Inkjet Injection of DNA Droplets for Microchannel Array Electrophoresis

Article pubs.acs.org/ac

Inkjet Injection of DNA Droplets for Microchannel Array Electrophoresis Takao Yasui,*,† Yosuke Inoue,†,¶ Toyohiro Naito,† Yukihiro Okamoto,† Noritada Kaji,† Manabu Tokeshi,†,‡ and Yoshinobu Baba†,§ †

Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, FIRST Research Center for Innovative Nanobiodevices, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan ‡ Division of Biotechnology and Macromolecular Chemistry, Faculty of Engineering, Hokkaido University, Kita 13 Nishi 8, Kita-ku, Sapporo 060-8628, Japan § Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14, Hayashi-cho, Takamatsu 761-0395, Japan S Supporting Information *

ABSTRACT: We demonstrated DNA droplets could be injected with an inkjet injector for microchannel array electrophoresis and attained high throughput analysis of biomolecules. This injection method greatly reduced both analysis time and sample amount, compared with a conventional microchip electrophoresis method, and allowed high parallelization of a microchannel array on a small substrate. Since we do not need to use complicated electric programs or microchannel design, our injection method should facilitate omics analyses and contribute to high performance clinical assays.

nkjet injectors have uses in a whole range of fields due to their desirable properties, such as droplet spatial controllability, droplet volume control (at nano- and picoliter levels of droplets), high speed, and accurate spotting. Since inkjet technology can accurately deposit very small quantities (tens of picoliters) of materials at desired spots on the surface of a wide variety of substrates, the technology has been used not only for office printing technology but also for various industrial fabrication processes. In particular, it has been used for plastic electronics1−3 and is a time-saving low-cost alternative method to photolithography4,5 because it allows direct patterning on substrates compared to other conventional multistep fabrication processes. Furthermore, inkjet printing has the potential to be an alternative arraying technique for biological samples.6,7 Although its versatility and possible use as a sensing platform have been recognized and its low fabrication cost and highly parallel mass production were achieved,8−11 the inkjet technology is still not fully used in lab-on-a-chip applications. Since conventional microchannel arrays for DNA electrophoresis have cross- or T-shaped microchannels for DNA injection,12,13 such microchannels require four reservoirs in a single chip, voltage programming (loading and separation processes), and large sample volumes for separation. These disadvantages have prevented researchers from attaining higher throughput and better ultratrace analysis results than at present. A straight microchannel is an ideal design because it requires only two reservoirs and allows simple voltage programming, which makes electrophoresis in the microchannel array straightforward. Therefore, new injection methods without the cross- or T-shaped microchannels are highly desired, and

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© 2012 American Chemical Society

electrophoresis with a straight microchannel can be enabled with the inkjet injection method. In this study, we developed DNA droplet formation with an inkjet injector for electrophoresis in microchannel array. The inkjet injector enables precise control of the injection volume of DNA samples and use of a simple straight microchannel for microchip electrophoresis. Compared with the conventional cross injection method in the cross- or T-shaped microchannels, the inkjet injector has many advantages: analysis of biomolecules in a minimum amount of samples, this is due to the extremely small volume of the droplets, such as nL (10−9 L) and pL (10−12 L); suitability for high density array of microchannels, this is due to the simple straight microchannel; ability to separate biomolecules by one voltage programming because of the inkjet injection method; and rapid analysis time because the inkjet injection method does not require any sample loading time. The injection and separation principle is shown in Figure 1a. The samples were injected into the fabricated small hole using the inkjet injector, and then, the sample was separated by electrophoresis; only 2 steps were required. Since we did not need any cross- or T-shaped microchannels and voltage programming, which are essential in the conventional microchip electrophoresis,14 we demonstrated DNA droplet injection into straight microchannels using our Received: July 20, 2012 Accepted: October 3, 2012 Published: October 3, 2012 9282

