Monolayers avidin-biotin

FIGURE 5.25. Avidin-biotin construction of a monolayer glucose oxidase electrode with an attached ferrocenium cosubstrate and cyclic voltammetric response in a phosphate buffer (pH 8) at 25°C and a scan rate of 0.04 V/s. a attached ferrocene alone, h In the presence of 0.5 M glucose, c Variation of the inverse of the plateau current with the inverse of substrate concentration. Adapted from Figure 1 in reference 24, with permission from the American Chemical Society. 

Fig. 12 Schematic illustration of immobilization strategy using affinity reactions via avidin-biotin coupling. Biotinylated mixed monolayers improve the availability of the surface-bound avidin or streptavidin to biotin coupled probe biomolecules...

Flowever, the focus of the major part of the chapters lies on the couphng chemistry used for DNA immobilization. Successful immobihzation techniques for DNA appear to either involve a multi-site attachment of DNA (preferentially by electrochemical and/or physical adsorption) or a single-point attachment of DNA (mainly by surface activation and covalent immobihzation or (strept)avidin-biotin linkage). Immobilization methods described here comprise physical or electrochemical adsorption, cross-linking or entrapment in polymeric films, (strept)avidin-biotin complexation, a surface activation via self-assembled monolayers using thiol linker chemistry or silanization procedures, and finally covalent coupling strategies. 

Avidin-Biotin Monolayer and Multilayer on LB Film Surface.149... 

A supramolecular complexation between avidin and biotin-labeled enzyme results in the formation of enzyme multilayers composed of avidin and enzyme monolayers, in which each monolayer is connected through avidin-biotin complexation with each other. The thickness of the multilayers can be precisely controlled by regulating the deposition number (the thickness of each avidin plus enzyme double layer is approximately 10 nm). The enzyme multilayers are useful in preparing high... 

In this part we will describe recent achievements in the development of biosensors based on DNA/RNA aptamers. These biosensors are usually prepared by immobilization of aptamer onto a solid support by various methods using chemisorption (aptamer is modified by thiol group) or by avidin-biotin technology (aptamer is modified by biotin) or by covalent attachment of amino group-labeled aptamer to a surface of self-assembly monolayer of 11-mercaptoundecanoic acid (11-MUA). Apart from the method of aptamer immobilization, the biosensors differ in the signal generation. To date, most extensively studied were the biosensors based on optical methods (fluorescence, SPR) and acoustic sensors based mostly on thickness shear mode (TSM) method. However, recently several investigators reported electrochemical sensors based on enzyme-labeled aptamers, electrochemical indicators and impedance spectroscopy methods of detection. 

Fig. 4. Immunocytochemistry of formalin fixed 147L) cell cultures using a-PR-A anti-receptor MAb (left) and a control MAb (right). Immunocytochemistry was performed with T47D breast cancer cells grown as monolayers in chamber slides. Cells were fixed for 15 min with 3.7% buffered formalin, followed by a permeabilization step with Triton X-100 (0.1%) for antibody penetration into the cell. Immunocytochemistry was performed by the indirect avidin-biotin immunoperoxidase method using diaminobenzi-dine as the chromagen.

A gold quartz crystal surface can be easily functionalized with an avidin monolayer (Fig. 7.3) that will then allow immobilization of any biotinylated biomolecule through strong avidin-biotin interaction. The only requirement for the development of a successful biosensor device is the availability of biotin-tagged molecules. We have used this strategy to achieve DNA electrode materials without the need for hybridization [44-46]. The dsDNA serial assembly was achieved by first linearizing circular plasmid... 

The avidin-biotin reaction can be used to link several molecular monolayers which lie on top of each other with avidin and biotin molecules arranged in alternating mode. A large variety of configurations results from different combinations of the simple reaction depicted schematically in Fig. 7.28. The stability of the avidin-biotin complex is extremely high. 

Patterning of enzyme monolayers on a solid surface was carried out by photoactivation of immobilized monolayer of caged -biotin derivatives in selected areas. Specific oriented binding of enzyme-avidin conjugates could be readily made to the photoactivated zones [42]. Oriented immobilization of G-protein-coupled receptors on a solid surface was also made possible on a biotinylated surface by first immobilizing streptavidin, followed by the immobilization of biotinylated G-protein-coupled receptor [43]. 

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