Method for measuring the size of DNA molecules, device for measuring the size of DNA molecules, and system for measuring the size of DNA molecules

Abstract

To provide a new technique capable of readily measuring the size of a DNA molecule in a short time.SOLUTION: An embodiment of a method for measuring the size of a DNA molecule includes extending one DNA molecule and measuring the time until the extended molecule becomes relaxed.SELECTED DRAWING: None

Description

Technical Field

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[0007] This disclosure provides a new technology that can easily and quickly measure the size of DNA molecules. [Brief explanation of the drawing]

[0008] [Figure 1] Figures 1(a) and 1(b) are schematic diagrams illustrating a DNA molecule size measurement device according to an embodiment. [Figure 2] This is a schematic diagram showing an example of the shape of the nanoslit channel as viewed from above the substrate of the device according to the embodiment. [Figure 3] This is a schematic diagram showing another example of the shape of the nanoslit channel as viewed from above the substrate of the device according to the embodiment. [Figure 4] Figure 4(a) is a schematic diagram showing the extension of DNA molecules within a nanoslit channel. Figures 4(b), 4(c), and 4(d) are schematic diagrams showing the relaxation of DNA molecules within a nanoslit channel. [Figure 5] This is a schematic diagram illustrating a DNA molecule size measurement system according to an embodiment. [Figure 6] This is a schematic diagram illustrating a DNA molecule size measurement system according to another embodiment. [Figure 7] We fabricated a device equipped with four types of nanoslit channels, as shown in Figures 7(a) to 7(d). [Figure 8] This is a schematic diagram of the experimental apparatus used in the example. [Figure 9] This figure shows typical results of fluorescence imaging of extended DNA. [Figure 10] This figure shows the brightness distribution along the extension direction of the molecule in a fluorescence image of an extended molecule. [Figure 11] This figure shows that DNA molecules, which were in a random coil shape at the entrance of the nanoslit, extend as they enter the nanoslit channel. [Figure 12]This is a diagram showing the results of plotting the migration speed and elongation rate of molecules measured in two nano-slit shapes (rectangular type and tapered type, FIGS. 7(b) and 7(c)). [Figure 13] This is a diagram showing the state of molecular relaxation of λDNA (48.5 kbp) observed at the center of the nano-slit flow channel. [Figure 14] This is a diagram showing the results of plotting the time change of the molecular elongation length of λDNA molecules. [Figure 15] This is a diagram showing the results of plotting the time change of the molecular extension length of T4 DNA (166 kbp). [Figure 16] This is a histogram of the relaxation time measurement results of two types of DNA molecules. [Figure 17] This is a diagram showing the relationship between the relaxation time measured with λDNA and the initial elongation rate (the elongation rate immediately after stopping the migration). [Figure 18] This is a diagram showing the relationship between the relaxation time measured with T4 DNA and the initial elongation rate (the elongation rate immediately after stopping the migration). [Figure 19] This is a diagram showing the results of measuring the elongation lengths of λDNA and T4 DNA in a nano-slit flow channel device and presenting the elongation lengths of each DNA molecule in a histogram. [Figure 20] This is a histogram of the relaxation times measured using a mixed sample of DNA samples of different sizes.

Mode for Carrying Out the Invention

[0009] The method for measuring the size of a DNA molecule according to an aspect of the present disclosure extends a single DNA molecule and measures the time it takes for the extended DNA molecule to relax. According to this aspect, the size of the DNA molecule can be simply measured in a short time. Here, DNA is a polymer chain and usually maintains an energetically stable random coil state. The relaxation of an extended DNA molecule refers to the phenomenon in which an extended DNA molecule, which has received external factors (such as mechanical tensile force, confinement effect, etc.), returns to its original random coil state when those external factors disappear. The time required for relaxation is called the relaxation time.

[0010] DNA molecules can be stretched by external factors such as mechanical tensile force and confinement effect. Examples of means for applying such external factors to DNA include electrophoresis, electroosmotic flow, and pressure flow.

[0011] The confinement effect refers to the confinement force exerted on a DNA molecule when it is confined in a nano-scale (1 nm to 1000 nm) channel. The entire confined DNA molecule is theoretically exposed to the same confinement force, enabling uniform stretching.

