MICROFLUIDIC PARALLEL STRETCHING AND STAMPING OF …
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MICROFLUIDIC PARALLEL STRETCHING AND STAMPING OF SINGLE DNA MOLECULES FOR SUPER RESOLUTION MICROSCOPE IMAGING Hirotoshi Yasaki 1, 2* , Daisuke Onoshima 2 , Takao Yasui 1, 2 , Toyohiro Naito 3 , Noritada Kaji 1, 2 , and Yoshinobu Baba 1, 2, 4 1 Department of Applied Chemistry, Nagoya University, JAPAN, 2 FIRST Research Center for Innovative Nanobiodevices, Nagoya University, JAPAN, 3 Department of Material Chemistry, Kyoto University, JAPAN, and 4 Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), JAPAN ABSTRACT We developed a method for making high-density arrayed DNA molecules on PDMS and glass substrates. Arrayed DNA could be applied to various observation methods, and were elongated and immobilized on cover plates without any modification to DNA molecules. The depth, size and shape of the devices were optimized. It enables researchers to make such a high-density array of DNA molecules in a simple way. KEYWORDS DNA Immobilization, DNA Array, Molecular Combing INTRODUCTION Super resolution imaging such as stimulated emission depletion (STED) microscopy has a great potential for exploring base sequence and epigenomic change of single DNA molecule [1]. In order to detect a signal variation caused by sequence specific dye binding or partial melting, it is crucial that the DNA molecules are arrayed in a parallel direction inside the narrow microscopic field. Since the observation area becomes small accompanied with the magnification increases in microscope, throughput for the number of analyzed DNA molecules relatively decreases. However, the previous methods for the DNA arrangement do not satisfy the demand. One of DNA elongation methods for DNA imaging of single molecule is using nonspecific adsorption of DNA molecules on a glass substrate [2]; glass substrate is picked up from DNA solution, and then DNA molecules are adsorbed and elongated. Other method is using single-terminal immobilizing of DNA molecule by chemical modification, such as a biotin-avidin binding [3]; DNA molecules are immobilized on single-terminal and elongated by flow. Former method is simple, but the positions of elongated DNA molecules are random and undesirable cleavage of DNA molecules occurs easily. Latter method can make a lot of arrayed DNA molecules because this method can define the starting point of DNA elongating, but the way to make single terminal immobilization of DNA molecules is relatively difficult and troublesome. Here we show that single DNA molecules can be stretched and arrayed on recessed grooves located in the center of a microfluidic channel [4]. The arrayed DNA molecules can be also transferred to the other substrate surface. The demonstrated combination of the DNA elongation and stamping enables a simultaneous imaging of DNA molecules. EXPERIMENTAL We used lambda DNA (48502 bp), which was stained with YOYO-1 intercalating dye at a 1:5 dye per base pair ratio for overnight in the dark at 4 ºC. The stained DNA solution was diluted to 20 ng/µL, 10 ng/µL, and 5 ng/µL with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The microfluidic device of polydimethylsiloxane (PDMS) consists of two parts; one is a microchannel part for flowing the YOYO-1 stained DNA molecules, and the other is a microstructure part for elongating and immobilizing the DNA molecules. The two parts were bonded each other without any plasma treatment. This was because we peeled off the channel part to observe DNA molecules after elongation and immobilization process. The microstructure part has a zigzag-shaped recessed groove (Fig. 1a). The pattern was made from line cavity and triangle structure at the edge of line. Those two PDMS parts was transferred from a SU-8 (negative photoresist) mold on silicon substrate. The PDMS and curing agent were mixed at a ratio of 10 to 1, poured onto the silicon substrate, and cured for 2 hours at 80 ºC. RESULTS and DISCUSSION The recessed grooves trap DNA molecules during the flowing process by a syringe pump, and then the DNA molecules are stretched and immobilized at the edge of the zigzag structure on the PDMS without any modification (Fig. 1c). The DNA molecules in grooves are forced to move and concentrate at the top of the zigzag structure. One of the DNA molecules in the solution is occasionally trapped, elongated, and immobilized when the DNA solution passes through the groove structure. The PDMS microstructure with the DNA molecules was peeled off and placed onto cover plates for the following microscopic observation. The arrayed DNA molecules were observed by using total internal reflection fluorescence (TIRF) microscope (Fig. 2a) and STED microscope (Fig. 2b). The DNA molecules were successfully trapped at the top of the zigzag 978-0-9798064-6-9/μTAS 2013/$20©13CBMS-0001 8 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences 27-31 October 2013, Freiburg, Germany
Transcript of MICROFLUIDIC PARALLEL STRETCHING AND STAMPING OF …
