compiled chip for all pretreatment processes of virus gene ...€¦ · compiled chip for all...

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: miyako@biorobotics.mech.nagoya-u.ac.jp, 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