, the PDMS and glass were bound together and any air bubbles removed from their contact using tweezers. The devices were placed in the oven for at least 10 minutes at 65 ºC. Such a process pro-duces irreversible bonding between glass and PDMS.

Ethylene-vinyl acetate (EVA) tubing (Cole-Palmer, USA) was cut to lengths of 25cm and inserted into the inlets and outlets of the devices, the tubing were held in place with epoxy glue (Selly’s, Australia).

3.2.2. Functionalization of the microfluidic devices The aim of the functionlization process is to prepare the surface of the device for antibody attachment. First, the microfluidic devices were connected to a syringe pump (NE-4000, Syringe Pump, USA) through 25 centimetre length tubing. While inlet tubing is connected to the sy-ringe pump, the outlet tubing was placed in different liquid/chemical containers during the experiments. All the functionalisation process before the antibody stage has been conducted under fume hood. From the antibody stage onwards the experimental set up was performed in a Biocabinet in sterile conditions. For each set of the experiments, two parallel devices have been conducted, one of them to act as an actual experiment with specific antibody functionalisation and the other as a control one, not functionalized with the specific Cryptosporidium an-tibody. Initially a series of experiments were performed with a new master pattern, in order to calculate the volume of the device (as this will dif-fer slightly between replicas). First, the syringes were filled with 5 ml of ethanol and connected to the inlet tubing while outlets were placed

Figure 3. A digital photo of oxygen plasma cleaner (PDC-002, Harrick Plasma, USA).

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in ethanol solutions. All the functionalization steps were completed at100 µl/min. The volume of the tubing is calculated by the time that ethanol requires to flow from the inlet of the tubing to the inlet of the device or from the outlet of the device to the outlet of the tubing at the speed of 100 µl/min. Similarly, the volume of the devices were calcu-lated using the time ethanol required to flow from the inlet of the de-vice to the outlet of the device at the speed of 100µl/min. These vol-umes provide the actual timing which is required for each step in the fictionalisation process with each chemical. For the functionalisation of the devices, three important chemicals are required; 3-Aminopropyl triethoxysilane (APTES) and glutaraldehyde (GA) and Antibody (cry104). Fig 4 depicts the schematic of the surface chemistry for the functionalisation of a microfluidic device.

APTES is toxic chemical and the solution is prepared in Ethanol with-in the fume hood. This step with APTES required the device to be pre and post washed by Ethanol. Likewise, the device requires pre and post wash by MilliQ Water for GA functionalisation by 1% GA solution in MilliQ water. The functionization process starts with stabilising the device in etha-nol; at least 3ml of ethanol was pumped through the devices as an ini-tial wash to remove any debris and to ensure the device is sufficiently sealed and no leaks are present. While the surface inside the devices is still activated from the prior plasma treatment, a 2% (3-Aminopropyl) triethoxysilane (APTES) so-lution in ethanol withdrawn through the devices for 30 minutes, leav-ing amine groups at the surface for further surface functionalisation, then it is rinsed in ethanol for 10 minutes to remove any unbound APTES residue. The buffer is then changed to MilliQ water and stabi-lized for 10 minutes prior to withdrawal of 1% GA in water for another 30 minutes. GA utilises the aldehydes homobifunctional characteris-tics, forming an imine bond with the available amine functional groups and leaving an exposed aldehyde functional group for further binding. Any residual GA solution was rinsed by withdrawing MQ wa-ter for 10 minutes. Figure 5 shows the microfluidic experimental set up after GA functionalisation.

Figure 4. A schematic representation of surface chemistry for the function-alisation of a microfluidic device.

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The Syringe pump along with devices then relocated to the biocabinet and the devices were equilibrated using at least 3ml of Phosphate Buffered Saline (PBS) withdrawn through the devices just before in-troducing 200 µl of 50 µg/ml cry104 in PBS that was left to react overnight in the fridge to ensure all binding sites were occupied. The next morning unreacted antibody was rinsed with PBS for 10 minutes and the surface blocked for any non specific binding with 2% Bovine Serum Albumin (BSA) in PBS. Figure 6 shows experimental set up un-der biocabinet. Control devices were functionalized following the same protocol, but no antibody underwent conjugation. Following this step different protocols were followed depending on the specific criteria of each experiment.