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Figure 1. (a) Illustration of inkjet injection of DNA droplets for microchannel array electrophoresis. There were two electrodes, a microchannel array, a detection part, and an inkjet injector. (b) Photograph of the PulseInjector with a cartridge. (c) Schematic illustration of the PulseInjector, which is a piezo element-based plastic drop-on-demand type inkjet head. Epoxy resin composite material was used as the structural material; this made it possible to integrate various components, such as a pressure chamber, nozzle with water repellent coating, flow channel, vibrating membrane, bracket, and PZT element into one body, resulting in superior chemical resistance. (d) Photograph of a COC microchip with three microchannels (size: 3 cm ×7 cm). (e) Magnified image of the injection ports for DNA samples; scale bar, 100 μm. The microchannel width was 100 μm. (f) Schematic diagram of a cross-sectional view of the COC microchip. The diameters of the reservoirs and injection ports were 1 mm and 100 μm, respectively. The height of the microchannels was 40 μm. Thicknesses of both the microchannel-patterned and nonpatterned COC substrate were 0.5 mm.

cm size, as shown in Figure 1d. COC microchips were fabricated using a conventional injection molding technique by Sumitomo Bakelite Co., Ltd.15 COC microchips are often employed for highly sensitive detection. The diameter of the injection ports for DNA droplets and the width of microchannel were each 100 μm (Figure 1e). The diameter of the reservoirs, the height of the microchannel, and the thickness of the patterned and nonpatterned COC substrate were 1 mm, 40 μm, and 0.5 mm, respectively (Figure 1f). DNA fragments of 10, 100, 200, 1000, and 3000 bp (NoLimits, Fermentas Inc.) were stained with bis-intercalating fluorescence dye, YOYO-1 (Invitrogen), at a dye-to-base pair ratio of 1:15 for DNA separation experiments. To dilute the concentration of all DNA fragments to 4.9 (for 10, 100, 1000, and 3000 bp) and 9.9 (for 200 bp) ng/μL, a concentrated buffer solution (1× TE; 10 mM Tris-HCl and 1 mM EDTA, pH 8.0, Nippon Gene Co., Ltd.) was used. A CCD camera (VH-D800, Keyence Corporation) equipped with an objective lens (VH-Z75, Keyence Corporation) was used to observe the dropping behaviors of DNA droplets and to capture those images. An LED device (Cluster Technology Co., Ltd.) was operated simultaneously with WaveBuilder, which led to turning on of the LED device soon after DNA droplets were generated (Figure 2a). Electrokinetic behaviors of DNA samples were simulated by the MEMS design software (CoventorWare, Coventor, Inc.). The Navier−Stokes equations, mass equation, and Poisson equation were used for the flow, mass, and electric field modeling, respectively (the details can be seen in the Supporting Information). The parameters for the simulation were as follows: the applied electric field was 160 V/cm; the electroosmotic flow mobility was 10900 μm2V−1s−1; the sample mobility was 25000 μm2V−1s−1; the substrate was COC; and the loading solution was water. Those values were obtained experimentally; 160 V/cm of the applied electric field was the actual value in the electrophoresis experiment. The electro-

novel inkjet injector for the electrophoresis in microchannel array.

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EXPERIMENTAL SECTION Inkjet injection of DNA droplets for microchannel array electrophoresis is illustrated in Figure 1a; it included the inkjet injector, two electrodes, a microchannel array, and a detection part. The piezo element-based inkjet injector was used for the DNA injector because a thermal-based inkjet injector will denature DNA molecules during the injection process. We used a PulseInjector (Cluster Technology Co., Ltd.) for the inkjet injector nozzle, a cartridge (PIJC-2CSET, Cluster Technology Co., Ltd.) to provide samples, and a WaveBuilder (Cluster Technology Co., Ltd.) for the inkjet injector. A photograph of the PulseInjector with the cartride and a schematic illustration of the PulseInjector were shown in Figure 1b,c, respectively. Epoxy resin composite material was used as the structural material for the PulseInjector which allowed integration of various components, including a pressure chamber, nozzle with water repellent coating, flow channel, vibrating membrane, bracket, and PZT element, into one body for high chemical resistance. Two platinum electrodes were inserted into the chip reservoirs. A HVS448 3000 V power supply (LabSmith) was connected to these electrodes for electrophoresis. The applied electric field was 160 V/cm. An inverted fluorescence microscope, Axivert 135TV, equipped with a 10×/0.3NA objective lens (both from Carl Zeiss) was used to observe DNA separation. Illumination for observations was provided by a 100 W mercury arc lamp (Carl Zeiss). An EB-CCD camera (C7190−43, Hamamatsu Photonics K.K.) was used to capture images of the separation process. The captured images were recorded on a DV tape (Sony) and then analyzed by image processing software (Cosmos32, Library Inc.). The microchannel was fabricated on a cyclic olefin copolymer (COC) substrate (Sumitomo Bakelite, Co., Ltd.), which was 3 cm × 7 9283

