Biomolecular Engineering 23 (2006) 213–231

www.elsevier.com/locate/geneanabioeng

Review
Merging microfluidics with microarray-based bioassays
Catherine Situma, Masahiko Hashimoto, Steven A. Soper *
Center for Bio-Modular Multi-Scale Systems, Department of Chemistry,
Louisiana State University, Baton Rouge, LA 70803, United States
Received 8 February 2006; received in revised form 10 March 2006; accepted 10 March 2006

Abstract
Microarray technologies provide powerful tools for biomedical researchers and medicine, since arrays can be configured to monitor the
presence of molecular signatures in a highly parallel fashion and can be configured to search either for nucleic acids (DNA microarrays) or proteins
(antibody-based microarrays) as well as different types of cells. Microfluidics on the other hand, provides the ability to analyze small volumes
(micro-, nano- or even pico-liters) of sample and minimize costly reagent consumption as well as automate sample preparation and reduce sample
processing time. The marriage of microarray technologies with the emerging field of microfluidics provides a number of advantages such as,
reduction in reagent cost, reductions in hybridization assay times, high-throughput sample processing, and integration and automation capabilities
of the front-end sample processing steps. However, this potential marriage is also fraught with some challenges as well, such as developing low-
cost manufacturing methods of the fluidic chips, providing good interfaces to the macro-world, minimizing non-specific analyte/wall interactions
due to the high surface-to-volume ratio associated with microfluidics, the development of materials that accommodate the optical readout phases of
the assay and complete integration of peripheral components (optical and electrical) to the microfluidic to produce autonomous systems
appropriate for point-of-care testing. In this review, we provide an overview and recent advances on the coupling of DNA, protein and cell
microarrays to microfluidics and discuss potential improvements required for the implementation of these technologies into biomedical and
clinical applications.
# 2006 Published by Elsevier B.V.

Keywords: Microfluidics; Microarrays; DNA diagnostics; Protein arrays; Cell arrays

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
1.1. What are microarrays? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
1.2. Microfluidics: basic fabrication and operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
1.3. Why merge microarray technology with microfluidics? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
2. Microfluidics and microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
2.1. Support materials for microarray production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
2.2. Probe array preparation techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
2.3. Hybridization speed enhancement in microfluidic devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
2.4. Applications of microfluidic-based microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
2.4.1. DNA microarray systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
2.4.2. Protein microarray systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
2.4.3. Cell-based microarray systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
3. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

* Corresponding author. Tel.: +1 225 578 1527; fax: +1 225 578 3458.
E-mail address: chsope@lsu.edu (S.A. Soper).

1389-0344/$ – see front matter # 2006 Published by Elsevier B.V.

doi:10.1016/j.bioeng.2006.03.002
214 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231
1. Introduction
The timely and correct expression of a large number of genes
and the products they produce, such as mRNAs and/or proteins,
is a critical component of normal development and main-
tenance of all living organisms. Any disruptions or changes in
genes and the products they express are responsible for many
diseases that possess a genetic basis. Biomedical and clinical
diagnosis/prognosis evolves and advances not only through the
compilation of knowledge accumulated by the discovery of new
biomarkers, but also through the development of new
technologies that can read the presence or absence of those
biomarkers as well as the amount of those markers found in a
clinical sample. Unfortunately, the traditional tools used to
assay gene expression, such as SAGE (serial analysis of gene
expression) requires steps that are not conducive to analyzing
large numbers of genes simultaneously, which is typically
required in many multi-gene related diseases, such as cancer.
Thus, the emergence of highly parallel processing techniques,
such as microarrays, has enabled researchers to study thousands
of genes simultaneously for both discovery and diagnostic
applications.
1.1. What are microarrays?
A microarray consists of a support onto which hundreds to
thousands of different molecular reporter probes are attached or
immobilized at fixed locations in either a two-dimensional or
three-dimensional format. The multiplexing capabilities of
microarray-based assays are produced by spatially encoding the
array, in which each location on the array is used as a reporter of
a specific analyte. Due to the similar nature in which each assay
at a location is carried out, highly reproducible and quantitative
information can be obtained from each address of the array. The
probes that can be used can consist of antibodies to recognize
specific proteins or oligonucleotides/PCR products that
recognize unique DNA sequences through Watson–Crick base
pairing.
DNA microarrays consist of oligonucleotides that are
attached onto supports and are used to interrogate the presence
or the amount of analyte (i.e., targets or DNAs) in sample
solutions. DNA microarray assays are based on the principle of
specific molecular recognition events that occur for nucleic acids
through interactions between complementary sequences. Selec-
tivity is imparted by targets and probes that are non-
complementary and thus do not form thermodynamically strong
hybrids, which can be selectively tuned out through stringent
washing with buffers or temperature regulation. Because
microarrays allow one to investigate large numbers of analytes
or targets rapidly and efficiently, researchers have applied this
technology not only in gene expression analysis (Zhang et al.,
1991), but also in drug screening/therapeutic studies (Lebrun,
2005), proteomics (MacBeath, 2002; MacBeath and Schreiber,
2000), cell analysis (McClain et al., 2003), and DNA sequencing/
fragment analysis (Pease et al., 1994).
Although microarray technology was initially fostered in
genomic applications, its basic operational concepts have been
applied in proteomics for the analysis and quantification of a
large number of proteins comprising a proteome. Microarray
applications in protein analyses includes screening binding
specificities of a protein expression library (Postier Bradley
et al., 2003), high-throughput antibody screening (Bussow
et al., 2000), or the detection of protein–protein interactions,
enzymatic targets and protein-small molecule interactions
(MacBeath and Schreiber, 2000).
Not only can DNAs and proteins be successfully printed
onto substrates in a microarray formats, but cells have also been
patterned onto substrates in a microarray format, which has
advanced the study of cellular functions and metabolism,
identification of the phenotype of a cell in a population of
heterogeneously transfected cells (Wu et al., 2002), the
detection of toxic agents and high-throughput screening of
combinatorial libraries/gene products (Kapur, 2005).
Many complex diseases and traits are caused by rare alleles,
which can only be detected by sequencing complete genomic
regions in multiple individuals first for the identification of the
variants and then for disease diagnostics. Progress in
microarray systems, which enable highly multiplexed genotyp-
ing, is therefore very useful for genome-wide single nucleotide
polymorphism (SNP) genotyping. There exists a database of
hundreds of SNPs/single base differences in the human
genome, which can be used as genetic markers. As such,
microarrays can be used in genotyping assays and also in
comparative genomic hybridization (CGH), a technique that
reveals how many copies of given genes are actually in a sample
being interrogated. Microarrays have been demonstrated
widely in SNP genotyping applications (Baner et al., 2003;
Erali et al., 2003; Grimm et al., 2004; Ishikawa et al., 2005; Lin
et al., 2004; Lovmar et al., 2003; Meaburn et al., 2006).
1.2. Microfluidics: basic fabrication and operation
Microfluidics is an emerging and relatively new technology
platform that typically requires a multi-disciplinary approach
for building effective systems through the interface of physics,
chemistry, engineering and biochemistry. These devices usually
consist of structures that are fabricated in the micrometer-scale
and can be designed to produce diagnostically useful systems
for potential point-of-care measurements. Systems based upon
microfluidic operation possess a unique set of potential
advantages, such as reduction in reagent cost, enhancement
of assay speed, potential for mass production of devices at low
cost and the ability to integrate several processing steps into a
single system (i.e., high levels of system automation).
Decreased reagent consumption eventually results in the
reduction of assay cost, which could translate into large-scale
molecular screening of certain diseases for early detection. The
accelerated analysis time is directly gained from the reduced
footprint of these devices. Other benefits of microfluidic-based
systems include high-throughput biological screening by
parallel processing of multiple samples achieved with multiple
fluidic paths and the minimization of cross contamination, since
different samples flow through separate fluidic processors in
closed architectures.
 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231
Microfluidic devices can be fabricated from a variety of
materials using different techniques. Based primarily on the
fabrication techniques developed in the microelectronics sector,
silicon and glass have traditionally been used to create
microfluidic devices (Li et al., 1999; McClain et al., 2003).
Microfabrication in glass/silicon substrates has been accom-
plished through the use of lithography, plasma etching or reactive
ion etching techniques (Boerner et al., 1996; He et al., 1999). The
particularly attractive advantages of glass and quartz include
their well defined surfaces and excellent optical properties,
which are highly desired for signal readout of microarrays by
fluorescence. However, the fabrication processes involved are
fairly time consuming, labor intensive and relatively expensive.
Polymers are rapidly evolving as alternative substrate
materials for many microfluidic applications due to their diverse
properties that can be selected to suite the particular application
need and the ability to microfabricate structures in a high
production mode and at low cost. The fabrication of the
prerequisite fluidic networks on polymer substrates can be
achieved by lithography, UV laser ablation, hot embossing,
injection molding, or direct micromilling techniques (Ford et al.,
1999; Friedrich et al., 1997; Grass et al., 2001; Ihlemann and
Rubahn, 2000; Martynova et al., 1997; Qi et al., 2002; Roberts
et al., 1997; Rossier et al., 1999; Soper et al., 2000). Hot
embossing and/or injection molding involve the use of mold
masters containing the desired microstructures to stamp (reverse-
duplicate) patterns into the required substrate. The patterns
poised on the mold masters can be made either by micro-
machining or by a lithography-based approach, such as LiGA;
German acronym for lithography (Lithograhie), electroplating
(Electroformung) and molding (Abformung). A schematic of the
LiGA processing steps using X-ray lithography to build the
microstructures on the mold master is shown in Fig. 1. The
important aspect of this technology is that the difficult fabrication
steps, lithography and electroplating, are performed but once and
then, parts are micro-replicated from this mold master using
either injection molding or hot embossing. For hot embossing,
three steps are required: (1) First, the polymer substrates are
placed between two metal plates, one containing the micro-
structure topology, and are heated above their glass transition
temperature. (2) A force is applied on the softened polymer by
pressing with the heated mold master under vacuum. (3) The
metal plates and polymer substrate are cooled below the
polymer’s glass transition temperature and the metal plates are
withdrawn from the polymer part. Due to the simplicity of the
process and the fact that the mold master only requires one
lithography step to fabricate it, the procedure is very cost
effective in that it can mass produce thousands of parts and the
whole production process can be completed in <5 min.
Examples of polymer parts embossed from a mold master are
depicted in Fig. 1. In addition, the process is conducive to
replicating parts in a variety of materials from the same mold
master as long as the glass transition temperature of the substrate
is below the melting point of the master. Once the fluidic network
is formed, a cover plate of the same material can be sealed to the
substrate to enclose the fluidic channels using a low-temperature
sealing process.
215
Using UV laser ablation or micromilling, a polymer
substrate can be exposed to laser radiation or a milling bit
resulting in the direct writing of the microstructures into the
polymer part. In the case of laser ablation, the laser photons,
typically from a pulsed excimer laser, is focused directly on the
polymer. Fluidic channels are produced either by moving the
polymer substrate or by moving a focused laser beam across the
surface. This method offers the ability to form complex features
with various geometries, even in the third dimension, because
the patterning beam can be moved both horizontally and
vertically on the substrates. In either case, fluidic prototypes
can be rapidly produced using direct write techniques to
optimize the device performance prior to mass production using
micro-replication techniques.
Poly(dimethylsiloxane) (PDMS) fabrication technology,
sometimes called soft lithography, was developed by White-
sides and co-workers and has been widely adopted for the
fabrication of different microfluidic networks (Anderson et al.,
2000a; Duffy et al., 1998, 1999; McDonald and Whitesides,
2002; Sia and Whitesides, 2003). Fabrication of microfluidic
structures using PDMS technology involves pouring a solution
containing the PDMS prepolymer and a curing agent over a
relief (mold) containing the prerequisite microstructures
followed by curing to cross-link the polymer. This technology
first requires creation of positive reliefs using a variety of
techniques (Duffy et al., 1998; Becker and Gartner, 2000; Love
et al., 2001), such as wet etching of silicon or glass following
photolithography, micromachining metals or reactive ion
etching of silicon. A solution containing the pre-polymer
and curing agent is then cast against the relief and the cross-
linked polymer conforms to the shape of the relief. After
casting, the polymer is simply removed from the relief resulting
in a replica that contains the network of microfluidic channels.
Finally, the PDMS replicate can be plasma oxidized and sealed
to other surfaces by conformal contact to enclose the fluidic
network. Plasma oxidation also renders the PDMS channels
hydrophilic so that they can easily be filled with aqueous
solutions. Besides plasma oxidation, Quake and coworkers has
also described a method for sealing fluidic networks created in
PDMS, which involves the use of two slabs (Unger et al., 2000);
one slab consists of an excess of base while the other contains
the curing agent. These two are then brought into conformal
contact followed by curing.
1.3. Why merge microarray technology with microfluidics?
For conventional microarrays, several shortcomings exist
including: (1) The hybridization (analyte binding) process on
supports requires long incubation times to produce the optimal
signal because of slow diffusional kinetics as the target
molecules must travel to the arrayed probes situated on a
surface. This process is typically diffusion-controlled and the
fact that reactions on the surface are slow due to local target
depletion near the probe environment. (2) The consumption of
large amounts of precious sample material for interrogating the
array due to the large area occupied by high density arrays
(1 cm2). In order to completely cover the array, the volume of
216 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231