3.2.3. Capture of Cryptosporidium parvum Oocysts

Different concentrations of oocyst solutions in PBS were prepared in Eppendorf tubes and mixed with a vortex mixer to maintain homoge-neity of the solutions before withdrawing into both the functionalized

Figure 6. A digital photo of experimental set up under biocabinet; Device connected to the syringe pump. A biocabinet was used to help maintain a sterile environment for the duration of the experiment.

Figure 5. A digital photo of microfluidic experimental set up after Glutaral-dehyde functionalisation in a fume hood. Note the yellow colour within the withdrawing waste syringes which indicates a reaction between ATPES and the Glutaraldehyde.

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device with Cry104 antibody (to allow recognition by their specific an-tibodies) and the control devices without antibody at flow rates of 0.5, 2 and 5 µl/min. Washing of un-bound Oocysts was conducted at the same flow rates by PBS. After the Oocysts were isolated into the de-vice, they were fixed with 4% formaldehyde, permeabilised with 0.5% Triton and stained with 1:10,000 DAPI to allow microscopic identifi-cation. After DAPI staining for 10 minutes and washing by PBS for 10 minutes, the devices were disconnected from the syringes and sealed from the atmosphere by putting the ending of the tubes in Eppendorf containing PBS solutions, this also prevented evaporation. It is worth mentioning that the first series of experiments with micro-fluidic devices, cold methanol was used instead of formaldehyde in or-der to fix the Cryptosporidium Oocysts within the device, but the methanol was problematic and caused difficulties to the process by in-troducing bubbles in to the devices. Bubble presence is one of the ma-jor problems while working with microfluidic devices. In order to fix the problem, formaldehyde was introduced in to the process for the rest of the experiments which reduced bubble formation and e en-trapment within the devices.

3.2.4. Fluorescence microscopy studies The microfluidic devices containing the isolated Oocysts were exam-ined under a Nikon Ti Eclipse inverted fluorescence microscope. The number of DAPI fluorescent bodies identified as Oocysts captured in-side the device was counted from 50 randomly chosen 10× low magni-fication fields of view at different sections of the device and used to de-termine the capture efficiency at different flow rates. Confirmation of the presence of Cryptosporidium Oocysts was achieved by staining the isolated bodies with the fluorescently tagged antibody FITC-cry104. Briefly, after capture, permeabilization and blocking was performed with 0.05% Triton X-100 and 10% fetal bovine serum for 10 minutes. Oocysts were incubated with FITC-cry104 primary antibody for 30 minutes asper the manufacturer’s instructions, before washing and imaging.

3.2.5. Imaging Flow Cytometry studies using Image Stream X Cytometer The state-of-the-art Imaging Flow Cytometer Image Stream X (ISX, AMNIS, Seattle, WA, USA) combines the high throughput quantitative nature of flow cytometry with high resolution imaging. Figure 7 illus-trates the configuration of the equipment at Nano and Biomaterial La-boratory at Ian Wark Reasearch Institute, University of South Austral-ia.

Figure 7. A digital photo showing configuration of the Imaging Flow Cy-tometer Image Stream X (ISX, AMNIS, Seattle, WA, USA).

