Microfluid Nanofluid (2007) 3:623–628
DOI 10.1007/s10404-007-0155-6
S H O R T C O M M U NI C A T I O N
Automated module for hybridization and staining
of commercially produced nucleic acid microarrays
J. S. Erickson Æ J. E. Hu Æ G. P. Anderson Æ
A. C. Salls Æ S. A. Gray Æ J. P. Golden Æ
B. Lin Æ F. S. Ligler
Received: 18 November 2006 / Accepted: 22 January 2007 / Published online: 17 February 2007

Springer-Verlag 2007
Abstract Nucleic acid microarrays are a rapidly
expanding technology that enables the detection of
pathogens at the genetic level. Currently, the process-
ing of commercially produced microarrays requires
cumbersome, expensive, and time-consuming benchtop
equipment, which is not practical for point-of-care
diagnostic applications. We demonstrate a portable
module that can perform the hybridization, wash, and
stain steps required for processing a nucleic acid
microarray; and it performs these steps in a timeline
significantly shorter than the standard commercial
protocol. This device is automated, has a small foot-
print, and serves as a replacement for two commercial
pieces of benchtop equipment. Furthermore, our de-
vice is designed to serve as a module in a portable
biosensor that performs automated sample preparation
and nucleic acid amplification. Results with Affymetrix
GeneChips show that our device performs as well as
non-portable equipment specifically manufactured to
process these microarrays.
Keywords Microarray
Hybridization
Automated

Diagnostics
Portable
J.S. Erickson and J.E. Hu contributed equally to this work.
J. S. Erickson
G. P. Anderson
A. C. Salls