Fig. 1. Processing steps required to prepare a mold master using X-ray LiGA and molding finished parts. The process starts with a lithography step using a mask that
transfers the desired pattern into a photoresist. Following development of the resist, the underlying metal layer is exposed, which serves as a plating base for another
metal that fills the voids left by the resist that was removed due to exposure to the patterning radiation. The deposited metal forms the desired microstructures. The
unexposed resist is then removed, forming the finished mold master. This mold master is then available for micro-replicating parts in polymers or other materials using
hot-embossing or injection molding. Shown on the right in this figure is a picture of the mold master and some parts that have been prepared from the mold master.
Adopted with permission from Ford et al. (1999).

sample analyzed must be such that the fluid covers every
element of the array. In addition, the large lateral distances that
must be traveled by low concentration species for high density
arrays in order to find their appropriate binding partners can add
to the incubation time, slowing down assay processing rate. (3)
The linkage chemistry of the probe to the support can undergo
solution-dependent cleavage over the extended incubation
times, lowering the reproducibility and sensitivity of the assay.
For example, siloxane-based chemistry is typically used to
tether molecules to glass, and this is susceptible to hydrolytic
 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231
cleavage, especially at high or low pH values. Therefore, any
process that can enhance the rate of producing the maximum
signal for surface-confined association events can provide a
number of important benefits, such as reducing the develop-
ment time and its associated consequences.
The use of microfluidic platforms can directly address many
of the issues cited above for microarrays and thus, the merging
of microfluidics with microarrays can provide some unique
opportunities that are not obtainable when arrays are configured
into conventional planar 2D formats. For example, microfluidic
addressing of the array can accelerate hybridization speeds due
to the enhanced mass transport of target molecules and reduced
diffusional distances required of targets when delivered in a
micro-scale flow-through hybridization chamber. In addition,
microfluidics can offer the potential for parallel processing of
multiple samples. Other advantages offered by the incorpora-
tion of microfluidics into the microarray includes reduced
reagent and sample volumes and the integration capabilities of
sample pre-processing prior to incubation with the array, which
can provide high levels of system automation and also, provide
closed architectures for sample processing minimizing con-
tamination effects that can give rise to false positives, which can
be a significant issue for diagnostic applications.
Since microfluidics is a relatively new field of research, there
are likely to be challenges when attempting to merge it to a
relatively mature technology platform, such as microarrays. For
instance, there is a need for the development or discovery of
new materials that are easily fabricated on the size-scale
demanded by the microfluidic application and at the same time,
offer modification chemistries that allow for the stable
attachment of probes. In addition, the fabrication technologies
as well as the material used for the device must be low cost if
intended for diagnostic screening using microarray-based
assays.
Even though miniaturized devices offer the advantages
associated with the small scale reactors and fluidic vias used in
their implementation, the high surface area to volume ratio
associated with these small scales produce surface effects that
dominate volume effects. As such, there is the potential for
increased surface adsorption of solution molecules that can lead
to high-levels of non-specific adsorption, providing poor
microarray spot definition or even fast positive results. Methods
to ameliorate these non-specific adsorption artifacts must be
employed, which can consist of selecting the appropriate
substrate for the microfluidic that demonstrates minimal non-
specific adsorption for the intended application or to use
dynamic or covalent coating procedures of the fluidic substrate
(Gong et al., 2005; Henry et al., 2000; Hu et al., 2004; Lee et al.,
2003; Lu et al., 2005; McCarley et al., 2005; Nuzzo, 2003;
Sebra et al., 2005).
While microfluidics can potentially provide the use of
reduced sample and reagent volumes due to the small size of
these elements, the detection of molecules in dilute solutions
will require highly sensitive detection devices, maybe even at
the single molecule level. This is so because the number of
molecules sampled scales as the cube of the volume analyzed
and hence, with low concentration samples the number of
217
molecules interrogated is low for microfluidic-based devices,
which typically have interrogation volumes that are <1 nL. In
addition, the substrate selected for the microfluidic must
accommodate the sensitivity requirements for readout. For
example, while polymers are attractive due to their low cost and
high production capabilities using micro-replication methods,
many polymers display poor optical properties that are not
conducive to the readout modality required for the array.
Judicious selection of the polymer must be undertaken, since
several materials do display excellent properties for optical
readout, especially when using visible or near-IR fluorescence
(Shadpour et al., 2006). In order to realize cost-savings in terms
of reagent consumption using microchips, precious reagents
can be distributed to multi-channel chips by using low-volume
fluidic distribution networks (Fu et al., 2002; Liu et al., 2003a).
Another issue that should be considered is that while the
footprint of microfluidic chips have been substantially reduced,
these devices must be interfaced to the macro-world. In most
cases, microfluidic chip loading, both with reagents and
samples, is accomplished using conventional pipettes with fluid
movement actuated either electrokinetically or hydrodynami-
cally. In addition, readout of molecular association events must
be accomplished using techniques such as laser-induced
fluorescence. In most cases, these operational processes are
carried out using off-chip apparati, such as mechanical pumps,
high voltage power supplies and optical readers, similar to
those used for reading conventional 2D microarrays. While
efforts have been initiated to package these components into a
complete and autonomous system with a footprint similar to
that of the fluidic elements, these efforts are still in their
infancy. In this review, we will focus primarily on miniaturiza-
tion efforts focused on the fluidic elements. For information on
packaging and integration of ancillary components to the fluidic
elements, the reader is referred to the following references
(Auroux et al., 2002; Burns et al., 1998, 1996; Liu et al., 2004a;
Pal et al., 2005; Reyes et al., 2002).
This review will detail the current research that involves
coupling microarrays to microfluidics with emphasis on DNA
arrays, protein arrays and cell-based arrays with concluding
remarks on potential improvements required for future
developments that will allow the benefits of microfluidics/
microarray devices to be fully utilized for applications in
diagnostics and the understanding of biological function for
discovery-based projects. Information will be presented on the
different types of substrates that can be used for microarrays
that are also conducive to microfluidic fabrication and
operation as well as the attachment chemistries that can be
employed for probe tethering to the surface. Novel topogra-
phical architectures for microarrays configured into micro-
fluidics will also be presented.
2. Microfluidics and microarrays
2.1. Support materials for microarray production
Microarrays require the use of substrates with well-
established surface modification/probe attachment chemistries
218 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231

Fig. 2. Various modification strategies that can be used to attach probes to different solid supports. (A) Glutardialdehyde linkage of amine-containing
oligonucleotides to glass surfaces. (B) Covalent immobilization of oligonucleotide probes to PC. (C) UV activation of PMMA with the subsequent attachment
of primary amine containing oligonucleotides. Adopted with permission from Joos et al. (1997), Lenigk et al. (2002a) and Situma et al. (2005).
 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231
Fig. 2. (Continued ).
that provide high probe densities on the surface of the substrate,
maximize probe accessibility by its binding partner, provide
robust attachment chemistries between the probe and substrate
that are stable and easy to implement, and minimize non-
specific interactions. Glass and quartz have been widely used as
typical support materials since their physical/chemical proper-
ties are well investigated including, surface modification
chemistries (Halliwell and Cass, 2001; Joos et al., 1997).
Glass is particularly well-established in the literature as a
substrate material for microarray applications due to its
superior optical properties and the presence of silanol groups
that can be easily functionalized using cross linking agents,
such as glutardialdehyde or an amino-silane reagent, to allow
stable attachment of DNA or protein probes directly to the glass
using siloxane-based chemistries (see Fig. 2A). In addition, the
functional group density can be relatively high, with levels
approaching 10 9 mol cm 2 (Horvath and Dolnik, 2006).
An example of microarrays fabricated on silicon is the work
reported by Ressine, who used a pore chip protein array (PCPA)
based on the use of micro-porous silicon as the matrix for
immobilizing antibodies (Ressine et al., 2003). The capture
antibodies were anchored onto the surface by physical
adsorption. The probes were micro-dispensed onto the chip
219
surface forming an array of spots with high affinity for target
analytes. The micro-porous silicon was fabricated by means of
anodic dissolution in hydrofluoric acid producing rigid sponge-
like structures with high uniformity and mechanical stability as
well as high surface area due to the three-dimensional network
formed following anodic dissolution. The microfluidic proper-
ties of these substrates were then utilized for arraying of
antibodies that were able to capture antigen binding partners
with detection levels of 70 pM.
Recently, polymers have generated attention from research-
ers as potential supports for the production of microarrays
because of their low cost and also, they offer a diverse range of
chemical structures to allow the implementation of different
immobilization strategies of probes to the support. Polymers
that have been used for microarray applications include;
poly(dimethylsiloxane) (PDMS), polycarbonate (PC), poly(-
methyl methacrylate) (PMMA), polystyrene, poly(ethylene
terephthalate) (PET) and cellulose acetate. PDMS has been
widely used because it is inexpensive, and has high optical
transparency. Other substrates that have been reported in the
literature as substrates for microarrays include gold (Csaki
et al., 2001; Steel et al., 2000), filter membranes (Liu et al.,
2004b), and optical fibers (Ahn and Walt, 2005).
220 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231

