Annurev Bioeng 4 020702 153438

19 Jun 2002 9:1 AR AR164-07.tex AR164-07.
SGM LaTeX2e(2002/01/18) P1: IKH

10.1146/annurev.bioeng.4.020702.153438
Annu. Rev. Biomed. Eng. 2002. 4:129–53

doi: 10.1146/annurev.bioeng.4.020702.153438
Copyright °
c 2002 by Annual Reviews. All rights reserved
DNA MICROARRAY TECHNOLOGY: Devices,

Systems, and Applications
Michael J. Heller
Departments of Bioengineering/Electronic and Computer Engineering, University of
California, San Diego, La Jolla, California 92093; e-mail: mheller@bioeng.ucsd.edu
Annu. Rev. Biomed. Eng. 2002.4:129-153. Downloaded from www.annualreviews.org
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Key Words DNA arrays, DNA chips, DNA microchips, DNA devices, DNA
hybridization
■ Abstract In this review, recent advances in DNA microarray technology and
their applications are examined. The many varieties of DNA microarray or DNA chip
devices and systems are described along with their methods for fabrication and their
use. This includes both high-density microarrays for high-throughput screening appli-
cations and lower-density microarrays for various diagnostic applications. The meth-
ods for microarray fabrication that are reviewed include various inkjet and microjet
deposition or spotting technologies and processes, in situ or on-chip photolithographic
oligonucleotide synthesis processes, and electronic DNA probe addressing processes.
The DNA microarray hybridization applications reviewed include the important ar-
eas of gene expression analysis and genotyping for point mutations, single nucleotide
polymorphisms (SNPs), and short tandem repeats (STRs). In addition to the many
molecular biological and genomic research uses, this review covers applications of
microarray devices and systems for pharmacogenomic research and drug discovery,
infectious and genetic disease and cancer diagnostics, and forensic and genetic iden-
tification purposes. Additionally, microarray technology being developed and applied
to new areas of proteomic and cellular analysis are reviewed.
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
REVIEWS ON MICROARRAY TECHNOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
MICROARRAY DEVICES AND SYSTEMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
High-Density Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Microelectronic Array Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
New Microarray Technologies, Chemistries, and
Detection Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
MICROARRAY INFORMATICS AND APPLICATIONS . . . . . . . . . . . . . . . . . . . . . 142
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
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130 HELLER
INTRODUCTION
A variety of DNA microarray and DNA chip devices and systems have now been
developed and commercialized. These devices allow DNA and/or RNA hybridiza-
tion analysis to be carried out in microminiaturized highly parallel formats. DNA
microarray hybridization applications are usually directed at gene expression anal-
ysis or screening samples for single nucleotide polymorphisms (SNPs). In addition
to the molecular biologically related analyses and genomic research applications,
such microarray systems are also being used for pharmacogenomic research, in-
fectious and genetic disease and cancer diagnostics, and forensic and genetic iden-
tification purposes. Microarray technology continues to improve in performance

aspects regarding sensitivity and selectivity and in becoming a more economical
research tool. The use of DNA microarrays will continue to revolutionize genetic
analysis and many important diagnostic areas. Additionally, microarray technol-
ogy that has been developed for DNA analysis is now also being applied to new
areas of proteomic and cellular analysis.
DNA hybridization microarrays are generally fabricated on glass, silicon, or
plastic substrates. The microarrays may have from a hundred to many thousands
of test sites that can range in size from 10 to 500 microns. High-density mi-
croarrays may have up to 106 test sites in a 1–2-cm2 area. DNA probes are se-
lectively spotted or addressed to individual test sites by a variety of techniques.
Probes can include synthetic oligonucleotides, amplicons, or larger DNA/RNA
fragments. The DNA probes can be either covalently or noncovalently attached to
support material. Depending on the array format, probes can be the target DNA
or RNA sequences to which other “reporter probes” would subsequently be hy-
bridized. The actual construction of microarrays involves the immobilization or
in situ synthesis of DNA probes onto the specific test sites of the solid support
or substrate material. High-density DNA arrays can be fabricated using physical
delivery techniques (e.g., inkjet or microjet deposition technology) that allow the
dispensing and spotting of nano/picoliter volumes onto the specific test site lo-
cations on the microarray. In some cases, the probes or oligonucleotides on the
microarrays are synthesized in situ using a photolithographic process. Microarray
devices with lower densities of test sites have been developed that provide direct
electronic detection of the hybridization reactions. Active electronic microarray
devices that provide electronic addressing or spotting of probes as well as rapid
high-performance hybridization analysis are now also available for research and
diagnostic applications.
The successful implementation of microarray technologies has required the de-
velopment of many methods and techniques for fabricating the microarrays and
spotting the probes, for carrying out and detecting the hybridization reactions, and
informatics for analyzing the data. DNA hybridization analysis on microarrays usu-
ally involves detecting the signal generated by the binding of a reporter probe (fluor-
escent, chemiluminescent, colorimetric, radioisotope, etc.) to the target DNA se-
quence. The microarray is scanned or imaged to obtain the complete hybridization
DNA MICROARRAY TECHNOLOGY AND APPLICATIONS 131
pattern. Fluorescence scanning/imaging or mass spectroscopy are two of the more

common methods used for the “reading” of the microarrays. For high-density type
microarrays, a variety of bioinformatic tools have been used to reduce the com-
plex data into useful information. The automation of DNA microarray systems
greatly facilitates their use and ease of operation and helps to eliminate many of
the human errors that would be involved in manually carrying out the multiplex
hybridization analyses. The development of microarray technology has depended
on the integration of many different disciplines such as molecular biology, gene-
tics, advanced microfabrication and micromachining technologies, nucleic acid
chemistry, surface chemistry, analytical chemistry, software, and robotics and auto-
mation. Microarray technology represents a truly successful synergy of these many

different scientific and engineering fields. The following sections include a com-
posite of recent general reviews and comments on microarray technologies and

