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MICROFLUIDIC MICROARRAY ASSEMBLY AND ITS
APPLICATIONS TO MULTI-SAMPLE DNA
HYBRIDIZATION
Xing Yue (Larry) Peng 1, 2, Paul C.H. Li 1, Lin Wang 1, Hua-Zhong (Hogan) Yu 1,
Ash M. Parameswaran 1, Wa Lok (Jacky) Chou 1
1
Simon Fraser University, CANADA, 2Xiamen University, CHINA
ABSTRACT
A microfluidic microarray assembly (MMA) concept has been developed for multi-sample DNA
hybridization. Both the creation of the microarray and the hybridization of DNA samples are achieved by
microfluidic flow in microchannels without the robotic spotting procedure. By centrifugal force pumping in
CD-like chips, 96 samples were tested simultaneously and the hybridization process could be performed in
3 minutes. The detection limit is down to one femtomole of oligonucleotide.
Keywords: DNA microarray; Spiral microchannels; Centrifugal pumping; Multi-sample DNA
hybridization.
1. INTRODUCTION
Currently, in the use of microarrays, only one sample can be applied on a microarray slide at a time [1].
However, in analysis such as clinical diagnostics, multiple samples were usually involved, and tens of
thousands of DNA probes may not always be needed. Therefore, a MMA method has been developed to create
a DNA probe microarray as well as to apply it to multiple DNA samples. In this concept, there were two
channels plates fabricated with polydimethylsilioxane (PDMS) while an aldehyde glass chip served as the
microarray substrate (Figure 1). In the first step, channel plate 1 was assembled with the glass chip via
reversible bonding between PDMS and glass. Aminated DNA probes were introduced into the microchannels
and were immobilized on the glass chip. A line microarray was thus created. After peeling off plate 1, channel
Peel off PDMS
channel plate 1
1) Sealed against an
aldehyde glass chip
2) Flow of aminated
DNA probe solution
6-Line microarray of DNA
probes immobilized on glass chip
Channel plate 1
with 6 microchannels
1) Sealed against the glass
chip with line microarray
1) Peel off PDMS
channel plate 2
2) Flow of DNA
sample solution
2) Scan with confocal
fluorescence scanner
Channel plate 2
with 6 microchannels
Hybridization with
probes in microchannels
Detection results
Figure 1. Schematic diagram for the MMA concept
plate 2 was then assembled with the same glass chip. The sample solution flowed through the line microarray
and hybridization could be fulfilled in a few minutes. Both the creation of probe microarray and hybridization
process in a microfluidic channel was capable of reducing the sample volume (down to 1.0 µL) and of
preventing from evaporation and cross-contamination. Based on the MMA concept, CD-like plates were
developed further to facilitate liquid transport as well as the rapid removal of non-specifically adsorbed
materials within the channels. Centrifugal pumping was used for liquid transport in a similar manner as other
research groups [2, 3]. What is new here is the two-time use of centrifugal pumping, first along radial
channels and then along spiral channels. As shown in Figure 2, 96 RADIAL and SPIRAL microchannels were
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cast on plate 1 and plate 2, respectively. The spiral pattern was specially designed to balance the centrifugal
force and viscous drag force to ensure a steady flow of sample solution. At room temperature, it has been
demonstrated that one femotomole DNA (1nM, 1nL) could be detected in three minutes. This concept allows
a much higher density than attained using parallel channels [4].
Figure 2. PDMS channel plates 1 and 2 each containing 96 channels
2. EXPERIMENTAL
Two aminated single-stranded DNA (AD6: H2N-CGCCGATGGACAAAACTTAAA; AB1:
H2N-CGCCAGAGAATACCAAAACTC) as probes and their complementary single-stranded DNA (D6' and
B1') labeled with fluorescein or Cy5 as samples were synthesized and purified by Sigma-Genosys. The glass
slides or wafers were chemically modified to give aldehyde surfaces. PDMS plates were made by molding on
silicon wafers with 96 microchannels of 100 µm width and 20 µm depth. The optimization of immobilization
and hybridization conditions was performed on glass slides. The fluorescent signal was then detected with a
scanner (Typhoon 9410). When using CD-like chips, channel plate 1 was assembled first on the glass wafer
and spun at 500 RPM. Solutions of different DNA probes flowed through the 96 RADIAL microchannels to
initiate the immobilization. After removing channel plate 1 and assembling the channel plate 2, different DNA
samples were applied into the 96 SPIRAL microchannels at 1800 RPM to complete the hybridization
reactions.