The use of microbeads as supports for the immobilization of
probes has also been reported (Marquette and Blum, 2004; Sato
et al., 2001). Bead arrays have been used extensively because
they are inexpensive, efficient, require mild operating condi-
tions and allow multiplexed assays when each bead is used to
capture a specific analyte. However, the challenges that occur in
these multiplexed formats hinge on the ability to decode the
specific identity of the probe on any particular microbead.
Thus, bead-based assays have focused predominately on their
use in low density array applications. Microbeads do, however,
have the advantage of enhanced surface area compared to a
planar support, which increases the binding capacity of the
probe and thus, increasing the dynamic range of the assay. In
work reported by Marquette et al., a method of arraying beads
bearing active enzymes, antibodies and oligonucleotides was
presented (Roberts et al., 1997). A picture of the bead-based
microarray system is shown in Fig. 3. The beads were spotted
and dried on a poly(vinylchloride) (PVC) master and
subsequently transferred to a PDMS interface by direct
molding of the polymer on the mask (Marquette and Blum,
2004). This technique enabled the development of low density
arrays that were read out using chemiluminescence detection.
Fan et al. have also described a dynamic DNA hybridization
protocol that incorporated paramagnetic beads as a transpor-
table support (Fan et al., 1999). Target molecules were
immobilized onto beads through streptavidin–biotin conjuga-
tion for interrogation with DNA probes that were transported
via pneumatic pumping. This dynamic DNA hybridization
offered flexibility such that the type/amount of beads, DNA
targets or probes could be changed as needed. Beads conjugated
with DNA probes have also been placed in a capillary tube
producing linear probe arrays (Kohara et al., 2002). Most
recently, Lilliehorn et al. have demonstrated a method of
generating arrays in microfluidic devices based on ultrasonic
trapping of microbeads using acoustic forces in standing waves
(Lilliehorn et al., 2005). They produced protein arrays for
simultaneous analysis of different sets of bead-based biotin–
avidin samples. The antibody/antigen interactions were
performed on chemically activated microbead clusters in a
laminar streaming microfluidic system. This involved the use of
different antigen-specific bead clusters trapped in an array
pattern followed by addressing each bead cluster with
individual sample streams using acoustic forces. A model
bioassay using fluorescein-labeled avidin was bound to biotin-
coated beads using this technique, which showed its potential
for parallel protein analysis in microfluidic devices.
Microbeads have also been used in conjunction with
quantum dots for readout. For example, the ‘‘Q bead
technology’’ uses multi-colored microbeads labeled with
quantum dots for specific biological assays. This technology

Fig. 3. Low density bead arrays fabricated in PDMS. The microbeads were made from Sepharose and were spotted onto a polyvinylchloride (PVC) master. (A) The
Sepharose beads could be derivatized with a variety of different probes, such as nucleic acids, antibodies or glucose oxidase (Gox). The readout of an association event
was performed using a chemiluminescent reaction with horseradish peroxidase (HRP) that was strapped to the target via a biotin/streptavidin linkage. (B) The beads
containing the appropriate probes were spotted onto the PVC master, which was used to form the fluidic network with the entrapped beads. A PDMS solution is poured
over the PVC master and following drying, it is peeled from the PVC master. The bottom image is generated by a chemiluminescence response following
hybridization of a poly d(T)22 oligonucleotide containing HRP. Adopted with permission from Marquette and Blum (2004).
 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231
involves the insertion of a combination of various quantum dots
into an array of beads with each bead serving as a recognition
substrate for specific molecules. Since each bead can be
individually prepared with different probes, there is an obvious
advantage of flexibility. An example of the use of quantum dot
encoded micro-sphere hybridization assays have been described
by Xu et al., who applied this technology for multiplexed SNP
genotyping (Xu et al., 2003). Several companies have also
developed special encoding techniques for bead-based hybridi-
zation assays, such as Illumina (http://www.illumina.com) and
Becton Dickinson (http://www.bdbiosciences.com).
2.2. Probe array preparation techniques
DNA microarrays, which utilize short (<25 bp) oligonu-
cleotide probes, can either be synthesized directly on the
substrate or pre-synthesized separately and then micro-printed
or spotted onto the support in an orderly or fixed manner,
typically in a two-dimensional format. Identification of the
presence of a particular DNA sequence is transduced by a
recognition signal produced through an association event
between the surface-tethered probe and its solution comple-
ment and locating the location of the signal in the two-
dimensional array, which allows for highly parallel processing
of multiple targets. High quality probe arrays are usually
desired to improve the sensitivity of the microarray assay. The
probe array quality depends intimately on the probe attachment
chemistry and deposition (spotting) technique used. Any non-
uniform surface morphology that might arise during surface
activation or spotting can lead to variances in the array element
shape and probe surface concentration uniformity across the
particular element, which eventually affects the data quality.
The availability of these surface attached probes to interact with
their complementary targets during hybridization is also highly
desirable. The surface probe density as well as its spacing from
the surface will determine the extent to which the immobilized
probes are able to capture their targets from solution because,
other than thermodynamic conditions, the immobilized probes
can also be kinetically or sterically inaccessible (Peterson et al.,
2001).
Direct on-chip probe synthesis is achieved by combining
solid-phase chemistry, photolabile protecting groups and
photolithography (Pease et al., 1994; Fodor et al., 1991;
Southern et al., 1994). An example of this is the Affymetrix
microarray technology, whose Genechips are produced by
photolithography (Kim, 2003). These on-chip probe synthesis
protocols allow for the manufacture of high density micro-
arrays. Moorcroft et al. have recently described an in situ
synthesis technique of oligonucleotide probes onto PDMS
surfaces, which is a common substrate frequently used in
microfluidic applications, using phosphoramidite chemistry
(Moorcroft et al., 2005). The PDMS surface was activated by
UV/ozone exposure followed by vapor phase deposition of
glycidoxypropyl trimethloxysilane, which was then reacted
with polyethylene glycol spacers that aided in oligonucleotide
coupling and the minimization of non-specific adsorption of
targets to the PDMS surface.
221
Pre-synthesized probes can be covalently or physically
arrayed onto a variety of chip surfaces using several different
chemistries (Gingeras et al., 1987; Okamoto et al., 2000).
Examples of several of these chemistries can be seen in Fig. 2.
Lenigk et al. (2002a) demonstrated the use of PC substrates
coated with bi-functional linker molecules for the immobiliza-
tion of pre-synthesized amine-terminated oligonucleotide
probes (see Fig. 2B). Probe immobilization procedures were
also studied on four different polymers (polystyrene, PC,
PMMA and polypropylene) as possible support candidates for
microarrays with three different immobilization procedures
(Surmodics, cetyltrimethylammonium bromide (CTAB) and
Reacti-bind procedures). These authors showed that different
polymers exhibited different immobilization efficiencies
depending on the immobilization procedure used (Liu and
Rauch, 2003). Soper and coworkers described a functionaliza-
tion protocol that yielded amine-terminated functionalities on
PMMA (Henry et al., 2000). This protocol was later used for the
covalent attachment of probe oligonucleotides for microarray
hybridization assays (Hashimoto et al., 2006; Waddell et al.,
2000; Wang et al., 2003). Recently, this same group has
described a simple UV photo-modification protocol for PMMA
and PC substrates that generate surface-confined carboxylate
groups, which can be subsequently used for the immobilization
of amine-terminated nucleic acid probes through an amide-
bond as the linking chemistry (Situma et al., 2005). The photo-
modification process for array fabrication involved only three
steps (see Fig. 2C): (1) broadband UV exposure of the polymer
surface; (2) carbodiimide coupling of amine-terminated
oligonucleotide probes to the surface (via an amide bond);
(3) washing of the surface. Other probe attachment techniques
on PMMA surfaces have been presented by Fixe et al.
(2004a,b). They developed a chemical procedure to functio-
nalize PMMA by reaction of the substrate with hexamethylene
diamine to yield an aminated surface for immobilizing DNA
probes, which was accomplished using a glutardialdehyde
cross-linking agent for the attachment of amino-modified
oligonucleotide probes via a Schiff base.
Probe arrays can also be generated by use of PDMS-assisted
techniques, which is based on the properties of PDMS to seal
both reversibly and irreversibly with other substrates. Lee et al.
constructed 1D and 2D DNA microarrays on gold films through
the use of parallel microfluidic channels fabricated in PDMS
(Lee et al., 2001). A PDMS polymer sheet containing parallel
microchannels was physically attached to a gold film modified
with self-assembled monolayers. Single stranded DNA
(ssDNA) probes were directed through these channels with
each channel containing a unique probe and allowed to
immobilize onto the gold surface after which the PDMS was
removed from the gold surface. These ssDNA probes were used
for the interrogation of complementary targets, which were
directed onto the probe array surface by using a second set of
PDMS fluidic channels placed perpendicular to the probe
arrays. This stenciling technique has been adopted for
patterning protein antigens on gold surfaces (Kanda et al.,
2004) and DNA probe arrays onto PMMA surfaces (Situma
et al., 2005).
222 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231
Su et al. demonstrated the use of microfluidic networks
fabricated in PDMS to prepare peptide microarrays on gold by
using self-assembled monolayers (SAMs) of alkanethiols (Su
et al., 2005). Microchannels fabricated in PDMS were used to
immobilize a linear array of cysteine-terminated peptides onto
the SAMs containing maleimide groups. The PDMS stencil was
then reapplied to the SAM in a perpendicular direction to
introduce enzyme solutions so that each solution could interact
with an identical linear array of immobilized peptides. The set
of peptides used enabled a multi-analyte assay selective for
kinase and phosphatase enzymes. Another example is that
demonstrated by Tani et al., who used PDMS microchannels
connected via perforated microwells to a silicon substrate for
the immobilization of Escherichia coli sensor strains (Tani
et al., 2004). The sensor strains were mixed with agarose and
then injected into the channels and immobilized by gelation.
Probe arrays have also been constructed in 3D ordered
microchannels (Benoit et al., 2001; Cheek et al., 2001). These
uniform porous substrates enhance probe immobilization
because the effective surface area available for probe
immobilization is increased compared to that of planar 2D
surfaces. They also enable uniform spot shapes to be achieved
since the evaporation of probe solutions are greatly reduced
during the immobilization process. Probes used in these flow-
through geometries eventually increase the sensitivity and
reduce hybridization assay analysis times as well. Hydrogel
microstructures can also provide an ideal 3D aqueous
microenvironment for the capture and encapsulation of
biomolecules. Koh et al. fabricated arrays of hydrogel
microstructures of different cell phenotypes using microfluidics
made from PDMS (Koh et al., 2003). They used three different
phenotypes of cells; fibroblasts, hepatocytes and macrophages,
for the fabrication of multi-phenotype cell microarrays. This
group was able to uniformly distribute and control the number
of cells in one hydrogel microstructure by changing the cell
density in the precursor conditions. This reduced the sample
volume of a cell containing precursor solutions when compared
to conventional cell patterning techniques, which involve the
use of spin coating procedures.
2.3. Hybridization speed enhancement in microfluidic
devices
In all microarray applications, whether they are DNA or
protein-based arrays, the target is typically found in solution
and must diffuse to the surface to interact with its probe, since
the probe is chemically tethered to the surface and thus,
immobile. In addition, it must move laterally to find the location
of its appropriate binding partner in 2D gridded arrays. As such,
in many cases, several hours of incubation time are required to
allow probe saturation to be obtained due to the slow mass
transfer of target to its appropriate probe. For example, with a
1 cm2 array area and a low target concentration in the solution,
depletion of target near the probe recognition element of the
two-dimensional array will require new target to diffuse
laterally to ‘‘find’’ its appropriate probe. As a reference, the
translational diffusion coefficient for a single-stranded DNA
molecule of 30 bases is approximately 2.4  10 7 cm2 s 1 at
room temperature and in free solution. Therefore, for a
molecule to travel laterally 1 cm across the array would require
approximately 289 h!
Several techniques have been employed to enhance mass
transport of analytes and reduce diffusional distances using
shallow microchannels (<100 mm) to reduce the hybridization
incubation time. One such technique is the use of continuous
flow of targets within microchannels by hydrodynamic
pumping (Hashimoto et al., 2006; Wang et al., 2003; Cheek
et al., 2001; Noerholm et al., 2004). Forced flow of the target
solution over the probe array surface provides enhanced mass
transport of targets to the surface-immobilized probes since the
transport does not depend solely on diffusion, but also on
convection leading to reduced hybridization times. In addition,
the use of shallow fluidic channels reduces the diffusional
distances that must be traveled to reach the probe. Hydro-
dynamic flow has been shown to reduce the hybridization time
to less than 1 min (Wang et al., 2003).
Introduction of high sample velocities can induce exten-
sional strain to solution targets reducing hybridization time and
efficiency as well (Chung et al., 2004, 2003; Vanderhoeven
et al., 2004). Chung et al. demonstrated that long DNA targets
tend to change into a super coil form in solution that reduces
hybridization efficiencies due to the inaccessibility of the
recognition sequence in the target. Therefore, extensional strain
can help to uncoil the long DNA molecules improving
hybridization efficiency (Chung et al., 2004). The authors
were able to show a nearly nine-fold increase in hybridization
signal due to strain-induced extension of a 1.4 kbp single-
stranded DNA target by introducing a taper within the fluidic
channel. Vanderhoeven et al. also developed a shear-driven flow
system that induced enhanced lateral transport rates in
hybridization chambers increasing hybridization efficiencies
as well (Vanderhoeven et al., 2004).
Another approach for reducing hybridization time is by
using active mixing of samples. While the use of devices with
small dimensions reduces the target diffusional lengths, the
fluidic flow within these devices are uniaxial and laminar due to
their low Reynolds numbers. Mixing in such flows is due
primarily to molecular diffusion (Yaralioglu et al., 2004). The
hybridization rate can be improved by active mixing because it
can replenish the target concentration in the vicinity of each
probe due to depletion created through binding. Active mixing
also relieves the dependence of only lateral diffusion to
replenish target near its probe.
Mixing-enhanced hybridization can be described as a three-
step process: (1) transport (diffusion and convective flow) of
targets in the solution to the diffusion (stagnant) boundary
layer; (2) transport (primarily diffusion) of target within the
diffusion boundary layer to the probes on the chip surface; (3)
reaction of target with probes on the surface (Liu et al., 2003b).
Conventional microstreaming has the ability to provide rapid
lateral mass transport and enhanced vertical mass transport of
the target DNA in solution. This combination results in rapid
transport of targets in solution to the diffusion boundary layer,
which allows a continuous replenishment of DNA targets
 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231 223