their applications; an overview of the important microarraying technologies, with
selected examples of microarray technologies that have utilized techniques from
the microelectronics industry; and a final overview of the numerous applications
of DNA microarrays in research and diagnostics.
REVIEWS ON MICROARRAY TECHNOLOGY
An early history and overview of the microarray field has been provided by Edwin
M. Southern (1). This review begins with the first simple DNA arrays, which were
called “dot blots,” and progresses through the development of inkjet and light-
directed fabrication of high-density microarrays. Ekins & Chu have provided an-
other review on the origins of microarrays and their applications (2). In 1998, Mark
Schena provided two reviews that describe using gene expression microarrays as
a discovery platform for functional genomics (3, 4). Robert Service provides an
interesting overview of microarray applications for gene expression and its po-
tential for revolutionizing drug discovery and diagnostics (5). Other early reviews
include state-of-the-art development of DNA chips (6) and use of microarrays for
gene expression applications (7, 8). Early in 1999, a series of reviews appeared in
Nature Genetics that included discussions of microarray fabrication and detection
techniques (9), the use of microarrays for gene expression analysis (10, 11), the
use of oligonucleotide arrays for resequencing and mutation analysis (12), and
the potential for microarray technology in drug discovery and pharmacogenomic
applications (13). The issue also contained a review on important molecular interac-
tions that can take place on microarrays (14). Thompson & Furtado provide another
review on the use of high-density oligonucleotide arrays for DNA analysis (15).
Further reviews include expression profiling in cancer (16), different techniques for
expression profiling on DNA microarrays (17), and the development of automated
microarray systems for mutation analysis (18). Freeman and coworkers provide a
review on carrying out gene expression analysis on single-cell samples (19).
Because the development and applications of DNA microarray technology be-
gan to expand during the late 1990s, a large number of reviews appear in the year
132 HELLER
2000. Wang provides an encompassing survey and summary of the area (20).
In addition to DNA microarrays, his review includes background on the closely
related areas of DNA sensors and lab-on-a-chip technology. Meldrum provides
another encompassing review on the automation of genomics (21). In addition
to a good overview of microarrays and future technologies, this review also con-
tains information on the advancement of important DNA sequencing technologies
and their role in the genome project. The sections on microarraying and future
technologies provide a compilation of the many companies that are now involved
in DNA microarrays and related areas. A further series of reviews give good cov-
erage of the many gene expression techniques and applications being carried out
(22–24). Freeman and coworkers provide another review on the fundamentals of

using microarrays for gene expression (25). A series of general reviews on microar-
ray technology and applications have been prepared by Bednar (26), Burke (27),
and Jain (28). Stewart provides a review on making and using microarrays derived
from a short course given at the Cold Spring Harbor Laboratory (29). Other, more
specific, reviews include the use of DNA microarrays and combinatorial chemical
libraries for drug discovery applications (30), the use of microchips as a specific
genetic tool in psychiatry (31), and the impact of functional genomics and micro-
array technology on the modern pathology laboratory (32). Zanders (33) and Jain
(34) review gene expression analysis as an aid to the identification of drug targets
and for applications in pharmacogenomics. Ivanov et al. (35) provide a review
of DNA microarray technology for antimicrobial drug discovery applications.
Cunningham (36) provides a good overview of the new genomics and proteomics
and its importance to drug discovery and development.
More recent general reviews on microarrays for gene expression include those
by Hughes & Shoemaker (37) and Deyholos & Galbraith (38) on high-density mi-
croarrays for gene expression analysis and by Nelson (39) on microarrays maturing
as a gene expression tool. More specialized reviews on microarrays and gene ex-
pression include the use of microarrays for studying the aging process in mice (40)
and genome-wide expression analysis for plant cell–modulated genes (41). A num-
ber of important reviews have appeared related to the use of microarrays for cancer
research and diagnostics. These include more general reviews by Kallioniemi (42)
on biochips in cancer research and Polyak & Riggins (43) on serial analysis of
gene expression techniques and the implications for cancer research. More specific
reviews include those on microarrays for breast cancer research (44), gene expres-
sion analysis for maturation and cell death in acute promyelocytic leukemia (45),
microarrays in pediatric cancer research (46), and using microarrays for identify-
ing prostate cancer markers (47). In the area of microarrays for microbiological
applications, several general reviews are available (48, 49). More specific reviews
are available on DNA microarray analyses of host-pathogen interactions (50) and
the use of microarrays for the molecular diagnosis of mycobacteria (51). More gen-
eralized reviews in the medical-related areas include microarrays for disease gene
discovery (52), microarrays in medicine (53), microarrays for molecular pathology
(54), and a review by Eyster & Lindahl on DNA microarrays and their application
to clinical medicine (55). Special reviews have been prepared on using microarrays
to study human alcoholism (56) and the potential for using microarrays for envi-
ronmental health applications (57). Some of the most recent reviews and overviews
include Kricka (58) on the potential of microarrays to be the personal laboratories
of the twenty-first century, Blohm & Guiseppi-Elie (59) on new developments
in microarray technology, Davenport (60) on data standards for microarrays, and
Becker (61) on issues related to sharing of cDNA microarray data.
A large number of companies have been involved in the development of DNA
microarrays and the associated systems and applications. Table 1 gives a compi-
lation of some the main companies involved in DNA microarray/DNA chip areas.
Meldrum provides a good overview of many of these companies and their products
in the sections on microarraying (21). Further descriptions of some of the compa-
nies are provided in his “future technologies” section. This section gives a good
review of those systems that utilize mass spectrometry for detection and analysis.
The section on new biochips reviews several of the more exotic approaches such
as micron-scale bead arrays, electronic DNA chips, and several of the so-called
TABLE 1 Producers of microarrays, Biochips, and lab-on-a-chip devices
Company Description of device or system References
ACLARA Bio Sciences, LabCard—device uses electric fields to move 64, 65