MICROFLUIDIC PARALLEL STRETCHING AND STAMPING OF SINGLE DNA MOLECULES FOR SUPER RESOLUTION MICROSCOPE IMAGING
Hirotoshi Yasaki1, 2*, Daisuke Onoshima2, Takao Yasui1, 2, Toyohiro Naito3, Noritada Kaji1, 2, and Yoshinobu Baba1, 2, 4
1 Department of Applied Chemistry, Nagoya University, JAPAN, 2FIRST Research Center for Innovative Nanobiodevices, Nagoya University, JAPAN,
3Department of Material Chemistry, Kyoto University, JAPAN, and 4Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), JAPAN
ABSTRACT We developed a method for making high-density arrayed DNA molecules on PDMS and glass substrates. Arrayed DNA could be applied to various observation methods, and were elongated and immobilized on cover plates without any modification to DNA molecules. The depth, size and shape of the devices were optimized. It enables researchers to make such a high-density array of DNA molecules in a simple way. KEYWORDS
DNA Immobilization, DNA Array, Molecular Combing INTRODUCTION
Super resolution imaging such as stimulated emission depletion (STED) microscopy has a great potential for exploring base sequence and epigenomic change of single DNA molecule [1]. In order to detect a signal variation caused by sequence specific dye binding or partial melting, it is crucial that the DNA molecules are arrayed in a parallel direction inside the narrow microscopic field. Since the observation area becomes small accompanied with the magnification increases in microscope, throughput for the number of analyzed DNA molecules relatively decreases. However, the previous methods for the DNA arrangement do not satisfy the demand. One of DNA elongation methods for DNA imaging of single molecule is using nonspecific adsorption of DNA molecules on a glass substrate [2]; glass substrate is picked up from DNA solution, and then DNA molecules are adsorbed and elongated. Other method is using single-terminal immobilizing of DNA molecule by chemical modification, such as a biotin-avidin binding [3]; DNA molecules are immobilized on single-terminal and elongated by flow. Former method is simple, but the positions of elongated DNA molecules are random and undesirable cleavage of DNA molecules occurs easily. Latter method can make a lot of arrayed DNA molecules because this method can define the starting point of DNA elongating, but the way to make single terminal immobilization of DNA molecules is relatively difficult and troublesome. Here we show that single DNA molecules can be stretched and arrayed on recessed grooves located in the center of a microfluidic channel [4]. The arrayed DNA molecules can be also transferred to the other substrate surface. The demonstrated combination of the DNA elongation and stamping enables a simultaneous imaging of DNA molecules. EXPERIMENTAL
We used lambda DNA (48502 bp), which was stained with YOYO-1 intercalating dye at a 1:5 dye per base pair ratio for overnight in the dark at 4 ºC. The stained DNA solution was diluted to 20 ng/µL, 10 ng/µL, and 5 ng/µL with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The microfluidic device of polydimethylsiloxane (PDMS) consists of two parts; one is a microchannel part for flowing the YOYO-1 stained DNA molecules, and the other is a microstructure part for elongating and immobilizing the DNA molecules. The two parts were bonded each other without any plasma treatment. This was because we peeled off the channel part to observe DNA molecules after elongation and immobilization process. The microstructure part has a zigzag-shaped recessed groove (Fig. 1a). The pattern was made from line cavity and triangle structure at the edge of line. Those two PDMS parts was transferred from a SU-8 (negative photoresist) mold on silicon substrate. The PDMS and curing agent were mixed at a ratio of 10 to 1, poured onto the silicon substrate, and cured for 2 hours at 80 ºC. RESULTS and DISCUSSION
The recessed grooves trap DNA molecules during the flowing process by a syringe pump, and then the DNA molecules are stretched and immobilized at the edge of the zigzag structure on the PDMS without any modification (Fig. 1c). The DNA molecules in grooves are forced to move and concentrate at the top of the zigzag structure. One of the DNA molecules in the solution is occasionally trapped, elongated, and immobilized when the DNA solution passes through the groove structure. The PDMS microstructure with the DNA molecules was peeled off and placed onto cover plates for the following microscopic observation. The arrayed DNA molecules were observed by using total internal reflection fluorescence (TIRF) microscope (Fig. 2a) and STED microscope (Fig. 2b). The DNA molecules were successfully trapped at the top of the zigzag
978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 8 17th International Conference on MiniaturizedSystems for Chemistry and Life Sciences27-31 October 2013, Freiburg, Germany
structure in the flow direction. Figure 2c shows the length of elongated DNA molecules. Narrow peak indicate that length of elongated molecules is 20 ±0.16µm on average (n=200).