S. A. Gray
J. P. Golden
B. Lin
F. S. Ligler (&)
Naval Research Laboratory, Code 6900,
Center for Bio/Molecular Science and Engineering,
Washington, DC 20375-5438, USA
e-mail: fligler@cbmse.nrl.navy.mil
J. E. Hu
Nova Research Inc., Alexandria, VA 22309, USA
1 Introduction
Nucleic acid microarrays can detect pathogens at the
genetic level and offer the potential for broad-spec-
trum surveillance and diagnostics, and for the investi-
gation of gene expression within a species. Our group
has adapted this technology and demonstrated that
multiple pathogens can be identified at the strain level
directly from clinical fluids using commercially pro-
duced high-density resequencing microarrays (Lin
et al. 2006). High-density nucleic acid microarrays can
have hundreds of thousands of spots, so that the
number of pathogens possible to detect becomes lim-
ited by the ability to perform generic or highly multi-
plexed PCR amplifications. In addition, nucleic acid
microarrays have demonstrated higher sensitivity than
traditional antibody-based assays, making the detec-
tion of pathogens down to 10 cfu/mL or 10 pfu/mL
possible (Lin et al. 2007). Indeed, several studies have
shown the utility of DNA microarrays for pathogen
detection (Call et al. 2003a, b; Chizhikov et al. 2001,
2002; Davignon et al. 2005; Gingeras et al. 1998; Lin
et al. 2007; Roth et al. 2004; Troesch et al. 1999; Wang
et al. 2002, 2003, 2006; Wilson et al. 2002a, b).
Although currently expensive, new manufacturing
processes have reduced the cost of DNA microarray
technology to the point that is practical to be used in
the clinical setting (Dobrowolski et al. 1999). Despite
falling production costs, the application of microarray
technology in the field is limited because of costly and
cumbersome equipment required to perform assays
and the expertise required of the technicians who must
operate it. In order to move microarray technology
from the lab to the point-of-care, conversion of the
bulky benchtop devices to portable, automated systems
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is the key. To integrate the microarray and sample
processing systems, several major hurdles will need to
be overcome. These include the process of automating
(1) cell lysis and nucleic acid extraction, (2) reverse
transcriptase and polymerase amplification, (3) frag-
mentation and biotinylation, (4) hybridization, (5)
washing and staining, (6) reading the image, and (7)
interpreting the data. In this study, we are focusing on
steps 4 and 5.
Other research groups have addressed this automa-
tion issue for commercially produced microarrays. In
2000, a microfluidic device the size of a credit card was
demonstrated for the processing of Affymetrix Gene-
Chips (Anderson et al. 2000). While the report did
demonstrate automated hybridization and staining, the
conditions under which the operations were performed
were designed for a very specific HIV analysis. The
demonstrated hybridization took place at only 37C
and for only 20 min; it would need to be modified and
tested for broad-spectrum diagnostic applications.
Furthermore, the device was designed around Gene-
Chips that were removed from their protective hous-
ing. Handling exposed GeneChips in the field can be
difficult, especially when transferring them outside of
the device between the hybridization and scanning
steps.
More recently, a microfluidic device was designed
for use with CombiMatrix microarrays (Liu et al.
2006). This device contained electrochemical pumps,
mixers, and reagent chambers, and was small enough
to be hand-held. While this device did demonstrate
automated hybridization, washing, and staining steps, it
is not suitable for the point-of-care diagnostic appli-
cations that we envision for a number of reasons. First,
the hybridization demonstrated by Liu et al. required
18 h, which is far too long for rapid diagnostics. Fur-
thermore, significant evaporative losses were incurred
by the mixing process used in this device, resulting in
the loss of hybridization stringency and requiring the
use of a high humidity atmosphere to minimize losses.
Finally, the feature size of the CombiMatrix chips is
limited. Clearly, a need still exists for automated sys-
tems to move microarray technology from the lab to
the point-of-care.
This communication reports a small automated de-
vice, called the ‘‘hyb–wash–stain device,’’ which we
demonstrate can expedite the processing of commer-
cial microarrays. Although our module could be used
with a wide variety of commercially produced micro-
array, we chose to demonstrate it with Affymetrix
Custom Seq Resequencing Arrays designed for the
detection of upper respiratory pathogens. We note that
the published Affymetrix protocol is highly involved
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Microfluid Nanofluid (2007) 3:623–628
and represents a worst-case scenario among commer-
cial microarray processing in regards to time require-
ments and solution handling. The normal procedure
for processing Affymetrix GeneChips is as follows: (1)
Inject a sample containing biotinylated DNA and
incubate in an Affymetrix rotisserie oven for 2–16 h.
Mixing is accomplished as an air bubble tumbles across
the surface of the microarray. (2) Remove the
hybridization mixture. (3) Stain and wash using a
three-step staining procedure in an Affymetrix wash
station for 1.5 h. (4) Image on a confocal scanner.
While these protocols are acceptable for large cen-
tralized laboratories with trained technicians, they are
not appropriate for portable devices for point-of-care
diagnostics. If commercially produced nucleic acid
microarrays are to be used in portable biosensors, it
will be necessary to design miniaturized and integrated
devices that can perform all of the hybridization,
staining, and washing steps necessary in an automated
fashion. The hyb–wash–stain device described here is
capable of performing these operations on Affymetrix
Custom Seq Resequencing Arrays without evapora-
tive losses and within a reasonable timeframe for a
point-of-care biosensor. The device is small, com-
pletely automated, and has been designed for modular
integration into a portable system that includes sample