[0012] The stretching of a DNA molecule by the confinement effect can be carried out, for example, by electrophoresing a single DNA molecule in a nano-slit channel using a DNA molecule size measurement device comprising a substrate, a nano-slit channel formed on one surface of the substrate, and a cover member covering the nano-slit channel.

[0013] The relaxation of a DNA molecule can be achieved by eliminating the external factors applied for stretching. When using the above-mentioned device, after stretching the DNA molecule, electrophoresis can be stopped and the DNA molecule can be relaxed in the nano-slit channel.

[0014] The stretching and relaxation of a DNA molecule can be detected by fluorescently labeling the DNA molecule and observing the fluorescence image using, for example, a fluorescence microscope.

[0015] Various methods for measuring the relaxation time of DNA molecules are known. For example, relaxation time can be measured from the time change in the degree of elongation associated with the relaxation phenomenon. Since the time change in the degree of elongation is exponential (Bakajin et al., Phys. Rev. Lett., 1998, 80, 12), after plotting the time change in the degree of elongation of the DNA molecule, the value corresponding to the time constant τ can be measured as the relaxation time by fitting it to the exponential function shown in equation (1). The time required for relaxation of a DNA molecule depends on the molecular size (A. Balducci et al., Phys. Rev. Lett., 2007, 99, 238102), and it is thought that the time constant τ (the slope of the time change in molecular elongation) differs depending on the molecular size. Therefore, the size of the DNA molecule can be calculated using the relaxation time. Details of the calculation of DNA molecule size will be explained in the examples below.

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[0016] For example, commercially available DNA ladders of known size can be flowed through the nanoslit channels of the device, the relaxation time for each type of DNA can be measured, a calibration curve can be drawn, and the size of an unknown DNA molecule can be measured using this calibration curve. Alternatively, two or more nanoslit channels can be formed on the same surface of a substrate, a DNA ladder can be flowed through one of them, and a DNA molecule of unknown size can be flowed through the other, and the size of the DNA molecule can be measured by comparing the relaxation times of both. It is also possible to flow DNA ladders with different labels and a DNA molecule of unknown size through a single nanoslit channel.

[0017] Another aspect of this disclosure is a DNA molecule size measuring device. The device comprises a substrate, a nanoslit channel formed on one surface of the substrate, and a cover member covering the nanoslit channel. A single DNA molecule is stretched within the nanoslit channel by electrophoresis, and the time it takes for the stretched DNA molecule to relax is measured. According to this aspect, the size of a DNA molecule can be measured quickly and easily.

[0018] The width of the entrance to the nanoslit channel into which one DNA molecule flows may be 1 to 1000 μm, and the depth of the nanoslit channel may be 1 to 1000 nm. According to this embodiment, one DNA molecule can be efficiently extended.

[0019] The nanoslit channel may have a tapered shape. This embodiment allows for more effective extension of the DNA.

[0020] A further aspect of this disclosure is a DNA molecule size measurement system. The system comprises a DNA molecule size measurement device, at least a pair of electrodes positioned on either side of a nanoslit channel within the device, and a power supply for applying a voltage to the electrodes. A single DNA molecule is stretched in the nanoslit channel by electrophoresis, and the time it takes for the DNA molecule to relax is measured. According to this aspect, the size of a DNA molecule can be measured quickly and easily.

[0021] The DNA molecule size measurement system may further include a detection unit that detects the stretching of a single DNA molecule by electrophoresis and its relaxation due to the cessation of electrophoresis.

[0022] Furthermore, any combination of the above components, as well as any conversion of the expressions of this disclosure between methods, apparatus, systems, etc., are also valid as aspects of this disclosure.

[0023] The embodiments for implementing this disclosure will be described in detail below with reference to the drawings and other figures. The embodiments are illustrative and not limiting to the invention, and not all features or combinations thereof described in the embodiments are necessarily essential to the invention.

[0024] (DNA molecule size measurement device) A DNA molecule size measuring device according to an embodiment will be described. Figures 1(a) and 1(b) are schematic diagrams illustrating the DNA molecule size measuring device according to an embodiment. As shown in Figures 1(a) and (b), the DNA molecule size measuring device 10 (hereinafter also simply referred to as the device) mainly comprises a substrate 12, a nanoslit channel 14, a first microchannel 16, a second microchannel 18, a cover member 20, a first reservoir 22, and a second reservoir 24.

