Taguchi Tomoyuki, Arakaki Atsushi, Takeyama Haruko, Haraguchi Satoshi, Yoshino Masato, Kaneko Masao, Ishimori Yoshio, Matsunaga Tadashi
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
Biotechnol Bioeng. 2007 Feb 1;96(2):272-80. doi: 10.1002/bit.21104.
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.
报道了一种配备微网的微流控装置用于检测微小隐孢子虫卵囊的研究进展。通过激光烧蚀在不锈钢板上微加工出一个由10×10个腔室组成的微网。每个腔室直径约2.7微米，用于捕获单个微小隐孢子虫卵囊。在负压操作下，含有微珠或微小隐孢子虫卵囊的悬浮液流入微通道。由于在进样过程中微珠在聚二甲基硅氧烷（PDMS）微通道表面存在强烈的非特异性吸附，因此对微通道表面进行了空气等离子体处理，随后用1%的十二烷基硫酸钠（SDS）溶液处理。与未处理的微通道相比，该过程将微珠在微通道上的非特异性吸附降低至10%或更低。这种配备不锈钢微网的微流控装置进一步用于捕获微小隐孢子虫卵囊。捕获的微小隐孢子虫卵囊通过在微网上用异硫氰酸荧光素（FITC）标记的抗微小隐孢子虫卵囊抗体染色进行可视化，并在荧光观察下计数。我们的方法得到的结果与直接免疫荧光分析结合免疫磁分离（DFA-IMS）方法得到的结果一致，表明不锈钢微网可用于微小隐孢子虫卵囊的计数。新设计的微流控装置采用了一种几何结构，该结构允许在微网上截留卵囊，同时通过微流控结构快速引入一系列试剂和冲洗液。我们的数据表明，这种微流控装置可用于对自来水样本中的微小隐孢子虫卵囊进行高通量计数。
