Bonding, immobilization methods covalent

The covalent attachment of enzymes to water-insoluble carriers is usually the preferred immobilization method for sensor manufacturing. Obviously, the selected procedure should avoid the loss of enzymatic activity and keep the accessibility of the binding site to the substrate molecules. Unfortunately, this is usually not the case and due to the severe conditions of many of these procedures, major activity losses and/or changes on the substrate selectivity are produced during immobilization. Some authors have pointed out that the enzyme activity decreases approximately one fifth per formed bond [66]. 

Covalently immobilized arrays are formatted by glycans with functionalized spacers that react with a complementary activated surface to form a covalent bond. Several different covalent interactions were reported to construct a specified glycan array. Amine chemistry and thiol chemistry are the two major methods to conjugate glycans to the reactive substrate in the array surface (Fig. 15.2). Thiol chemistry was first adapted by Injae Shin in 2002 to react with the maleimide functional group (Fig. 15.2a, b) [7,41 ]. Disulfide bond formation was then reported for the fabrication... 

Enzyme immobilization methods are classified as chemical or physical. Chemical methods involve the formation of covalent bonds between functional groups on the... 

Physical immobilization methods do not involve covalent bond formation with the enzyme, so that the native composition of the enzyme remains unaltered. Physical immobilization methods are subclassified as adsorption, entrapment, and encapsulation methods. Adsorption of proteins to the surface of a carrier is, in principle, reversible, but careful selection of the carrier material and the immobilization conditions can render desorption negligible. Entrapment of enzymes in a cross-linked polymer is accomplished by carrying out the polymerization reaction in the presence of enzyme the enzyme becomes trapped in interstitial spaces in the polymer matrix. Encapsulation of enzymes results in regions of high enzyme concentration being separated from the bulk solvent system by a semipermeable membrane, through which substrate, but not enzyme, may diffuse. Physical immobilization methods are represented in Figure 4.1 (c-e). 

Covalent immobilization methods rely on functional groups on both the enzyme and the support material for the formation of stable covalent bonds. For this reason, the choice of a support is crucial in that it determines the immobilization chemistry... 

Chemical immobilization methods may alter the local and net charges of enzymes, through covalent modification of charged residues such as lysine (NH4), aspartate, and glutamate (COO-). Conformational changes in secondary and tertiary protein structure may occur as a result of this covalent modification, or as a result of electrostatic, hydrogen-bonding or hydrophobic interactions with the support material. Finally, activity losses may occur as a result of the chemical transformation of catalytically essential amino acid residues. 

The immobilization methods ate identified either as chemical or physical procedures depending on whether covalent bonds ate established or not. °... 

It is obvious that the immobilization protocol depends on the transducer characteristics, nevertheless it is preferred to use robust immobilization methods in order to avoid the probe desorption from the sensor [57]. Thus, the retention in pol5nneric matrix, covalent bonds on a functionalized surface, SAMs, and immobilization through affinity reactions are the most successful methods at the moment, because these strategies give place to an immobilization across the ends of the probes in a tidy and orientated way. In addition, these strategies allow to control the conformational freedom of the probes and the space between chains by means of the control of the superficial covering obtaining hybridization efficiencies up to 100%. 

In order to make a useful biosensor, enzyme has to be properly attached to the transducer with maintained enzyme activity. This process is known as enzyme immobilization. The choice of immobilization method depends on many factors such as the nature of the enzyme, the type of transducer used, the physiochemical properties of analyte, and the operating conditions [73]. The major requirement out of all these is its maximum activity in immobilized microenvironment. Enzyme-based electrodes provide a tool to combine selectivity of enzyme toward particular analyte and the analytical power of electrochemical devices. The amperometric transducers are highly compatible when enzymes such as urease, generating electro-oxidizable ions, are used [74]. The effective fabrication of enzyme biosensor based on how well the enzyme bounds to the transducer surface and remains there during use. The enzyme molecules dispersed in solutions will have a freedom of their movement randomly. Enzyme immobilization is a technique that prohibits this freedom of movement of enzyme molecules. There are four basic methods of immobilizing enzymes on support materials [75] and they are physical adsorption, entrapment, covalent bonding, and cross-linking, as shown in the Fig. 36. 

Fig. 6 Non-specific immobilization methods with covalent bond formation. Chemoselec-tive immobilization will yield a SMM with a defined orientation (top) whereas unselective immobilization will allow different orientations of the small molecule on the array (bottom)...

Pinto et al. reported the PDMS surface functionalization for the development of an immune-sensor for salivary cortisol analysis, using three different antibodies immobilization methods immobilization by passive adsorption on pristine PDMS silaniza-tion of PDMS surface with (3-aminopropyl)-triethoxysilane (APTES) to generate amino groups and posterior covalent immobilization of antibodies on APTES-PDMS using crosslinker glutaraldehyde (GA) coating the PDMS surface with BSA to block nonspecific protein adsorption, and then covalent bond of the protein A via GA (Pinto etal.,2015). 

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