Biomedical Engineering Reference
In-Depth Information
fluorescence amplitude values. The standard expression matrix format also means that it's possible
to spot a microarray with an arbitrary pattern of genes-condition cDNA without regard to how the
data will eventually be displayed. Note that the data used for analysis is actually based on the
digitized (numerical) value of the relative red and green fluorescence of the spots on the microarray.
In many respects, the spotting process, which was developed at Stanford University, has many
parallels with the early digital electronic computers. The first commercially successful digital
computers, such as the UNIVAC line, used discrete components and mechanical means—including
punched paper cards—to work with the system. Individual components were soldered by hand to
create the thousands of circuits. Because of variability in the tubes and components, the circuits had
to be tuned by hand. There were often failures of individual components because of device failure or
because the solder joints of components and cables eventually failed. Because construction was done
by hand and because every computer was built with thousands of components—each of which varied
somewhat from their ideal values and performance—it took a month or more to produce a computer
system. This investment in time was well worth it. Compared to earlier computational methods, the
early digital electronic computers shaved countless hours off the time required to compile a census or
compute the trajectory of a projectile.
Even though the first discrete-component electronic digital computers worked well, because of the
time required to create and test each computer, customers were limited to large corporations, the
military, and the government. The situation changed with the introduction of the integrated circuit
(IC). Not only did the development of the IC allow for much smaller computers, but component count
and variability dropped precipitously. As a result, reliability increased, prices dropped, and computers
became affordable to a mass market.
The process used to make ICs is based on photolithography. Instead of soldering discrete
components by hand or with mechanical jigs, transistors, diodes, resistors, and capacitors are formed
by a process in which multiple layers of semiconductor material are alternatively laid down on a
ceramic or silicon substrate. Masks or barriers block the light used to sensitize the surface, allowing it
to accept the next layer of semiconductor, insulator, or resistive material. As a result, tens of
thousands of ICs can be produced in days. Furthermore, because most of the process is performed
with high-tolerance, mostly non-mechanical methods, failure rates are low and performance is
consistent from one IC to the next.
The approach used in IC fabrication has been applied to microarray preparation and analysis. For
example, the process of microarray preparation based on photolithography and solid-phase chemistry
commercialized by Affymetrix
®
is illustrated in
Figures 6-6
and
6-7
. The overall process depicted in
Figure 6-6
illustrates how commercial process begins with a 5-inch square quartz wafer, similar to
the quartz discs used to create ICs. The wafer is washed and then placed in a silane bath that forms
a matrix of covalently linked molecules on the surface of the wafer. Linker molecules on the silane
matrix provide a surface that may be light-activated.
Figure 6-6. Affymetrix Microarray Preparation Process. The process
parallels that used in the microcomputer industry used to create ICs. The
technology offers much higher capacity and more quantitative results
compared to microarray spotting.