3. RESULTS AND DISCUSSION
Replicates of sample DNA
concentration: 1 µM D6'-F
50 µM
20 µM
10 µM
5 µM
Replicates of 30 µM of
probe DNA (AD6)
Probe Concentration
100 µM
1 µM DNA sample (D6'-F) solution
Without SDS
With 0.15% SDS
2 µM
Figure 3. Effect of DNA probe (AD6) conc.
Figure 4. Effect of SDS in sample solution
Replicates of
30µM of probe
DNA (AD6)
As shown in Figure 3, different concentrations of AD6 probes were immobilized on an aldehyde glass chip
(with the first channel plate) for hybridizing with 1 µM of D6'-F. The use of 20 µM of immobilized probe
DNA was found to be enough to achieve the hybridization results.
Hybridization for 3 min Hybridization for 45 min
The effect of sodium dodecylsulfonate (SDS) on hybridization
was also studied. From Figure 4 it can be seen that the 0.15%
SDS in sample solution can effectively prevent non-specific
adsorption of DNA samples to the glass substrate. Without SDS,
streak of D6'-F was seen along the vertical channels due to
non-specific adsorption. Figure 5 shows the effect of
1
0.1 0.01
1
0.1 0.01
Sample DNA (D6'-F ) concentration (µM)
hybridization time on the signal intensity of microarray detection.
Figure 5. Effect of hybridization time
A hybridization time of 3 minutes is enough to achieve results
for 3 different sample concentrations. Full 96-probe-96-sample
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hybridization was achieved on a CD-like glass chip. The circular microarray generated by the MMA is
mathematically transformed into a 96×96 rectangular array (Figure 6). Samples with 2 different volumes
and 8 concentrations were tested. The signal increased with the sample volume. Specific hybridization can
be seen both at reduced image sensitivity (bottom inset) and enhanced image sensitivity (top inset).The
detection limit was one femtomole of oligonucleotide, that is, both 1µL of 1 nM DNA sample and 10 µL of
0.1 nM DNA sample can be detected.
A: amino CGCCGATGGACAAAACTTAAA.
B: amino CGCCAGAGAATACCAAAACTC.
A': The complementary of A labeled by Cy5.
B': The complementary of B labeled by Cy5.
Figure 6. The fluorescent image of the hybridization results on CD-like chips
5. CONCLUSIONS
In the MMA method, both probe immobilization and DNA hybridization were achieved within microchannels.
No expensive probe spotting equipment is needed and a low-volume (1 µL) of samples can be applied without
a lengthy incubation step. Compared with the routine microfluidic pumping methods (pressure-driven flow,
electroosmotic flow), centrifugal pumping is easy to implement simply by spinning the CD-like chip at a high
speed. 96 samples could thus be detected simultaneously with a detection limit of one femtomole.
REFERENCES
[1] G. H. W. Sanders and A. Manz, Trends in Analytical Chemistry, 2000, 19, 364-378.
[2] M. J. Felton, Modern Drug Discovery, 2003, 6(11), 35-39.
[3] G. Jia, K. Ma, J. Kim, S. K. Deo, S. Daunert, R. Peytavi, M. G. Bergeron, J.V. Zoval and M.J. Madou,
Proceedings of SPIE International Symposium-Photonics Europe, April 26-30, 2004, Strasbourg,
France.
[4] R. H. Liu, H. Chen, K. R. Luehrsen, D. Ganser, D. Weston, J. Blackwell and P. Grodzinski, MEMS 2001.
The 14th IEEE International Conference, January 21-25, 2001, 439 - 442
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