The development of biochips
is a major thrust of the rapidly
growing
biotechnology industry, which
encompasses a very diverse range
of research efforts including
genomics,
proteomics,
computational biology, and
pharmaceuticals, among other
activities. Advances in these
areas are giving scientists new
methods for unraveling the complex
biochemical processes occurring
inside cells, with the larger goal
of understanding and treating
human diseases. At the same time,
the
semiconductor industry has
been steadily perfecting the
science of microminiaturization.
The merging of these two fields in
recent years has enabled
biotechnologists to begin packing
their traditionally bulky sensing
tools into smaller and smaller
spaces, onto so-called biochips.
These chips are essentially
miniaturized laboratories that can
perform hundreds or thousands of
simultaneous biochemical
reactions. Biochips enable
researchers to quickly screen
large numbers of biological
analytes for a variety of
purposes, from disease diagnosis
to detection of bioterrorism
agents.
The development of biochips has
a long history, starting with
early work on the underlying
sensor technology. One of the
first portable, chemistry-based
sensors was the
glass pH electrode, invented
in 1922 by Hughes (Hughes, 1922).
Measurement of
pH was accomplished by
detecting the potential difference
developed across a thin glass
membrane selective to the
permeation of hydrogen ions; this
selectivity was achieved by
exchanges between H+
and SiO sites in the glass. The
basic concept of using exchange
sites to create permselective
membranes was used to develop
other
ion sensors in subsequent
years. For example, a K+
sensor was produced by
incorporating
valinomycin into a thin
membrane (Schultz, 1996). Over
thirty years elapsed before the
first true biosensor (i.e.
a sensor utilizing biological
molecules) emerged. In 1956,
Leland Clark published a paper on
an
oxygen sensing electrode
(Clark, 1956_41). This device
became the basis for a
glucose sensor developed in
1962 by Clark and colleague Lyons
which utilized
glucose oxidase molecules
embedded in a
dialysis membrane (Clark,
1962). The
enzyme functioned in the
presence of glucose to decrease
the amount of oxygen available to
the oxygen electrode, thereby
relating oxygen levels to glucose
concentration. This and similar
biosensors became known as enzyme
electrodes, and are still in use
today.
In 1953,
Watson and Crick announced
their discovery of the now
familiar
double helix structure of
DNA molecules and set the
stage for
genetics research that
continues to the present day
(Nelson, 2000). The development of
sequencing techniques in 1977
by
Gilbert (Maxam, 1977) and
Sanger (Sanger, 1977) (working
separately) enabled researchers to
directly read the genetic codes
that provide instructions for
protein synthesis. This
research showed how
hybridization of complementary
single
oligonucleotide strands could
be used as a basis for DNA
sensing. Two additional
developments enabled the
technology used in modern
DNA-based biosensors. First, in
1983
Kary Mullis invented the
polymerase chain reaction (PCR)
technique (Nelson, 2000), a method
for amplifying DNA concentrations.
This discovery made possible the
detection of extremely small
quantities of DNA in samples.
Second, in 1986 Hood and coworkers
devised a method to label DNA
molecules with
fluorescent tags instead of
radiolabels (Smith, 1986), thus
enabling hybridization experiments
to be observed optically.
The rapid technological
advances of the
biochemistry and
semiconductor fields in the
1980's led to the large scale
development of biochips in the
1990's. At this time, it became
clear that biochips were largely a
"platform" technology which
consisted of several separate, yet
integrated components. Figure 1
shows the makeup of a typical
biochip platform. The actual
sensing component (or "chip") is
just one piece of a complete
analysis system.
Transduction must be done to
translate the actual sensing event
(DNA binding,
oxidation/reduction, etc.)
into a format understandable by a
computer (voltage,
light intensity, mass, etc.),
which then enables additional
analysis and processing to produce
a final, human-readable output.
The multiple technologies needed
to make a successful biochip --
from sensing chemistry, to
microarraying, to signal
processing -- require a true
multidisciplinary approach, making
the barrier to entry steep. One of
the first commercial biochips was
introduced by
Affymetrix. Their "GeneChip"
products contain thousands of
individual DNA sensors for use in
sensing defects, or single
nucleotide polymorphisms (SNPs),
in genes such as
p53 (a tumor suppressor) and
BRCA1 and
BRCA2 (related to breast
cancer) (Cheng, 2001). The chips
are produced using
microlithography techniques
traditionally used to fabricate
integrated circuits (see
below).
Figure 1. Biochips are a
platform that require, in
addition to microarray
technology, transduction
and signal processing
technologies to output the
results of sensing
experiments.
Today, a large variety of
biochip technologies are either in
development or being
commercialized. Numerous
advancements continue to be made
in sensing research that enable
new platforms to be developed for
new applications. Cancer diagnosis
through DNA typing is just one
market opportunity. A variety of
industries currently desire the
ability to simultaneously screen
for a wide range of chemical and
biological agents, with purposes
ranging from testing public water
systems for disease agents to
screening airline cargo for
explosives. Pharmaceutical
companies wish to combinatorially
screen drug candidates against
target enzymes. To achieve these
ends,
DNA,
RNA,
proteins, and even living
cells are being employed as
sensing mediators on biochips.