around probes depleted of complementary targets. This
phenomenon was later used to perform DNA hybridizations
with electrochemical detection on a fully integrated biochip,
which consisted of fluidic components that were used to
enhance target cell capture and accelerate hybridization
reactions (Liu et al., 2004a).
Yaralioglu et al. demonstrated the use of ultrasonic
transducers for mixing by an acoustic streaming mechanism
(Yaralioglu et al., 2004). They showed that when reagents were
localized within discrete droplets in a microchannel with a low
Reynolds number, they could be mixed quickly by re-circulating
flows caused by the shearing interaction of the fluid in the plug
with the walls of the microchannel.
Lenigk et al. demonstrated the use of an integrated cartridge,
which was comprised of fluidic channel networks and a
micropump for the oscillation of the hybridization mixture
(Lenigk et al., 2002b). Accelerated hybridization times were
realized in these channels due to the ability to pass a large
number of target molecules per unit time over a probe spot
during filling of the channel. This model was then used for the
simultaneous detection of four pathogenic bacteria surrogate
strains in a parallel format.

Fig. 4. (A) Schematic of an array of electrodes fabricated on Si. The 25 electrodes, made from Pt, are 80 mm in diameter and cover an area of 1 cm2. The insulating
layer consists of silicon nitride layer and over the Pt electrodes was a gel permeation layer consisting of agarose gel loaded with streptavidin. The bottom panel shows
a cross-section of one element of the array with the immobilized DNA probes. The probes are attached to the permeation layer via a biotin–streptavidin association.
(B) Electric field enhanced transport of a poly d(T)12 target to the Pt electrode surface with different bias voltages applied to the Pt electrode: C1: positive potential;
C2: negative potential; C3: no potential. R3 and R5 correspond to biotinylated and non-biotinylated probes, respectively. Adopted with permission from Sosnowski
et al. (1997).
 224
Table 1
Abbreviated list of literature reports highlighting the coupling of DNA microarrays to microfluidics
Reference Sample Support Analysis speed Detection Functions Applications/achievements
volume material (min) limits integrated
Lilliehorn et al. (2005) 1 ml Glass n/a n/a Ultrasonic microtransducers A model bioassay of a multiplexed bead-trapping
microarray consisting of piezoelectric microtranducers
integrated in microfluidic channels
Lenigk et al. (2002a) 25 ml PC 30 n/a Integrated air pump for (i) Accelerated hybridization process by sample oscillation;
sample oscillation (ii) application in single nucleotide polymorphism detection,
detection of pathogen bacteria surrogate
Wei et al. (2005) 1 ml PMMA 8 19 amol n/a Enhanced microarray processing speed in
microfluidics by ‘‘shuttle hybridization’’

C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231

Anderson et al. (2000b) 15 ml PC 20 n/a Multiple enzymatic reactions Parallel multi-step molecular operations and hybridization
and reagent handling assays integrated on a miniaturized device
Dimalanta et al. (2004) ml PDMS n/a 25 pg/ml DNA elongation, deposition Restriction enzyme mapping and identification
and digestion of human genomic DNA molecules
13
Ali et al. (2003) 1.5 ml Silicon <10 10 M Fluid transport (i) Chip-based sensor array incorporated DNA
capture probes into microporous microbeads;
(ii) multiplexed oligonucleotide microarray assays
Wang et al. (2003) 0.2 ml PMMA 5 1 mutant in 10,000 n/a Detection of point mutations in certain gene fragments
wild type copies using universal zipcode arrays
Hashimoto et al. (2006) 0.1 ml PMMA/PC <20 1 mutant in 100 Ligase detection reaction/ Flow throughput biochip for mutational analysis
wild type copies hybridization assay
Liu et al. (2003b) 1 ml PC 60 n/a Fully integrated for cell capture/ Pathogenic bacterial DNA detection/single nucleotide
cell preconcentration/purification/ polymorphism genetic analysis
cell lysis and PCR amplification/
DNA hybridization
Erickson et al. (2004) nl Glass/PDMS 5 50 pM Synchronized online DNA (i) Reduced sample volumes; (ii) rapid removal
hybridization, washing of nonspecifically adsorbed materials;
and detection (iii) enhanced hybridization
Benoit et al. (2001) 0.5 ml Glass 180 40 pM n/a Multiplexed DNA detection
Lee et al. (2001) 1 ml Gold/PDMS 60 20 fmol n/a Parallel detection of specific adsorptions of DNA /RNA.
Cheek et al. (2001) 50 ml Glass 180 250 amol n/a Flow-through chemiluminescence detection of multiple
enzymes in a microarray format
Dimalanta et al. (2004) 10 ml PMMA/glass/ 120 n/a n/a (i) Hybrid multiplexed detection of campylobacter DNA;
PDMS (ii) effective mechanical protection and reduced
sample evaporation
Noerholm et al. (2004) 10 ml PC 300 10 nM Temperature control Online monitoring of DNA hybridization assays
and liquid handling
Yuen et al. (2003) 50 ml PDMS/glass 120 1.25 pg/ml n/a Enhanced hybridizations by two- to five-fold through
sample fluid circulation and mixing
Chung et al. (2004) n/a PMMA/glass 30 n/a n/a Enhanced hybridization efficiencies via strain rate
n/a: not available.
 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231 225

Immunoassays based on solid supported lipid bilayers for multivalent

Bynum and Gordon used a microfluidic platform consisting
of a two-axis centrifuge, whose fluidic chambers rotated in a

(i) Fabrication of arrays of microwells for patterning proteins;

Polyethylene glycol-based microstructures used to immobilize

binding between surface-bound ligands and aqueous receptors

planetary relationship to produce a radial gravitational field to
produce effective mixing based on the premise that this process

cells with potential applications in cell-based biosensors

would overcome surface and viscous forces in microchambers

On chip bioassays for carrying out genotoxicity tests

(Bynum and Gordon, 2004). Yuen and co-workers have also

Low density protein arrays patterned on gold by

On-chip transformation of Escherichia coli cells

(i) Multiplexed fabrication of cell phenotypes;

developed a microchip device for closed loop fluidic circulation

(ii) selective adhering of cells onto the wells

and mixing (Liu et al., 2004b). The device consisted of two

using fluidic networks containing SAMs

interconnected reaction chambers molded in PDMS with a
standard microscope glass slide. Mixing and circulation of fluid
samples was accomplished by rotation of a magnetic stirring

Profiling expression dynamics

(ii) reduced sample volumes

bar driven by a standard magnetic stirrer (Yuen et al., 2003).

