Biomedical Engineering Reference
In-Depth Information
11.3.2.3 Fabrication of gel pad, ELISA, and SELDI protein biochips
Different fabrication technologies have been reported in the literature [34, 57-64]. The
most straightforward way to construct the protein biochip is to follow the approach
which has already been successfully applied in DNA biochip, namely, gel pad. Actually,
gel-pad technology provides three-dimensional (3D) structure for protein immobilization
which includes larger surface area and greater probe density. As we know, the functional
properties of a protein are highly dependent on its conformation and structure. Therefore,
it is possible that delicate proteins may not be able to survive the harsh conditions of 2D
surfaces. Since gel pad closely mimics the biological environments of various proteins,
the conformation and activity of immobilized protein are preserved in the homogeneous
surrounding water. In the past years, researchers in this fi eld have mainly focused on
polyacrylamide gels by virtue of its unique properties such as low fl uorescence back-
ground, low non-specifi c absorption, high immobilization density and high porosity for
easy access of macromolecules into the gel pad [65]. Normally, polyacrylamide gels depos-
ited on certain substrate (for example, microscope slides) are synthesized by exposing
acrylamide solution to UV radiation and then washing to remove non-polymerized
monomer. After that, polyacylamide gels are further activated with glutaraldehyde
or partially substituting the amide groups with hydrazide groups. Hydrazine-activated
microchips demonstrated higher capacity than glutaraldhyde-activated microchips and
gave signals 1.8-fold higher after immobilization of the same antibodies.
Protein array chips can be fabricated by using well-established protein detection
methods such as enzyme-linked immunosorbent assay (ELISA). Since the signals can
be dramatically amplifi ed by enzyme, this method can detect very low abundance of
antigen or antibody. In general, ELISA contains direct and secondary detection. The
direct detection directly labels the target proteins for measurements while secondary
detection recognizes the analyte proteins through a secondary labeled antibody bind-
ing to the captured target proteins in a sandwich mode after an initial incubation. Due
to its highly inherent specifi city and sensitivity, secondary detection is preferable. In
sandwich ELISA mode (Fig. 11.25), the secondary antibodies are labeled either with
a fl uorophore or with an enzyme that produces a detectable luminescent product. The
sandwich ELISA allows an assay with little or no purifi cation. The primary drawback,
however, is that well-characterized antibodies generated against a single antigen are
necessary to confer a high degree of specifi city. If an antibody binds a common antigen
across multiple protein species, a “composite” signal will be generated and additional
assays are necessary to decipher the binding event.
Nevertheless, methodologies that combine genomic arraying techniques with ELISA-
based methodologies are now gaining considerable attention [66]. This approach strives
to deposit nanoliter quantities of bait reagents, such as antibodies, recombinant peptides
or small drug libraries on an addressable, high density (potentially thousands of fea-
tures) microarray in a process analogous to genomic microarrays, which is now possible
due to advances in recombinant protein technologies that allow the rapid production and
purifi cation of affi nity-based reagents such as antibodies, ScFv, aptamers, and peptide
libraries. Although many groups have demonstrated the utilities of protein microarrays,
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