Disposable microfluidic micromixers for effective capture of Cryptosporidium parvum oocysts from water samples
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Protecting drinking water supplies from pathogens such as Cryptosporidium parvum is a major concern for water utilities worldwide. The sensitivity and specificity of current detection methods are largely determined by the effectiveness of the concentration and separation methods used. The purpose of this study is to develop micromixers able to specifically isolate and concentrate Cryptosporidium, while allowing in situ analysis.
In this study, disposable microfluidic micromixers were fabricated to effectively isolate Cryptosporidium parvum oocysts from water samples, while allowing direct observation and enabling quantification of oocysts captured in the device using high quality immunofluorescence 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, capture efficiencies up to 96% were achieved in spiked samples.
Scaled microfluidic isolation and detection of Cryptosporidium parvum will provide a faster and more efficient detection method for Cryptosporidium compared to other available laboratory-scale technologies.
KeywordsCryptosporidium parvum oocysts Disposable microfluidic micromixers Imaging flow cytometry Water quality Immunocytochemistry Fluorescence microscopy
Bovine Serum Albumin
Enzyme-Linked Immunosorbent Assay
Fetal Bovine Serum
Imaging Flow Cytometer
Microfluidic Impedance Cytometry
Phosphate Buffered Saline
Cryptosporidium is a highly resistant protozoan commonly encountered in surface waters. Although Cryptosporidium infections are self-limiting in healthy individuals, the consequences can be far more serious in infants and young children causing moderate to severe diarrhea . 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 . Procedures for Cryptosporidium detection typically includes collection of a large volume of water sample (10 - 1000 L), followed 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 immunomagnetic separation to isolate oocysts from debris. Isolated oocysts are stained with specific fluorescent antibodies and nuclear stain for detection by immunofluorescence and microscopy. This method has demonstrated a recovery yield of 61% in spiked pure water, 48.8% in spiked filter tap samples and 19.5% in raw sources, with a limit of detection of approximately 10 oocysts/L . The final steps in the EPA 1623 method require skilled technicians and are time- and resource-consuming. While immunofluorescence microscopy is the gold standard for the detection of the oocysts, other antigen based detection methods including ELISA (enzyme-linked immunosorbent assay) and immunochromatographic assays are also commercially available .
A number of alternative approaches have been reported to detect waterborne pathogens , including those based on Surface Plasmon Resonance , nucleic acid detection [7, 8, 9, 10, 11], immunocantilevers , Surface Enhanced Raman Scattering , dielectrophoresis  and impedance spectroscopy . But all detection methods rely on efficient concentration of large volume of water sample and isolation of the pathogen. A number of microfabricated filters [16, 17, 18] have been described and yielded oocysts capture efficiencies as high as 97% in spiked pure water samples. Microfluidic methods have also been actively investigated in recent years for oocysts detection due to their excellent reliability and efficiency. Owing to the inability of microfluidic systems to deal with very large volumes, these approaches have typically focused on treating samples that have already been subjected to the initial concentration step. Microfluidic filters achieved an efficiency of 86% when capturing Cryptosporidium oocysts from spiked concentrated water samples . Inertial microfluidics has also been applied with some success to enrich water and food pathogens, yielding 100% efficiency sorting Cryptosporidium from concentrated water  and 68.4% when recovering Giardia from food samples . The Nano-DEP enrichment system provided a 10 times concentration of the raw sample . These microfluidic systems rely on physical filtration or separation of oocysts from a pre-concentrated sample to deliver the sample for the final detection step. The pre-concentrated samples still need to be further processed to be analyzed using for example fluorescence microscopy. In addition, approaches based on physical features lack specificity in comparison to standard immunomagnetic separation. With this in mind, the integration of immunomagnetic separation with microfluidics has been advanced . In addition, good recovery yield using microfabricated microwells functionalized with antibodies has also been reported, although the static nature of this approach limits its application to only a small volume of sample . Recently, McGrath et al. reported on the interesting concept of high throughput Microfluidic Impedance Cytometry (MIC) for rapid enumeration and identification of different types of Cryptosporidium spiked in saline buffer and achieved over 92% accuracy in discriminating Cryptosporidium parvum, Cryptosporidium muris and Giardia lamblia .