[0025] The substrate 12 is a material capable of forming nanoslits, such as a silicon substrate. The size of the substrate is, for example, 40 mm in length and 9 mm in width.

[0026] A nanoslit channel 14, a first microchannel 16, and a second microchannel 18 are formed on one surface of the substrate 12. Figure 1(b) is an enlarged cross-sectional view of part A of the device 10 shown in Figure 1(a). As shown in Figure 1(b), the first microchannel 16 and the second microchannel 18 are connected to both sides of the nanoslit channel 14. Here, "nanoslit channel" refers to a channel whose depth is on the order of nanometers (1 nm to 1000 nm), and the width and length of the nanoslit channel do not necessarily have to be on the order of nanometers.

[0027] The depth of the nanoslit channel 14 can be appropriately set according to the size of a single random coil-shaped DNA molecule. The depth is preferably 1 nm to 1000 nm, and more preferably 10 nm to 50 nm, in order to allow for greater stretching of the DNA. The width of the nanoslit channel 14 can also be appropriately set according to the size of a single random coil-shaped DNA molecule. The width is preferably 1 μm to 1000 μm, and more preferably 10 μm to 50 μm, in order to facilitate entry of the DNA molecule into the nanoslit channel. The length of the nanoslit channel 14 should be sufficient for the DNA molecule to stretch by electrophoresis. For example, the length is 100 μm to 1000 μm, and preferably 100 μm to 200 μm. The nanoslit channel 14 can be formed, for example, by focused ion beam processing (FIB).

[0028] Here, "microchannel" refers to a channel with a depth on the order of micrometers (1 μm to 1000 μm), but the width and length of the microchannel do not necessarily have to be on the order of micrometers. For example, the width of the first microchannel 16 and the second microchannel 18 is, for example, 1 μm to 1000 μm, preferably 50 μm to 200 μm. Their length is, for example, 10 mm to 100 mm, preferably 20 mm to 50 mm. The first microchannel 16 and the second microchannel 18 can be formed, for example, by photolithography.

[0029] The device according to this embodiment has one nanoslit channel, but the number of nanoslit channels may be two or more. By providing multiple nanoslit channels, multiple samples can be passed through simultaneously, and the size of the DNA molecules contained in each sample can be measured.

[0030] The cover member 20 covers the nanoslit channel 14, the first microchannel 16, and the second microchannel 18, sealing these channels. To facilitate the detection of DNA molecule extension and relaxation within the nanoslit channel 14, the cover member 20 is preferably made of a transparent material. Examples of materials for the cover member 20 include glass and resin.

[0031] The first reservoir 22 is located at the end of the first microchannel 16 opposite the nanoslit channel 14. The second reservoir 24 is located at the end of the second microchannel 18 opposite the nanoslit channel 14. The first reservoir 22 and the second reservoir 24 are the locations for sample introduction and electrode placement. The first reservoir 22 and the second reservoir 24 are formed from, for example, glass tubes.

[0032] Figure 2 is a schematic diagram showing an example of the shape of a nanoslit channel as viewed from above the substrate of the device according to the embodiment. The nanoslit channel 14 shown in Figure 2 has a rectangular shape. Figure 3 is a schematic diagram showing another example of the shape of a nanoslit channel as viewed from above the substrate of the device according to the embodiment. The nanoslit channel 14 shown in Figure 3 has a tapered shape. This tapered nanoslit channel 14 is formed so that its width narrows in the direction of electrophoresis. With this configuration, DNA molecules can be stretched more effectively.

[0033] Next, we will explain how DNA molecules are stretched and relaxed using the device according to the embodiment. Figure 4(a) is a schematic diagram showing the stretching of DNA molecules in the nanoslit channel. Figures 4(b), 4(c), and 4(d) are schematic diagrams showing the relaxation of DNA molecules in the nanoslit channel. As shown in Figure 4(a), random coil-shaped DNA molecules M1 introduced into the device 10 move to the nanoslit channel 14 by electrophoresis in the buffer. DNA molecules M2 that reach the entrance of the nanoslit channel 14 enter the nanoslit channel 14 one molecule at a time. DNA molecules M3 that have entered the nanoslit channel stretch because the viscous force increases due to the nanoslit channel 14 and the entropy force is suppressed by the confinement effect. When electrophoresis is stopped, the stretched DNA molecules M3 gradually relax as shown by DNA molecules M4 in Figure 4(b) and M5 in Figure 4(c), and finally become random coil-shaped as shown by M6 in Figure 4(d).