Inc. (Mountain View, CA) fluids through capillaries on chips for the
miniaturization and integration of complex,
multistep biochemistry processes.
Affymetrix, Inc. GeneChip—high-density arrays produced by 66, 67, 68,
(Santa Clara, CA) photolithographic process for gene expression 69, 70, 71
and genotyping.
Caliper Technologies Corp. LabChip—microfluidic devices with active fluid 72
(Mountain View, CA) control and parallel processing for variety of
genome analysis procedures including molecular
purification and high-speed DNA separations.
Clinical Micro Sensors Low-density array with electrochemical sensing
(Pasadena, CA) for point-of-care diagnostic applications.
Gamera Bioscience Corp. LabCD System—“lab-on-a-disc” microfluidic
(Medford, MA) platform uses a modified CD player and
disposable compact disc to perform a variety of
microscale analytical processes.
Gene Machines Omni Grid is a high-performance, multi-axis
(San Carlos, CA) robot for arraying samples onto glass slides
and membranes.
Genetix Ltd. Q Array is a dedicated microarrayer with 16-pin
(Christchurch, UK) microarraying head for spotting slides.
(Continued )
134 HELLER
TABLE 1 (Continued)
Company Description of device or system References
Gene Trace Systems, Inc. High-throughput, automated mass spectrometry 73, 74, 75
(Alameda, CA) systems with liquid-phase expression
technology for gene discovery, gene expression
analysis, genotyping, and mutation scanning.
Hewlett Packard Company A thermal jet spotting system to produce cDNA
(Palo Alto, CA) microarrays.
Hyseq, Inc. Hyseq HyChip—a universal sequencing chip 76, 77
(Sunnyvale, CA) based on sequencing-by-hybridization (SBH)

technology.
Illumina, Inc. BeadArray—fiber-optic self-assembled 78

(San Diego, CA) addressable arrays are filled with optically
encoded libraries of 3–5-µm diameter
beads to simultaneously process millions
of assays.
Incyte Microarray GEM or gene expression microarrays.
Systems (Fremont, CA)
Intelligent Bio-Instruments High-throughput microarraying system that can
(Cambridge, MA) dispense from 12 to 64 spots onto multiple
glass slides.
Nanogen, Inc. Molecular Biology Workstation and NanoChip— 79, 80, 81,
(San Diego, CA) an active programmable electronic matrix chip 82, 83
that has addressable locations for genotypying
applications.
Orchid BioSciences, Inc. Developing microfluidic glass chips to enable 84
(Princeton, NJ) high-throughput chemical synthesis, genomics,
DNA analysis, screening, and diagnostics.
Packard Instrument BioChip Arrayer with four PiezoTip piezoelectric
Company (Meriden, CT) drop-on-demand tips provides noncontact
dispensing for arraying onto glass, filters,
and HydroGel substrates.
Rosetta Inpharmatics, Inc. FlexJet inkjet-based microarrays available only 85
(Kirkland, WA) through scientific collaborations.
Sequnome High-throughput resequencing array that uses a 86
(San Diego, CA) MALDI-TOF mass spectrometer for subsequent
detection and identification of DNA fragments,
used for SNP and genotyping.
TeleChem International, ChipMaker 2 and ChipMaker 3 microspotting
Inc. (Sunnyvale, CA) devices with noncontact, inkjet-type dispensers.
Vysis, Inc. GenoSensor—a genomic array system for
(Downers Grove, IL) addressing gene copy number.
lab-on-a-chip technologies. Wang also provides another encompassing review that

includes good sections on DNA biosensors, DNA microarrays, and lab-on-a-chip
technologies (20). Additionally, several new monographs on DNA microarray tech-
nologies are now available. Mark Schena has edited a volume titled DNA Micro-
arrays: A Practical Approach (85), and Jang B. Rampal has edited another volume
titled DNA Arrays: Methods and Protocols (86). A short review on some recent
patents in the microarray area has appeared in Nature Biotechnology (87). Although
microarray patent coverage is not the intent of this chapter, it is strongly recom-
mended that anyone who plans to do active research and development in the micro-
array area carry out a good patent search. This is particularly important before
investing time and money in development work. Considerable research and de-
velopment in the microarray area is now carried out by the biotechnology
industry, which will generally patent the technology before publishing the work.
MICROARRAY DEVICES AND SYSTEMS

In the past several years, many different microarray technologies, devices, and
instrument systems have been developed and are now commercially available for
producing DNA microarrays. These microarrays and systems are being used for
gene expression, genotyping, and other applications. A number of different meth-
ods have been developed for creating microarrays, including various techniques for
using them (88). Many microarray spotting technologies and techniques now exist.
Two of the more important spotting techniques used are the pin-based fluid trans-
fer systems (3, 4, 89–93) and the piezo-based inkjet dispenser systems (94–96).
Other methods for preparing DNA arrays include the use of photolithography for
the in situ synthesis of high-density DNA microarrays, developed by Affymetrix,
as well as the electronic-based addressing of microarrays developed by Nanogen.
Both of these methods are discussed below. For further reviews and discussions
on other methods for preparing microarrays and the associated techniques and
technologies, see References (24, 97–99).
High-Density Microarrays
High-density DNA arrays manufactured by Affymetrix provide a massively par-
allel approach to DNA hybridization analysis that is having a significant impact
on genomic research and diagnostics. In the early 1990s, Steve Fodor and col-
leagues at Affymetrix began to develop photolithographic techniques (used by
computer chip manufacturers) to carry out the parallel synthesis of large numbers
of oligonucleotides on solid surfaces (64, 65). This light-directed synthesis has
enabled the large-scale manufacture of arrays containing hundreds of thousands
of oligonucleotide probe sequences on glass slides, or “chips,” less than 2 cm2 in
size (100). This method is now used by Affymetrix to produce the high-density
GeneChip® probe arrays, which are finding use for the detection and analysis of
point mutations and SNPs and for gene expression studies (66–69).
136 HELLER
The Affymetrix in situ process combines DNA synthesis chemistry with pho-
tolithography techniques from the microelectronic industry. In this process, 50 -
terminal protecting groups are selectively removed from growing oligonucleotide
chains in predefined regions of a glass substrate by controlled exposure of light
through photolithographic masks (64, 65, 100). The glass substrate, or chip, is first
covalently modified with a silane reagent to provide hydroxyalkyl groups, which
serve as the initial synthesis sites. These sites are then extended with linker groups
protected with special photolabile protecting groups. When specific regions of
the surface are exposed to light (through masks), the protecting groups are selec-
tively removed, allowing the sites to now be coupled with the next appropriate
nucleoside phosphoramidite monomer. The monomers, which are also protected