Microfluidic devices bioconjugated with specific molecular probes have been used with remarkable success in the isolation of circulating tumor cells from the blood of cancer patients . To perform efficient immunocapture of cells in functionalized microfluidic devices, it is necessary to maximize the surface to volume ratio and to create appropriate mixing to optimize the chances of the target cells coming into contact with the immunoconjugated surface. To our knowledge, the application of a biofunctionalised micromixer for the immunospecific capture of Cryptosporidium has not been investigated thus far. Towards simplifying the workflow of Cryptosporidium oocysts detection from a pre-concentrated water sample with increasing accuracy, the aim of this work is to evaluate a streamlined microfluidic technology for its ability to specifically isolate Cryptosporidium oocysts from concentrated water samples while simultaneously allowing for high quality immunofluorescence observation. In this way, oocysts present in pre-concentrated samples (using any of the above mentioned approaches) can be specifically isolated and quantified in a single system. A positive enrichment strategy has been used due to sample/technical specificities including multiple contamination source in water samples and the existence of an antibody that binds the target oocysts with high specificity and affinity. For this purpose, disposable microfluidic devices were fabricated and functionalized with an antibody (Cry 104) specific against antigens expressed on the oocyst surface. The microfluidic device consists of an array of 25 μm thin micromixers designed to enhance the surface interactions between the Cryptosporidium and the channel walls. Once isolated within the micromixer, the oocysts can be readily stained using standard fluorescent tags and observed in situ under fluorescence microscopy, providing a simpler alternative to the current method based on immunomagnetic separation. Under optimal conditions, a recovery yield of up to 96% could be obtained for Cryptosporidium oocysts spiked in water.
The design of this microfluidic device enhances mixing and hence binding of the Cryptosporodium with its many parallel channels which not only increase the area for binding but also provides some redundancy for blockage as would be expected with environmental water.
The micromixers were fabricated in SU8 on a silicon wafer. The design consists of an array of 25 μm high microchannels favoring chaotic mixing. Standard soft lithography was used to produce disposable microfluidic devices, sealed with oxygen plasma. Using a silane-based functionalization strategy, antibodies were immobilized on the channel surface. Known numbers of Cryptosporidium were spiked in saline buffer and introduced through the microfluidic devices at different flow rates. The oocysts captured in the device were washed, fixed, permeabilized and stained prior to microscope examination. The waste solution recovered from the devices was also analyzed with an Imaging Flow Cytometer Image Stream X (ISX, AMNIS, Seattle, WA, USA).
Phosphate buffered saline (PBS), (3-aminopropyl) trimethoxysilane (APTMS), trichloro 1,1,2,2-perfluorooctyl-silane, bovine serum albumin (BSA), fetal bovine serum (FBS), glutaraldehyde, formaldehyde, Triton™ X-100, and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma Aldrich (USA). γ-irradiated Cryptosporidium parvum were kindly donated from SA Water (Australia). Specific monoclonal antibody Cry104 was obtained from BTF Biomerieux (Australia). 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 purchased 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.
Fabrication of microfluidic devices
Standard photolithography was used to fabricate chaotic mixing silicon masters for PDMS molding, as previously described . Briefly, silicon substrates were cleaned with acetone in a sonic bath for 5 min and then with isopropanol for 5 min. After rinsing with isopropanol, substrates were dried with a nitrogen gun and activated with oxygen plasma for 10 min. The negative photoresist, SU-8 10, was then spin-coated onto the wafer. The SU-8 was then patterned using a film photomask (JD tools) with a UV dose of 225 mJ/cm2 and post-baked at 65 °C for 1 min and at 95 °C for 2 min. SU-8 was then developed during 2 min to form a template and hard-baked with a ramping temperature from 65 °C to 200 °C.
The template was hydrophobized submitting it to a trichloro 1,1,2,2-perfluorooctyl-silane vapor in a desiccator for 1 h at 80 °C, and covered with liquid PDMS (the PDMS prepolymer was mixed with the cross-linker at a 10:1 ratio and degassed). PDMS was then degassed, cured at 80 °C for 1 h and unmolded from the silicon master. Inlet and outlet were punched in the PDMS replica that was then irreversibly sealed against a clean glass slide upon treatment in oxygen plasma at low power for 15 s.
Functionalization of the microfluidic devices
After attaching tubing to the inlet and outlet ports, the micromixers were connected to a syringe pump and filled with ethanol at a flow rate of 100 μl/min. Once the devices were stabilized, 2% APTMS in ethanol was withdrawn into the device for 30 min and rinsed with ethanol for 10 min. The buffer solution was then changed with MilliQ water and stabilized for 10 min prior to withdrawal of 1% glutaraldehyde in water for another 30 min and rinsing in ultrapure water for 10 min. PBS was then withdrawn into the device and equilibrated for 10 min just before introducing 200 μl of 50 μg/ml Cry104 in PBS that was left to react overnight at 4 °C. Unreacted antibodies were rinsed with PBS and the surface blocked with 2% BSA in PBS. All the functionalization steps were done at the same flow rate. Control devices were functionalized following the same protocol, but without the antibody conjugation.
Capture of Cryptosporidium parvum oocysts
To optimize the parameters for the isolation of Cryptosporidium, small volumes (50 μl) of high concentrated spiked samples in PBS (1.5 × 106 and 1.5 × 104 oocysts/ml) were injected into the functionalized micromixer at flow rates of 0.5, 2 or 5 μl/min to allow binding of the oocysts to the specific antibodies and explore the effect of flow rate. Washing of unbound oocysts was conducted at the same flow as for binding of oocysts, i.e. 0.5, 2 or 5 μl/min.
