Protein microarray
The chip consists of a support surface such as a glass slide, nitrocellulose membrane, bead, or microtitre plate, to which an array of capture proteins is bound.
[3] Protein microarrays are rapid, automated, economical, and highly sensitive, consuming small quantities of samples and reagents.
Additionally post-translational modifications, which are often critical for determining protein function, are not visible on DNA microarrays.
The proteins are arrayed onto a solid surface such as microscope slides, membranes, beads or microtitre plates.
Microscope slides made of glass or silicon are a popular choice since they are compatible with the easily obtained robotic arrayers and laser scanners that have been developed for DNA microarray technology.
Immobilising agents include layers of aluminium or gold, hydrophilic polymers, and polyacrylamide gels, or treatment with amines, aldehyde or epoxy.
An aqueous environment is essential at all stages of array manufacture and operation to prevent protein denaturation.
Therefore, sample buffers contain a high percent of glycerol (to lower the freezing point), and the humidity of the manufacturing environment is carefully regulated.
The printhead moves across the array, and at each spot uses electric stimulation to deliver the protein molecules onto the surface via tiny jets.
A series of chemical treatments then enables deposition of the protein in the desired pattern upon the material underneath the photomask.
Many of these methods can be automated for high throughput production but care must be taken to avoid conditions of synthesis or extraction that result in a denatured protein which, since it no longer recognizes its binding partner, renders the array useless.
This presents a challenge in maintaining protein arrays in a stable condition over extended periods of time.
Reverse phase protein microarray (RPPA) involve complex samples, such as tissue lysates.
The most common and widely used method for detection is fluorescence labeling which is highly sensitive, safe and compatible with readily available microarray laser scanners.
Immunoassays on thiol-ene "synthetic paper" micropillar scaffolds have shown to generate a superior fluorescence signal.
This limits interferences due to auto-fluorescence of the nitrocellulose at the UV wavelengths used for standard fluorescent detection probes.
A cost-effective fabrication platform (using OSTE polymers) for such microwell arrays has been recently demonstrated and the bio-assay model system has been successfully characterised.
The former approach is attractive in its simplicity and is compatible with purified proteins derived from native or recombinant sources[21][22] but suffers from a number of risks.
