Detection of Cryptosporidium parvum oocysts using a microfluidic device equipped with the SUS micromesh and FITC-labeled antibody.
Development of a microfluidic device equipped with micromesh for detection of Cryptosporidium parvum oocyst was reported. A micromesh consisting of 10 x 10 cavities was microfabricated on the stainless steel plate by laser ablation. Each cavity size, approximately 2.7 microm in diameter, was adopted to capture a single C. parvum oocyst. Under negative pressure operation, suspensions containing microbeads or C. parvum oocysts flowed into the microchannel. Due to strong non-specific adsorption of microbeads onto the PDMS microchannel surface during sample injection, the surface was treated with air plasma, followed by treatment with 1% sodium dodecyl sulfate (SDS) solution. This process reduced the non-specific adsorption of microbeads on the microchannel to 10% or less in comparison to a non-treated microchannel. This microfluidic device equipped with the SUS micromesh was further applied for the capture of C. parvum oocysts. Trapped C. parvum oocysts were visualized by staining with FITC-labeled anti-C. parvum oocyst antibody on a micromesh and counted under fluoroscopic observation. The result obtained by our method was consistent with that obtained by direct immunofluorescence assay coupled with immunomagnetic separation (DFA-IMS) method, indicating that the SUS micromesh is useful for counting of C. parvum oocysts. The newly designed microfluidic device exploits a geometry that allowed for the entrapment of oocysts on the micromesh while providing the rapid introduction of a series of reagents and washes through the microfluidic structure. Our data indicate that this microfluidic device is useful for high-throughput counting of C. parvum oocysts from tap water sample.
Taguchi
Tomoyuki
T
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
Arakaki
Atsushi
A
Takeyama
Haruko
H
Haraguchi
Satoshi
S
Yoshino
Masato
M
Kaneko
Masao
M
Ishimori
Yoshio
Y
Matsunaga
Tadashi
T
D000818:Animals; D000913:Antibodies, Protozoan / Q000737:chemistry*; D016785:Cryptosporidium parvum / Q000276:immunology / Q000302:isolation & purification*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D044085:Microfluidics / Q000295:instrumentation; D033661:Oocysts / Q000276:immunology*; D016062:Porosity; D014867:Water / Q000469:parasitology*; D014881:Water Supply / Q000032:analysis
D000913:Antibodies, Protozoan; D014867:Water; D016650:Fluorescein-5-isothiocyanate