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Image Stream X is an excellent tool for not only high throughput de-tection of Cryptosporidium parvum within water samples but also al-lows for cytomorphologic analysis with brightfield & darkfield chan-nels and specific identification with fluorescent channels. The brightfield channel demonstrates the morphology of Cryptosporidium parvum Oocysts with their distinct internal structure. The darkfield channel shows the amount of laser side scatter from the Cryptosporid-ium particles and measures the granularity/complexity of the parti-cles. Cryptosporidium parvum oocytes have distinct morphology fea-tures which can be identified enumerated and characterised with the Image Stream X analysis system. Therefore, all waste solutions recov-ered from microfluidic devices were analysed with the Image Steam X. The solutions were placed in 1.5ml Eppendorf Tubes and centrifuged at 10,000 RCF for 15 minutes at 4ºC (Fig. 8). Note: centrifugation speed was optimized to avoid particle cell loss but also not result in damage. The supernatants were then removed and the pellets resus-pended with PBS to 100 µl for Imaging Flow Analysis. Since Cryptos-poridium particles were already stained with 4',6-diamidino-2-phenylindole (DAPI) during the microfluidic experiments, channel 1 of the flow cytometer was set to record all the DAPI stained events, with the 405 laser. Channel 4 and 6 were set for brightfield and darkfield images, respectively. In order to discriminate the Oocysts from speed beads (small polystyrene beads used to focus the ISX camera), a size classification was applied. All laser intensities were optimized for de-tection without creating pixel saturation. Morphological characterisa-tion was then used to separate the Cryptosporidium from any auto-fluorescent debris. In order to confirm that the DAPI-stained bodies identified as oocytes are indeed Cryptosporidium parvum, a Fluores-cein isothiocyanate (FITC) secondary antibody was used. 25 µl of a concentrated Oocysts solution was resuspended in 300µl cold perme-abilisation block (1xPBS with 0.5% Sodium Azide, 0.05% Trition X-100, 10% FBS) and incubated for 10 minutes on ice. Then the sample was spun down, the supernatant removed and the pellet resuspended in 100µl of permeabilisation buffer (1xPBS with 0.5% Sodium Azide, 0.05% Trition X-100, 1% FBS) and 2µg/ml of cry104 mAb was added to the solution as primary antibody. After incubation for 60 minutes, the sample was centrifuged, the supernatant removed and the pellet washed with 500µl of permeabilisation buffer. 1µl of 1:50 FITC sec-ondary antibody was added to the pellet. A control sample was pro-duced adding the secondary antibody to a sample not conjugated with the primary to identify any possible non-specific staining. At each sample acquisition set up, Image Stream X takes about 50 µl of the solution from Eppendorf tube and introduces a stream of beads to the sample while processing. Beads are 1 µm spherical polystyrene which assists the machine in focussing the camera. The machine takes

Figure 8. A digital photo of high speed centrifuge for eppendorf tubes (left) and a centrifuge for larger tubes 10 and 50ml Falcon tubes;(right).

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hundreds of images of the particles per-second as the sample flows in front of the camera. The number of the images acquired depends on the time frame applied for the data acquisition stage. Two computers are connected to the cytometer equipped with specific Software, for Data Acquisition and Data Analysis called “INSPIRE” and “IDEAS”, respectively. Image Stream X enables classification of pathogens based on their morphology and can be used to identify cells. Cryptosporidium Par-vum Oocysts have special morphology, features and characteristics which is visible in the image stream analysis.

4. Results and discussion 4.1. Microscope Imaging

4.1.1. Evaluating the effect of device pattern on capturing Cryptosporidium Oocysts Two different patterns were considered in order to fabricate the devic-es. One type called “Post” Device and the other called ‘Serpantine’, ‘F-shaped’,’ Chaotic mixer’ or ‘micromixer’ device. All series of experi-ments contained two separate parallel experiments. The first series of experiments were both control, meaning that no functionalization process with antibody has been undertaken on the devices. The only difference between them was the pattern used for the master fabrica-tion of the devices. The aim of conducting these experiments was comparing the suitability of the geometry of the devices for Cryptos-poridium oocyst capture. Figure 9 illustrates a “post” microfluidic device after running the control experiment at Cryptosporidium con-centration of 1500 000 per ml and flow rate of 2 µl/min. Examining both devices under the microscope showed that the “post” device was not capable of capturing the Cryptosporidium Oocysts, whereas a number of Cryptosporidium Oocysts were detected with control Mi-cromixer device under microscope.

It is therefore concluded that the distance between the posts and con-figuration of the posts is not ideal for capturing the Oocysts with post device. While, the micromixer device with numerous array of mixers designed to enhance the surface interaction between Cryptosporidium

Figure 9. Photomicrograph of the microfluidic “Post” device after running the control experiment at Cryptosporidium concentration of 1500 000 per ml and flow rate of 2 µl/min. The posts are 50 µm in size, the image was taken at100x magnification.

