VIRUS PURIFICATION, RNA EXTRACTION, AND TARGETED GENOME CAPTURE IN ONE CHIP Miyako Niimi1*, Taisuke Masuda1, Kunihiro Kaihatsu2, Nobuo Kato2, and Fumihito Arai1 1Nagoya University, JAPAN and 2Osaka 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. (1) Hydroxyapatite chromatography, Boom method, and PNA (2) (Peptide Nucleic Acid) were used for the above three (3) 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. KEYWORDS: Virus purification, RNA extraction and detection, Infectious disease diagnosis INTRODUCTION For the purpose of diagnosing the infectious diseases Figure 1. Concept of the microfluidic chip. The microflu- quickly and accurately, DNA sequencers for gene analysis idic chip consists of the three parts: (1) hydroxyapatite- of infectious viruses have been developed rapidly. The packed microcolumn for virus purification, (2) silica- latest DNA sequencers can treat the massive numbers of packed microcolumn for viral RNA extraction, and (3) samples such as saliva and nasal at one time. However, it PNA immobilized glass for capture of the targeted virus is necessary to purify and enrich the virus and extract the genome. viral RNA in the sample as the pretreatments before gene (1) (2) analysis. Hydroxyapatite chromatography[1] have been Sample Elution Buffer used extensively for purification and fractionation of Hydroxy- various biochemical substances such as protein and virus. apatite Viral RNA is specifically adsorbed to silica when the Protein solution contains chaotropic agent such as guanidine salt. Virus 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 (3) (4) kinds of commercial kit for RNA extraction. However, Lysis Buffer both of hydroxyapatite chromatography and conventional commercial kit for RNA extraction require the large and Silica expensive equipments. They also need cumbersome RNA 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. Elution Buffer In this research, we demonstrated a microfluidic chip Figure 2. The each process carried out in the microfluidic to pretreat the samples. The microfluidic chip utilizes two chip. (1)Introduce the sample into the hydroxyapatite- microcolumns; one is a hydroxyapatite-packed packed microcolumn. (2)Elute the impurities such as pro- microcolumn for virus purification and the other is a teins in the sample. (3)Introduce the lysis buffer into the silica-packed microcolumn for viral RNA extraction. hydroxyapatite-packed microcolumn so that the lysate is Furthermore PNA[3] immobilized on the glass is integrated introduced into the silica-packed microcolumn. on the microfluidic chip to detect the targeted virus genome (4)Introduce the elution buffer into the silica-packed so that in situ virus detection would be possible by anybody, microcolumn to extract the viral RNA. anywhere, anytime. 978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 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. Virus genome 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. PNA 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 Virus Protein purified viruses are subsequently introduced into the Luminol Substrate silica-packed column for viral RNA extraction by Boom HRP nucleic acid extraction method. The extracted viral RNAs 3-aminophthalic acid dianion (Ex; 430 nm, Em; 460 nm) are subsequently introduced into the detection port and the targeted virus genomes are captured by PNA as Figure 3. Concept of capture and detection of the tar- shown in figure 3. PNA is an RNA/DNA mimic in which geted virus genome by PNA. Virus genome is captured the phosphate deoxyribose backbone is replaced by a by PNA immobilized on the glass. The HRP is ad- neutral amide backbone composed of N-(2-aminoethyl) sorbed to the viral protein by antigen-antibody reac- glycine linkage as shown in figure 4. Base pairing by tion. The Luminol substrate is captured by the HRP PNAs is not affected by intrastrand electrostatic and 3-aminophthalic acid dianion is generated. 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 3- aminophthalic acid dianion is generated. The supernatant is collected and the fluorescence intensity of 3- aminophthalic 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 Figure 4: Structure of PNA. PNA is an RNA/DNA mimic in was produced by replica molding using a master mold which the phosphate deoxyribose backbone is replaced by a fabricated by photolithography. The negative-type neutral amide backbone composed of N-(2-aminoethyl) gly- photoresist (SU-8 3050, Kayaku Microchem, Co., Ltd.) was spin coated on the silicon substrate. After prebaking, cine linkage ultraviolet light was exposed through a photomask to 1. SU-8 Coating 3. Development 6. Assembly produce a microchannel pattern using a mask aligner. After exposure, the substrate was developed and rinsed. SU-8 Then the PDMS was molded by patterned substrate. 4. Molding Plasma Finally, the PDMS microchannel and the substrate were Si-Wafer bonded by air plasma. The height of channel was 100 m. PDMS Figure 6 shows the both of the two microcolumns. In the 2. Exposure upstream and downstream parts of the microcolumns, the Heating cylindrical micropillars 50 m in diameter were included UV 5. Removing 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 Figure 5: Fabrication process of the proposed microfluidic diameter and the silica particles 30 m in diameter can be chip. The PDMS microchannel was produced by replica hold in the microculumn. molding using a master mold fabricated by photolithogra- Targeted genome capture by PNA was demonstrated phy. 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 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 Hydroxyapatite Silica A/H1N1, A/H3N2, and B were applied to the PNA immobilized glass substrate to demonstrate the specificity of the designed PNA. Micropillar Micropillar 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 be- came stronger as the virus titer increased. And in figure 8, Both of the columns were fabricated using it was found that PNA selectively captured influenza photolithography. A/H1N1 virus genome. while it didn’t capture influenza A/H3N2 and influenza B virus. From these results, it was 400 Virus titer [pfu/mL] suggested that PNA can diagnose the virus titer and subtype. 0 7.0x103 CONCLUSION 4 In this research we proposed a microfluidic chip for 7.0x10 the pretreatment of samples before gene analysis. All of the pretreatment processes for virus gene analysis can be [a.u.] carried out in one microfluidic chip. With our microfluid- ic chip, it would be possible to detect the virus genome in bodily fluid in situ. If several kinds of PNA that capture Fluorescence intensity the representative virus genome such as influenza A, in- 0 fluenza B, and norovirus is immobilized on the glass sub- 400 450 500 strate, parallel diagnosis for different diseases would be Wavelength [nm] possible. Therefore the rapid diagnosis of infectious dis- ease 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 extract- ed viral RNA using DNA sequencers. We are sure that 400 rapid, easy, and accurate diagnosis of infectious diseases would be possible using a combination of our microfluid- 0 ic chip and DNA sequencers. H1N1 ACKNOWLEDGEMENTS H3N2 This work was supported in part by the Management [a.u.] Expenses Grants for National Universities Corporations B from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT). Fluorescence intensity 0 REFERENCES 400 450 500 [1] “Virus Detection by On-chip Hydroxyapatite Chro- Wavelength [nm] matography”, M. Niimi et al., Proc. of MicroTAS, 605 (2011) Figure 8.