Fluorescence microscopy studies
The microfluidic devices were examined under a Nikon Ti Eclipse inverted fluorescence microscope. The oocysts isolated in the device were fixed with 4% formaldehyde, permeabilised with 0.05% Triton X-100 and stained with 1:10,000 DAPI at 0.5, 2 or 5 μl/min (same as flow rate used for oocyte binding) to allow identification by fluorescence microscopy as a proof of concept. The presence of DAPI fluorescent bodies identified as oocysts captured inside the device was quantified in situ by imaging 50 randomly chosen low magnification fields of view in different sections of the device, using a 10× objective.
Towards demonstrating the relevance of this approach for environmental samples, the presence of Cryptosporidium oocysts was also confirmed using the Cry104 antibody. Briefly, after capture, the device was incubated with the Cry104 antibody (1/50) for 60 min. After washing with PBS, permeabilisation and blocking was performed with 0.05% Triton X-100 and 2% BSA for 10 min. The devices were then incubated with the FITC goat anti mouse IgG secondary antibody for 30 min as per the manufacturer’s instructions, before washing and imaging.
Imaging flow cytometry studies
To quantitatively characterise the performance of the device and calculate the isolation yield of the micromixer, the input solution and the solution eluted from the device were recovered and analyzed with an Imaging Flow Cytometer (IFC) Image Stream X (AMNIS, Seattle, WA, USA). Solutions were centrifuged at 10,000 RCF for 15 min at 4 °C. The supernatants were removed and the pellets resuspended to 100 μl for Imaging Flow analysis. Since Cryptosporidium particles were already stained with DAPI during the microfluidic procedure, 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 autofluorescent debris, a size classification was applied.
To confirm that the DAPI-stained bodies identified as oocysts were indeed Cryptosporidium parvum, the recovered sample was labelled with the specific Cry104 antibody and a goat anti mouse FITC IgG secondary antibody was used. Briefly, after blocking with 10% fetal bovine serum in PBS for 10 min on ice, the sample was incubated with the Cry104 antibody (1/50) for 60 min. After washing with PBS, 0.05% Triton X-100 and 1% FBS, the sample was incubated with the FITC secondary antibody (1/5000) for 30 min. After washing with PBS samples were run through the IFC. The FITC events were recorded in Channel 2 of the IFC with the 488 laser. Controls were performed to discriminate non-specific staining.
Statistical analysis was performed in Matlab (2017b, Mathworks) and Excel. Student’s t-tests were used for directly comparing two variables. A 1-way ANOVA (confidence interval 0.05) adjusted using Bonferroni correlation was used for multi-variable analysis.
Results and discussion
Capture of Cryptosporidium in the micromixer microfluidic devices
The detection of pathogenic oocysts in water is an important health and environmental issue. A microfluidic micromixer device was developed to capture Cryptosporidium and enable direct in situ high quality microscopic observation. Imaging flow cytometry and fluorescence microscopy demonstrated that the oocysts had a strong binding affinity to the antibody-functionalized micromixers. Only minimal non-specific binding was observed in control devices, confirming the specific nature of the binding. A capture efficiency of 96% was determined under optimal conditions. Further studies are warranted beyond this proof of principle work to validate the proposed approach for its analytical potential for quantification in real samples. The main limitation of this microfluidic technology is its limited throughput, which restricts its application to water concentrates. However, it is anticipated that a minor redesign and/or multiplexing of the microfluidic mixers could lead to higher throughputs that would be readily compatible with volumes of water typically obtained from standard concentration methods as described in the EPA guideline. Besides its excellent efficiency, the main advantage of this technology is that its application would have strong potential to simplify the overall workflow, leading to notable time and resource savings. In summary, the microfluidic micromixer approach can capture Cryptosporidium in water concentrates and has the potential to accelerate and simplify the detection of Cryptosporidium and other microorganisms of concern in surface or drinking water.
This work was performed in part at the South Australian node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano and micro-fabrication facilities for Australia’s researchers.
This work has been supported by the NHMRC Project grant APP1045841 and by the NORTE-45-2015-02 program under grant NORTE-01-0145-FEDER-000029. Thierry is supported by a NHMRC CDA Fellowship.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
LD designed and fabricated the microfluidic masters and devices, designed the surface functionalization strategy and the experimental protocol for cryptosporidium isolation. She was involved in the planning of all experiments and was the primary responsible of manuscript writing. MW participated in the fluorescent labelling of the cryptospodirium, was in charge of the imaging flow cytometry and participated extensively in manuscript writing. SM fabricated the disposable PDMS microfluidic devices, and carried out all the experiments for the optimisation of oocytes isolation, labelling and quantification. PM and BK contributed to the design of the isolation strategy, provided the irradiated oocytes and developed the antibody used for the isolation. They also participated in the manuscript writing. BT directed the work, had the initial idea of the project and followed closely the experimental procedures. He also supported the work financially and was involved in the manuscript writing. All authors read and approved the final manuscript.
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The authors declare that they have no competing interests.
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