Disposable microfluidic micromixers for effective capture of Cryptosporidium parvum oocysts from water samples

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Cryptosporidium parvum oocysts; Disposable microfluidic micromixers; Fluorescence microscopy; Imaging flow cytometry; Immunocytochemistry; Water quality.

Fig. 1

Cryptosporidium images from the different channels in the Imaging Flow Cytometer: Brightfield (BF), Darkfield, nuclear staining in DAPI and the Cryptosporidium specific Cry104 antibody (goat anti mouse FITC IgG secondary antibody), and the combined image. Images taken at 400× magnification and have been adjusted to enhance visual appearance

Fig. 2

Capture efficiency of Cryptosporidium oocysts in the microfluidic devices at different flow rates calculated with Imaging Flow Cytometry. Light grey bars are the functionalized experimental results and the dark grey bars are the non-functionalized control devices. The average capture efficiency was 92%, 96% and 40% at 0.5, 2 and 5 μl/min respectively. Capture efficiency in the control devices was 18%, 10% and 9% for 0.5, 2 and 5 μL/min, respectively

Fig. 3

Microscopic images at 100× magnification of Cryptosporidium oocysts, stained in blue (DAPI), captured at the entrance of the micromixers, from PBS samples spiked with 1500 000 oocysts per ml

Fig. 4

Microscopic images of the Cryptosporidium oocysts captured inside the functionalized microfluidic devices at the flow rate of 2 μl/min. Cryptosporidium oocysts are specifically recognized by Cry104 antibody, stained with a secondary FITC antibody and imaged at 100×, scale bar 100 μm (1). Composite image with brightfield (BF), FITC and DAPI channels (1a), DAPI channel (1b), FITC channel (1c) and BF image (1d). Images were also taken at higher magnification, 400×, scale bar 50 μm (2, 3). Composite images with BF and FITC (2a) or BF and DAPI (3a), and individual FITC channel (2b), and DAPI channel (3b)

Fig. 5

The surface density of Cryptosporidium oocysts in the microfluidic micromixers decreased from inlet to outlet. a Merged fluorescent microscopy images show the capture of oocysts in regions of the device at different distances from the inlet. (Left to right are distances of 0, 4, 8, and 12 mm from the inlet). b Average number of Cryptosporidium oocysts captured in a micromixer at 2 μl/min at different penetration lengths from inlet to outlet. The black line shows a functionalized experiment device and the red is the non-functionalized control device

Fig. 6

Dependence of the capture efficiency of Cryptosporidium oocysts in microfluidic micromixers at 2 μl/min at concentrations 15,000 and 1,500,000 crypto/ml, using imaging flow cytometry. Light grey bars are the functionalized experimental results and the dark grey bars are the non-functionalized control devices