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occysts and the channel walls demonstrating that the Geometry of the device is an important factor in capturing Cryptosporidium particles.

4.1.2. Evaluating the effect of flow rate on capturing Cryptosporidium Oocysts The Cryptosporidium oocyte solutions were withdrawn at a very high concentration into the micromixers at 3 different flow rates. A high concentration of oocytes than would be seen in real world situations was used to ensure adequate concentrations within the device and en-able valid microscopic analysis. Each experiment was accompanied by a control and all the experiments were performed in triplicate. As shown by microscopic observations (Fig.10), the higher number of Cryptosporidium was retained in the functionalized devices at 2 µl/min and minimal non-specific binding in the control (non-functionalized) devices was observed, confirming the specific nature of the binding.

The results show that the mixing of Oocysts and surface interaction between the Oocysts and device surface is best achieved in the flow rate of 2 µl/min within the micromixer device. In order to observe such a chaotic mixing process, videos have been recorded from one device under microscope at different flow rates of 0.5, 2 and 5 µl/min. The recording proved that neither 0.5 nor 5 µl/min flow rates shows the chaotic mixing for the moving particles through the micromixer channels of the device, which supports our initial finding that the op-timum flow rate is 2 µl/min. In fact, at the 0.5 and 5 µl/min flow rate Oocysts from separate channels do not interact with the Oocysts from other channel stream which decreases the chance of attaching to the chanel surface or walls However, the flow rate of 2 µl/min provides the particle with the ideal speed for the interaction and mixing with the particles from the other stream all contributing to increase the proba-bility of being captured by the device. Figure 11 illustrates images from video records of 2 µl/min flow rate experiment, showing mixing pro-cess of Oocysts from different channels.

Figure 10. Microscopic images of Micromixer microfluidic devices at 10x magnification with captured Cryptosporidium (red circles), stained in blue (DAPI). The devices were run at the concentration of 1500 000 Cryptosporidium per ml.

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Cryptosporidium Oocysts captured inside the microfluidic devices are shown at higher magnifications (Figure 12), 40x (a) and (b) and 60x (c) are stained in blue with DAPI while Figure 12(d) illustrates the Oo-cysts at 40x magnification stained with both DAPI and FITC. FITC secondary antibody is specific to Cryptosporidium but it is an expen-sive chemical, staining process with FITC is also more complicated comparing to DAPI staining. Therefore, it is beneficial to target the Oocysts with DAPI staining first and confirm their presence with sec-ondary antibody. Cryptosporidium Oocysts captured inside the micro-fluidic devices are analysed by microscope at different magnifications (Figure 12). Cryptosporidium Oocysts which are stained with DAPI are blue in colour (Figures (a) to (c)), while Oocysts stained with both DAPI and FITC are visible in a combined blue and green colours (Fig-ure 12(d)). This dual staining confirms that the features which are stained as Cryptosporidium Oocysts by DAPI staining are actual oo-cysyts.

Figure 11. Microscopic images of microfluidic serpentine devices with Cryptosporidium run at the flow rate of 2 µl/min. These images show the mixing process (a-c) of oocysts from different microfluidic channels within the microfluidic device (Images are extracted from a video record).

(a)

(b)

(c)

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4.1.3. Distribution of Cryptosporidium Oocysts into the microfluidic devices

The Oocysts had a strong binding affinity to antibody-functionalized devices as shown by preferential binding at the inlet side. Figure 13 shows four different microscopy images of a device at different pene-tration lengths, where Cryptosporidium parvum Oocysts at a concen-tration of 1.5 106/ml were captured at the flow rate of 2 µl/min. As it is clear from the images, the number of Oocysts captured decreases to-wards the outlet of the device. To confirm this observation, the number of Oocysts captured at differ-ent lengths into the device was systematically counted using the fol-lowing protocol: the number of Oocysts captured was counted at 10 randomly chosen areas at different lengths into the device (0, 4, 8, 12 and 16 mm), 0 and 16 being the inlet and the outlet of the micromixer, respectively. Then the number of Oocysts per area was averaged at each length and plotted in Figure 14. It is observed that the number of Oocysts found in the microfluidic device decreases with the penetra-tion length, while the number remains constant in the control device, proving that the Cryptosporidium Oocysts have specific and strong binding affinity to the functionalised surface and the Cry104 antibody.

