Recovery of schistosome eggs from the microfiltration device.

<p>(A) Micrographs of the microfluidic chamber before egg capture. (B) Schistosome eggs were captured in the microfluidic trap. (C) Recovery of schistosome eggs by pipetting the sample out from the inlet. Scale bars, 500 μm.</p

Schistosome eggs capture and enumeration.

<p>(A) Trapping efficiency of the microfiltration device by withdrawing samples from the outlet or infusing samples through inlet. (B) Trapping efficiency of the device at different flow rates. (C) Calibration of the trapping efficiency by spiking various number of egg into the sample. (D) Capture of schistosome eggs in urine and normal saline.</p

A Microfiltration Device for Urogenital Schistosomiasis Diagnostics

<div><p>Schistosomiasis is a parasitic disease affecting over 200 million people worldwide. This study reports the design and development of a microfiltration device for isolating schistosome eggs in urine for rapid diagnostics of urogenital schistosomiasis. The design of the device comprises a linear array of microfluidic traps to immobilize and separate schistosome eggs. Sequential loading of individual eggs is achieved autonomously by flow resistance, which facilitates observation and enumeration of samples with low-abundance targets. Computational fluid dynamics modeling and experimental characterization are performed to optimize the trapping performance. By optimizing the capture strategy, the trapping efficiency could be achieved at 100% with 300 μl/min and 83% with 3000 μl/min, and the filtration procedure could be finished within 10 min. The trapped eggs can be either recovered for downstream analysis or preserved <i>in situ</i> for whole-mount staining. On-chip phenotyping using confocal laser fluorescence microscopy identifies the microstructure of the trapped schistosome eggs. The device provides a novel microfluidic approach for trapping, counting and on-chip fluorescence characterization of urinal <i>Schistosoma haematobium</i> eggs for clinical and investigative application.</p></div

Bright-field images of <i>Schistosoma haematobium</i> eggs.

<p>Scale bars, 500 μm (A) and 100 μm (B).</p

Computational fluid dynamics simulation.

<p>(A) Streamline velocity of the fluid flow in the microfiltration device. Color bar represents the fluid velocity. (B) Two-dimensional profiles of streamline velocity before and after trapping an egg. (C) 2D simulation of the sequential loading. After capture of the first egg, the following egg is diverted to the adjacent channel. The color represents the magnitude of velocity. (D) The pressure field in the cross-section in depth 50 μm of the chamber. Color bar represents the pressure. (E) Pressure drop across a trapping channel as a function of the volumetric flow rate.</p

On-chip fluorescence characterization of schistosome eggs.

<p>(A) Fluorescence, brightfield and combined images of an egg capture in the microfluidic array. Scale bar: 100 μm. (B-D) Confocal fluorescence characterization of eggs of Schistosoma haematobium. Arrow and stars indicate the neural mass primordium and blastomere pairs. Arrow heads represent the flame cells. The eggs were double-labelled with phalloidin (red) and Sytox Green (green). Scale bars, 50 μm.</p

A microfiltration device for trapping and analysis of Schistosoma haematobium eggs.

<p>(A) Schematic of the microfiltration device. (B) Schematic representation of the experimental setup with a syringe pump withdrawing fluid from the outlet of the microchip. (C) Image of a fabricated microfiltration device. Scale bar, 1 mm. (D) Image of the microchip with a reservoir assembled. (E) Brightfield image of a schistosome egg captured in the microfluidic trap. Scale bar, 100 μm.</p

Biosensor-based molecular detection of urinary pathogen.

<p>The biosensor is composed of three planar gold electrodes (working, auxiliary, and reference). For the biosensor assay, capture probes are bound to the surface of the working electrode via a thiol linkage. Cells in the sample are lysed and mixed with a buffered solution of detector probe, then applied to the sensor surface. If the target rRNA is present, a hybridization complex of target, capture, and detector probes forms. This complex is detected by binding of horseradish peroxidase (HRP)-conjugated antifluorescein binding to a fluorescein tag on the detector probe and addition of tetramethylbenzidine (TMB) substrate. The electron transport mediated by the HRP is measured amperometerically, and the signal is proportional to the quantity of the target.</p

Sequences of capture and detector probe pairs tested for detection of <i>S. hematobium</i>^a.

<p>^<i>a</i> The capture probes were modified with 5’ thiol, and detector probes were modified with 3’ fluorescein.</p><p>^<i>b</i> The degenerate base “R” represents either A or G.</p><p>Sequences of capture and detector probe pairs tested for detection of <i>S. hematobium</i><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003845#t001fn001" target="_blank">^a</a>.</p

Detection of <i>S</i>. <i>haematobium</i> eggs.

<p>A. <i>S</i>. <i>haematobium</i> eggs spiked in human urine were lysed by agitation with glass beads or sonication and the crude lysate tested in the biosensor assay with the 28S495 probe set with positive and negative controls as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003845#pntd.0003845.g002" target="_blank">Fig 2</a>. The higher signal with sonication compared to glass beads indicates more efficient cell lysis. B. To determine the LOD for detection of <i>S</i>. <i>haematobium</i> eggs in urine in the biosensor assay, a sample of 10⁶ egg/ml was lysed by sonication and serially diluted in urine. Biosensor assay detection of <i>S</i>. <i>haematobium</i> eggs with the 28S495 probe set indicated an LOD of approximately 30 eggs/ml.</p