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Protein Attachment to Microarray Chips

Protein and Antibody Microarrays

Creation and Manufacture of Protein and Antibody Microarray Chips

           

Attachment
            To attach proteins to a solid surface, the surface of the substrate has to be modified to achieve maximum binding capacity (8,27).  The proteins are attached to the chip on a protein attachment layer (see Figure 3).  This layer is typically an organic film which varies with the nature of the application.  A variety of materials have been studied including agarose (39), dextran-based hydrogel (40), porous polyacrylamide hydrogel  hydrophilic polymers and polyamino acids (41).
            A convenient attachment method used nitrocellulose-membrane or poly-L-Lysine coated glass such that proteins could be passively absorbed onto the surface through non-specific interactions (34,42,43).  The attached proteins bind onto the surface in random orientations and can be washed off under stringent washing conditions.  However, the noise level is usually higher because of the non-specific absorption/adsorption. 
            A more specific and stronger attachment is achieved by creating reactive surfaces on glass that can covalently cross-link to proteins (31,36,26).  A bifunctional silane cross-linker is used to form a self-assembled monolayer (SAM), which has one functional group that reacts with the hydroxyl groups on glass the glass surface, and another group which is free to react with primary amine groups of proteins or can be further chemically modified to reach maximum specificity (44,45).  Another variation is gold-coated glass (46,47).  The advantage of gold-coated chips is that SPR and mass spectrometry can be integrated as detection methods to monitor the dynamics of the reaction, and to identify the captured molecules.
            The above-mentioned covalent cross-linking approaches however have a disadvantage. Due to the fact that reactive ligands also exist in the side chains of proteins it is possible that their random attachment may alter the native confirmation of proteins, reduce the activity of proteins, or make them inaccessible to probes (8,27).
            In order to orient proteins uniformily away from the surface of the chip, proteins may be fused with a high-affinity tag at their amino or carboxy termini.  With this method, immobilized proteins/antibodies are more likely to remain in their native conformation, thus allowing the analytes efficient access to the active sites of the proteins.  This method was first successfully demonstrated with the attachment of 5800 fusion proteins containing a His tag onto a nickel-coated glass slide (26).  Other affinity methods such as glutathione/GST have also been used (48).
Streptavidin based immobilization methods have been also widely employed to attach any biotinylated biological element to the array surface (49).
            The chip support material is important because proteins are highly sensitive to physiochemical properties.  For example, polar arrays are chemically treated to bind to hydrophilic proteins however such surfaces are unsuitable for cell membrane proteins (e.g. G-protein coupled receptors) as they possess hydrophobic domains (9). 
            Proteins do not behave like nucleic acids, and different proteins will behave in different ways when exposed to the same surface chemistry.  Different types a surface chemistries will thus promote the retention of some proteins and cause denaturation or loss of activity of others.  Therefore, the proper choice of surface chemistry is important as this will allow immobilized proteins of diverse types to retain their secondary and tertiary structures, and thus their biological activity.  This problem is magnified when the number of different spots on the chip increases, as there may be 100 different ways to immobilize 100 different proteins in order to obtain proper folding and function of all the proteins.  Also a significant problem is because the functions of most proteins are currently unknown, so there is no method to actually test whether they are still functional on the chip (7).

Next: Protein Chip Delivery Methods

References for Protein and Antibody Microarrays

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Introduction and Background to Protein Chips and Antibody Chips.

Types of Antibody and Protein Chips

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