Coordination Complexes as Molecular Glue for Immobilization of Antibodies on Cyclic Olefin Copolymer Surfaces.
Analytical Biochemistry (Impact Factor: 2.22). 04/2014; 456(1). DOI: 10.1016/j.ab.2014.03.023
A novel metal-based chelating method has been used to provide an order of magnitude increase in immunoassay performance on COC plastics compared to passive binding. COCs are hydrophobic and without surface modification are often unsuitable for applications where protein adhesion is desired. When interacting with the bare plastic, the majority of the bound proteins will be denatured and become non-functional. Many of the surface modification techniques reported to-date require costly equipment setup or the use of harsh reaction conditions. Here, we have successfully demonstrated the use of a simple and quick metal chelation method to increase the sensitivity, activity and efficiency of protein binding to COC surfaces. A detailed analysis of the COC surfaces after activation with the metal complexes is presented, and the immunoassay performance was studied using three different antibody pairs.
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ABSTRACT: Rapid preparation of high quality capture surfaces is a major challenge for surface-based single-molecule protein binding assays. Here we introduce a simple method to activate microfluidic chambers made from cyclic olefin copolymer for single-molecule imaging with total internal reflection fluorescence microscopy. We describe a surface coating protocol and demonstrate single-molecule imaging in off-the-shelf microfluidic parts that can be activated for binding assays within a few minutes. As the first example, biotinylated protein directly captured on the neutravidin-coated surface was detected using fluorescently labeled antibody. We then showed detection of a fusion construct containing green fluorescence protein and verified its single fluorophore behavior by observing stepwise photobleaching events. Finally, a target protein was identified in the crude cell lysate using antibody-sandwich complex formation. In all experiments, controls were completed to ensure that nonspecific binding to the surface was minimal. Based on our results, we conclude that the simple surface preparation described in this paper enables single-molecule imaging assays without time-consuming coating procedures. Microsc. Res. Tech., 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.
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ABSTRACT: An electrochemical immunosensor for adiponectin (APN) using screen printed carbon electrodes (SPCEs) modified with functionalized double-walled carbon nanotubes (DWCNTs) as platforms for immobilization of the specific antibodies is reported. DWCNTs were functionalized by treatment with 4-aminobenzoic acid (HOOC-Phe) in the presence of isoamylnitrite resulting in the formation of 4-carboxyphenyl-DWCNTs. The oriented binding of specific antibodies toward adiponectin was accomplished by using the metallic-complex chelating polymer Mix&Go™. The HOOC-Phe-DWCNTs-modified SPCEs were characterized by cyclic voltammetry and compared with HOOC-Phe-SWCNTs/SPCE. The different variables affecting the performance of the developed immunosensor were optimized. Under the selected conditions, a calibration plot for APN was constructed showing a range of linearity extending between 0.05 and 10.0μg/mL which is adequate for the determination of the cytokine in real samples. A detection limit of 14.5ng/mL was achieved. The so prepared immunosensor exhibited a good reproducibility for the APN measurements, excellent storage stability and selectivity, and a much shorter assay time than the available ELISA kits. The usefulness of the immunosensor for the analysis of real samples was demonstrated by analyzing human serum from female or male healthy patients. Copyright © 2015 Elsevier B.V. All rights reserved.
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