title: Implementation of microfluidic sandwich ELISA for superior detection of plant pathogens.
abstract: Rapid and economical screening of plant pathogens is a high-priority need in the seed industry. Crop quality control and disease surveillance demand early and accurate detection in addition to robustness, scalability, and cost efficiency typically required for selective breeding and certification programs. Compared to conventional bench-top detection techniques routinely employed, a microfluidic-based approach offers unique benefits to address these needs simultaneously. To our knowledge, this work reports the first attempt to perform microfluidic sandwich ELISA for Acidovorax citrulli (Ac), watermelon silver mottle virus (WSMoV), and melon yellow spot virus (MYSV) screening. The immunoassay occurs on the surface of a reaction chamber represented by a microfluidic channel. The capillary force within the microchannel draws a reagent into the reaction chamber as well as facilitates assay incubation. Because the underlying pad automatically absorbs excess fluid, the only operation required is sequential loading of buffers/reagents. Buffer selection, antibody concentrations, and sample loading scheme were optimized for each pathogen. Assay optimization reveals that the 20-folds lower sample volume demanded by the microchannel structure outweighs the 2- to 4-folds higher antibody concentrations required, resulting in overall 5-10 folds of reagent savings. In addition to cutting the assay time by more than 50%, the new platform offers 65% cost savings from less reagent consumption and labor cost. Our study also shows 12.5-, 2-, and 4-fold improvement in assay sensitivity for Ac, WSMoV, and MYSV, respectively. Practical feasibility is demonstrated using 19 real plant samples. Given a standard 96-well plate format, the developed assay is compatible with commercial fluorescent plate readers and readily amendable to robotic liquid handling systems for completely hand-free assay automation.
authors.affiliation: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Klong Luang, Pathum Thani, Thailand.
authors.forename: Numrin
authors.initials: N
authors.lastname: Thaitrong
authors.affiliation: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Klong Luang, Pathum Thani, Thailand.
authors.forename: Ratthaphol
authors.initials: R
authors.lastname: Charlermroj
authors.affiliation: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Klong Luang, Pathum Thani, Thailand.
authors.forename: Orawan
authors.initials: O
authors.lastname: Himananto
authors.affiliation: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Klong Luang, Pathum Thani, Thailand.
authors.forename: Channarong
authors.initials: C
authors.lastname: Seepiban
authors.affiliation: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Klong Luang, Pathum Thani, Thailand.
authors.forename: Nitsara
authors.initials: N
authors.lastname: Karoonuthaisiri
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis / Q000737:chemistry*; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000737:chemistry*; D042621:Comamonadaceae / Q000276:immunology / Q000302:isolation & purification* / Q000472:pathogenicity; D004797:Enzyme-Linked Immunosorbent Assay / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D051379:Mice; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D010935:Plant Diseases / Q000382:microbiology / Q000821:virology; D010944:Plants / Q000382:microbiology / Q000821:virology; D011817:Rabbits; D012371:Robotics; D012680:Sensitivity and Specificity; D013997:Time Factors; D017865:Tospovirus / Q000276:immunology / Q000302:isolation & purification* / Q000472:pathogenicity
chemical_list: D000907:Antibodies, Bacterial; D000914:Antibodies, Viral