Numerous transduction methods can
be employed including
surface plasmon resonance,
fluorescence, and
chemiluminescence. The
particular sensing and
transduction techniques chosen
depend on factors such as price,
sensitivity, and reusability.
The microarray -- the dense,
two-dimensional grid of biosensors
-- is the critical component of a
biochip platform. Typically, the
sensors are deposited on a flat
substrate, which may either be
passive (e.g. silicon or
glass) or active, the latter
consisting of integrated
electronics or
micromechanical devices that
perform or assist signal
transduction.
Surface chemistry is used to
covalently bind the sensor
molecules to the substrate medium.
The fabrication of microarrays is
non-trivial and is a major
economic and technological hurdle
that may ultimately decide the
success of future biochip
platforms. The primary
manufacturing challenge is the
process of placing each sensor at
a specific position (typically on
a
Cartesian grid) on the
substrate. Various means exist to
achieve the placement, but
typically robotic micro-pipetting
(Schena, 1995) or micro-printing (MacBeath,
1999) systems are used to place
tiny spots of sensor material on
the chip surface. Because each
sensor is unique, only a few spots
can be placed at a time. The
low-throughput nature of this
process results in high
manufacturing costs.
Figure 2. Method for
combinatorially producing
single-stranded
oligonucleotides using
photolithography (from
Pease, 1994). Each of the
four nucleotides (A, G, C,
and T) requires a separate
lithography step.
Fodor and colleagues developed
a unique fabrication process
(later used by
Affymetrix) in which a series
of microlithography steps is used
to
combinatorially synthesize
hundreds of thousands of unique,
single-stranded DNA sensors on a
substrate one
nucleotide at a time (Fodor,
1991; Pease, 1994) (see Figure 2).
As the figure shows, one
lithography step is needed per
base type; thus, a total of four
steps is required per nucleotide
level. Although this technique is
very powerful in that many sensors
can be created simultaneously, it
is currently only feasible for
creating short DNA strands (15-25
nucleotides). Reliability and cost
factors limit the number of
photolithography steps that can be
done. Furthermore, light-directed
combinatorial synthesis techniques
are not currently possible for
proteins or other sensing
molecules.
As noted above, most
microarrays consist of a Cartesian
grid of sensors. This approach is
used chiefly to map or "encode"
the coordinate of each sensor to
its function. Sensors in these
arrays typically use a universal
signaling technique (e.g.
fluorescence), thus making
coordinates their only identifying
feature. These arrays must be made
using a serial process (i.e.
requiring multiple, sequential
steps) to ensure that each sensor
is placed at the correct position.
"Random" fabrication, in which
the sensors are placed at
arbitrary positions on the chip,
is an alternative to the serial
method (see Figure 3). The tedious
and expensive positioning process
is not required, enabling the use
of parallelized self-assembly
techniques. In this approach,
large batches of identical sensors
can be produced; sensors from each
batch are then combined and
assembled into an array. A
non-coordinate based encoding
scheme must be used to identify
each sensor. As the figure shows,
such a design was first
demonstrated (and later
commercialized by Illumina) using
functionalized beads placed
randomly in the wells of an etched
fiber optic cable (Steemers,
2000; Michael, 1998) Each bead was
uniquely encoded with a
fluorescent signature. However,
this encoding scheme is limited in
the number of unique dye
combinations that be can be used
and successfully differentiated.
Figure 3. Randomly
assembled microarrays use
an alternative "encoding"
technique to differentiate
the sensors. Sensors are
made in batches from which
small lots are taken to
create the array. (Adapted
from Gunderson, 2004.)
Microarrays are not limited to
DNA analysis;
protein microarrays can also
be produced using biochips.
Randox Laboratories Ltd.
launched Evidence®, the first
protein Biochip Array Technology
analyzer in 2003. In protein
Biochip Array Technology, the
biochip replaces the
ELISA plate or
cuvette as the reaction
platform. The biochip is used to
simultaneously analyze a panel of
related tests in a single sample,
producing a
patient profile. The patient
profile can be used in disease
screening,
diagnosis, monitoring disease
progression or monitoring
treatment. Performing multiple
analyses simultaneously, described
as multiplexing, allows a
significant reduction in
processing time and the amount of
patient sample required. Biochip
Array Technology is a novel
application of a familiar
methodology, using sandwich,
competitive and antibody-capture
immunoassays. The difference
from conventional immunoassays is
that the capture ligands are
covalently attached to the surface
of the biochip in an ordered array
rather than in solution.
In sandwich assays an enzyme-labelled
antibody is used; in competitive
assays an enzyme-labelled antigen
is used. On antibody-antigen
binding a
chemiluminescence reaction
produces light. Detection is by a
charge-coupled device (CCD)
camera. The CCD camera is a
sensitive and high-resolution
sensor able to accurately detect
and quantify very low levels of
light. The test regions are
located using a grid pattern then
the chemiluminescence signals are
analysed by imaging software to
rapidly and simultaneously
quantify the individual analytes.
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