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.

Microfluidics: A New Tool for Modeling Cancer–Immune Interactions

“…The low cell number can also be a drawback if a large number of cells is need for downstream biochemical studies (e.g., by western blotting) or if secreted proteins need to be quantified (e.g., by ELISA). Of note, microfluidic platforms that include such miniaturized assays on-chip are currently being developed[7, 8]. The mechanical properties of the device materials (e.g.…”

“…They are particularly relevant for studying the metastatic cascade, since metastatic spread occurs through several steps that are often difficult to resolve in vivo . While intravital imaging has proven extremely useful in the study of metastasis[6–7], it requires specialized expertise and equipment, and high image resolution is harder to attain in some visceral organs due to the depth of their location. Furthermore, some events of the metastatic cascade (e.g.…”

A microscale approach for simple and rapid monitoring of cell growth and lipid accumulation in Neochloris oleoabundans

“…In addition, N. oleoabundans reached a maximum value of l max of 2.13 day -1 in the microreactor, followed by 1.6 day -1 in the 6-well plates and 1.34 day -1 in the flask. The growth rate of N. oleoabundans in microreactor was 1.58-fold faster than the flask, because the available surface area, mass transfer and permeability of the PDMS-based microreactor are larger and higher than those of the 6-well plates and the flask [28,29,31,32]. The 6-well plates showed faster growth (l max = 1.60 day -1 ) in the early phase than the flask (l max = 1.34 day -1 ) due to the large surface area and low shading effect (Fig.…”

Rapid Detection of Food Allergens by Microfluidics ELISA-Based Optical Sensor

“…Microfluidics has become an increasingly popular and powerful tool in biochemistry applications [ 18 ] and presents distinctive advantages, such as faster reaction times, lower sample consumption, greater sensitivity, small dimensions, short diffusion distance, and high surface tension [ 19 , 20 ]. Many studies have described immunoassays in microfluidic devices employing various detection methods such as fluorometric and colorimetric measurement and SPR, but few have been applied to the detection of food allergens [ 21 , 22 , 23 ].…”