Figure 12. Microscopic images at 40x (a, b, d) and 60x (c) of the Cryptosporidium oocysts stained in blue with DAPI (a,b,c), captured inside the functionalized microfluidic devices at the optimal flow rate of 2 µl/min. , stained with both DAPI and FITC (d)

(d)

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4.1.4. Flow Cytometry image analysis

Imaging Flow Cytometry, Image Steam X (ISX, AMNIS, Seattle, WA, USA) studies of the samples provided precise information about the Cryptosporidium parvum Oocysts in the sample. The unbound oo-cytes were recovered from the outlet of the microfluidic devices and prepared for analysis in the ISX as explained in section 3.2.5. Figure 15, illustrates one example of the overall screenshot from the IDEAS software for image analysis, categorising and interpreting the data.

Figure 14. Average number of Cryptosporidium oocysts captured in a micromixer with a flow rate of 2 µl/min at different penetration lengths from inlet to outlet. The dark grey line shows a functionalized experiment device and the light grey is the non-functionalised con-trol device.

Figure 13. The distribution of Cryptosporidium oocysts in the microfluidic device decreas-es from inlet to outlet (left to right). This decrease in oocysts towards to end of the microflu-idic device demonstrates the strong specific binding of the oocysts to the functionalised device.

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Figure 16 shows single Cryptosporidium Oocysts recorded from dif-ferent channels in the ISX: Brightfield, darkfield and stained only with DAPI –blue(a), or (b) with DAPI and a Cry104 antibody bound to a FITC secondary antibody –blue and green, respectivly. It is clear from the images that both staining protocols for DAPI and FITC are appro-priate for Cryptosporidium detection with Image Stream X analysis. Figure 16b shows that all the DAPI identified events also show green fluorescence when stained with the Cry104 primary and FITC second-ary antibodies, while there is no non-specific binding of the secondary in the control sample. Therefore, confirming that in this specific case (with no other contaminating cellular material) DAPI staining is enough to properly identify the Cryptosporidium oocytes.

Figure 15. Example of the overall snapshot from the IDEAS software for image analysis.

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Figure 17 shows the efficiency of staining and detection of a clump of Oocysts. Figure 17b also shows the combination of three channels- Brightfiled, DAPI and FITC of a cluster of Cryptosproidium Oocysts. Therefore demonstrating that not only single Cryptosporidium are de-tected with this method.

Figure 17. (a) Images of Cryptosporidium clusters from different channels in the Imaging Flow Cy-tometer (b) Composite image -combination of three channels, Brightfield, DAPI and FITC- for a cluster of Cryptosporidium Otocysts. Images were taken at 40× magnification.

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DAPI/FITC/BF DAPI FITC Darkfield BF

4181

DAPI/FITC/BF DAPI FITC Darkfield BF

10 µm

2976

DAPI/FITC/BF

10 µm

Figure 16. Cryptosporidium images from the different channels in the Imaging Flow Cytometer (Brightfield (BF), Darkfield) and the combined image with (a) only DAPI staining, and (b) DAPI and secondary FITC antibody (binding to the Cry104 primary antibody). Images were taken at 40× magnification.

(a)

(b)

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Figure 18 depicts images of beads, and debris with images of the dif-ferent channels. As it is clear from the Figure 18a, beads present a strong darkfield effect while debris (Fig. 18b) presents both strong au-tofluorescence and darkfield effects. At this image, it is even possible to easily differentiate between debris and Cryptosporidium Oocysts from their different appearances in Brightfield channel, however, in some other cases there is a strong resemblance between autofluores-cent particles and Cryptosporidium oocytes in brightfield. Moreover, the intensity of the darkfield channel helps in differentiating between the particles. The application of primary Cry104 and FITC secondary antibodies also assists in accurate detection of the Cryptosporidium Oocysts in complicated cases such as in water samples.