[0034] (DNA molecule size measurement system) A DNA molecule size measurement system according to an embodiment will be described. Figure 5 is a schematic diagram illustrating the DNA molecule size measurement system according to an embodiment. As shown in Figure 5, the DNA molecule size measurement system 100 (hereinafter also simply referred to as the system) mainly comprises a DNA molecule size measurement device 10, at least a pair of electrodes 110 and 112, and a power supply 120.

[0035] The DNA molecule size measurement device 10 has the configuration shown in Figures 1(a) and 1(b), and its detailed configuration is as described above. The configuration of the system 100 will be described below, referring to the configuration of the device 10 shown in Figures 1(a) and 1(b) as needed. Electrodes 110 and 112 are positioned so as to sandwich the device 10. For example, electrodes 110 and 112 can be placed in the first reservoir 22 and second reservoir 24 of the device 10, respectively. Electrodes 110 and 112 can be formed from electrode materials commonly used in electrophoresis (such as platinum). The power supply 120 is connected to electrodes 110 and 112 via a wire 130 and applies a voltage between these electrodes.

[0036] Next, a method for measuring DNA molecule size using the system 100 according to the embodiment will be described. A buffer containing DNA molecules is introduced into the first reservoir 22 of the device 10. A voltage is applied between electrodes 110 and 112 by power supply 120, and one DNA molecule is extended by electrophoresis in the nanoslit channel 14 within the device 10. After that, the application of voltage is stopped, and the time it takes for the DNA molecule to relax in the nanoslit channel 14 is measured.

[0037] Figure 6 is a schematic diagram illustrating a DNA molecule size measurement system according to another embodiment. The system 200 shown in Figure 6 has the same configuration as the system 100 shown in Figure 5, except that it further includes a detection unit 140 that detects the extension of a single DNA molecule by electrophoresis and the relaxation caused by the cessation of electrophoresis. Therefore, the same reference numerals are used for components common to the system 100 shown in Figure 5, and redundant explanations are omitted as appropriate.

[0038] The detection unit 140 is positioned above the device 10, in a location where DNA molecules flowing through the nanoslit channel 14 of the device 10 can be observed. The detection unit 140 may include, for example, a fluorescence microscope and a camera attached to the fluorescence microscope to detect the fluorescence of fluorescently labeled DNA molecules. With such a configuration, the extension and relaxation of DNA molecules and the relaxation time can be efficiently detected and measured. A computer may also be installed to process the image data acquired by the camera.

[0039] The present disclosure will be described in more detail below with reference to examples, but the present disclosure is not limited to these examples. [Examples]

[0040] (Device fabrication) Devices 10, as shown in Figures 1(a) and 1(b), were fabricated. A cover glass was used as a cover member 20 to seal the entire channel. Glass tubes were used as the first reservoir 22 and the second reservoir 24. Devices equipped with four types of nanoslit channels 14, as shown in Figures 7(a) to 7(d), were fabricated. In all figures, the direction of DNA molecule electrophoresis is from microchannel 16 to microchannel 18, and the length of the nanoslit channel 14 is 100 μm. The nanoslit channel 14 in Figure 7(a) is rectangular in shape with a width of 10 μm. The nanoslit channel 14 in Figure 7(b) is rectangular in shape with a width of 50 μm. The nanoslit channels 14 in Figures 7(c) and 7(d) are tapered in shape, narrowing in the direction of electrophoresis. The one in Figure 7(c) has an inlet width of 50 μm and an outlet width of 10 μm, while the one in Figure 7(d) has an inlet width of 20 μm and an outlet width of 2 μm. The device was fabricated by microfabrication on a silicon substrate. This consists of two main processes: photolithography to create microchannels and focused ion beam (FIB) fabrication to create nanoslit channels. The sequence of processes is as follows [1] to