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Table 1. Comparison of the Inkjet Injection Method with the Conventional Injection Method in the PMMA Microchips inkjet injection method

conventional injection method analysis time sample amount peak width for 250 bp peak width for 1000 bp resolution between 250 and 1000 bp (Rs250−1000) number of electrodes (number of reservoirs) voltage programming a

20 pL 0.37 sb 1.66 sb 11.8b

4

2

necessary

According to Figure S2, Supporting Information. Figure S3, Supporting Information.

Figure 2. (a) Photograph of the DNA droplet observation system equipped with a CCD camera and LED device. Optical micrographs of DNA droplets ejected from the nozzle at a 1000 Hz frequency (scale bar, 100 μm), and the applied voltages of (b) 7 V and (c) 10 V. The droplets were captured at 10 ms intervals, and a sequence of photographs was investigated. The effective diameter of the nozzle was 25 μm. The droplet diameters were about 30 μm, corresponding to a 20 pL volume. (b) While the DNA droplets ejected at 7 V were stable, (c) the DNA droplets ejected at 10 V were split into two droplets; a main droplet (marked by red arrows) and a subdroplet (blue arrows). (d) Dropping velocity of DNA droplets vs applied voltage. Circles and triangles show the velocity of main droplets and subdroplets, respectively.

20 sa

a couple of hundred seconds (including sample loading time) ∼μL 0.75 sb 0.79 sb 12.1b

unnecessary b

According to

Figure 3. Sample distributions derived from numerical simulations (a) in the whole COC microchannel with separation buffer and (b) in the COC microchannel with separation buffer except the area under the injection hole; scale bar, 200 μm. Each image shows cross sections in the microchannel. As sample concentrations increased, the color was shifted from blue to red.

osmotic flow mobility (1.09 ± 0.10) × 10−4 cm2V−1s−1 was measured using the current monitoring method, as described elsewhere;16,17 the sample mobility 2.5 × 10−4 cm2V−1s−1 was calculated using the measured sample velocity 0.04 cm/s and the applied electric field 160 V/cm.

a main droplet and a subdroplet (Figure 2c). We observed the droplet formation behaviors at each applied voltage and measured droplet velocity. There was a good correlation between droplet velocity and the applied voltage (Figure 2d); the velocity was proportional to the applied voltage, but the DNA droplets generated at more than 7 V caused disruption of a droplet formation. Therefore, an applied voltage and frequency of 7 V and 1000 Hz, respectively, was used in all the experiments. Since the inkjet injection process generated surface tension wave and shear forces, which triggered the droplet formation of DNA samples from the inkjet nozzle,18 DNA molecules may be subject to be cleaved by the shear forces in the inkjet injection process.19 The DNA cleavage generated with the inkjet injection process was confirmed in Figure S1, Supporting Information. It was clear that the DNA cleavage with the inkjet injection process occurred for long DNA molecules, such as λDNA, but several thousand basepairs of DNA samples were not cleaved by the inkjet injection process. Generally, longer size DNA molecules are much easier to be cleaved than smaller size one, and in terms of the DNA cleavage, our inkjet injection method would be adopted to less than 10 or 20 kbp DNA