Figure 19 depicts the classified population of both single oocytes and a cluster of Oocysts based on their size. Apart from differentiating be-tween single and multiple oocyte populations, it is possible to distin-guish between circular and non circular Oocysts

Figure 20 demonstrates the circularity graph of the single Cryptospor-idium particles. Two single oocytes with circular and non circular

Figure 19. A graph of the normalised frequency against the area of bright-field demonstrating the area for classified population of both single oocytes and cluster of oocysts.

Figure 18. Images of (a) beads, and (b) debris from different channels (composite, DAPI, FITC, Darkfield and Brightfield) in the Imaging Flow Cytometer. Images were taken at 40× magnification.

3618

DAPI/FITC/BF DAPI FITC Darkfield BF

10 µm

1427

DAPI/FITC/BF DAPI FITC Darkfield BF

10 µm

2991

DAPI/FITC/BF DAPI FITC Darkfield BF

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(b)

(a)

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shapes are also illustrated in Figure 21. In general, the Cryptosporidi-um particles are extremely homogenious in size and circularity, how-ever, a small number of particles present in a different orientation or have a slightly elongated shape and are therefore classified as non-circular. The use of the circularity feature may help in the characteri-sation of these particles and indeed provide another variable to sepa-rate these cells from debris and other particles.

4399

DAPI/FITC/BF DAPI FITC Darkfield BF

10 µm

5913

DAPI/FITC/BF DAPI FITC Darkfield BF

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Figure 22a-c shows three stages for categorizing and identifying the Cryptosporidium oocyte population from beads and other particles. Figure 22a shows the frequency of the particles at different brightfield gradients, the area of peak in this graph is selected and the intensity of darkfield versus DAPI is plotted for the selected particles (Fig. 22.b). This plot depicts two distinct populations of the particles. One popula-tion with higher darkfield intensity and lower DAPI intensity describes the characteristics of debris/impurities in the sample. The other popu-lation- circled and named as R2, represents higher DAPI intensity and low darkfield intensity which represents Cryptosporidium Oocysts to-gether with any autofluorescent particles with the similar brightfield gradient of the Cryptosporidium oocytes. Cryptosporidium oocytes were then selected from this population based on their brightfield morphology or stained with Cry104 primary and secondary FITC anti-bodies, then the intensity of FITC plotted versus DAPI (Fig. 22c), in order to filter out such autofluorescent particles. The population with high intensities in both FITC and DAPI are identified as definite Cryp-tosporidium Oocysts which is shown as the R3 population in Fig. 22.c. After defining the precise area around Cryptosporidium particles, the

Figure 21. Single oocytes with (a) circular and (b) non circular shapes at different channels of Flow Cytometry. (b) demonstrates the change when oocysts present in a different orientation and there-fore are seen as less circular. Images were taken at 40× magnification.

Figure 20. The circularity graph of single Cryptosporidium particles based on the brightfield image of each particle. The particles can be seen to be fairly homogenous with a small number presenting as less circular, poten-tially due to their orientation or due to their shape.

(a)

(b)

21

statistical information on the number of particles in each population has been extracted. The number of Cryptosporidium Oocysts then used to calculate the capture efficiency of the microfluidic devices.

4.1.5. Capture efficiency by Imaging Flow Cytometry

The results of quantitative analysis from Imaging Flow Cytometry, was used to assess the capture efficiency of Cryptosporidium in the micro-fluidic devices. Once the protocol to properly identify and count the oocytes with the ISX was defined, the Cryptosporidium Oocysts were counted for all the waste solutions of the experiments and controls as well as a pre-sample and used to calculate the capture efficiently of the microfluidic devices. These numbers combined with the total number of introduced Cryptosporidium Oocytes to the microfluidic device at each specific

Figure 22. Three stages of categorizing and differentiating the Cryptosporidium oocyte popu-lation from the other particles, the frequency of the particles at different brightfield gradients (a) the intensity of darkfield versus DAPI (b) and the intensity of FITC versus DAPI (c).

