Coordination Complexes as Molecular Glue for Immobilization of Antibodies on Cyclic Olefin Copolymer Surfaces.
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.
- [Show abstract] [Hide abstract] 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.
- [Show abstract] [Hide abstract] ABSTRACT: Reduced graphene oxide–carboxymethylcellulose hybrid (CMC–rGO) was used for the development of a novel electrochemical immunosensor for the determination of adiponectin (APN) cytokine. The hybrid material was synthesized by covalent binding of oxidized CMC to GO layers followed by chemical reduction with sodium borohydride. A sandwich-type immunoassay was employed involving the commercial metal-complexes based polymer Mix & GoTM for the stable and oriented immobilization of anti-APN capture antibody. Biotinylated-anti-APN and HRP-Strept were used for the assay configuration. The APN quantification was performed by amperometry at −200mV using the hydrogen peroxide/hydroquinone enzyme substrate/mediator system. A sigmoidal calibration plot for APN in the 0.1–50�g/mL range, with a linear portion in the 0.5–10.0�g/mL APN concentration range was obtained. The calculated limit of detection (3sb/m) was 61 ng/mL APN. The usefulness of the immunosensor was evaluated by analyzing human serum from hypercholesterolemia or diabetes patients
- [Show abstract] [Hide abstract] ABSTRACT: Cyclic Olefin Copolymer (COC) was grafted with aryl layers from aryldiazonium salts, and then we combined infrared spectrometry, atomic force microscopy (AFM) and ion mobility mass spectrometry with atmospheric solid analysis probe ionization (ASAP-IM-MS) to characterize the aryl layers. ASAP is a recent atmospheric ionization method dedicated to the direct analysis of solid samples. We demonstrated that ASAP-IM-MS was complementary to other techniques characterizing of bromide and sulfur derivates on surfaces. ASAP-IM-MS was useful to optimize experimental grafting conditions and to elucidate hypotheses around aryl layer formation during the grafting process. Thus, ASAP-IM-MS is a good candidate tool to characterize covalent grafting on COC surfaces.