[10] . [1]: Photoresist coating by spin coating [2]: Transfer of photomask pattern by exposure [3]: Dissolution of the photosensitive area by development [4]: Silicon etching by reactive ion etching (RIE) [5]: Removal of residual resist film [6]: Cutting out microchips with a dicing saw [7] Formation of oxide film by wet thermal oxidation [8]: Fabrication of nanoslit channels by FIB processing [9]: Sealing of the flow channel by anodic bonding of the cover glass

[10] : Adhesion of the reservoir (liquid reservoir)

[0041] Details of each step are as follows. [1] A 4-inch (10.16 cm) silicon substrate was vacuum-chucked in a spin coater (Mikasa, MS-A150), and primer OAP (Tokyo Ohka) was dropped onto it. A uniform film was then coated by continuously rotating at 500 rpm for 5 seconds and then at 3000 rpm for 20 seconds. Next, photoresist OFPR-800, 34 cP (Tokyo Ohka) was dropped onto the substrate and coated by rotating in the same manner. After that, the resist film was heated in a 90°C bake oven for 30 minutes to evaporate the organic solvent and improve adhesion to the substrate.

[0042] [2] As a photomask, a 2x6 arrangement of the channel shapes shown in Figures 1(a) and 1(b) was used and attached to the exposure apparatus (SussMicro Tec, MA-6). The alignment gap was set to 20 μm, and ultraviolet irradiation was performed in contact mode for 50 s.

[0043] [3] To dissolve the photoresist portion exposed to ultraviolet light, the substrate was immersed in developer NMD-3 (Tokyo Ohka). After immersion for about 10 minutes until the pattern stabilized, it was rinsed with pure water. Subsequently, a post-bake treatment (120°C, 30 min) was performed to improve the adhesion between the resist and the substrate, which had been reduced by development.

[0044] [4] The channel shapes patterned in [1] to [3] were etched onto a silicon substrate using RIE (Sumitomo Precision Products, Multiplex-ASE). Microchannels with a depth of 1.3 μm were formed by etching for 20 s.

[0045] [5] The remaining resist film that was not exposed in [2] was removed by ultrasonic cleaning in acetone.

[0046] [6] To cut 12 substrates (40mm x 9mm silicon chips) shown in Figure 1(a) from a 4-inch silicon wafer, a dicing saw (DISCO, DAD522) was used. The blade height was set to 0.1mm, the blade rotation speed to 30,000 rpm, and the feed rate to 5mm / s.

[0047] [7] To improve the wettability of the channel surface, thermal oxidation (1057°C, 20 min) was performed under wet conditions to form an oxide film on the surface.

[0048] [8] Using JEM-9320 (JEOL) and NX-5000 ETHOS (HITACHI) as FIB instruments, processing rate conditions were determined to fabricate arbitrary nanoslit shapes. For each instrument, parameters related to the milling depth (dose amount and target depth) were varied, and the processing rate was obtained by measuring the actual milled depth with an atomic force microscope (AFM). Based on this processing rate, values ​​were set according to the desired nanoslit depth, and nanoslit structures were fabricated.

[0049] [9] Anode bonding was used to bond the silicon chip and the cover glass. A voltage of 1 kV was applied for approximately 30 to 60 minutes at a temperature of 400°C.

[0050]

[10] A reservoir (a glass tube with an outer diameter of 5 mm, an inner diameter of 3.5 mm, and a length of 5 mm) for introducing the DNA sample was attached with araldite adhesive.

[0051] The cross-section of the nanoslit in the depth direction was measured using an atomic force microscope (AFM). The average depth was 25 nm, and the surface roughness (arithmetic mean roughness) was 0.9 nm.

