Covalent immobilization site-nonspecific

To date, several tactics and strategies have been described for the fabrication of chemical arrays. These strategies can be classified in the following manner based on how small molecules are introduced into the array (1) site-specific covalent immobilization, (2) site-nonspecific covalent immobilization, (3) site-specific... 

Physical adsorption and covalent attachment of both native and reconibinant OPH onto various supports such as nylon membranes, porous glass and nanometer size silica beads have been enqiloyed (3,21,22).. Unfortunately, physical adsorption offers poor and nonspecific binding, vdiile coval modifications to OPH often results in reduction of enzyme activity and kinetic properties (22,23). In addition to reducing catalytic activity, there is no controlled orientation of the immobilized enzymes, leading to inaccessibility of the substrate to the enzyme active site. In the case of covalent bondii, the immobilization support is not reusable since the formed covalent bond is irreversible. In addition, the tedious and costly protocol for purification of OPH limits its use in large-scale enzymatic degradation. 

Not only the components of the sensing layer are important but also the strategies of integration between them and with the primary transducer are of paramount importance. Passive adsorption is one of the simplest and most frequently used immobilization techniques, based mainly on weak noncovalent bindings. However, random protein orientation can lead to the obstruction of functional binding sites and can result in the loss of affinity or activity. Thus, covalent attachment of a protein layer on a chemically functionalized surface produces a more stable layer with correct orientation. This influences sensitivity and specificity of the immunoassay as density of immobilized protein can be better controlled and nonspecific adsorption may be decreased [155]. On the other hand, covalent procedures are usually longer and more tedious and are less justified when disposable surfaces can be used. 

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