Download virus purification, rna extraction, and targeted genome capture in

VIRUS PURIFICATION, RNA EXTRACTION,
AND TARGETED GENOME CAPTURE IN ONE CHIP
Miyako Niimi1*, Taisuke Masuda1, Kunihiro Kaihatsu2, Nobuo Kato2, and Fumihito Arai1
1
Nagoya University, JAPAN and
2
Osaka University, JAPAN
ABSTRACT
In this research, we demonstrated a microfluidic chip to
pretreat the samples for viral genome assay. The
microfluidic chip has the following three functions; (1)
Virus purification and enrichment, (2) Viral RNA
extraction, and (3) Capture of the targeted virus genome.
Hydroxyapatite chromatography, Boom method, and PNA
(Peptide Nucleic Acid) were used for the above three
functions, respectively. These three functions were
integrated in one chip. Furthermore PNA immobilized on
the glass can detect the targeted virus genome so that in situ
virus detection would be possible by anybody, anywhere,
anytime.
(1)
(2)
(3)
KEYWORDS: Virus purification, RNA extraction and
detection, Infectious disease diagnosis
INTRODUCTION
For the purpose of diagnosing the infectious diseases
quickly and accurately, DNA sequencers for gene analysis
of infectious viruses have been developed rapidly. The
latest DNA sequencers can treat the massive numbers of
samples such as saliva and nasal at one time. However, it
is necessary to purify and enrich the virus and extract the
viral RNA in the sample as the pretreatments before gene
analysis. Hydroxyapatite chromatography[1] have been
used extensively for purification and fractionation of
various biochemical substances such as protein and virus.
Viral RNA is specifically adsorbed to silica when the
solution contains chaotropic agent such as guanidine salt.
The adsorbed RNA can be eluted by low-salt buffer such
as nuclease-free water. This method is called Boom
method[2] and has been used very extensively in many
kinds of commercial kit for RNA extraction. However,
both of hydroxyapatite chromatography and conventional
commercial kit for RNA extraction require the large and
expensive equipments. They also need cumbersome
processes by human hand. Thus it takes very long time to
complete the pretreatment processes for viral gene
analysis although the throughput of DNA sequencers have
been getting higher and higher.
In this research, we demonstrated a microfluidic chip
to pretreat the samples. The microfluidic chip utilizes two
microcolumns; one is a hydroxyapatite-packed
microcolumn for virus purification and the other is a
silica-packed microcolumn for viral RNA extraction.
Furthermore PNA[3] immobilized on the glass is integrated
on the microfluidic chip to detect the targeted virus genome
so that in situ virus detection would be possible by anybody,
anywhere, anytime.
978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001
Figure 1. Concept of the microfluidic chip. The microfluidic chip consists of the three parts: (1) hydroxyapatitepacked microcolumn for virus purification, (2) silicapacked microcolumn for viral RNA extraction, and (3)
PNA immobilized glass for capture of the targeted virus
genome.
(1)
Sample
(3)
Lysis Buffer
(2)
Elution Buffer
Hydroxyapatite
Protein
Virus
(4)
Silica
RNA
Elution Buffer
Figure 2. The each process carried out in the microfluidic
chip. (1)Introduce the sample into the hydroxyapatitepacked microcolumn. (2)Elute the impurities such as proteins in the sample. (3)Introduce the lysis buffer into the
hydroxyapatite-packed microcolumn so that the lysate is
introduced into the silica-packed microcolumn.
(4)Introduce the elution buffer into the silica-packed
microcolumn to extract the viral RNA.
482
17th International Conference on Miniaturized
Systems for Chemistry and Life Sciences
27-31 October 2013, Freiburg, Germany
THEORY
Figure 1 shows the concept of our microfluidic chip.
The microfluidic chip consists of the three parts;
(1)hydroxyapatite-packed column for virus purification,
(2)silica-packed column for viral RNA extraction, and
(3)PNA-immobilized glass for capture of virus genome.
Figure 2 shows the each process carried out in the
microfluidic chip. The sample is introduced into the
hydroxyapatite-packed column for virus purification. The
impurities such as proteins are removed and the viruses
are purified by hydroxyapatite chromatography. The
purified viruses are subsequently introduced into the
silica-packed column for viral RNA extraction by Boom
nucleic acid extraction method. The extracted viral RNAs
are subsequently introduced into the detection port and
the targeted virus genomes are captured by PNA as
shown in figure 3. PNA is an RNA/DNA mimic in which
the phosphate deoxyribose backbone is replaced by a
neutral amide backbone composed of N-(2-aminoethyl)
glycine linkage as shown in figure 4. Base pairing by
PNAs is not affected by intrastrand electrostatic
repulsion and occurs with high affinity and enhanced
rates of association with strict sequence specificity. As
shown in figure 3, horseradish peroxidase(HRP) is
adsorbed to the viral protein by antigen-antibody reaction.
Luminol substrate is captured by the enzyme and 3aminophthalic acid dianion is generated. The supernatant
is collected and the fluorescence intensity of 3aminophthalic acid dianion is measured by the
fluorescence spectrometer.
