compiled chip for all pretreatment processes of virus gene ...€¦ · compiled chip for all...
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Compiled Chip for All Pretreatment Processes of Virus Gene Analysis
○Miyako Niimi1 Taisuke Masuda1 Kunihiro Kaihatsu2 Nobuo Kato2 Fumihito Arai11Nagoya University, JAPAN
2Osaka University, JAPAN
Contact Person：Miyako NiimiE-mail: [email protected], URL: http://www.biorobotics.mech.nagoya-u.ac.jp/TEL: 052-789-5220, FAX : 052-789-5027
Acknowledgements：This work was supported in part by the Nano-Macro Materials,Devices and System Research Alliance Project from theMinistry of Education, Culture, Sports, Science andTechnology of Japan (MEXT).
Abstract: In this research, we proposed a microfluidic chip to pretreat the samples for genetic analysis of infectious viruses. The microfluidic chip has the followingthree functions; (1) Virus purification by hydroxyapatite-packed microcolumn, (2) Viral RNA extraction by silica-packed microcolumn, and (3) Capture of the targeted virusgenome by PNA-immobilized glass substrate. Each function has been demonstrated separately using microfluidic chips.
1.Background Genetic Analysis of Infectious VirusesDNA SequencerHigh throughputDiagnosis of multiple diseases
Pretreatment of clinical sampleVirus Purification and EnrichmentViral DNA/RNA Extraction
Cumbersome processes by human hand
On-chip Sample Pretreatment2. Concept
1. Virus Purification
2. Viral RNA Extraction
3. Virus Detection (Targeted Genome Capture)
Silica
Micropillar
Sample Elution Buffer
1. Virus Purificationby Hydroxyapatite-packed Microcolumn
100 m
1. Virus Purification
PNA selectively captured influenza
A/H1N1 virus genome.
2. RNA Extraction
Hydroxyapatite
Micropillar
00.20.40.60.81.01.21.41.6
0 10 20 30 40 50Flu
ores
cenc
e S
igna
l
Cycle Number
Virus Titer [pfu/mL]104
5x103
1x103
Experimental Result
RNA Collection Rate: 70.8 %
1.Lyse a 104 pfu/mL NDV suspension2.Introduce the lysate. 3.Introduce the wash buffer.4.Introduce the elution buffer.
1.Introduce a mixture of NDV(Newcastle Disease Virus) and FBS proteins
2.Introduce 500 mM KCl to elute the FBS proteins
3.Introduce 1 M Phosphate buffer to elute the NDVs
4.Hemagglutination Reaction
Before After
1 mm
Succeeded in Removal of FBS
Virus Protein
Horseradish peroxidase (HRP)
Luminol substrate
3-aminophthalic acid dianion(Ex: 430, Em: 460 nm)
Virus genome
PNA
3. Virus Detection
3. Virus Detection
Three different functions of the microfluidic chip have been demonstrated separately. In our future work, we will integrate all the functions in one chip.
Red blood cell
Viruswithout virus with virus
Ref.) M. Niimi et al., Proc. of MHS2012, pp.12-15
3. Results
The fluorescence intensity became
stronger as the virus titer increased.F
luor
esce
nce
Inte
nsity
a.u.
Wavelength nm
0
200
400
400 450 500 550
0H1N1(Target)H3N2B
Flu
ores
cenc
e In
tens
itya.
u.
Wavelength nm
0
200
400
400 450 500 550
07.0x103
7.0x104
Virus Titer [pfu/mL]
4. Conclusion
2. Viral RNA Extraction by Silica-packed Microcolumn
Elution Buffer
Lysis Buffer
References：1. M. Niimi, et. al., 17th International Conference on Miniaturized Systems for
Chemistry and Life Sciences (Micro TAS 2013), pp. 482-484, 20132. M. Niimi, et. al., 24th Micro-NanoMechatronics and Human Science (MHS 2013),
pp. 248-249, 2013