[0052] (DNA elongation) As a buffer, 1× Tris-borate-EDTA (TBE) (TaKaRa Bio), commonly used as a buffer for DNA electrophoresis, was used. In addition, Lipidure®-BL203 (5 wt% Aq. Solution, NOF Corporation), an aqueous solution of MPC polymer, was used to prevent electroosmotic flow that occurs when voltage is applied. The mixing ratio was 100 μL of 10× TBE, 20 μL of Lipidure®-BL203, and 880 μL of pure water. The buffer was pre-filled into the channel before introducing the DNA sample. The procedure for introducing the buffer into the channel involved injecting 50 μL of buffer into one reservoir and confirming that it filled the microchannel and nanoslit channel. Then, the same amount of buffer was introduced into the other reservoir. λDNA (48.5 kbp) (Thermo Fisher) and T4 DNA (Thermo Fisher) were used as DNA samples. YOYO-1 was also used as a fluorescent label (excitation wavelength 491 nm, fluorescence wavelength 509 nm) (Thermo Fisher). Generally, DNA is stained so that one YOYO-1 molecule is intercalated for every five bases (Reisner et al., Phys. Rev. Lett., 2007, 99, 058302). This ratio corresponds to 3 μL of 0.1 μM YOYO-1 aqueous solution per 1 ng of DNA. The mixing ratio is 10 μL of λDNA or T4 DNA (10 ng / μl) and 300 μL of YOYO-1 (0.1 μL).

[0053] The above mixed sample is designated as DNA Sample I. DNA Sample II is obtained by diluting I 750-fold. The DNA sample introduced into the chip device was a mixture of DNA Sample II, water, and Lipidure®-BL203 (200 μL of DNA Sample II, 20 μL of 10×TBE, and 8 μL of Lipidure®-BL203). With the entire fluidic device filled with buffer solution, the reservoir on the nanoslit channel inlet side was emptied, and this DNA sample was injected. Then, a platinum electrode was inserted between the reservoirs at both ends, and a voltage of 20 V was applied for about 30 minutes to electrophores the DNA molecules up to the nanoslit channel.

[0054] A schematic diagram of the experimental apparatus is shown in Figure 8. The experimental apparatus consists of a chip device, a temperature controller, a voltage / current source, and a microscope fluorescence detection system. A Peltier-type cooling and heating stage for microscopes (Japan Hitec, T95-HS and LNP95) was used to control the temperature of the chip device. A platinum electrode was connected to the reservoir of the chip device placed on this stage, and this was connected to the voltage / current source via a wire. The voltage / current source used was a multifunction generator (NF Circuit Design Block, WF1948) and an amplifier (MESS-TEK, M-2617). An arbitrary waveform was set using the function generator, and the voltage value was amplified using the amplifier. By controlling the voltage in this way, the electrophoretic rate of DNA molecules was changed, and the movement of the DNA molecules was controlled.

[0055] The microscopic fluorescence detection system consists of a fluorescence microscope, an EMCCD camera, and a PC. Fluorescence observation was performed using an epifluorescence microscope (OLYMPUS, BX51N-33FL-2-SP) and an LED light source (LUMEN DYNAMICS, XLED1). YOYO-1, used as the fluorescent label for DNA molecules, has an absorption spectrum of 491 nm and an emission spectrum up to 509 nm; therefore, a wavelength of 460 nm was output from the light source, and the fluorescence mirror unit MMNIBA3 was selected as the filter. Furthermore, the fluorescently labeled DNA molecules were observed using an EMCCD camera (Andor, iXonEM+897). Data acquired by the camera was transmitted to the PC and imported into imaging software Micro Manager 2.0 and Image J, outputting as images and videos.

[0056] A DC voltage (~10V) was applied to induce electrophoresis and extension of DNA molecules within the nanoslit channel. The DNA migrating from the nanoslit was observed using a fluorescence microscope. The observation and imaging environment is shown in Table 1 below. [Table 1]

[0057] This section describes a method for evaluating molecular elongation. Representative results of fluorescence imaging of elongated DNA are shown in Figure 9. ImageJ was used for video processing. For the fluorescence images of the elongated molecules, the brightness distribution in the elongation direction was obtained along the molecule. This brightness distribution is shown in Figure 10. After fitting this brightness distribution with the function represented by equation (2) below, the difference between the fitting parameters m3 and m2 was defined as the actual elongation length l (Tegenfeldt et al., PNAS, 2004, 101, 30).

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[0058] As shown in equation (3) below, the molecular elongation rate S was calculated as a percentage by dividing the elongation length l by the contour length L and multiplying by 100. In the case of the λDNA (48.5 kbp) DNA sample used in this study, L = 21.8 μm (Kim et al., Lab on a Chip, 2011, 11, p. 1721).