at their 50 position with a photolabile group, are coupled to the substrate using
standard phosphoramidite DNA synthesis protocols. The current methodology

employs nucleoside monomers protected with a photo-removable 50 -(O-methyl-
6-nitropiperonyloxycarbonyl) (MeNPOC) group (64, 65, 100). The cycles of pho-
todeprotection and nucleotide addition are repeated to build any given array of
oligonucleotide sequences. The average stepwise efficiency of oligonucleotide
synthesis is approximately 90%–95%. This value is the result of the average yield
of the photochemical deprotection step after exhaustive photolysis (102). Many
of the semi-automated manufacturing techniques and lithography tools used in
the GeneChip® array production process were adapted from the microelectronics
industry.
The Affymetrix method for highly parallel synthesis of oligonucleotide (probe)
sequences provides an excellent process for making high-density microarrays.
With this process, a complete set, or subset, of all probe sequences of length n
requires 4n synthesis steps. By way of example, a complete array of all possi-
ble 10-mer probes (1,048,576 sequences) would require only 40 process cycles.
Currently, arrays made using photolithographic synthesis have individual probe
features 24 × 24 microns on a 1.6-cm2 size chip. The technology will ultimately al-
low arrays to be fabricated with densities >106 sequences/cm2, which corresponds
to a feature size of less than 102 microns. Typical arrays comprise customized sets
of probes that may range from 14 to 25 bases in length. Factors such as high probe
density (∼120 pmol/cm2), special hybridization stringency conditions, and the
use of comparative-intensity algorithms for data analysis allow accurate sequence
information to be “read” from these arrays with single-base resolution (68).
Although other photolabile protecting groups and processes have been de-
scribed that may be applicable for light-directed DNA array synthesis, they have not
been used extensively (103–105). McGall and coworkers at Affymetrix continue to
achieve higher photolysis rates, synthesis yields, and spatial resolution, and have
also developed photolithographic methods for fabricating DNA arrays that utilize
polymeric photoresist films as the photoimageable component (100, 106, 107). One
such polymer film contains a chemically amplified photoacid generator, which
creates localized acid development adjacent to the substrate surface when ex-
posed to light. This localized acid results in direct removal of 4,40 -dimethoxytrityl
(DMT) protecting groups from the oligonucleotide chains. This high-resolution

photoresist-based process may enable the production of oligonucleotide arrays
with features on the order of five microns in size.
McGall & Fidanza also describe other methods (100) for the synthesis of pho-
tolabile MeNPOC nucleoside phosphoramidite building blocks for standard 30 –50
as well as 50 –30 photolithographic synthesis, and for using “modified” 20 -0-methyl
and 2,6-diaminopurine nucleosides. These methods can be used to synthesize
arrays with improved hybridization affinities for A/T-rich probe sequences. Sev-
eral nonstandard probe chemistries may be used to impart specific properties to
an array. For example, the “reverse” (50 –30 ) synthesis to the support at the 50 end
leaves the 30 end available for later enzymatic extension or ligation reactions (108).
These “reverse” arrays can be synthesized using 30 -MeNPOC 50 -phosphoramidate

monomers and have characteristics nearly equivalent to the standard 30 –50 arrays.
McGall & Fidanza (100) outline the photolithographic synthesis method, general
protocols for determining photochemical deprotection rates and yields for oligonu-
cleotide synthesis based on surface fluorescence, and procedures based on hybrid-
ization for comparing the array performance characteristics of new chemistries
and protocols.
Oligonucleotide arrays with many thousands of probe sequences will have a
fairly broad range of hybridization thermodynamics. This can produce a corre-
sponding range of signal intensities when they are hybridized with labeled target
sequences (100). A/T-rich probe sequences frequently display lower hybridiza-
tion intensities than do more stable sequences with high G/C content. This is a
particular concern when very stringent hybridization conditions are required to
break up secondary structure in certain RNA targets or to increase the discrimi-
nation of arrays when used for the analysis of high-complexity mRNA samples
(66, 109). The use of TMAC1 has been explored as a means of improving hy-
bridization to A/T-rich probe sequences, but has limited use due to its toxicity
and corrosiveness (110). Nevertheless, the sensitivity of A/T-rich probe sequences
in arrays can be improved by the introduction of certain nucleotide analogs that
improve the overall duplex stability. The replacement of deoxyadenosine (dA)
with analogs such as 20 -deoxy-2,6-diaminopurine (dD) has been suggested as a
means of enhancing the stability of A-rich sequences (111–115); however, the sta-
bilizing effect is not entirely uniform for all sequences. The use of dD in 10-mer
arrays was found to have only a small impact on hybridization of a complemen-
tary oligonucleotide, 50 -fluorescein-rCTGAACGGTA-30 . However, it was found to
stabilize duplexes in RNA targets and probes with poly-dA tracts (100, 114, 115).
Good overall improvement in RNA hybridization intensities is observed when
both the deoxyadenosine (dA) and Thymidine (T) in the probe sequences are
replaced with 20 -O-methyl-2,6-diaminopurine (20 -OMe-D) and the 20 -O-methyl-
5-methyluridine (20 -OMe-5-Me-U) analogs, respectively. This is primarily due to
their stabilizing effect on duplex formation with complementary RNA target se-
quences (116). 20 -0-alkyl-modified oligonucleotides that were used as antisense
therapeutic agents have more recently been used to improve probes for RNA
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138 HELLER
detection (117). McGall & Fidanza (100, p. 97) have shown that the substitution
of dA and T in 10-mer probe arrays with the 20 -OMe-D and 20 -OMe-5-Me-U
analogs can increase the relative hybridization intensity two- to threefold. Im-
proved hybridization sensitivity for A/T-rich RNA targets has been shown on
human immunodeficiency virus (HIV) PRT 440 resequencing GeneChip® probe
arrays with 20-mer dD and 20 -OMe-5-Me-U substituted probe array. In these ex-
periments, the analogs increased the “base-call” accuracy of the array from 88.6%
to 98.6% (100). In this work, sample preparation, labeling, and the microarray
hybridizations were carried out according to published protocols (67, 118). The
use of these nucleotide analogs has also led to a reduction in the length of the
probes (12- to 14-mers) that are needed to obtain performance characteristics pre-
viously obtained using arrays with longer (>20-mer) probes. The value of using
such improved high-density GeneChip® probe arrays has been demonstrated for
analyzing drug resistance traits in HIV (119). Finally, the further improvement of
hybridization performance of short probe arrays is also needed for the develop-
ment of generic mutation detection microarrays based on using sets of all n-mer
sequences (67).
Microelectronic Array Technology

