Digital microfluidic platform for high-throughput monoclonal antibodies screening and isotyping analysis (original) (raw)
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Microfluidics and Nanofluidics, 2009
Although the reaction time for antigen-antibody binding has been greatly reduced in microchannels, other processes in heterogeneous immunoassays (HEIs), such as blocking and antigen adsorption have not benefited from miniaturization as a reduction in size to micro dimensions does not increase the speed of these processes significantly. The overall assay time of reported microfluidic HEIs has continued to be limited by these processes. In this study, we successfully develop an ultrafast quantitative HEI with pre-functionalized microfluidic poly(dimethylsiloxane) (PDMS) chips. The protein A functionalized PDMS surface is found to be highly effective in reducing the antigen adsorption time in microchannels. The functionalized surfaces can be stable at least for 2.5 months when stored at 4°C in a buffer solution consisting of 10 mM Tris, 0.05% bovine serum albumin, 0.05% Proclin 300, and 5% glycerol. In addition, the immunosorption process, which is substantially accelerated in micro scale, results in a significant reduction in nonspecific binding.
Analytical Chemistry, 2001
This article presents the first example of a microfluidic chip for heterogeneous bioassays using a locally immobilized biospecific layer and operated electrokinetically. The reaction chamber has picoliter dimensions and is integrated into a network of microchannels etched in glass. The high affinity of protein A (PA) for rabbit immunoglobulin G (rIgG) was exploited for chip testing, with PA being immobilized on microchannel walls and fluorescently labeled (Cy5) rIgG serving as sample. It was possible to operate the chip in an immunoaffinity chromatographic manner, using electrokinetically pumped solutions. Concentration of antibody from dilute solution onto the solid phase was demonstrated, with signal gains of ∼30 possible. A dose-response curve for Cy5-rIgG was obtained for concentrations down to 50 nM, for an incubation time of 200 s. The flexibility of chip layout was demonstrated for competitive immunoassay of rIgG, using both a combined sample/tracer incubation and sequential addition of these solutions. With assay times generally below 5 min for this unoptimized device, the microfluidic approach described shows great potential for many highthroughput screening applications.
Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay
Journal of Visualized Experiments
Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H 2 O 2 /Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized. Video Link The video component of this article can be found at https://www.jove.com/video/60489/ 15. Recently, in-field diagnostic against measles and rubella IgG has been demonstrated in remote Northwestern Kenya's
Target delivery in a microfluidic immunosensor
Biosensors and Bioelectronics, 2007
A study is presented that examines the effect of microfluidic mixing elements on direct and sandwich assays performed in microchannels. Patterned grooves were embossed in the top of microchannels made in PDMS using soft lithography. The grooves redirected the fluid flowing in the channel, enhancing delivery of the target from the bulk fluid to the surface and preventing the formation of a depletion layer at the surface. Comparing assays in grooved and plain channels demonstrated that the mixers improved assay results by 26-46%. A computational flow analysis showed that the grooves caused virtual particles in the bulk flow to come close to the surface (∼11 m) which is consistent with the signal increase seen experimentally. Direct assays for several concentrations of CY5-labeled biotin were performed in the microchannels. The mixers also improved signal intensity in sandwich assays for botulinum toxin which required mixing of the reagents as well as the direction of the target to the surface.
Microfluidic analysis of antibody specificity in a compact disk format
2006
A new and flexible technology for high throughput analysis of antibody specificity and affinity is presented. The method is based on microfluidics and takes advantage of compact disks (CDs) in which the centrifugal force moves fluids through microstructures containing immobilized metal affinity chromatography columns. Analyses are performed as a sandwich assay, where antigen is captured to the column via a genetically attached His 6 -tag. The antibodies to be analyzed are applied onto the columns. Thereafter, fluorescently labeled secondary antibodies recognize the bound primary antibodies, and detection is carried out by laser-induced fluorescence. The CDs contain 104 microstructures enabling analysis of antibodies against more than 100 different proteins using a single CD. Importantly, through the three-dimensional visualization of the binding patterns in a column it is possible to separate high affinity from low affinity binding. The method presented here is shown to be very sensitive, flexible and reproducible.