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RESULTS AND DISCUSSION Due to the balance between the applied voltage and the surface tension, the high applied voltage made the liquid column long to enlarge the droplet velocity, resulting in disruption of a DNA droplet formation, division into two droplets. We adjusted the applied voltage and frequency of WaveBuilder to discharge a different volume of DNA droplets from PulseInjector; the applied voltages were 7 (Figure 2b) and 10 V (Figure 2c), and the applied frequency was 1000 Hz. A sequence of optical micrographs of DNA droplets ejected from the nozzle was investigated at 10 ms intervals. Although 25 μm was the nozzle effective diameter, due to the surface tension of DNA liquids, the diameter of the DNA droplets generated was about 30 μm, corresponding to 20 pL volume. While the applied voltage of 7 V made the formation of DNA droplet stable as shown in Figure 2b, that of 10 V divided DNA droplet into two droplets: 9284

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3 as: (a) sample distributions in the whole COC microchannel with separation buffer and (b) sample distributions in the COC microchannel with separation buffer except the area under the injection hole. On the basis of the simulations that the separation buffer was introduced into the whole microchannel (Figure 3a), samples floated onto the separation buffer and only a small amount of injected samples was loaded onto the microchannel wall under the applied electric field. On the other hand, when the separation buffer was introduced into the whole microchannel except the area under the injection hole (Figure 3b), almost all the injected samples were loaded into the microchannel for separation. We concluded our injection method was valid for separating DNA samples when a separation buffer was introduced in the whole microchannel except the area under the injection hole. The potential of our injection method for high throughput analysis was performed through the separation of DNA samples in a microchip with three straight microchannels in Figure 4. We fabricated a microchip with three straight microchannels (Figure 1d) on a COC substrate; the distance between two microchannels was 100 μm. Each channel in the microchip had two sample reservoirs at both ends and one sample injection hole between the reservoirs (Figure 1e). DNA samples were injected into the sample hole with the inkjet injector. For the high throughput analysis, we attached the DNA injector on a stage equipped with a stepping motor (Sigma Koki, Co., Ltd.). The DNA injector was operated simultaneously with the stepping motor, and DNA samples were injected at equal intervals of 50 μm when we set the velocity of the stepping motor to 83.3 μm/s (Figure S4, Supporting Information). Thus, we increased the velocity of the stepping motor to 166.7 μm/s for the simultaneous separation in the microchip, which had 100 μm distances between two microchannels. Separation results of the mixture of DNA samples in the microchip are shown in Figure 4, and the comparison of the plug width between experimental and simulated results are shown in Figure S5, Supporting Information. DNA samples in three microchannels were simultaneously separated and detected within 30 s, and the plug width in the experimental result showed a good agreement with that in the simulated one. In Table 2, we summarized the reproducibility of migration time and resolution for the three microchannels. These results indicated that high reproducibility was achieved, and thus, our injection approach had good potential for realizing versatile applications to the high throughput analysis of biomolecules (Figure S6, Supporting Information).

Figure 4. Simultaneous separation of the mixture of DNA samples, 10 bp (4.9 ng/mL), 100 bp (4.9 ng/mL), 200 bp (9.9 ng/mL), 1000 bp (4.9 ng/mL), 3000 bp (4.9 ng/mL), in the COC microchip with three microchannels. Two nL of the mixture was injected with the inkjet injector into the microchannels which were filled with 0.2% methylcellulose in 0.5× TBE buffer. Red, blue, and black electropherograms were taken at the separation length of 1 cm in channels 1, 2, and 3, respectively, at the applied electric field of 160 V/cm. An inset showed the magnified electropherogram of channel 1 from 18 to 23 s.