processing and nucleic acid amplification. Eppendorf
1.5 ml tubes containing sample and reagents are in-
serted into holders on the device, and the result after
processing is a hybridized and labeled microarray,
ready for scanning. Results show that hybridization
intensities and base calls are comparable to those ob-
tained on the commercially available (benchtop)
microarray processing equipment, which we chose as a
gold-standard.
2 Materials and methods
2.1 Reagents and solution compositions
Tris, Tween-20, and normal goat IgG were purchased
from Sigma (St Louis, MO, USA). SSPE (saline/sodium
phosphate/EDTA) was purchased from Ambion
(Austin, TX, USA). Phycoerythrin was supplied by
Molecular Probes (Eugene, OR, USA). Herring sperm
DNA was obtained from Promega (Madison, WI,
USA). All other reagents were purchased from Af-
fymetrix (Santa Clara, CA, USA). Pre-hybridization
buffer is a 10-mM Tris buffer at pH 7.8, with 0.01%
Tween-20. The hybridization buffer is a 10-mM Tris
buffer, with added control oligos, herring sperm DNA,
and surfactants. Wash A solution contains 6· SSPE
Microfluid Nanofluid (2007) 3:623–628
and 0.01% Tween-20. Wash B solution contains 0.6·
SSPE and 0.01% Tween-20. Stain 1 contains phycoer-
ythrin dye, while stain 2 contains a biotinylated anti-
body. All solutions were made up in nuclease free
water. Purified DNA from a field strain of adenovirus
types 4 and 5 was kindly provided by Dr Kevin L.
Russell at the Naval Health Research Center (San
Diego, CA, USA). Microarray composition and
hybridization details are described in a previous pub-
lication (Lin et al. 2007).
2.2 Experimental
The microarrays used in this study were Affymetrix
Custom Seq Resequencing Arrays designed for 20
common upper respiratory pathogens and six CDC
category A bio-threat agents. In a typical hybridization
experiment run on our hyb–wash–stain device, the chip
was activated by circulating a pre-hybridization solu-
tion for 10 min at 45C. The pre-hybridization buffer
was then removed and discarded. Next, a sample con-
taining adenovirus types 4 and 5 was introduced to fill
the resequencing GeneChip and tubing, and was
allowed to hybridize to the microarray under re-
circulating flow in the automated device for 2 h at
45C. A sample concentration of 125 pg/mL was cho-
sen because it can be realistically obtained from PCR
amplification. Finally, a series of wash and staining
steps using two separate washes and stains was per-
formed in the following order: wash A, stain 1, wash A,
wash B, stain 2, stain 1, wash A. In our hyb–wash–stain
device, each washing step uses 2 mL of buffer and lasts
for 2 min. In each staining step, the resequencing
GeneChip and tubing are filled with the stain and re-
circulating flow is introduced for 15 min. All washing
and staining steps take place at room temperature, with
the exception of wash B, which takes place at 45C.
In order to evaluate the efficiency of our hyb–wash–
stain device, hybridizations were also performed using
commercially available Affymetrix equipment. We
chose this benchtop system as a gold-standard because
it was specifically designed to optimize the perfor-
mance of Affymetrix GeneChips. GeneChips were pre-
hybridized for 10 min, and then sufficient sample was
introduced into the chip to leave an air bubble
approximately 50 lL in size. Hybridizations were per-
formed in an Affymetrix GeneChip Hybridization
Oven, Model 640 for 2 h at 45C. The overall sample
volume required for hybridization in commercial
equipment was about half that required for our hyb–
wash–stain device, since the commercial hybridization
equipment had no associated tubing that needed to be
filled. In this work, we chose to keep the DNA con-
625
centration constant between the two devices (at
125 pg/mL), although we observed in other experi-
ments that small concentration differences such as a
factor of 2 had very little effect on the hybridization
efficiency (data not shown). In cases where staining
and washing on commercially available equipment was
also desired, an Affymetrix GeneChip Fluidics Station
Model 450 was used. The sequence times and volumes
of reagents used by the different pieces of equipment
are shown in Table 1. Regardless of the hybridization
or washing equipment used, all chips were scanned on
an Affymetrix GeneChip Scanner 3000.
2.3 Device design
Our hyb–wash–stain device is shown in Fig. 1. It con-
sists of a single Instech peristaltic pump (Plymouth
Meeting, PA, USA), two commercially available
Minco foil heaters (Minneapolis, MN, USA), two Lee
Company miniature three-way valves (Westbrook, CT,
USA), and a six-way Hamilton chromatography valve
(Reno, NV, USA). All components are interfaced to a
computer via RS-232 communications using an Ontrak
Control Systems A/D board (Sudbury, Ontario,
Canada). Power is provided by a power supply from
Sunpower Technologies, USA (Union City, CA,
USA), and a custom electronics control board. Control
software written in ANSI C using Lab Windows from
National Instruments Company (Austin, TX, USA)
provided automatic timing and control of all the
components, allowing operation without user inter-
vention.
The device that holds the Affymetrix GeneChip it-
self, as well as the pump, the two Lee valves, and the
two heaters, is contained inside an insulating Plexiglas
box. The dimensions of the insulated box are
2.5† · 3† · 3.5†. The chip is inserted into a window in
the front of the box and secured using a pair of screws.
3 Results and discussion
Obtaining adequate mixing within the Affymetrix
GeneChip itself is necessary in order to minimize
hybridization times, as well as to obtain uniform
hybridization efficiency across the surface of the
microarray. Affymetrix suggests an overnight hybrid-
ization (16–24 h) in a rotisserie oven, where even
mixing is facilitated through the motion of an internal
bubble. We have found that the hybridization time can
be reduced to 4 h with good results, and in some cases,
can be made as short as 2 h (data not shown). How-
ever, the rotating mixer provided by Affymetrix would
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Table 1 A comparison of the solution volumes and circulation