Figure 1: Elongation and immobilization of DNA molecules. (a) Image of pattern for elongation and immobilization of DNA molecules. The pattern was made from line cavity and triangle structure at the edge of line. (b) After bonding of a microchannel part and a microstructure part, DNA molecules were introduced into the PDMS device. After flowing the DNA molecules, the DNA molecules on the microstructure part were placed on cover plates. (c) Overviews of schematic illustration of the microstructure and DNA molecules (black lines). (d) Cross-section views in Fig. c.
We investigated the effect of depth, size, and shape of the zigzag structure on the number of DNA molecules in the array. The occupancy, which represents the ratio of the number of trapped DNA molecule to the total number of zigzag vertices, was used to evaluate the arrayed condition quantitatively.
𝑂𝑐𝑐𝑢𝑝𝑎𝑛𝑐𝑦 [%] =𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑡𝑟𝑎𝑝𝑝𝑒𝑑 𝐷𝑁𝐴 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒 𝑎𝑡 𝑡ℎ𝑒 𝑒𝑑𝑔𝑒 𝑜𝑓 𝑧𝑖𝑔𝑧𝑎𝑔 𝑣𝑒𝑟𝑡𝑖𝑐𝑙𝑒
𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑧𝑖𝑔𝑧𝑎𝑔 𝑣𝑒𝑟𝑡𝑖𝑐𝑒𝑠×100
There were two factors to increase the occupancy; one is the depth of groove structure, and the other is the surface area of zigzag structure. In the former, we used three types of depth, 3 µm, 9.5 µm, and 27 µm. The occupancy of these three types were about 84%, 37%, and 19%.The shallower structure showed higher occupancy. In the latter, we used three types of patterns. The pattern of zigzag structure was made from line cavity and triangle structure at the edge of line. In the optimization, we made three size of triangle pattern. One side of these triangle patterns are 5 µm, 10 µm, and 15 µm. The occupancy of these three types were about 40%, 87%, and 72%. The larger the area showed higher occupancy. We considered that is because larger area made high concentration of DNA molecules at the edge of zigzag structure when DNA solution was flown.
CONCLUSION
We achieved elongation and immobilization of DNA molecules using PDMS device. This method is simpler and can be applied to many observation conditions compared with any other elongation and immobilization method of DNA molecules. Our device might give us a chance for molecular mapping and analysis with spatial resolution sufficient to identify multiple epigenetic marks on DNA molecules.
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Figure 2: (a) TIRF microscope image of arrayed DNA molecules (b) STED microscope image. DNA molecules are elongated and immobilized at the edge of zigzag structure. Concentration of flown DNA solution was 10 ng/µL. (c) Length of elongated DNA molecules. Narrow peak indicate that length of elongated molecules is 20 ±0.16µm on average (n=200). REFERENCES 1) F. Persson, P. Bingen, T. Staudt, J. Engelhardt, J. O. Tegenfeldt, Stefan W. Hell, Fluorescence Nanoscopy of
Single DNA Molecules by Using Stimulated Emission Depletion (STED), Angew. Chem. Int. Ed., 50, 5581-5583 (2011).
2) A. Bensimon, A. Simon, A. Chiffaudel, V. Croquette, F. Heslot, D. Bensimon., Alignment and sensitive detection of DNA by a moving interface, Science, 265, 2096-2098 (1994).
3) E. C. Greene, S. Wind, T. Fazio, J. Gorman, M. L. Visnapuu, DNA curtains for high-throughput single-molecule optical imaging., Methods Enzymol., 472, 293-315 (2010).
4) A. Cerf, B. R. Cipriany, J. J. Benítez, H. G. Craighead, Single DNA Molecule Patterning for High-Throughput Epigenetic Mapping, Anal. Chem., 83(21), 8073-8077 (2011).
ACKNOWLEDGEMENT
This research is granted by the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program),” initiated by the Council for Science and Technology Policy (CSTP). CONTACT Hirotoshi Yasaki, +81-52-789-3560; [email protected]
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