22

experiment were used to determine the efficiency applying the follow-ing formula. E = (( C T – C W) / CT) × 100 where E is capture efficiency of microfluidic device, CT is total number of Cryptosporidium Oocysts introduced to the device and CW is num-ber of Cryptosporidium Oocytes detected by flow Cytometry. A maximum capture efficiency of 96% was determined at 2 µL/min (Fig. 23) while all the control experiments show a low non-specific binding, which was in good agreement with the qualitative microscop-ic observation.

To further confirm the validity of our system to isolate and quantify the Cryptosporidium presence in water samples, the capture efficiency of Cryptosporidium Oocysts was also studied at lower concentrations. For this purpose, two different concentrations of Cryptosporidium, 1.5×106 and 1.5×104 Oocysts/ml, were introduced at the same flow rate, 2 µl/min, in the micromixers and the capture efficiency was ana-lysed with means of Imaging Flow Cytometry. As shown in Figure 24, the capture efficiency for the concentrations tested remains constant independently of the dilution of the working solution.

Figure 23. Capture efficiency of Cryptosporidium oocysts in the microfluidic devices at different flow rates calculated with Imaging Flow Cytometry. Lined bars are the functionalised experimental results and the checked bars are the non-functionalised control devices.

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5. Conclusion

Identifying rapid and automatable systems for Cryptosporidium mon-itoring at water treatment plants are of paramount importance. The aim of this research was to capture and detection of Cryptosporidium Oocysts spiked in water applying novel microfluidic devices and Imag-ing Flow Cytometry. The aim was achieved by setting a few objectives to evaluate the effect of (1) geometrically different microfluidic devic-es, (2) different flow rates and (3) different concentration of the ocysts on capture efficiency of Cryptosporidium Oocysts in microfluidic de-vices. In order to confirm the hypothesis of this research study both functionalised and control devices have been examined for each exper-imental set of the study. Flow cytometry equipment was also tested for its ability in both detecting the oosists and determining the capture ef-ficiency of Oocysts in the microfluidic devices by analysing the waste solution from the devices. The results showed that geometry of the device is an important factor in capturing Cryptosporidium particles. Serpentine or F- shaped devices were more effective in capturing the Oocysts comparing to Post devices. A capture efficiency of 96% was achieved at the optimal flow rate of 2 µl/min. It was also concluded that capture efficiency of the functionalised devices remain constant at lower concentration of the Oocysts. Imaging Flow Cytometer was used to determine the capture efficiency of the Cryptosporidium in the devices which were also successfully applied in detecting the Cryp-tosporidium particles. These flow cytometer observations were in close agreement with microscopy analysis. Microscopic observations revealed that a high number of Cryptosporidium Oocysts were re-tained in the functionalized devices compared to control devices. Therefore, the Oocysts had a strong binding affinity to the antibody-functionalized devices as shown by preferential binding at the inlet side.

Figure 24. Dependence of the capture efficiency of Cryptosporidium oo-cysts in microfluidic micromixers at 2 µl/min at concentrations 15,000 and 1,500,000 Cryptosprodium/ml, using Imaging Flow Cytometry. Lined bars are the functionalised experimental results and the checked bars are the non-functionalised control devices.

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The main limitation of the proposed approach is its limited through-put, which restricts its application to water concentrates. However, it is anticipated that a minor redesign and/or multiplexing of the micro-fluidic mixers could lead to higher throughputs. Minimal non-specific binding of Cryptosporidium in control devices was observed, confirm-ing the specific nature of the binding in PDMS devices. Even though the concentrations of Cryptosporidium in this study were high, we ex-pect that the results are scalable for low concentrations. In summary, the microfluidic micromixer approach has the potential to accelerate and simplify the detection of Cryptosporidium and other microorganisms of concern in surface or drinking water. Moreover, this research demonstrated that imaging flow cytometry is a useful technology to identify and quantify the Cryptosporidium pathogen in water samples. Therefore, this combined novel approach of applying microfluidic devices and flow cytometry equipment in detection of Cryptosporidium in drinking water proved a high potential to acceler-ate and simplify the detection process of Cryptosporidium in drinking water treatment plants.

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