Applications/achievements

in agarose hydrogel wells

Other examples of convective mixing involve cavitational
microstreaming, which has been reported to accelerate
hybridization kinetics by nearly five-fold (Liu et al., 2003b).
In this process, tiny air-filled chambers were added to a backing
slide; the air bubbles resting on a surface were set into vibration
by a sound field generating steady circulatory flows that
resulted in convective flows producing rapid mixing and
therefore, enhancing hybridization kinetics.

cell monitoring of expression events

Accelerated hybridization achieved by mixing has also been

stimulation and non-destructive

described by Wei et al., who used fluidic channels that were

High-throughput molecular
scrambled into discrete plugs to induce droplet mixing (Wei
et al., 2005). The plugs were thoroughly mixed by the natural
re-circulating flows shuttled back and forth along the channels
sweeping over the array probes, which reduced the hybridiza-
tion times to 500 s with reduced sample volumes of 1 ml.
integrated
Functions

DNA hybridizations can also be accelerated by electro-

kinetic delivery of samples (Edman et al., 1997; Erickson et al.,
n/a

n/a

n/a

n/a
n/a

n/a

n/a
2004; Sosnowski et al., 1997). Electrokinetic transport of
targets offers the advantages of controlled fluidic flow and the
2  107 cells/ml
Abbreviated list publications outlining the coupling of protein/cell microarrays to microfluidics

capability of miniaturization without having to use pumps or

1–106 cells/ml
10–30 ng/ml
active valves. The electrokinetic control allows rapid and
Detection

230 pmol
105 cells

efficient mass transfer of samples and therefore, hybridization

1 ng/ml
limits

equilibrium is rapidly attained leading to reduced reaction

n/a

n/a
times. Fig. 4 shows an example of electrokinetic addressing of a
Analysis speed

DNA microarray.

2.4. Applications of microfluidic-based microarrays

(min)
360

n/a

n/a

n/a

n/a

n/a
60

10

A comprehensive description of applications involving the

Glass/silicon/PDMS

use of microfluidics coupled to DNA microarrays (Liu et al.,

2004a; Lilliehorn et al., 2005; Lenigk et al., 2002a; Hashimoto
Silicon/PDMS
Silicon/PDMS

et al., 2006; Wang et al., 2003; Lee et al., 2001; Benoit et al.,
Gold/PDMS

2001; Cheek et al., 2001; Koh et al., 2003; Noerholm et al.,

material
Support

PDMS

PDMS

PDMS

PDMS

2004; Chung et al., 2003; Yuen et al., 2003; Wei et al., 2005;
Erickson et al., 2004; Ali et al., 2003; Anderson et al., 2000b;
Dimalanta et al., 2004; Keramas et al., 2004) is summarized in
Sample
volume

0.2 ml

10 ml

Table 1 while those of protein and cell microarrays (Kanda

5 ml

2 ml

3 ml
n/a

n/a

n/a

et al., 2004; Tani et al., 2004; Koh et al., 2003; Khademhosseini

et al., 2004; Nagamine et al., 2005; Ostuni et al., 2001;
Thompson et al. (2004)
Nagamine et al. (2005)

Thompson et al., 2004; Yang et al., 2001) are summarized in

Ostuni et al. (2001)

Kanda et al. (2004)

Yang et al. (2001)

n/a: not available.

Tani et al. (2004)

Koh et al. (2003)

Table 2. The information provided illustrates a full range of

Khademhosseini
et al. (2004)

microarrays performed in microfluidic devices detailing the

Reference

important achievements obtained in terms of volume reduction,

Table 2

analysis speed, applications, integration, automation and limit

of detection.
226 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231

A standard microarray experiment typically involves
several steps: (1) extraction/purification of the targets from
the cells following lysis; (2) in the case of mRNAs, reverse
transcription into cDNAs; (3) labeling of the solution targets
with reporter molecules, such as fluorescent dyes, which can be
added during PCR amplification in the case of DNAs; (4)
incubation of the targets with the arrayed probes. The probes
are patterned onto derivatized/treated substrates either by
printing or direct synthesis as mentioned previously. Printing
or spotting is accomplished through the use of robotic arrayers
that are designed to dispense small volumes of solutions
(Cheung et al., 1999; Duggan et al., 1999) or by ink-jet printing
(Okamoto et al., 2000). Other than spotting/printing, micro-
fluidics can also be used for deposition of pre-synthesized
probes onto supports as well via the stenciling techniques
described above. In most cases, printing, spotting and
stenciling techniques are effective in building low to medium
density arrays. In the case of direct in situ synthesis, high
density arrays can be produced (300,000 features on a
1.28 cm  1.28 cm chip of 25-mer oligonucleotide probes)
(Lipshutz et al., 1999).
The advantages exhibited by microfluidics, including their
ability to integrate several processing steps into a fully
functional system, has attracted several examples where the
biological sample is fully processed in the microfluidic prior to
hybridization interrogation. Systems have been reported that
utilize microfluidics to process the biological sample and
prepare it for structure analysis using either DNA, protein or
cell-based microarrays. In the following sections, examples
will be given from the literature in which sample-preprocessing
using microfluidics was invoked prior to interrogation of the
targets using a microarray.

