Detection of Cryptosporidium parvum oocysts using a microfluidic device equipped with the SUS micromesh and FITC‐labeled antibody

Abstract

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.

Rapid and Specific Detection of Escherichia coli O157:H7 in Ground Beef Using Immunomagnetic Separation Combined with Loop-Mediated Isothermal Amplification

“…Some previously published reports from our lab and elsewhere A large number of fi eld applications showed that the O157 immunomagnetic method greatly improved the isolation rate of bacteria [Buesa et al, 2002;Wolf et al, 2007]. The clinical samples for diagnosis are initially treated with a specifi c antibody-coated beads, and the target bacteria after reaction with their specifi c antibody are separated from other bacteria by immunomagnetic separation (IMS) [Bapanpally et al, 2014;Taguchi et al, 2007]. Loop-mediated isothermal amplifi cation (LAMP) is a sensitive, specifi c, convenient and fast nucleic acid amplifi cation technology, which obviates the need for expensive thermal cyclers used in conventional PCR [Saito et al, 2005].…”

Miniaturized Detection Systems

“…The LOD was reported as 36 oocysts mL −1 , above the desired ability to detect single oocysts. 69 In 2001 Stokes and coworkers developed a sandwich immunoassay in a 0.4 mL reaction chamber on-chip, demonstrating E. coli detection. In the same year McClain et al employed a microfluidic flow cytometry setup for E. coli detection.…”

“…69 The maximum flow rate tested was 350 µL min −1 , so 5 mL could be processed in under 15 min. When loading a 0.5 mL test sample (spiked oocysts in PBS) at a concentration of 36 oocysts mL −1 a recovery rate of 93% from the mesh was reported, comparable to that achieved by off-chip IMS.…”

“…Magnetic separation [21][22][23][24][25][26][27][28][29][30][31] Electrical separation Fluidic-only separation [59][60][61][62][63][64][65][66][67][68][69][70][71] Other separation [72][73][74][75][76][77] Optical separation [9][10][11][12][13][14][15][16][17][18][19] Optical Giardia, developed by Ramadan and colleagues in 2010, 60 was described in Chapter 4 (see Figure 4.5). In 2012, Agrawal et al described a PDMS 3D circular microfluidic system with imbedded permanent magnets, designed for multiplex pathogen detection, where the first stage is capture of the bacteria with immunomagnetic nanoparticles.…”

Immunomagnetic separation and rapid detection of bacteria using bioluminescence and microfluidics

“…The antibody coated on the surface of the IMB could capture the target organism when they are close to IMB. So IMS is a rapid, specific, efficient and technically simple method that can be used for separation of target organism directly from non-target organism and other particles without any need for centrifugation or filtration [14][15][16].…”