Microelectronic chip/array devices developed by Nanogen again combine many
aspects of technology from the microelectronics industry to uniquely improve
molecular biology techniques such as DNA hybridization. Microelectronic arrays
provide high-performance hybridization that overcomes many of the problems
of passive array hybridization techniques. These active microelectronic array de-
vices have the ability to produce reconfigurable electric fields (more specifically
electrophoretic fields) on the microarray surface that allow rapid and controlled
transport of charged DNA/RNA molecules to any test site (77–80). These mi-
croelectronic array devices are able to increase the DNA hybridization rate by
concentration of probe/target DNA at the test site. Reversing the electric field on
the test site produces an electronic stringency effect, which can greatly improve
hybridization specificity and ability to discriminate point mutation and SNPs. In
addition to DNA and RNA molecules, these devices have the ability to selectively
transport any charged entity, which can include proteins (antibodies, enzymes,
etc.), cells, nanoparticles, and semiconductor microstructures.
The manufacture of microelectronic array devices utilizes a variety of micro-
fabrication, photolithography, and other microelectronic technologies. Microelec-
tronic arrays have been designed and fabricated with 25, 100, 400, and 10,000
test-sites or microlocations. The 100-test-site chip, which has been commercial-
ized by Nanogen, has 80-micron diameter test sites/microlocations with under-
lying platinum microelectrodes, and 20 auxiliary outer microelectrodes
(Figure 1A). The outer group of microelectrodes provides encompassing elec-
tric fields for concentrating DNA from the bulk sample solution to specific test-
sites/microlocations. The 100-test-site DNA chip is less than 1 cm in size, with the
active test-site array area being approximately 2 mm in size. The microelectronic
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arrays or chips are fabricated on silicon wafers, having a base structure of sili-
con with an insulating layer of silicon dioxide. The microelectrode structures are
platinum with connecting gold wires. Silicon dioxide or silicon nitride is used to
cover and insulate the conducting wires, but not the surface of the platinum micro-
electrode structures. Important to the device function is the permeation layer that
overcoats the underlying microelectrodes (77–81). The permeation layer, com-
posed of a porous hydrogel material (agarose or polyacrylamide), allows the array
to be run at current levels (>100 nanoamperes) and voltages (>1.2 volts). This
layer serves to protect the sensitive DNA hybridization reactions from the adverse
electrochemical oxidation and reduction effects that occur on the actual micro-
electrode (platinum) surface during active operation. The permeation layer also
ameliorates the adverse effects of electrolysis products (H+, OH−, O2, H2, free
radicals, etc.) on the sensitive hybridization and affinity reactions. The perme-
ation layer is impregnated with a coupling agent (streptavidin, chemical agent,
etc.) that allows for the subsequent attachment of DNA probes. The permeation
layer and its proper manufacture are important to the performance of the device
(78–81). The ability to use silicon and microlithography for fabrication of the
DNA chips allows a wide variety of devices to be designed and tested. Higher-
density arrays with 400 and 10,000 test-sites have been developed (Figure 1B,C).
These higher-density devices have on-chip CMOS control elements that can reg-
ulate the currents and voltages to each of the test sites on the array (120). The
semiconductor control elements are located in the underlying silicon structure
and are not exposed to the aqueous samples that are applied to the chip surface.
To facilitate both the rapid electrophoretic transport of DNA molecules and their
efficient hybridization, special zwitterionic buffer species with low conductivity
properties have been utilized. Histidine has been found to be particularly effec-
tive for electronic hybridization (79–81). The 100-test-site microelectronic chip
or array device is incorporated into a cartridge package (NanoChipTM cartridge)
that provides for the electronic, optical, and fluidic interfacing. A complete in-
strument system (NanoChipTM Molecular Biology Workstation) provides a chip
loader component, fluorescent detection/reader component, and computer control
interface and data display screen component. The probe-loading component al-
lows oligonucleotide probes or target molecules (DNA, RNA, PCR amplicons,
etc.) to be selectively addressed to the array test-sites, providing the end-user
with “make your own chip” capabilities (80, 81). When the electric field is ad-
justed to the appropriate level, it can be used to affect the selective dehybridization
of the hybridized DNA sequences from the attached complementary probe. This
novel parameter is called electronic stringency and it provides a powerful and
rapid method for single-base mismatch discrimination analysis of point mutations
and SNPs (77–81, 121). Electronic hybridization allows most genotyping appli-
cations, which can include point mutations, SNPs, and STRs, to be carried out
rapidly with high accuracy and reliability. Target DNA can be basically any size,
with sequences up to 1000 nucleotides having been genotyped. Gilles and other
workers have demonstrated the advantages of electronic stringency for genotyping
human mannose binding protein (MBP) SNPs (121). In this work, the electronic
140 HELLER
array-based genotyping results for 22 blinded MBP quadra-allelic samples, and