Fig. 5. (A) Conceptual schematic of the PCR/LDR/universal array assay format. The zip code probes and complements contain no sequence homology to the DNA
target being interrogated for point mutations. In the absence of a successful ligation event, LDR primers bearing zip code complements do hybridize to their zip code
probes, but produce no fluorescence signature since the LDR primer containing the fluorescent label is not present. (B) Schematic representation of the CFPCR/
CFLDR and zip code array biochips. The CFPCR/CFLDR microchip possessed channels that were 100 mm in width and 50 mm in depth with a 400 mm inter-channel
spacing. The total length of the thermal cycling channel was 2.28 m and consisted of a 30-cycle PCR (1.57 m long) and a 13-cycle LDR (0.71 m long). The top
inset represents the PMMA zip code array microfluidic chip. The bottom inset is an enlarged schematic of the Y-shaped passive micromixer for mixing the PCR
product with the LDR cocktail. Adopted with permission from Hashimoto et al. (2006).
 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231
Fig. 5. (Continued ).
2.4.1. DNA microarray systems
An attractive feature of microfluidic systems is their ability to
process the sample in a close architecture and with high levels of
automation. This provides the ability to not only reduce
processing time, but also minimize sample contamination and
simplify assay implementation and cost. Recently, a flow through
microchip assembly containing two biochips was fabricated to
carry out a sequential ligase detection reaction (LDR) followed
by an hybridization assay (Hashimoto et al., 2006). The system
(see Fig. 5) was used to detect rare point mutations in genomic
DNA that possess high diagnostic value for colorectal cancers.
The LDR generates products that bear a fluorescence label and
also, a short sequence appended to the DNA primers involved in
the ligation reaction that directs a successfully formed ligation
product to a location on the array. The tethered probes are
complements to the short sequence found on one of the LDR
primers. This ‘‘universal array format’’ consisting of zip code
probes and zip code complements allows for the hybridization
detection of a variety of biologically derived products. PC and
227
PMMA chips were integrated into a single unit by using PDMS-
assisted interconnect technology. The PC chip was used for
performing the LDR on chip while the PMMA chip was used for
detection of these LDR products in a microarray format.
Hydrodynamic pumping of fluids on this 3D microfluidic
network enabled an LDR/hybridization assay to be carried out
with applications in the detection of low-abundant point
mutations in K-ras genes in less that 20 min (total processing
time). This processing time was significantly reduced compared
to previous work performed, which involved carrying out a PCR/
LDR with array readout using conventional bench-top equip-
ment, which required approximately 270 min.
2.4.2. Protein microarray systems
Protein arrays are based on biospecific binding, e.g. antibody/
antigen interactions. Yang et al. designed a multivalent immuno-
assay for monitoring the binding of bivalent anti-dinitrophenyl
(anti-DNP) antibodies to phospholipid bilayer surfaces contain-
ing dinitrophenyl haptens (Yang et al., 2001). These assays were
228 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231
conducted inside linear arrays of bilayer-coated microchannels
formed by placing patterned PDMS molds into conformal
contact with borosilicate cover slips. Twelve microchannels were
arrayed onto a single chip to allow heterogeneous assays to be
performed rapidly with reduced protein sample volumes.
2.4.3. Cell-based microarray systems
The ability of microfluidics to enhance controlled cellular
interactions and increase response times has also resulted in
their incorporation into cell studies. Cell studies provide
information on the dynamics of gene expression or their
morphological characteristics. Arrays of different phenotypes
of cells can be useful in clinical diagnostics as well. Thompson
et al. reported a living cell array platform for the simultaneous
stimulation and monitoring of gene expression in living cells
(Thompson et al., 2004). They used a PDMS/glass chamber
coated with fibronectin to promote cell attachment. This
platform was used to control dynamic inputs and measure the
outputs from adherent mammalian cells. One advantage offered
by this device over conventional cell microarrays was its ability
to simultaneously screen many conditions and efficiently
explore the large parameter space relating to stimulation of
certain cell responses.
Protein and cell microarrays have allowed the reduction in
sample and reagent volumes and analysis speed to be achieved,
however, they are still in their infancy in terms of development
and as such, are faced with several operational challenges. For
example, it is difficult to observe low abundant targets by direct
molecular association assays because the trace amount of target
has to compete with the mixed population of higher abundant
interferents for binding onto the recognition element situated
on the support (MacBeath, 2002). Also, high content analysis of
cell microarrays in living cells require the imaging time of an
entire slide at sufficient time-resolution so as not to obscure
transitory phenotypes of interest (Wheeler et al., 2005).
3. Conclusions
Microfluidics has advanced the rapid growth in biochip
applications due in part to their ability to pre-process the sample
in an automated fashion prior to incubation with the array and
the fact that the operational characteristics of the array can be
improved when scaled to the micrometer domain. One
particularly compelling advantage associated with the marriage
of microfluidics with microarrays is the reduction in reaction
times exhibited in all applications ranging from DNA arrays,
protein arrays and cell bioassays. The reduced reaction times
are attributed to improved kinetics compared to conventional
non-microfluidic techniques and often times is a consequence
of enhanced mass transport to the surface tethered recognition
elements by the target and small diffusional distances.
Successful integration of sample pre-treatment processes, such
as cell culture/lysis, sample purification, pre-concentration and
amplification, with the hybridization reaction on a single
microfluidic platform has shown significant reduction in
analysis time, improved sensitivities, fully automated sample
processing, and the reduction in hardware demands for the
intended assay, which are very useful for transitioning high end
molecular analysis to a point-of-care format. Parallelization
capability of microfluidics on a single chip is also important,
since different samples can be simultaneously processed with
highly controlled reaction conditions allowing high-throughput
analysis as well as reducing inter-sample variability. Also, the
closed architecture of the microfluidic provides reduction in
possible sample contamination, which will have important
clinical ramifications in the area of DNA forensics. The use of
inexpensive polymers as the substrate for microfluidic devices
allows one to employ these systems as single-use devices,
which will be particularly attractive to eliminate cross-
contaminations during operation, eliminating false positive
results due to sample carryover. Coupling microfluidics to DNA
arrays, protein arrays or cell arrays therefore will have a major
impact on understanding many fundamental biological
activities, since these technology platforms make high end
technologies for studying these processes available to a large
research community due to the simplicity of their operation and
the low cost of their production.
Another challenge associated with coupling microfluidics and
microarrays is the ability to manipulate microfluidics in a
complicated fully integrated fluidic network. With this, many
multi-step microarray hybridization processes that require
manual handling can be eliminated when all the processes are
integrated into a single system. These automated platforms may
eventually lead to improved reproducibility of microarray results
with acceptable array-to-array coefficients of variation. Micro-
fluidics has also improved microarray sensitivities enabling
detection of even low expressed genes, however, array analyses
still lacks an efficient data analysis tool (bioinformatics) for the
generated microarray information. Improved bioinformatics
tools and fully integrated microfluidic devices will therefore
enable microarrays to be fully exploited in gene expression
analysis and clinical diagnostics with reduced analysis time.
While microfluidics can offer some attractive advantages for
microarray-based assays, these systems still depend on
conventional peripheral equipment for their operation, such
as sample/reagent loading, fluid actuation and readout. It is
clear that complete packaging of these peripherals into a system
that is on the same size as the fluidic element will allow the
creation of instruments that can be envisioned for potential
point-of-care testing. This realization will provide increased
accessibility of microarray technologies to a potentially larger
user base and expand on their capabilities.
Acknowledgements
The authors would like to thank the financial support of this
work through the US National Institutes of Health (R01-
EB00215), the US National Science Foundation (EPS-
0346411) and the State of Louisiana Board of Regents.
References
Ahn, S., Walt, D.R., 2005. Detection of Salmonella spp. Using microsphere-
based, fiber-optic DNA microarrays. Anal. Chem. 77 (15), 5041–5047.
 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231
Ali, M.F., Kirby, R., Goodey, A.P., Rodriguez, M.D., Ellington, A.D., Neikirk,
D.P., McDevitt, J.T., 2003. DNA hybridization and discrimination of single-
nucleotide mismatches using chip-based microbead arrays. Anal. Chem. 75
(18), 4732–4739.
Anderson, J.R., Chiu, D.T., Jackman, R.J., Cherniavskaya, O., McDonald, J.C.,
Wu, H., Whitesides, S.H., Whitesides, G.M., 2000a. Fabrication of topo-
logically complex three-dimensional microfluidic systems in PDMS by
rapid prototyping. Anal. Chem. 72 (14), 3158–3164.
Anderson, R.C., Su, X., Bogdan, G.J., Fenton, J., 2000b. A miniature integrated
device for automated multistep genetic assays. Nucleic Acids Res. 28 (12)
e60, ii–vi.
Auroux, P.-A., Iossifidis, D., Reyes, D.R., Manz, A., 2002. Micro-total analysis
systems. 2. Analytical standard operations and applications. Anal. Chem. 74
(12), 2637–2652.
Baner, J., Isaksson, A., Waldenstroem, E., Jarvius, J., Landegren, U., Nilsson,
M., 2003. Parallel gene analysis with allele-specific padlock probes and tag
microarrays. Nucleic Acids Res. 31 (17) e103/1-e03/7.
