Covalent immobilization site-specific

Lee M, Shin I. Eacile preparation of carbohydrate microarrays by site-specific, covalent immobilization of unmodified carbohydrates on hydrazide-coated glass slides. Org. Lett. 2005 7 4269-4272. 

Hemoproteins are a broad class of redox-proteins that act as cofactors, e.g. cytochrome c, or as biocatalysts, e.g. peroxidases. Direct ET between peroxidases such as horseradish peroxidase, lactoperoxidase," or chloropcroxidasc"" and electrode surfaces, mainly carbonaceous materials, were extensively studied. The mechanistic aspects related with the immobilized peroxidases on electrode surfaces and their utilization in developing biosensor devices were reviewed in detail. The direct electrical contact of peroxidases with electrodes was attributed to the location of the heme site at the exterior of the protein that yields close contact with the electrode surface even though the biocatalyst is randomly deposited on the electrode. For example, it was reported " that non-oriented randomly deposited horseradish peroxidase on a graphite electrode resulted in 40-50% of the adsorbed biocatalyst in an electrically contacted configuration. For other hemoproteins such as cytochrome c it was found that the surface modification of the electrodes with promoter units such as pyridine units induced the binding of the hemoproteins in an orientation that facilitated direct electron transfer. By this method, the promoter sites induce a binding-ET process-desorption mechanism at the modified electrode. Alternatively, the site-specific covalent attachment of hemoproteins such as cytochrome c resulted in the orientation of the protein on the electrode surfaces and direct ET communication. ... 

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... 

Zhi ZL, Powell AK, Turnbull JE. Fabrication of carbohydrate microarrays on gold surfaces direct attachment of nonderivatized oligosaccharides to hydra-zide-modified self-assembled monolayers. Anal Chem 2006 78 4786-4793. Lee M, Shin I. Facile preparation of carbohydrate microarrays by site-specific, covalent immobilization of unmodified carbohydrates on hydrazide-coated glass slides. Org Lett 2005 7 4269-4272. 

With the exception of wired enzyme electrodes discussed above, the enzymatically produced cofactor needs an available electron transfer site for the electrochemical reaction to take place to be monitored. As has been discussed, the procedures used for the derivatization of the electrode surface can reduce the rate of electron transfer for the redox mediator (NADH) [8,15], decreasing the sensitivity of the measurement dramatically. Thus, at least two principal factors influence the response of an enzyme-modified electrode the amount of enzyme present near the electrode surface and the availability of electron transfer sites. Therefore, to get a more general protocol for the immobilization of any enzyme to an electrode surface, it would be advantageous to covalently modify only specific parts of the microelectrode surface, leaving other (directly adjacent) regions available for facile electron transfer. 

For laccase, orientation can be guided by different attachment strategies that aim specifically to attach the enzyme in a particular position [77,78]. As an example, in 2011, Pita et al. developed and optimized a strategy for oriented covalent immobilization of Trametes hirsuta laccase on gold electrodes [72]. After optimizing the immobilization for DET via the type 1 (Tl) copper site, they were able to measure current density values up to 40 pA cm for the electrocatalytic reduction of O2 in the absence of redox mediators. 

Another label-free optical detection method—FTIR-ATR—has been applied for detection of thrombin by means of DNA aptamers [73], The antithrombin DNA aptamer previously developed by Tasset et al. [17] was immobilized covalently onto Si surface using UV irradiation method. As a quantitative measure, the area of N-H and CH2 bands was used. This method allowed to detect thrombin with a sensitivity around 10 nmol/L. The specificity of binding of protein to aptamer was also investigated using DNA with no binding site for thrombin. It has been noted that for effective binding study by FTIR-ATR method, the concentration of protein should be kept lower than 100 nmol/L. 

Polyclonal antibodies of different types are known to show affinity for specific compounds. Thus, antibodies that can bind to a specific substance to be analyzed are immobilized to the walls of the test tubes, plates, or microwells. Such test tubes and plates are commercially available and supplied in the test kit. A measured amount (between 10 and 50 pL) of sample or sample extract is added to one such test tube containing an assay diluent (a phosphate buffer). An equal volume of analyte-enzyme conjugate (commercially available and supplied in the kit) is then added to the test tube. The enzyme conjugate is a solution containing the same analytes covalently bound to an enzyme. The solution mixture is incubated or allowed to stand for a specific amount of time. During this period, the enzyme conjugate competes with the analyte molecules for a limited number of antibody binding sites in the test tube. 

---