A New Game Changer for Immunoassays and IVD: Microfluidics and Polymer Lab-on-a-Chips
Procedia Engineering, 2011
Immunoassays and enzyme-linked immunosorbent assays (ELISA) are one of the key technologies for the quantification of a wide range of analytes for serum, plasma and cell lysates. The protocols of the immunoassays have not changed significantly for over 6 decades, as used extensively in both research and clinical applications. However, there is a large demand for the development of new protocols or platforms for immunoassays with rapid turnaround time (TAT), high sensitivity and low cost for high throughput drug screening, point-of-care clinical diagnostics, or rapid food safety monitoring. Recently, microfluidics and lab-on-a-chips (LOC) have been emerging as one of the most promising platforms for better immunoassays due to its distinctive natures enabling to meet the demands. In this talk, the distinctive natures of microfluidics and lab-on-a-chips are discussed as a new game changer for immunoassays and in vitro diagnostics (IVD), and then its applications to ELISA, IVD and point-of-care testing (POCT) clinical diagnostics are introduced
An ELISA Chip Based on an EWOD Microfluidic Platform
We have developed an enzyme-linked immunosorbent assay (ELISA) chip based on a digital microfluidic platform, in which droplet generation and manipulation are implemented using the electrowetting on dielectrics (EWOD) effect. First, droplet transportability was experimentally tested on silicon test chips with different control electrodes for optimizing the EWOD-based droplet manipulation, and then tested on transparent indium-tin-oxide (ITO) chips. The latter were then used for the development of an ELISA chip, on which we carried out immunoassay with rat immunoglobulin G (IgG) and goat anti-rat IgG marked with horseradish peroxidase as an example. In driving the reagent solutions, the average velocity of droplets is up to 5–7 mm/s on the ELISA chip, and a chip-scaled immunoassay can be finished within 20 min by using colorimetric detection, with the volume of the sample and reagents of only 0.5–1 μl.
Production of monoclonal antibodies in microfluidic devices
Integrative biology : quantitative biosciences from nano to macro, 2018
Herein, a microfluidic device with cistern design for cultivation of adherent eukaryotic cells for the production of recombinant proteins is presented. The geometric configuration of the microchannels in the device provided laminar flow with reduced velocity profiles in the cisterns, resulting in an adequate microenvironment for long-term adherent cell growth with passive pumping flow cycles of 24 hours. CHO-ahIFNα2b and HEK-ahIFNα2b adherent cell lines expressing a novel anti-hIFN-α2b recombinant monoclonal antibody (MAb) for the treatment of systemic lupus erythematosus were cultured on the surface of PDMS/glass microchannels coated with poly-d-lysine. A 24 day culture of CHO-ahIFNα2b cells resulted in MAb concentrations up to 166.4 μg mL per day. The productivity of CHO-ahIFNα2b and HEK-ahIFNα2b cell lines was higher in the microdevice compared to that obtained using the adherent cell culture method (T-flask), with a 5.89- and 7.31-fold increase, respectively. Moreover, biologica...
2007
for introducing me to the concept of droplet based transport using electrowetting and for their guidance through out the project. I extend my heartfelt gratitude to Dr. Allen Echkhardt for his guidance and support through out the research. His enthusiasm for my results was one of the main driving forces for my research. I would also like to express my special thanks to Dr. Phil Paik and Dr. Dwayne Allen for assisting me in setting up experiments and for their insightful discussions. I am grateful to each and everyone at Advanced Liquid Logic, Inc. for making my dissertation project a pleasant experience. Last but not the least, I would like to thank my family and friends for their continuous support and encouragement through out the process. v