13 blinded D allele samples were in 100% agreement with conventional DNA
sequencing results. In another type of assay called base stacking, the captured
target DNA is first hybridized with a stabilized or helper probe and then with a
set of fluorescent reporter probes (122). STRs represent another important class of
polymorphisms with applications in forensics, human identification, and diagnos-
tics. The typical STR loci are selected groups of four nucleotide repeats that are
represented in the human population by 4 to 15 alleles (123, 124). Unlike SNPs,
STRs are more difficult to analyze by hybridization techniques and are generally
not done by hybridization technologies that rely on passive techniques (125, 126).
However, electronic hybridization techniques have been proven to overcome these

problems and allow STR analysis to be reliably performed on microelectronic ar-
rays (122). In addition to SNP and STR genotyping applications already described,
other applications include gene expression analysis, on-chip or in-situ DNA am-
plification (127–129), immunoassays and protein-protein interactions (130, 131),
sample-to-answer systems, electronic cell separations, viral and bacterial identifi-
cation, biowarfare/bioterrorism agent detection devices (132–134), use of electric
fields for particle manipulations, semiconductor device pick, and place and for
potential micro/nanofabrication applications (77, 135–137).
New Microarray Technologies, Chemistries, and

Detection Methods
A variety of additional technologies are being developed for making microarrays
and techniques for improving their performance. Considerable efforts are being
carried out related to better methods for preparing oligonucleotides and other types
of DNA probes (also discussed in the section on high density microarrays), and new
procedures are being created for their attachment or immobilization onto support
materials. There are also new advancements relating to the detection of hybridiza-
tion reactions that occur on the surface of the arrays. LeProust and coworkers have
reported a new digitally light-directed synthesis method that permits microar-
ray platforms to be designed that can carry out rapid reaction optimization on a
combinatorial basis (138). Galbraith and coworkers have described techniques for
printing DNA microarrays using the Biomek 2000 laboratory automation worksta-
tion (139), Stillman & Tonkinson have described new “FAST” slides that provide
novel surface characteristics for preparing microarrays (140), and Thompson and
coworkers have described techniques that allow researchers to build their own
microarrayer from readily available materials and components (141). With regard
to methods for deposition of DNA and oligonucleotides, Hicks and coworkers
describe methods for the modification of an automated liquid-handling system
for reagent-jet nanoliter-level dispensing (142), Okamoto and coworkers describe
methods for the fabrication microarrays with covalent attachment of DNA using
bubble jet technology (143), and Hughes and coworkers describe methods for
carrying out expression profiling using microarrays fabricated by an ink-jet oligonu-
cleotide synthesizer (144). Regarding quality control issues, Diehl and coworkers
discuss methods for manufacturing DNA microarrays with high spot homogeneity
and reduced background signal (145).
With regard to hybridization techniques on microarrays, Mir & Southern pro-
vide guidance on how to determine the influence of DNA structure on hybridiza-
tion using oligonucleotide arrays (146). Maldonado-Rodriguez & Beattie have
described new methods for the analysis of nucleic acids using a tandem hybridiza-
tion technique on oligonucleotide microarrays (147). In other work on hybridiza-
tion methodology, Belosludtsev and coworkers describe how near-instantaneous
cation-independent nucleic acid hybridization can be carried out on DNA microar-
rays (148). Additionally, considerable effort has gone into methods and techniques
related to immobilization and attachment of probes and oligonucleotides onto

various support or substrate materials. Zammatteo and coworkers have carried
out comparison studies between different strategies for the covalent attachment of
DNA to glass surfaces in order to improve DNA microarray performance (149), and
Lindroos and coworkers have carried out comparison studies of immobilization
chemistries for minisequencing applications on oligonucleotide microarrays (150).
Work on polyacrylamide gel-immobilized microarrays for nucleic acids and pro-
teins analysis has been carried out by Zlatanova & Mirzabekov (151). Belosludtsev
and coworkers have developed DNA microarrays based on noncovalent oligonu-
cleotide attachment for hybridization in two dimensions (152). Procedures for the
fabrication of DNA microarrays using unmodified oligonucleotide probes have
been developed by Call and coworkers (153). Special techniques for the character-
ization of oligodeoxyribonucleotide synthesis on glass plates have been developed
by LeProust and coworkers (154). Zhang and coworkers have developed repro-
ducible and inexpensive procedures for the preparation of oligonucleotide probe
arrays (155). Vernon and coworkers have demonstrated reproducibility for alterna-
tive probe synthesis approaches for gene expression profiling with arrays (156), and
Kane and coworkers have developed assessment techniques for determining the
sensitivity and specificity of 50-mer oligonucleotide probes on microarrays (157).
Detection is another important area in which improvements are being made and
new technologies are being developed. Because it is so widely used for microarray
analysis, considerable efforts are always being carried out on fluorescent detection.
Dixon & Damaskinos have reported on improvements in confocal scanning devices
for microarrays (158). New high-resolution near-infrared imaging techniques for
DNA microarrays with time-resolved acquisition of fluorescence lifetimes have
been reported by Waddell and coworkers (159). Marti and coworkers have reported
on new developments in quantitative fluorescence calibration for analyzing cells
and microarrays (160). Battaglia and coworkers have developed techniques for
using SYBR green for nondestructive fluorescent staining on microarrays (161).
This technique may prove useful as it eliminates the need for using labeled probes,
which adds expense to the overall cost of hybridization assays. The use of surface
plasmon resonance imaging for DNA and RNA hybridization adsorption on DNA
microarrays has been carried out by Nelson and coworkers (162). This technique
may allow the microarray detection system to be reduced in overall size. van De
Rijke and coworkers have developed methods for using up-converting phosphor
142 HELLER
reporters for nucleic acid microarray analysis (163). This technology has the poten-
tial to provide more detection sensitivity. Another novel detection technique that
may improve sensitivity by utilizing dendrimer technology has been described by
Stears and coworkers (164). Numerous advancements in detection technology have
also been made using nonfluorescent techniques. Whitney and coworkers describe
using radioactive 33-P probes to carry out hybridization to glass cDNA microar-
rays for DNA samples from neural tissue (165). The detection of mitochondrial
single nucleotide polymorphisms (SNPs) using a primer elongation reaction on
oligonucleotide microarrays has been reported by Erdogan and coworkers (166).
In the area of direct hybridization sensing, Umek and coworkers report on using
electronic detection of nucleic acids for molecular diagnostics (167). In the area of
mass-spectrometry-based detection, Stomakhin and coworkers report on DNA se-
quence analysis by hybridization using MALDI mass spectrometry identification