Becker, H., Gartner, C., 2000. Polymer microfabrication methods for micro-
fluidic analytical applications. Electrophoresis 21 (1), 12–26.
Benoit, V., Steel, A., Torres, M., Yu, Y.-Y., Yang, H., Cooper, J., 2001.
Evaluation of three-dimensional microchannel glass biochips for multi-
plexed nucleic acid fluorescence hybridization assays. Anal. Chem. 73 (11),
2412–2420.
Boerner, M.W., Kohl, M., Pantenburg, F.J., Bacher, W., Hein, H., Schomburg,
W.K., 1996. Sub-micron LIGA process for movable microstructures.
Microelectron. Eng. 30 (1–4), 505–508.
Burns, M.A., Johnson, B.N., Brahmasandra, S.N., Handique, K., Webster, J.R.,
Krishnan, M., Sammarco, T.S., Man, P.M., Jones, D., Heldsinger, D.,
Mastrangelo, C.H., Burke, D.T., 1998. An integrated nanoliter DNA
analysis device. Science 282 (5388), 484–487.
Burns, M.A., Mastrangelo, C.H., Sammarco, T.S., Man, F.P., Webster, J.R.,
Johnsons, B.N., Foerster, B., Jones, D., Fields, Y., Kaiser, A.R., Burke, D.T.,
1996. Microfabricated structures for integrated DNA analysis. Proc. Natl.
Acad. Sci. U.S.A. 93 (11), 5556–5561.
Bussow, K., Nordhoff, E., Lubbert, C., Lehrach, H., Walter, G., 2000. A human
cDNA library for high-throughput protein expression screening. Genomics
65 (1), 1–8.
Bynum, M.A., Gordon, G.B., 2004. Hybridization enhancement using micro-
fluidic planetary centrifugal mixing. Anal. Chem. 76 (23), 7039–7044.
Cheek, B.J., Steel, A.B., Torres, M.P., Yu, Y.-Y., Yang, H., 2001. Chemilumi-
nescence detection for hybridization assays on the flow-thru chip, a three-
dimensional microchannel biochip. Anal. Chem. 73 (24), 5777–5783.
Cheung, V.G., Morley, M., Aguilar, F., Massimi, A., Kucherlapati, R., Childs,
G., 1999. Making and reading microarrays. Nat. Genet. 21 (Suppl. 1), 15–
19.
Chung, Y.-C., Lin, Y.-C., Hsu, Y.-L., Chang, W.-N.T., Shiu, M.-Z., 2004. The
effect of velocity and extensional strain rate on enhancing DNA hybridiza-
tion. J. Micromech. Microeng. 14 (10), 1376–1383.
Chung, Y.-C., Lin, Y.-C., Shiu, M.-Z., Chang, W.-N.T., 2003. Microfluidic chip
for fast nucleic acid hybridization. Lab. Chip 3 (4), 228–233.
Csaki, A., Moller, R., Straube, W., Kohler, J.M., Fritzsche, W., 2001. DNA
monolayer on gold substrates characterized by nanoparticle labeling and
scanning force microscopy. Nucleic Acids Res. 29 (16), e81/1–e81/5.
Dimalanta, E.T., Lim, A., Runnheim, R., Lamers, C., Churas, C., Forrest, D.K.,
de Pablo, J.J., Graham, M.D., Coppersmith, S.N., Goldstein, S., Schwartz,
D.C., 2004. A microfluidic system for large DNA molecule arrays. Anal.
Chem. 76 (18), 5293–5301.
Duffy, D.C., McDonald, J.C., Schueller, O.J.A., Whitesides, G.M., 1998. Rapid
prototyping of microfluidic systems in poly(dimethylsiloxane). Anal.
Chem. 70 (23), 4974–4984.
Duffy, D.C., Schueller, O.J.A., Brittain, S.T., Whitesides, G.M., 1999. Rapid
prototyping of microfluidic switches in poly(dimethylsiloxane) and their
actuation by electro-osmotic flow. J. Micromech. Microeng. 9 (3), 211–217.
Duggan, D.J., Bittner, M., Chen, Y., Meltzer, P., Trent, J.M., 1999. Expression
profiling using cDNA microarrays. Nat. Genet. 21 (Suppl 1), 10–14.
Edman, C.F., Raymond, D.E., Wu, D.J., Tu, E., Sosnowski, R.G., Butler, W.F.,
Nerenberg, M., Heller, M., 1997. Electric field directed nucleic acid
hybridization on microchips. Nucleic Acids Res. 25 (24), 4907–4914.
229
Erali, M., Schmidt, B., Lyon, E., Wittwer, C., 2003. Evaluation of electronic
microarrays for genotyping factor V, factor II, and MTHFR. Clin. Chem. 49
(5), 732–739.
Erickson, D., Liu, X., Krull, U., Li, D., 2004. Electrokinetically controlled DNA
hybridization microfluidic chip enabling rapid target analysis. Anal. Chem.
76 (24), 7269–7277.
Fan, Z.H., Mangru, S., Granzow, R., Heaney, P., Ho, W., Dong, Q., Kumar, R.,
1999. Dynamic DNA hybridization on a chip using paramagnetic beads.
Anal. Chem. 71 (21), 4851–4859.
Fixe, F., Dufva, M., Telleman, P., Christensen, C.B.V., 2004a. Functionalization
of poly(methyl methacrylate) (PMMA) as a substrate for DNA microarrays.
Nucleic Acids Res. 32 (1), e9/1–e9/8.
Fixe, F., Dufva, M., Telleman, P., Christensen, C.B.V., 2004b. One-step
immobilization of aminated and thiolated DNA onto poly(methylmetha-
crylate) (PMMA) substrates. Lab. Chip 4 (3), 191–195.
Fodor, S.P.A., Read, J.L., Pirrung, M.C., Stryer, L., Lu, A.T., Solas, D., 1991.
Light-directed, spatially addressable parallel chemical synthesis. Science
(Washington, DC, United States) 251 (24), 767–773.
Ford, S.M., Davies, J., Kar, B., Qi, S.D., McWhorter, S., Soper, S.A., Malek,
C.K., 1999. Micromachining in plastics using X-ray lithography for the
fabrication of microelectrophoresis devices. J. Biomech. Eng. 121 (1), 13–
21.
Friedrich, C.R., Coane, P.J., Vasile, M.J., 1997. Micromilling development and
applications for microfabrication. Microelectron. Eng. 35 (1-4, Micro- and
Nano-Engineering 96), 367–372.
Fu, A.Y., Chou, H.-P., Spence, C., Arnold, F.H., Quake, S.R., 2002.
An integrated microfabricated cell sorter. Anal. Chem. 74 (11), 2451–
2457.
Gingeras, T.R., Kwoh, D.Y., Davis, G.R., 1987. Hybridization properties of
immobilized nucleic acids. Nucleic Acids Res. 15 (13), 5373–5390.
Gong, Y.-K., Luo, L., Petit, A., Zukor, D.J., Huk, O.L., Antoniou, J., Winnik,
F.M., Mwale, F., 2005. Adhesion of human U937 macrophages to phos-
phorylcholine-coated surfaces. J. Biomed. Mater. Res. 72A (1), 1–9.
Grass, B., Neyer, A., Johnck, M., Siepe, D., Eisenbeiss, F., Weber, G.,
Hergenroder, R., 2001. A new PMMA-microchip device for isotachophor-
esis with integrated conductivity detector. Sens. Actuators B 72 (3), 249–
258.
Grimm, V., Ezaki, S., Susa, M., Knabbe, C., Schmid, R.D., Bachmann, T.T.,
2004. Use of DNA microarrays for rapid genotyping of TEM beta-lacta-
mases that confer resistance. J. Clin. Microbiol. 42 (10), 4918 [Erratum to
document cited in CA142:087118].
Halliwell, C.M., Cass, A.E.G., 2001. A factorial analysis of silanization
conditions for the immobilization of oligonucleotides on glass surfaces.
Anal. Chem. 73 (11), 2476–2483.
Hashimoto, M., Barany, F., Soper, S.A., 2006. Polymerase chain reaction/ligase
detection reaction/hybridization assays using flow-through microfluidic
devices for the detection of low-abundant DNA point mutations. Biosens.
Bioelectron. 21, 1915–1923.
He, B., Tan, L., Regnier, F., 1999. Microfabricated filters for microfluidic
analytical systems. Anal. Chem. 71 (7), 1464–1468.
Henry, A.C., Tutt, T.J., Galloway, M., Davidson, Y.Y., McWhorter, C.S., Soper,
S.A., McCarley, R.L., 2000. Surface modification of poly(methyl metha-
crylate) used in the fabrication of microanalytical devices. Anal. Chem. 72
(21), 5331–5337.
Horvath, J., Dolnik, V., 2006. Polymer wall coatings for capillary electrophor-
esis. Electrophoresis 22, 644–655.
Hu, S., Ren, X., Bachman, M., Sims, C.E., Li, G.P., Allbritton, N.L., 2004.
Surface-directed, graft polymerization within microfluidic channels. Anal.
Chem. 76 (7), 1865–1870.
Ihlemann, J., Rubahn, K., 2000. Excimer laser micro-machining: fabrica-
tion and applications of dielectric masks. Appl. Surf. Sci. 154–155,
587–592.
Ishikawa, S., Komura, D., Tsuji, S., Nishimura, K., Yamamoto, S., Panda, B.,
Huang, J., Fukayama, M., Jones, K.W., Aburatani, H., 2005. Allelic dosage
analysis with genotyping microarrays. Biochem. Biophys. Res. Commun.
333 (4), 1309–1314.
Joos, B., Kuster, H., Cone, R., 1997. Covalent attachment of hybridizable
oligonucleotides to glass supports. Anal. Biochem. 247 (1), 96–101.
230 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231
Kanda, V., Kariuki, J.K., Harrison, D.J., McDermott, M.T., 2004. Label-free
reading of microarray-based immunoassays with surface plasmon reso-
nance imaging. Anal. Chem. 76 (24), 7257–7262.
Kapur, R., 2005. Whole cell microarrays. Microarray Technol. Appl. 335–343.
Keramas, G., Perozziello, G., Geschke, O., Christensen, C.B.V., 2004. Devel-
opment of a multiplex microarray microsystem. Lab. Chip 4 (2), 152–158.
Khademhosseini, A., Yeh, J., Jon, S., Eng, G., Suh, K.Y., Burdick, J.A., Langer,
R., 2004. Molded polyethylene glycol microstructures for capturing cells
within microfluidic channels. Lab. Chip 4 (5), 425–430.
Kim, H.-L., 2003. Comparison of oligonucleotide-microarray and serial ana-
lysis of gene expression (SAGE) in transcript profiling analysis of mega-
karyocytes derived from CD34+ cells. Exp. Mol. Med. 35 (5), 460–466.
Koh, W.-G., Itle, L.J., Pishko, M.V., 2003. Molding of hydrogel microstructures
to create multiphenotype cell microarrays. Anal. Chem. 75 (21), 5783–
5789.
Kohara, Y., Noda, H., Okano, K., Kambara, H., 2002. DNA probes on beads
arrayed in a capillary, bead-array, exhibited high hybridization perfor-
mance. Nucleic Acids Res. 30 (16), e87/1–e87/7.
Lebrun, S.J., 2005. The use of microarrays for highly sensitive, multiplex
immunoassays in biomarker discovery and drug efficacy studies. Pharma-
ceut. Discov. Suppl. 53–56.
Lee, H.J., Goodrich, T.T., Corn, R.M., 2001. SPR imaging measurements of 1-D
and 2-D DNA microarrays created from microfluidic channels on gold thin
films. Anal. Chem. 73 (22), 5525–5531.
Lee, K.-B., Yoon, K.R., Woo, S.I., Choi, I.S., 2003. Surface modification of
poly(glycolic acid) (PGA) for biomedical applications. J. Pharm. Sci. 92 (5),
933–937.
Lenigk, R., Liu, R.H., Athavale, M., Chen, Z., Ganser, D., Yang, J., Rauch, C.,
Liu, Y., Chan, B., Yu, H., Ray, M., Marrero, R., Grodzinski, P., 2002a.
Plastic biochannel hybridization devices: a new concept for microfluidic
DNA arrays. Anal. Biochem. 311 (1), 40–49.
Lenigk, R., Liu, R.H., Athavale, M., Chen, Z.J., Ganser, D., Yang, J.N., Rauch,
C., Liu, Y.J., Chan, B., Yu, H.N., Ray, M., Marrero, R., Grodzinski, P.,
2002b. Plastic biochannel hybridization devices: a new concept for micro-
fluidic DNA arrays. Anal. Biochem. 311 (1), 40–49.
Li, J., Thibault, P., Bings, N.H., Skinner, C.D., Wang, C., Colyer, C., Harrison,
J., 1999. Integration of microfabricated devices to capillary electrophoresis–
electrospray mass spectrometry using a low dead volume connection:
application to rapid analyses of proteolytic digests. Anal. Chem. 71 (15),
3036–3045.
Lilliehorn, T., Nilsson, M., Simu, U., Johansson, S., Almqvist, M., Nilsson, J.,
Laurell, T., 2005. Dynamic arraying of microbeads for bioassays in micro-
fluidic channels. Sens. Actuators B 106 (2), 851–858.
Lin, M., Wei, L.-J., Sellers, W.R., Lieberfarb, M., Wong, W.H., Li, C., 2004.
dChipSNP: significance curve and clustering of SNP-array-based loss-of-
heterozygosity data. Bioinformatics 20 (8), 1233–1240.
Lipshutz, R.J., Fodor, S.P.A., Gingeras, T.R., Lockhart, D.J., 1999. High density
synthetic oligonucleotide arrays. Nat. Genet. 21 (Suppl. 1), 20–24.
Liu, J., Hansen, C., Quake, S.R., 2003a. Solving the ‘‘world-to-chip’’ interface
problem with a microfluidic matrix. Anal. Chem. 75 (18), 4718–4723.
Liu, R.H., Lenigk, R., Druyor-Sanchez, R.L., Yang, J., Grodzinski, P., 2003b.
