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
, computational biology
, 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
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.
A biochip is a collection of miniaturized test sites (microarrays) arranged on a solid substrate that permits many tests to be performed at the same time in order to achieve higher output and speed.
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+
produced by incorporating valinomycin
into a thin membrane (Schultz, 1996).
Over thirty years elapsed before the first true biosensor
sensor utilizing biological molecules) emerged. In 1956, Leland Clark
published a paper on an oxygen sensing electrode
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
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
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 1980s led to the large scale development of biochips in the 1990s.
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).
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,
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.
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). 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. 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
Protein biochip array and other microarray technologies
are not limited to DNA
analysis; protein microarrays
, antibody microarray
, chemical compound microarray
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.
Details about other array technologies can be found in the following pages: Antibody microarray and chemical compound microarray.
- W. S. Hughes, “The potential difference between glass and electrolytes in contact with water,” J. Am. Chem. Soc. 44, pp. 2860–2866, 1922.
- J. S. Schultz and R. F. Taylor in Handbook of Chemical and Biological Sensors, J. S. Schultz and R. F. Taylor, eds., ch. Introduction to Chemical and Biological Sensors, pp. 1–10, Institute of Physics Publishing, Philadelphia, 1996.
- L. C. Clark, Jr., “Monitor and control of blood tissue O2 tensions,” Transactions of the American Society for Artificial Internal Organs 2, pp. 41–84, 1956.
- L. C. Clark, Jr. and C. Lyons, “Electrode system for continuous monitoring in cardiovascular surgery,” Annals of the New York Academy of Sciences 148, pp. 133–153, 1962.
- D. L. Nelson and M. M. Cox, Lehninger Principles of Biochemistry, Worth Publishers, New York, 2000.
- A. M. Maxam and W. Gilbert, “A new method for sequencing DNA,” Proc. Nat. Acad. Sci. 74, pp. 560–564, 1977.
- F. Sanger, S. Nicklen, and A. R. Coulson, “DNA sequencing with chainterminating inhibitors,” Proc. Nat. Acad. Sci. 74, pp. 5463–5467, 1977.
- L. M. Smith, J. Z. Sanders, R. J. Kaiser, P. Hughes, C. Dodd, C. R. Connell, C. Heiner, S. B. H. Kent, and L. E. Hood, “Fluorescence detection in automated DNA sequence analysis,” Nature 321, pp. 61–67, 1986.
- P. Fortina, D. Graves, C. Stoeckert, Jr., S. McKenzie, and S. Surrey in Biochip Technology, J. Cheng and L. J. Kricka, eds., ch. Technology Options and Applications of DNA Microarrays, pp. 185–216, Harwood Academic Publishers, Philadelphia, 2001.
- M. Schena, D. Shalon, R. W. Davis, and P. O. Brown, “Quantitative monitoring of gene expression patterns with a complementary DNA microarray,” Science 270, pp. 467–470, 1995.
- G. MacBeath, A. N. Koehler, and S. L. Schreiber, “Printing small molecules as microarrays and detecting protein-ligand interactions en masse,” J. Am. Chem. Soc. 121, pp. 7967–7968, 1999.
- S. P. Fodor, J. L. Read, M. C. Pirrung, L. Stryer, A. T. Lu, and D. Solas, “Light-directed, spatially addressable parallel chemical analysis,” Science 251, pp. 767–773, 1991.
- A. C. Pease, D. Solas, E. J. Sullivan, M. T. Cronin, C. P. Holmes, and S. P. Fodor, “Light-generated oligonucleotide arrays for rapid DNA sequence analysis,” Proc. Natl. Acad. Sci. 91, pp. 5022–5026, 1994.
- F. J. Steemers, J. A. Ferguson, and D. R. Walt, “Screening unlabeled DNA targets with randomly-ordered fiber-optic gene arrays,” Nature Biotechnology 18, pp. 91–94, 2000.
- K. L. Michael, L. C. Taylor, S. L. Schultz, and D. R. Walt, “Randomly ordered addressable high-density optical sensor arrays,” Analytical Chemistry 70, pp. 1242–1248, 1998.
- K. L. Gunderson, S. Kruglyak, M. S. Graige, F. Garcia, B. G. Kermani, C. Zhao, D. Che, T. Dickinson, E. Wickham, J. Bierle, D. Doucet, M. Milewski, R. Yang, C. Siegmund, J. Haas, L. Zhou, A. Oliphant, J.-B. Fan, S. Barnard, and M. S. Chee, “Decoding randomly ordered DNA arrays,” Genome Research 14(5), pp. 870–877, 2004.
- C. Roberts, C. S. Chen, M. Mrksich, V. Martichonok, D. E. Ingber, and G. M. Whitesides, “Using mixed self-assembled monolayers presenting RGD and (EG)3OH groups to characterize long-term attachment of bovine capillary endothelial cells to surfaces,” J. Am. Chem. Soc. 120, pp. 6548–6555, 1998.
- H. Schmeck, "Blazing the Genetic Trail." Bethesda, MD: Howard Hughes Medical Institute, 1991.
- Interview of A. Caplan, "Should We or Shouldn't We?" http://web.reed.edu/reed_magazine/spring06/features/life_in_venice/should_we.html
- Vahid Bemanian, Frøydis D. Blystad, Live Bruseth, Gunn A. Hildrestrand, Lise Holden, Endre Kjærland, Pål Puntervoll, Hanne Ravneberg and Morten Ruud, "What is Bioethics?" Dec 1998.
- M. Burnham, R. Mitchell, " Bioethics — An Introduction" 1992.
- NBIAP NEWS REPORT, U.S. Department of Agriculture, "To Regulate or Not to Regulate" Forum: To Rationalize U.S. Biotech Regs. June 1994