of 5-mers contiguously stacked to microchip oligonucleotides (168).
MICROARRAY INFORMATICS AND APPLICATIONS
In general, DNA microarray hybridization applications are usually directed at

gene expression analysis or for point mutation/SNP analysis. In addition to these
important molecular biological and genomic research applications, microarray
systems are also used for pharmacogenomic research, for infectious and genetic
disease and cancer diagnostics, and for forensic and genetic identification purposes.
Additionally, microarray technologies are now used for many proteomic and cell-
based applications. These devices and systems that carry out various multiplex
analyses in highly parallel formats produce enormous amounts of data. This is
particularly true in the case of the high-density microarrays. Thus, considerable
efforts have gone into data acquisition and bioinformatic tools that are able to
reduce the complex data into useful information.
With regard to the issue of managing large amounts of information, Rao &
Bond have provided an appropriate review titled Microarrays: Managing the Data
Deluge (169). Troyanskaya provides information on missing value estimation
methods for DNA microarrays (170), and Baldi & Long discuss a Bayesian frame-
work for the analysis of microarray expression data and for regularized t-test and
managing statistical inferences of gene changes (171). Reviews are provided on the
analysis of variance in gene expression microarray data (172) and methods for iden-
tification of novel small RNAs using comparative genomics on microarrays (173).
Ideker and coworkers have discussed testing methods for differentially expressed
genes by maximum-likelihood analysis of microarray data (174), and Kerr &
Churchill discuss statistical designs for gene expression analysis of microarray
data (175). Further discussions on bioinformatic methods for gene expression
analysis are provided by Newton and coworkers (176), Lee and coworkers (177),
and Scherf and coworkers (178).
An enormous number of publications have now appeared on the uses of micro-
arrays for gene expression, point mutations, SNPs, and for pharmacogenomic and
diagnostic applications. The following is a compilation of microarray publications

on some of the broad areas of application: general applications of microarrays
for gene expression analysis (179–181); gene expression analysis on high-density
microarrays (182, 183); gene expression analysis for cancer (184–191); gene ex-
pression analysis for drug discovery, metabolism, and toxicity (192, 193); gene
expression analysis for neuroscience applications (194–196); gene expression
analysis for microbiological and infectious disease (197–200); use of micro-
arrays for genotyping (201–204); general articles on use of microarrays for can-
cer (205, 206); infectious disease diagnostics (207–209); use of microarrays for
disease diagnostics (210–212); and applications of microarrays for plant biology
(213–215). In addition to microarrays for DNA hybridization analysis, consider-

able efforts are now being directed at the development and use of microarrays for
proteomic applications (216–220) and microarrays for the immobilization of cell

and tissue samples (221, 222).
SUMMARY
The development of DNA microarray, biochip, and lab-on-a-chip technologies

continues at a very rapid pace. These new technologies represent a highly inter-
disciplinary and synergist effort, with contributions from a range of scientific and
engineering fields that have previously not worked together as closely. This in-
cludes molecular biologists, geneticists, chemists, physicists, mathematicians, mi-
crofabrication scientists, computer scientists, and various types of engineers. The
miniaturization of this technology has been achieved by using many of the micro-
fabrication processes that have revolutionized the microelectronics and computer
industry. Microarray technologies are rapidly advancing with numerous applica-
tions in gene expression, genotyping, and pharmacogenomics. The technology is
now also making an impact in the area of medical diagnostics, in particular, for
cancer, genetic, and infectious disease applications. As the technology continues
to advance, it will most certainly lead to the development of a new generation of
“point of care” diagnostic devices and systems. Finally, DNA microarray technol-
ogy is also forming the basis for a new generation of devices for proteomic and
cell biology applications.
The Annual Review of Biomedical Engineering is online at

http://bioeng.annualreviews.org
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3 Jul 2002
10:48
AR
AR164-07-COLOR.tex
AR164-07-COLOR.SGM
Figure 1 (A) An active microelectronic DNA array device with 100 test-sites or microlocations. The test-sites are approximately
80 microns in diameter, with an underlying platinum electrode. A ring of another twenty microelectrodes that can be used as counter electrodes
surrounds the test-sites. (B) Wafer containing 400 test-site active microelectronic array devices. These devices contain underlying CMOS
LaTeX2e(2002/01/18)
control elements for on-chip current and voltage control and regulation. (C ) View of an individual 10,000 test-site active microelectronic
array device. These devices contain underlying CMOS control elements for on-chip current and voltage control and regulation.
P1: GDL
P1: FRK
June 20, 2002 10:12 Annual Reviews AR164-FM
Annual Review of Biomedical Engineering

Volume 4, 2002
CONTENTS
Frontispiece—Kenneth R. Foster xii
HERMAN P. SCHWAN: A SCIENTIST AND PIONEER IN BIOMEDICAL
ENGINEERING, Kenneth R. Foster 1