EXPERIMENTAL
The proposed microfluidic chip consists of a PDMS
(polydimethylsiloxane) microchannel and a PDMS
substrate. Figure 5 shows the fabrication process of the
PDMS microchannel and assembly method of the
microchannel and the substrate. The PDMS microchannel
was produced by replica molding using a master mold
fabricated by photolithography. The negative-type
photoresist (SU-8 3050, Kayaku Microchem, Co., Ltd.)
was spin coated on the silicon substrate. After prebaking,
ultraviolet light was exposed through a photomask to
produce a microchannel pattern using a mask aligner.
After exposure, the substrate was developed and rinsed.
Then the PDMS was molded by patterned substrate.
Finally, the PDMS microchannel and the substrate were
bonded by air plasma. The height of channel was 100 m.
Figure 6 shows the both of the two microcolumns. In the
upstream and downstream parts of the microcolumns, the
cylindrical micropillars 50 m in diameter were included
to hold the hydroxyapatite particles and silica particles in
the column. The distance between the micropillars was
20 m so that the hydroxyapatite particles 40 m in
diameter and the silica particles 30 m in diameter can be
hold in the microculumn.
Targeted genome capture by PNA was demonstrated
using a glass substrate on which PNA was immobilized.
The PNA base sequence was designed to capture
influenza A/H1N1 virus genome selectively. Three
samples 0, 7.0x103, 7.0x104 pfu/mL in virus titer were
Virus genome
PNA
Virus Protein
Luminol Substrate
HRP
3-aminophthalic acid dianion
(Ex; 430 nm, Em; 460 nm)
Figure 3. Concept of capture and detection of the targeted virus genome by PNA. Virus genome is captured
by PNA immobilized on the glass. The HRP is adsorbed to the viral protein by antigen-antibody reaction. The Luminol substrate is captured by the HRP
and 3-aminophthalic acid dianion is generated.
Figure 4: Structure of PNA. PNA is an RNA/DNA mimic in
which the phosphate deoxyribose backbone is replaced by a
neutral amide backbone composed of N-(2-aminoethyl) glycine linkage
1. SU-8 Coating
3. Development
6. Assembly
SU-8
Si-Wafer
4. Molding
Plasma
PDMS
2. Exposure
UV
5. Removing
Heating
Figure 5: Fabrication process of the proposed microfluidic
chip. The PDMS microchannel was produced by replica
molding using a master mold fabricated by photolithography.
483
applied to the PNA immobilized glass substrate to
demonstrate the effect of the virus titer on detection
sensitivity. Furthermore the virus genomes of influenza
A/H1N1, A/H3N2, and B were applied to the PNA
immobilized glass substrate to demonstrate the specificity
of the designed PNA.
Hydroxyapatite
Silica
Micropillar
Micropillar
Fluorescence intensity
[a.u.]
RESULTS AND DISCUSSION
Figure 7 and figure 8 show the results of capture of
100 m
influenza virus genome by PNA. The fluorescence inten- Figure 6: Left: Hydroxyapatite-packed microcolumn.
sity was measured by the fluorescence spectrometer. In
Right: Silica-packed microcolumn.
figure 7, it was found that the fluorescence intensity beBoth of the columns were fabricated using
came stronger as the virus titer increased. And in figure 8,
it was found that PNA selectively captured influenza photolithography.
A/H1N1 virus genome. while it didn’t capture influenza
Virus titer [pfu/mL]
A/H3N2 and influenza B virus. From these results, it was
400
0
suggested that PNA can diagnose the virus titer and subtype.
Fluorescence intensity
[a.u.]
7.0x103
CONCLUSION
In this research we proposed a microfluidic chip for
7.0x104
the pretreatment of samples before gene analysis. All of
the pretreatment processes for virus gene analysis can be
carried out in one microfluidic chip. With our microfluidic chip, it would be possible to detect the virus genome in
bodily fluid in situ. If several kinds of PNA that capture
the representative virus genome such as influenza A, in0
fluenza B, and norovirus is immobilized on the glass sub450
400
500
strate, parallel diagnosis for different diseases would be
Wavelength [nm]
possible. Therefore the rapid diagnosis of infectious disease would be possible. Furthermore, even though the all Figure 7. Results of capture of influenza virus genome
PNA immobilized on the microfluidic chip don’t capture by PNA. The fluorescence intensity became stronger as
the virus genome in a sample and infectious cause cannot the virus titer increased.
be identified, it would be possible to analyze the extracted viral RNA using DNA sequencers. We are sure that
400
rapid, easy, and accurate diagnosis of infectious diseases
0
would be possible using a combination of our microfluidic chip and DNA sequencers.
H1N1
ACKNOWLEDGEMENTS
This work was supported in part by the Management
Expenses Grants for National Universities Corporations
from the Ministry of Education, Culture, Sports, Science
and Technology of Japan (MEXT).
REFERENCES
[1] “Virus Detection by On-chip Hydroxyapatite Chromatography”, M. Niimi et al., Proc. of MicroTAS,
605 (2011)
[2] “Rapid and simple method for purification of nucleic
acids”, R. Boom et al., J. Clin. Microbiol.,
28(3):495 (1990)
[3] “Recognition of Chromosomal DNA Review by
PNAs”, K. Kaihatsu et al., Chemistry & Biology,
Vol. 11, 749 (2004)
H3N2
B
0
400
450
500
Wavelength [nm]
Figure 8. Results of capture of influenza virus genome
by PNA. PNA selectively captured influenza A/H1N1
virus genome.
CONTACT
*M. Niimi, tel: +81-52-789-5220
[email protected]
484