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Types of Protein Microarray and Antibody Chips

Protein and Antibody Microarrays

Protein Chips and Antibody Microarrays - Glass Slides, Microwell/Nanowell

            Two major protein chip formats are now used, including glass slides and nanowells (8,27).  Due to the large amounts of slide adherence methods only the most common and major types will be mentioned here.
            It is important that protein chips retain proteins in an active state at high densities, are compatible with most commercial arrayers and scanners, and can be printed in such a fashion that the proteins remain in a moisturized environment (8,27). (See Figure 2).

Glass Slide Chips
Glass slides have the advantage that they are compatible with standard microarray equipment and detection equipment used for DNA chips.  They are also inexpensive.  The majority of studies are now using glass slides.  However, they have a high evaporation rate and are susceptible to possible cross-contamination (8,27). 
            The first strategies for the creation of protein arrays on glass were developed by Mirzabekov et al..  Arrays were produced by immobilizing proteins in tiny gel pockets that were attached to the glass surface.  A variety of immunoassays, antigen detection, and enzymatic assays were carried out.  Due to the 3D matrix structure, protein immobilization was very efficient (28-30). 
In another study, proteins were attached to a glass surface activated with a crosslinking agent that reacts with primary amines (31).  Proteins were spotted in a 40% glycerol solution which keeps proteins in a wet environment and prevents dehydration.  To determine that their protein microarrays were feasible for biochemical assays, they tested three known protein-protein interactions, three known kinase-substrate reactions, and three known protein-ligand interactions using fluorophore-tagged proteins, radiolabeled ATP, and synthetic ligands coupled to fluorescently labeled bovine serum albumin (BSA), respectively.  In most of the experiments, small numbers of proteins were spotted however in one experiment they identified a single spot within an array of 10,000 spots containing one other protein.  This work demonstrated the potential of protein microarrays in large-scale biochemical assays however their study analyzed a very small number of proteins and novel activities were not identified.
Another study used glass slides for the detection of several protein antigens.  Analytes were spotted onto a treated glass surface at high density using a hand-spotting device or a microarray robot.  The spotted analytes were detected using antibodies attached to an oligonucleotide primer and a rolling circle amplification reaction.  The technique had a high sensitivity, a wide dynamic range, and excellent spot-to-spot reproducibility
(32).
Most groups now directly array proteins and antibodies onto plain glass slides (31,33-35) and spotting is carried out in a humidity-controlled environment (8).

Microwell/Nanowell Chips
            Compatible with standard microarray and detection equipment however alignment is required.  This method is versatible for solution-based assays and multiple-component reactions.  Evaporation is reduced and there is no cross-contamination.  Also these chips are relatively inexpensive (8,27).
Zhu et al., fabricated an open structure, namely nanowells on a polydimethylsiloxane (PDMS) surface supported by the standard glass slides (36). 
These chips consist of an array of microwells in a disposable silicone elastomer, poly(dimethylsiloxane) (PDMS) (37). Microwell arrays allow small volumes of different analytes to be densely packed on a single chip, yet remain physically segregated during subsequent batch processing. Proteins were covalently attached to the wells using a crosslinker 3-glycidoxypropyltrimethoxysilane (GPTS) (38).
Captured molecules can be easily recovered from the nanowells.  When covered with gold in the nanowells, it is expected that high-throughput mass spectrometry and surface plasmon resonance analyses can be performed.  The greatest disadvantage of this technique is that specialzed equipment is required to load the nanowells at high density (8,27). 

Next: Protein and Antibody Attachment Methods - Creation of a Microarray Chip

 

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Introduction and Background to Protein Chips and Antibody Chips.

References for Protein and Antibody Microarrays

 

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