Hybridization enhancement using cavitation microstreaming. Anal. Chem.
75 (8), 1911–1917.
Liu, Y., Rauch, C.B., 2003. DNA probe attachment on plastic surfaces and
microfluidic hybridization array channel devices with sample oscillation.
Anal. Biochem. 317 (1), 76–84.
Liu, R.H., Yang, J., Lenigk, R., Bonanno, J., Grodzinski, P., 2004a. Self-
contained, fully integrated biochip for sample preparation, polymerase
chain reaction amplification, and DNA microarray detection. Anal. Chem.
76 (7), 1824–1831.
Liu, J., Walker, N., Waalkes, M.P., 2004b. Hybridization buffer systems impact
the quality of filter array data. J. Pharmacol. Toxicol. Methods 50 (1), 67–
71.
Love, J.C., Wolfe, D.B., Jacobs, H.O., Whitesides, G.M., 2001. Microscope
projection photolithography for rapid prototyping of masters with micron-
scale features for use in soft lithography. Langmuir 17 (19), 6005–6012.
Lovmar, L., Fock, C., Espinoza, F., Bucardo, F., Syvanen, A.-C., Bondeson, K.,
2003. Microarrays for genotyping human group A rotavirus by multiplex
capture and type-specific primer extension. J. Clin. Microbiol. 41 (11),
5153–5158.
Lu, H.H., Cooper, J.A., Manuel, S., Freeman, J.W., Attawia, M.A., Ko, F.K.,
Laurencin, C.T., 2005. Anterior cruciate ligament regeneration using
braided biodegradable scaffolds: in vitro optimization studies. Biomaterials
26 (23), 4805–4816.
MacBeath, G., 2002. Protein microarrays and proteomics. Nat. Genet. Suppl.
32, 526–532.
MacBeath, G., Schreiber, S.L., 2000. Printing proteins as microarrays for high-
throughput function determination. Science (Washington, DC) 289 (5485),
1760–1763.
Marquette, C.A., Blum, L.J., 2004. Direct immobilization in poly(dimethylsi-
loxane) for DNA, protein and enzyme fluidic biochips. Anal. Chim. Acta
506 (2), 127–132.
Martynova, L., Locascio, L.E., Gaitan, M., Kramer, G.W., Christensen, R.G.,
MacCrehan, W.A., 1997. Fabrication of plastic microfluid channels by
imprinting methods. Anal. Chem. 69 (23), 4783–4789.
McCarley, R.L., Vaidya, B., Wei, S., Smith, A.F., Patel, A.B., Feng, J., Murphy,
M.C., Soper, S.A., 2005. Resist-free patterning of surface architectures in
polymer-based microanalytical devices. J. Am. Chem. Soc. 127 (3), 842–
843.
McClain, M.A., Culbertson, C.T., Jacobson, S.C., Allbritton, N.L., Sims, C.E.,
Ramsey, J.M., 2003. Microfluidic devices for the high-throughput chemical
analysis of cells. Anal. Chem. 75 (21), 5646–5655.
McDonald, J.C., Whitesides, G.M., 2002. Poly(dimethylsiloxane) as a material
for fabricating microfluidic devices. Acc. Chem. Res. 35 (7), 491–499.
Meaburn, E., Butcher, L.M., Schalkwyk, L.C., Plomin, R., 2006. Genotyping
pooled DNA using 100 K SNP microarrays: a step towards genome wide
association scans. Nucleic Acids Res. 34 (4), e27.
Moorcroft, M.J., Meuleman, W.R.A., Latham, S.G., Nicholls, T.J., Egeland,
R.D., Southern, E.M., 2005. In situ oligonucleotide synthesis on poly(-
dimethylsiloxane): a flexible substrate for microarray fabrication. Nucleic
Acids Res. 33 (8), e75/1–e75/10.
Nagamine, K., Onodera, S., Torisawa, Y., Yasukawa, T., Shiku, H., Matsue, T.,
2005. On-chip transformation of bacteria. Anal. Chem. 77 (13), 4278–4281.
Noerholm, M., Bruus, H., Jakobsen, M.H., Telleman, P., Ramsing, N.B., 2004.
Polymer microfluidic chip for online monitoring of microarray hybridiza-
tions. Lab. Chip 4 (1), 28–37.
Nuzzo, R.G., 2003. Biomaterials: stable antifouling surfaces. Nat. Mater. 2 (4),
207–208.
Okamoto, T., Suzuki, T., Yamamoto, N., 2000. Microarray fabrication with
covalent attachment of DNA using bubble jet technology. Nat. Biotechnol.
18 (4), 438–441.
Ostuni, E., Chen, C.S., Ingber, D.E., Whitesides, G.M., 2001. Selective
deposition of proteins and cells in arrays of microwells. Langmuir 17
(9), 2828–2834.
Pal, R., Yang, M., Lin, R., Johnson, B.N., Srivastava, N., Razzacki, S.Z.,
Chomistek, K.J., Heldsinger, D.C., Haque, R.M., Ugaz, V.M., Thwar,
P.K., Chen, Z., Alfano, K., Yim, M.B., Krishnan, M., Fuller, A.O.,
Larson, R.G., Burke, D.T., Burns, M.A., 2005. An integrated micro-
fluidic device for influenza and other genetic analyses. Lab. Chip 5 (10),
1024–1032.
Pease, A.C., Solas, D., Sullivan, E.J., Cronin, M.T., Holmes, C.P., Fodor, S.P.,
1994. Light-generated oligonucleotide arrays for rapid DNA sequence
analysis. Proc. Natl. Acad. Sci. U.S.A. 91 (11), 5022–5026.
Peterson, A.W., Heaton, R.J., Georgiadis, R.M., 2001. The effect of surface
probe density on DNA hybridization. Nucleic Acids Res. 29 (24), 5163–
5168.
Postier Bradley, L., Wang, H.-L., Singh, A., Impson, L., Andrews Heather, L.,
Klahn, J., Li, H., Risinger, G., Pesta, D., Deyholos, M., Galbraith David, W.,
Sherman Louis, A., Burnap Robert, L., 2003. The construction and use of
bacterial DNA microarrays based on an optimized two-stage PCR strategy.
BMC Genom. Electr. Resour. 4 (1), 23.
Qi, S., Liu, X., Ford, S., Barrows, J., Thomas, G., Kelly, K., McCandless, A.,
Lian, K., Goettert, J., Soper, S.A., 2002. Microfluidic devices fabricated
in poly(methyl methacrylate) using hot-embossing with integrated sam-
pling capillary and fiber optics for fluorescence detection. Lab. Chip 2
(2), 88–95.
 C. Situma et al. / Biomolecular Engineering 23 (2006) 213–231
Ressine, A., Ekstrom, S., Marko-Varga, G., Laurell, T., 2003. Macro-/nanopor-
ous silicon as a support for high-performance protein microarrays. Anal.
Chem. 75 (24), 6968–6974.
Reyes, D.R., Iossifidis, D., Auroux, P.-A., Manz, A., 2002. Micro-total analysis
systems. 1. Introduction, theory, and technology. Anal. Chem. 74 (12),
2623–2636.
Roberts, M.A., Rossier, J.S., Bercier, P., Girault, H., 1997. UV laser machined
polymer substrates for the development of microdiagnostic systems. Anal.
Chem. 69 (11), 2035–2042.
Rossier, J.S., Schwarz, A., Reymond, F., Ferrigno, R., Bianchi, F., Girault, H.H.,
1999. Microchannel networks for electrophoretic separations. Electrophor-
esis 20 (4–5), 727–731.
Sato, K., Tokeshi, M., Kimura, H., Kitamori, T., 2001. Determination of
carcinoembryonic antigen in human sera by integrated bead-bed immu-
noassay in a microchip for cancer diagnosis. Anal. Chem. 73 (6), 1213–
1218.
Sebra, R.P., Masters, K.S., Bowman, C.N., Anseth, K.S., 2005. Surface grafted
antibodies: controlled architecture permits enhanced antigen detection.
Langmuir 21 (24), 10907–10911.
Shadpour, H., Musyimi, H., Chen, J., Soper, S.A., 2006. Physiochemical
properties of various polymer substrates and their effects on microchip
electrophoresis performance. J. Chromatogr. A 1111 (2), 238–251.
Sia, S.K., Whitesides, G.M., 2003. Microfluidic devices fabricated in poly(-
dimethylsiloxane) for biological studies. Electrophoresis 24 (21), 3563–
3576.
Situma, C., Wang, Y., Hupert, M., Barany, F., McCarley, R.L., Soper, S.A.,
2005. Fabrication of DNA microarrays onto poly(methyl methacrylate) with
ultraviolet patterning and microfluidics for the detection of low-abundant
point mutations. Anal. Biochem. 340 (1), 123–135.
Soper, S.A., Ford, S.M., Qi, S., McCarley, R.L., Kelly, K., Murphy, M.C., 2000.
Polymeric microelectromechanical systems. Anal. Chem. 72 (19), 643A–
651A.
Sosnowski, R.G., Tu, E., Butlter, W.F., O’Connell, J.P., Heller, M.J., 1997.
Rapid determination of single base mismatch mutations in DNA hybrids by
direct electric field control. Proc. Natl. Acad. Sci. U.S.A. 94 (4), 1119–1123.
Southern, E.M., Case-Green, S.C., Elder, J.K., Johnson, M., Mir, K.U., Wang,
L., Williams, J.C., 1994. Arrays of complementary oligonucleotides for
analyzing the hybridization behavior of nucleic acids. Nucleic Acids Res.
22 (8), 1368–1373.
Steel, A.B., Levicky, R.L., Herne, T.M., Tarlov, M.J., 2000. Immobilization of
nucleic acids at solid surfaces: effect of oligonucleotide length on layer
assembly. Biophys. J. 79 (2), 975–981.
Su, J., Bringer, M.R., Ismagilov, R.F., Mrksich, M., 2005. Combining micro-
fluidic networks and peptide arrays for multi-enzyme assays. J. Am. Chem.
Soc. 127 (20), 7280–7281.
231
Tani, H., Maehana, K., Kamidate, T., 2004. Chip-based bioassay using bacterial
sensor strains immobilized in three-dimensional microfluidic network.
Anal. Chem. 76 (22), 6693–6697.
Thompson, D.M., King, K.R., Wieder, K.J., Toner, M., Yarmush, M.L., Jayara-
man, A., 2004. Dynamic gene expression profiling using a microfabricated
living cell array. Anal. Chem. 76 (14), 4098–5103.
Unger, M.A., Chou, H.-P., Thorsen, T., Scherer, A., Quake, S.R., 2000.
Monolithic microfabricated valves and pumps by multilayer soft lithogra-
phy. Science (Washington, DC) 288 (5463), 113–116.
Vanderhoeven, J., Pappaert, K., Dutta, B., Vanhummelen, P., Baron, G.V.,
Desmet, G., 2004. Exploiting the benefits of miniaturization for the
enhancement of DNA microarrays. Electrophoresis 25 (21–22), 3677–3686.
Waddell, E., Wang, Y., Stryjewski, W., McWhorter, S., Henry, A.C., Evans, D.,
McCarley, R.L., Soper, S.A., 2000. High-resolution near-infrared imaging
of DNA microarrays with time-resolved acquisition of fluorescence life-
times. Anal. Chem. 72 (24), 5907–5917.
Wang, Y., Vaidya, B., Farquar, H.D., Stryjewski, W., Hammer, R.P., McCarley,
R.L., Soper, S.A., Cheng, Y.-W., Barany, F., 2003. Microarrays assembled in
microfluidic chips fabricated from poly(methyl methacrylate) for the detec-
tion of low-abundant DNA mutations. Anal. Chem. 75 (5), 1130–1140.
Wei, C.-W., Cheng, J.-Y., Huang, C.-T., Yen, M.-H., Young, T.-H., 2005. Using
a microfluidic device for 1 ml DNA microarray hybridization in 500 s.
Nucleic Acids Res. 33 (8), e78.
Wheeler, D.B., Carpenter, A.E., Sabatini, D.M., 2005. Cell microarrays and
RNA interference chip away at gene function. Nat. Genet. 37 (Suppl.), S25–
S30.
Wu, R.Z., Bailey, S.N., Sabatini, D.M., 2002. Cell-biological applications of
transfected-cell microarrays. Trends Cell Biol. 12 (10), 485–488.
Xu, H., Sha, M.Y., Wong, E.Y., Uphoff, J., Xu, Y., Treadway, J.A., Truong, A.,
O’Brien, E., Asquith, S., Stubbins, M., Spurr, N.K., Lai, E.H., Mahoney, W.,
2003. Multiplexed SNP genotyping using the Qbead system: a quantum dot-
encoded microsphere-based assay. Nucleic Acids Res. 31 (8), e43/1–e43/10.
Yang, T., Jung, S.-Y., Mao, H., Cremer, P.S., 2001. Fabrication of phospholipid
bilayer-coated microchannels for on-chip immunoassays. Anal. Chem. 73
(2), 165–169.
Yaralioglu, Goksen, G., Wygant, Ira, O., Marentis, Theodore, C., Khuri-Yakub,
Butrus, T., 2004. Ultrasonic mixing in microfluidic channels using inte-
grated transducers. Anal. Chem. 76 (13), 3694–3698.
Yuen, P.K., Li, G., Bao, Y., Mueller, U.R., 2003. Microfluidic devices for fluidic
circulation and mixing improve hybridization signal intensity on DNA
arrays. Lab. Chip 3 (1), 46–50.
Zhang, Y., Coyne, M.Y., Will, S.G., Levenson, C.H., Kawasaki, E.S., 1991.
Single-base mutational analysis of cancer and genetic diseases using
membrane bound modified oligonucleotides. Nucleic Acids Res. 19 (14),
3929–3933.