times used by the commercially available equipment and our
hyb–wash–stain device
Commercial Hyb–wash–stain
equipment device

Sample volume About 250 lL About 400 lL

Wash volume About 8 mL 2 mL
Stain 1 volume 600 lL 600 lL
Stain 2 volume 600 lL 600 lL
Hybridization time 2h 2h
Wash time 7 min (·4) 2 min (·4)
Stain time 10 min (·3) 15 min (·3)
There are a total of four washing steps and three staining steps in
the protocol

be difficult to miniaturize due to the large number of

moving parts that would be required.
As an alternative to a rotisserie hybridization oven,
our hyb–wash–stain device uses a continuously re-cir-
culating fluid flow through the GeneChip to facilitate
hybridization. Although hybridizations performed un-
der fluid flow have been shown to be effective for
spotted arrays (Benn et al. 2006), our study is more
limited due to the fact that the internal chamber of the
GeneChip cannot be modified. The large internal
volume of the chip (approximately 300 lL) is partic-
ularly disadvantageous, because the sample must be
sufficiently diluted to fill both the chamber and any
accompanying tubing, which compromises hybridiza-
tion time. In addition, the internal geometry is fixed,
so that the shape of the flow profile cannot be ad-
justed.
It is important to ensure that the sample is well
mixed and flows continuously over the entire micro-
array surface, because the probes for different patho-
gens are located on different areas of the microarray.
In a well-designed flow hybridization chamber, con-
vective transport of the nucleic acids to the target sites
will result in a fast hybridization. For this reason, it is Fig. 1 a An image of our hyb–wash–stain device, with pump,
important to verify that there are no stagnant regions valves, and heaters. Not shown are electronics boards, the
six-way Hamilton chromatography valve, reagent and waste
in the flow profile under realistic experimental condi-
collection tubes, the computer interface, or the insulating
tions. Stagnant regions would rely on diffusive trans- Plexiglas box that surrounds the apparatus. b Our hyb–wash–
port of the nucleic acids to the probe sites, resulting in stain device (circled) is shown next to a commercial rotisserie
a very slow hybridization. Using fluorescence imaging, hybridization oven (top shelf) and a Affymetrix washing station
(sitting on tabletop). All components of the hyb–wash–stain
we have visualized the steady-state flow profile of the
device are present except for the laptop computer. For
sample through a GeneChip. An image of this profile is comparison, our hyb–wash–stain device has single chip capacity,
shown in Fig. 2. Observations show that flow rates are while the Affymetrix equipment can wash and stain up to four
largest along the middle of the chip in a path con- chips at once
necting the inlet (bottom center) and outlet (top cen-
ter) of the chip. However, flow does move radially tion times will be slower near the corners of the
outwards from the entrance of the GeneChip, and will microarray and faster near the center. Our experiments
eventually reaches all areas in the microarray. With demonstrate that despite this flow profile, sufficient
this type of flow profile, it is expected that hybridiza- exposure and mixing take place to produce hybridiza-

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were performed using our hyb–wash–stain device. In