ROLES FOR LEARNING SCIENCES AND LEARNING TECHNOLOGIES IN

BIOMEDICAL ENGINEERING EDUCATION: A REVIEW OF RECENT
ADVANCES, Thomas R. Harris, John D. Bransford, and Sean P. Brophy 29
SPINE ERGONOMICS, Malcolm H. Pope, Kheng Lim Goh,
and Marianne L. Magnusson 49
THREE-DIMENSIONAL CONFOCAL MICROSCOPY OF THE LIVING
HUMAN EYE, Barry R. Masters and Matthias Böhnke 69
BIOENGINEERING OF THERAPEUTIC AEROSOLS, David A. Edwards
and Craig Dunbar 93
DENATURATION OF COLLAGEN VIA HEATING: AN IRREVERSIBLE
RATE PROCESS, N.T. Wright and J.D. Humphrey 109
DNA MICROARRAY TECHNOLOGY: DEVICES, SYSTEMS, AND
APPLICATIONS, Michael J. Heller 129
PEPTIDE AGGREGATION IN NEURODEGENERATIVE DISEASE,
Regina M. Murphy 155
MECHANO-ELECTROCHEMICAL PROPERTIES OF ARTICULAR
CARTILAGE: THEIR INHOMOGENEITIES AND ANISOTROPIES,
Van C. Mow and X. Edward Guo 175
ELECTROMAGNETIC FIELDS: HUMAN SAFETY ISSUES, Om P. Gandhi 211
ADVANCES IN IN VIVO BIOLUMINESCENCE IMAGING OF GENE
EXPRESSION, Christopher H. Contag and Michael H. Bachmann 235
PHYSICS AND APPLICATIONS OF MICROFLUIDICS IN BIOLOGY,
David J. Beebe, Glennys A. Mensing, and Glenn M. Walker 261
TELEREHABILITATION RESEARCH: EMERGING OPPORTUNITIES,
Jack M. Winters 287
BIOMECHANICAL DYNAMICS OF THE HEART WITH MRI, Leon Axel 321
ADVANCES IN PROTEOMIC TECHNOLOGIES, Martin L. Yarmush
and Arul Jayaraman 349
v
P1: FRK
June 20, 2002 10:12 Annual Reviews AR164-FM
vi CONTENTS
ON THE METRICS AND EULER-LAGRANGE EQUATIONS OF

COMPUTATIONAL ANATOMY, Michael I. Miller, Alain Trouvé,
and Laurent Younes 375
SELECTIVE ELECTRICAL INTERFACES WITH THE NERVOUS SYSTEM,
Wim L.C. Rutten 407
INDEXES
Subject Index 453
Cumulative Index of Contributing Authors, Volumes 1–4 475
Cumulative Index of Chapter Titles, Volumes 1–4 477
ERRATA
An online log of corrections to Annual Review of Biomedical

Engineering chapters (if any, 1997 to the present) may be found at
http://bioeng.annualreviews.org/

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Annu. Rev. Biomed. Eng. 2002. 4:129–53

DNA MICROARRAY TECHNOLOGY: Devices,

tification purposes. Microarray technology continues to improve in performance

DNA MICROARRAY TECHNOLOGY AND APPLICATIONS 131

pattern. Fluorescence scanning/imaging or mass spectroscopy are two of the more

mation. Microarray technology represents a truly successful synergy of these many

posite of recent general reviews and comments on microarray technologies and

REVIEWS ON MICROARRAY TECHNOLOGY

(22–24). Freeman and coworkers provide another review on the fundamentals of

DNA MICROARRAY TECHNOLOGY AND APPLICATIONS 133

TABLE 1 Producers of microarrays, Biochips, and lab-on-a-chip devices

Company Description of device or system References

ACLARA Bio Sciences, LabCard—device uses electric fields to move 64, 65

Company Description of device or system References

(Sunnyvale, CA) based on sequencing-by-hybridization (SBH)

Illumina, Inc. BeadArray—fiber-optic self-assembled 78

DNA MICROARRAY TECHNOLOGY AND APPLICATIONS 135

lab-on-a-chip technologies. Wang also provides another encompassing review that

MICROARRAY DEVICES AND SYSTEMS

nucleoside phosphoramidite monomer. The monomers, which are also protected

standard phosphoramidite DNA synthesis protocols. The current methodology

DNA MICROARRAY TECHNOLOGY AND APPLICATIONS 137

(DMT) protecting groups from the oligonucleotide chains. This high-resolution

These “reverse” arrays can be synthesized using 30 -MeNPOC 50 -phosphoramidate

Microelectronic Array Technology

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array-based genotyping results for 22 blinded MBP quadra-allelic samples, and

However, electronic hybridization techniques have been proven to overcome these

New Microarray Technologies, Chemistries, and

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related to immobilization and attachment of probes and oligonucleotides onto

quence analysis by hybridization using MALDI mass spectrometry identification

MICROARRAY INFORMATICS AND APPLICATIONS

In general, DNA microarray hybridization applications are usually directed at

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diagnostic applications. The following is a compilation of microarray publications

(213–215). In addition to microarrays for DNA hybridization analysis, consider-

proteomic applications (216–220) and microarrays for the immobilization of cell

The development of DNA microarray, biochip, and lab-on-a-chip technologies

The Annual Review of Biomedical Engineering is online at

Curr. Opin. Biotechnol. 9(6):609–14 microarray-based expression profiling.

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oligonucleotide arrays. Am. Chem. Soc. 1998. Advantages of 20 -O-methyl oligori-

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microarrays. Nat. Biotechnol. 21(Suppl): directed pick-and-place assembly. High

Nat. Biotechnol. (18):199–204 (36):8589–94

selectivity nucleic acid hybridization to (50mer) microarrays. Nucleic Acids Res.

Res. Commun. 282(5):1263–67 158. Dixon AE, Damaskinos S. 2001. Confocal

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168. Stomakhin AA, Vasiliskov V, Timofeev E, Sci. USA 97(18):9834–39

Moroni C. 2001. Global analysis of differ- gene discovery to target identification.

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technology. Mol. Carcinog. 30(2):79– 216. Moerman R, Frank J, Marijnissen JC,

207. Chizhikov V, Rasooly A, Chumakov K, 2001. Miniaturized electrospraying as a

Annual Review of Biomedical Engineering