Biomedical Engineering Reference
In-Depth Information
derive amino acid sequence information, which can then be used to search protein
or genome databases to identify the protein in the original gel spot.
The interest in protein microarray technology also stems to a great extent from
the hugely successful DNA microarray technology. The tremendous variability in
the nature of proteins and consequently in the requirement of their detection and
identification also makes the development of protein chips a particularly challeng-
ing task. Typically, protein microarrays are developed using robotic dispensers such
as those developed for creating DNA microarrays. The protein samples are made
to adhere to the glass slides by various immobilization strategies described above.
Soft substrates such as polystryrene, poly(vinylidene fluoride), and nitrocellulose
membranes, which have been used to attach proteins in traditional biochemical
analyses (e.g., immunoblot and phage display analysis), are often not compatible
for protein microarrays. These surfaces often do not allow a suitable high protein
density, the spotted material may spread on the surface, and/or they may not allow
optimal signal-to-noise ratios. Thus, glass microscope slides or other materials that
have been derivatized to attach proteins on their surface at high density are cho-
sen. These slides have low fluorescence background and are compatible with most
assays. The detection of bound targets to proteins is considerably more complex
than that of DNA microarray detection. Among a variety of detection methods, the
preferred method of detection is the fluorescence, because it is generally safe, ex-
tremely sensitive, and simple, can have very high resolution and is compatible with
standard microarray scanners. Four major barriers in protein microarray develop-
ment are: (1) minimizing background noise; (2) preserving protein native state and
orientation; (3) protein detection and identification; and (4) speed of protein or
antibody production and purification.
Fluorescence detection methods are generally the preferred detection method
because they are simple, safe, and extremely sensitive and can have very high reso-
lution. They are also compatible with standard DNA microarray scanners. Typi-
cally, a chip is either directly probed with a fluorescent molecule (e.g., protein or
small molecule) or in two steps by first using a tagged probe (e.g., biotin), which
is then detected in a second step using a fluorescently labeled affinity reagent (e.g.,
streptavidin). One of the challenges that present itself for direct protein detection
in matrices such as whole blood, serum, or plasma is the range of pathogen load
and protein concentrations within the sample. Protein levels differ by as much as
nine orders of magnitude in these conditions, but the detection range is only three
to four orders of magnitude (i.e., only a fraction of the proteome is examined by a
given experiment). As antibody-binding constants are commonly in the nanomolar
range, they are ill equipped to directly measure low abundance targets, which may
be present in picomolar range, without sample preconcentration.
When measuring low abundance targets such as virus particles, alternative
strategies are required. In this case, diagnostic methods take advantage of either
surrogate markers or signal amplification by the immune system and measure the
antibodies expressed against the pathogen. Thus, constructing an array of antigens
is a convenient method of measuring antibody concentration and relating it back to
infection. Another alternative is to use probe molecules with higher binding affinity.
An excellent candidate for this approach is aptamers, which are single-stranded oli-
gomers of DNA or RNA that exhibit extremely high affinity for binding molecules