Fig. 2 Flow profile visualized in an Affymetrix GeneChip. A
20-lL plug of food dye was injected into the flow over the
GeneChip and imaged using a cooled CCD camera. The edges of
the GeneChip window are highlighted with a white square for
clarification. A video of this flow profile is provided in the
supplemental information
tion results comparable to those obtained using the
commercially available rotisserie mixer.
In order to demonstrate that our hyb–wash–stain
device can duplicate the results of the commercially
available Affymetrix equipment, a series of experi-
ments was set up as follows. Each experiment involved
hybridizing targets from adenovirus types 4 and 5 to
three separate microarrays. In run A, all operations
run B, hybridization took place on the commercial
equipment, but staining and washing took place in
our hyb–wash–stain device. In run C, the entire
hybridization, staining, and wash procedure was run
on commercial equipment. This experiment was per-
formed in triplicate. A blank run was also performed
where the chip was hybridized on the commercially
available equipment, but with the sample replaced by
hybridization buffer. All hybridizations took place over
a 2-h period.
Representative images of scanned chips are pre-
sented in Fig. 3, showing that comparable regions of
the chips have been hybridized by all of the methods
described above. The average base call percentages for
each run are presented in Table 2. There were three
sets of tiles for adenovirus type 4, and two for adeno-
virus type 5. In all cases, the percentage of bases called
were comparable for the three hybridization methods,
proving that our hybridization and staining device is as
effective as commercially available equipment.
In addition to achieving comparable results, our
hyb–wash–stain device has a number of advantages
over commercial equipment. First, the device is com-
pletely integrated, so that the nucleic acid microarray
never has to be manually moved between pieces of
equipment during the protocol. This helps prevent
inadvertent contamination as well as operator mis-
takes, and makes the use of our device less labor
intensive. Furthermore, the size of our hyb–wash–stain
device is significantly smaller than that of the com-

Fig. 3 Sample images of the

region of interest of a
hybridized Affymetrix
GeneChip. Run A was
performed completely on our
hyb–wash–stain device, run B
was hybridized on
commercial equipment and
stained on our hyb–wash–
stain device, and run C was
performed completely on the
commercial system. The light
spots and streaks are areas
where nucleic acid fragments
have hybridized. The light
spots on the blank chip are
control oligos. The figure
shows that comparable
hybridizations are obtained
by each of the three methods

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Table 2 Average base call percentages for adenovirus type 4
and adenovirus type 5 tiles
Run Adenovirus type 4 Adenovirus type 5
(tile 1/tile 2/tile 3) (tile 1/tile 2)
A 37%/86%/62% 73%/91%
B 36%/88%/63% 82%/91%
C 37%/87%/64% 82%/94%
mercial equipment, which has the footprint of a typical
laboratory benchtop. Finally, our device uses smaller
amounts of the washing buffers, and washing times are
shorter. No evaporative or other losses of solution
were observed in the system during hybridization runs.
In the future, it should be possible to integrate other
steps in the Affymetrix protocol with our hyb–wash–
stain device, including an automated sample prepara-
tion step, a nucleic acid amplification step, and a device
to incorporate the fragmentation and labeling required
immediately prior to hybridization. Such a modular
device would form a complete biosensor that could be
used for surveillance applications or point-of-care
diagnostics. The software, electronics, and fluidic ele-
ments of the device were designed in such a way as to
make integration as a module into a portable system
easy.
4 Conclusions
An automated, portable device that is capable of
hybridizing, washing, and staining Affymetrix Gene-
Chips has been fabricated. The hyb–wash–stain device
is designed in a modular fashion such that it could
potentially be included as part of a larger, complete
biosensor. Experimental data show that the device is as
effective as commercially available equipment.
Acknowledgments J.S.E. was supported by a National Re-
search Council Fellowship. A.C.S. was supported by an NSF-
SEAP fellowship. We would like to thank Dr Kevin L. Russell of
NHRC for providing the adenovirus types 4 and 5 DNA that was
used in this study. This project was supported by NRL 6.2 work
unit 6006 and by the Joint Program Executive Office for
Chemical and Biodefense.
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