molecules (approximately), which could not be cleaved by the shear forces. Our injection method (Figure 1a) did not require any complex microchannels and sample loading processes, and small enough samples could be detected quantitatively (∼20 pL) in Figure S2, Supporting Information, which meant that using our developed inkjet injection approach could greatly reduce sample amount, compared to the conventional analysis method. We summarized qualitative or quantitative differences in separation performance (Figure S3, Supporting Information) and characteristics between inkjet injection method and conventional injection method in Table 1. Besides reducing sample amount, our injection method could significantly shorten analysis time due to the lack of sample loading time. Resolution in the inkjet injection method slightly degraded because of relatively wider peak width for 1000 bp in the inkjet injection. Moreover, the method using the inkjet injection could reduce the number of electrodes (or reservoirs) and eliminate voltage programming. In order to investigate the sample loading process, the sample distribution of vertical cross-sectional views was investigated by numerical simulation (the details can be seen in the Supporting Information). The simulation results are displayed as cross-sectional views of the microchannel in Figure

Table 2. Reproducibility of Migration Time and Resolution for the Three Microchannelsa DNA size/bp migration time/s

resolution

a

channel 1 channel 2 channel 3 interchannel deviation channel 1 channel 2 channel 3 interchannel deviation

10 20.3 20.2 19.4 20.0

± ± ± ±

0.4 0.3 0.2 0.5 Rs10−100 0.4 0.9 0.9 0.7

± ± ± ±

100 20.4 20.8 19.9 20.4

0.1 0.2 0.1 0.3

± ± ± ±

200 21.2 21.8 21.0 21.3

0.4 0.5 0.2 0.5

Rs100−200 1.6 1.2 1.3 1.4

± ± ± ±

1.0 0.4 0.2 0.2

± ± ± ±

0.9 0.5 0.2 0.4

1000 25.0 25.0 24.3 24.8 Rs200−1000 2.2 2.9 2.4 2.5

± ± ± ±

0.3 0.3 0.1 0.4

± ± ± ±

0.9 1.1 0.2 0.4

3000 29.9 ± 1.5 28.9 ± 1.4 27.9 ± 0.5 28.9 ± 1.0 Rs1000−3000 2.0 2.4 1.8 2.1

± ± ± ±

0.1 0.4 0.3 0.3

Each standard deviation was obtained by three consecutive runs. 9285

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(13) Yasui, T.; Kaji, N.; Ogawa, R.; Hashioka, S.; Tokeshi, M.; Horiike, Y.; Baba, Y. Anal. Chem. 2011, 83, 6635−6640. (14) Jacobson, S. C.; Hergenroder, R.; Koutny, L. B.; Warmack, R. J.; Ramsey, J. M. Anal. Chem. 1994, 66, 1107−1113. (15) Geschke, O.; Klank, H.; Tellemann, P. Microsystem engineering of lab-on-a-chip devices; Wiley-VCH: Weinheim, 2004. (16) Huang, X. H.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1988, 60, 1837−1838. (17) Yasui, T.; Kaji, N.; Mohamadi, M. R.; Okamoto, Y.; Tokeshi, M.; Horiike, Y.; Baba, Y. ACS Nano 2011, 5, 7775−7780. (18) Homma, S.; Akimoto, K.; Koga, J.; Matsumoto, S. J. Chem. Eng. Jpn. 2007, 40, 920−927. (19) Adam, R. E.; Zimm, B. H. Nucleic Acids Res. 1977, 4, 1513− 1537.

CONCLUSIONS We demonstrated DNA droplets could be injected with an inkjet injector for microchip-based electrophoresis. Quantitative and accurate injection of DNA samples was possible with the adjustment of the applied voltage and frequency. Our inkjet injector eliminated the conventional sample loading process, allowed quantitative injection of DNA droplets, and simplified the voltage programming. Therefore, our inkjet injector, which needs only an ultrasmall amount (20 pL), will provide high throughput analysis for easy (one step) analysis using multiple inkjet nozzles and highly parallelized microchannels in a microchip, compared to the conventional method.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text and Figures S1−S6. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Fax: +81-52-789-4666. Tel: +81-52-789-4611. E-mail: yasui@ apchem.nagoya-u.ac.jp. Present Address ¶

Analytical Science Research Laboratories, Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-3497, Japan.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was partially supported by the Japan Society for the Promotion of Science (JSPS) through its “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program)”. Thanks are extended to Cluster Technology Co., Ltd. for technical support.

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