Noncovalent immobilization methods

DNA oligonucleotide arrays to profile mRNA expression patterns. In these spotted arrays, purified cDNA clones or PCR products are micropipetted as an array on a substrate surface and immobilized by one of a variety of covalent or noncovalent methods.(72 74)... 

Noncovalent attachment is a popular method of immobilization, and numerous different support materials have been employed, ranging from organic supports, like cellulose. 

In summary, enzyme immobilization is extremely important in the scale-up of many biocatalytic processes. The preferred method for pharmaceutical production involves covalent binding through cross-linking or attachment to a support. Noncovalent attachment is less attractive, but it is heavily utihzed owing to the commercial availabihty of industrial quantities of some enzymes immobilized using this technique. 

A third item to consider in using affinity chromatography is the way in which the ligand is attached to the solid support, or the immobilization method. Several techniques are available for this, including both covalent and noncovalent coupling methods [25,36]. For a protein or peptide, this generally... 

Other Immobilization Techniques Along with noncovalent and covalent immobilization methods, other techniques have been developed for the preperation of affinity supports. Such methods include entrapment, molecular imprinting, and the use of the ligands as both the support and stationary phase. Although these methods are not as common as the approaches already examined, they have important advantages in some applications [8]. 

Immobilization techniques can be classified by basically two methods, the chemical and the physical method. The former is covalent bond formation dependent and the latter is noncovalent bond formation dependent.1... 

Most biocatalytic conversions are performed with the enzyme immobilized in the microreactor. Miyazaki et al. [426] developed a simple noncovalent immobilization method for His-tagged enzymes on a microchannel surface. These enzymes contain a polyhistidine-tag motif that consists of at least six histidine residues, often located at the N- or C-terminus. The H is-tag has a strong affinity for nickel and can be reversibly immobilized by a nickel-nitrilotriacetic acid (Ni-NTA) complex (Scheme 4.103), a strategy commonly used in affinity chromatography. 

FIGURE 15.1 Noncovalent immobilization methods to construct a glycan array. 

However, the main disadvantage of this method of probe immobilization is that stringent washing steps (necessary to insure a high selectivity against mismatched sequences) cannot be performed, because of the desorption of the noncovalently bound hybrid. 

The methods of attachment to the solid support can be divided into two broad categories covalent and noncovalent. Several factors should be considered. First, the ligands should be stable to immobilization, storage, and assay conditions. Second, the immobilized ligands should be accessible to the receptors, which are usually in solution. Third, the method should be efficient. 

An elegant alternative approach for noncovalent interaction relies on fluorous-lluorous interactions. A glycan array of monosaccharides and disaccharides bearing anomeric fluorous tags was noncovalently immobilized on fluorous-derivatized glass slides (19, 20). The attachment method is compatible with a wide range of functional groups and has been successfully used to probe carbohydrate-protein interactions. 

LSD analogs and metabolites were used as model compoimds by Cai and Henion [73] in the development of on-hne immimoaffinity extraction coupled to capillary-colutrm LC-MS-MS. The system consists of a 2.1-mm-lD protein G iimmmoaffinity colutrm with a noncovalently immobilized antibody, which can be operated at flow-rates of up to 4 ml/min, a short packed-capillary trapping column, and a packedcapillary analytical column, operated at 3.5 pl/mia The method enables low ng/1 determination of LSD and related compoimds in urine, which is twenty-fold better than previous methods based on SPE and LC-MS-MS [73]. 

---