Immunosensors orientation

Lee et al. utilized the self-assembled layer of thiol group-modified protein A for the oriented immobilization of antibodies [64], An increased binding capacity was further observed. As another illustrative instance, a protein A-based orientation-controlled immobilization strategy for antibodies was proposed for the fabrication of a QCM immunosensor using nanometer-sized gold particles and amine-terminated PPF [65], Moreover, in recent years, there has emerged another oriented immobilization... 

One of the most important features in the immunosensor design is the proper choice of the immobilization method for keeping the affinity of the antibodies. As was previously demonstrated for Protein A, when the antibodies are immobilized through their Fc fragment to Protein A (or G), their Fab binding sites are mostly oriented away from the solid phase. As Protein A is able to link the Fc region of different antibodies, there is no need to modify the antibody with biotin. As an antecedent, we have previously demonstrated the utility of Protein A biocomposite (ProtA-GEB) for the universal attachment of antibodies with different specificities [54]. 

Those procedures that allow an oriented immobilization of intact antibodies, or antibody fragments, on solid supports have been the most applied in the fabrication of different immunosensors. 

McLean MA, Stayton PS, Sligar SG (1993) Engineering protein orientation at surfaces to control macromolecular recognition events. Anal Chem 65 2676-2678 Shen Z, Tan H, Zhang Y et al (2008) Engineering peptide linkers for scFv immunosensors. Anal Chem 80 1910-1917... 

The selectivity of immunosensors for steroid analytes is achieved with the use of appropriately selected monoclonal antibodies. The carbon working electrode provides a suitable surface for passive adsorption of proteins, and can therefore be tailored with an appropriate antibody, so that it will act as an immunoactive surface upon which an immunoaffinity assay can be performed an electrochemical signal can then be generated by monitoring the production of an electroactive species at the underlying electrode surface. We and other workers have found that to retain maximum monoclonal antibody activity, it is desirable to use a primary antibody (rabbit IgG), which serves both to capture (e.g., from a culture medium) and to orientate the mAb. Hence this approach... 

Combining immobilisation methods seems to be the best option, such as incorporating the stability and reproducibility of covalent methods such as SAMs with the orientated binding of protein A/G. The first example of this was in 1987 by Muramatsu et al. [4]. The authors cross-linked protein A to a layer of APTES, although it is unhkely that it was done to combine the advantages of both immobihsation methods since this was one of the earhest Pz immunosensors and more basic fundamental problems were being overcome at this stage. 

This section describes a modified electrochemical immunosensor where a boronic acid approach is employed for attaching antibodies onto the surfaces of assay devices. It is commonly observed that antibodies often adopt random orientations and therefore fail to display their original immunoaffinity toward their corresponding antigen when they are directly adsorbed onto sensing surfaces. 

Ferreira, N.S., Sales, M.G.F., 2014. Disposable immunosensor using a simple method for oriented antibody immobilization for label-free real-time detection of an oxidative stress biomarker implicated in cancer diseases. Biosens. Bioelectron. 53, 193-199. Available at http //linkinghub.elsevier.com/retrieve/pii/S095656631300674X. 

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