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[0059] As shown in Figure 11, we were able to observe how DNA molecules, which were in a random coil shape at the entrance of the nanoslit, extended as they entered the nanoslit channel. From this result, we confirmed that extension of a single DNA molecule can be achieved.

[0060] (Nano-slit shape) Focusing on the fact that the electric field gradient within the channel can be changed by altering the shape of the nanoslits, we fabricated a rectangular shape that forms a uniform electric field gradient, as well as a tapered shape where the electric field gradient increases in the direction of electrophoresis. We measured how the presence or absence of this electric field gradient affects the degree of molecular elongation. Figure 12 plots the electrophoretic velocity and elongation rate of molecules measured for the two nanoslit shapes (rectangular and tapered, Figures 7(b) and 7(c)). The tapered shape achieved a higher elongation rate at the same electrophoretic velocity than the rectangular shape. From these results, it was found that, in addition to molecular elongation due to viscous force, the presence of an electric field gradient within the channel in the tapered shape allows for more efficient molecular elongation than in the rectangular shape.

[0061] (Measuring DNA relaxation time) Two DNA samples with different molecular weights were used: λDNA (48.5 kbp) (Thermo Fisher) and T4 DNA (166 kbp) (Thermo Fisher). YOYO-1 was used as a fluorescent label (excitation wavelength 491 nm, fluorescence wavelength 509 nm) (Thermo Fisher). The apparatus for DNA sample preparation and observation was as described above.

[0062] A pulsed voltage was applied to induce electrophoresis and extension of DNA molecules within a nanoslit channel, followed by in-situ coiling. The pulsed voltage was set to a voltage value of 0-15V, a frequency of 0.4Hz, and a duty cycle of 0.5. The reason for setting the voltage value in this way was to increase the electrophoresis speed and ensure sufficient extension, and the reason for setting the frequency and duty cycle in this way was to ensure sufficient time to observe the relaxation phenomenon.

[0063] The area above the center of the nanoslit was observed at a fixed point, and the coiling of DNA molecules when the pulse voltage became zero was observed. The observation and imaging conditions were the same as in Table 1. The device used was the tapered nanoslit channel shown in Figure 7(b).

[0064] Figure 13 shows the molecular relaxation of λDNA (48.5 kbp) observed in the center of the nanoslit channel. We successfully captured the moment the pulse voltage was cut off (t=t0) as the extended molecule returned to a random coil shape.

[0065] The relaxation time was measured based on these observations. As shown in Figure 14, the time change in molecular elongation was plotted and fitted to the exponential function represented by equation (4) below, and the value corresponding to the time constant was obtained as the relaxation time τ.

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[0066] Figure 16 shows histograms of the relaxation time measurements for two types of DNA molecules. There were 15 and 17 data points for λDNA and T4 DNA, respectively. Note that these results were obtained by independently measuring λDNA and T4 DNA without mixing them. Since only one type of DNA is present in the channel, all measured relaxation times represent a single size. The average relaxation time τ1 for λDNA was measured at 0.075 s, and the average relaxation time τ2 for T4 was measured at 0.116 s, with standard deviations σ1 and σ2 being 0.004 s and 0.008 s, respectively. From the histogram results, we attempted to estimate the degree of peak separation if the two types of DNA molecules were mixed. The Gaussian distribution function of equation (5) below was fitted to the histogram.

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[0067] Figure 17 shows the relationship between relaxation time measured for λDNA and initial elongation rate (elongation rate immediately after stopping electrophoresis). Figure 18 shows the relationship between relaxation time measured for T4 DNA and initial elongation rate (elongation rate immediately after stopping electrophoresis). Since relaxation time is a unique value for a DNA molecule of a certain molecular weight, it does not depend on the initial elongation rate in principle. Experimental results also confirmed that even if the initial elongation rate differed by around 10%, the measured relaxation time was similar, indicating no dependence. This is thought to be because the molecules measured in this experimental system are within the range of a linear force regime at an elongation rate of around 30%, and are represented by a single relaxation time. However, if the initial elongation rate is too small, there is a possibility that an incorrect relaxation time may be measured due to fitting accuracy. Therefore, in order to guarantee the accuracy of size analysis, it is necessary to achieve an elongation rate above a certain value.

[0068] The small dependence of the measured relaxation time on the initial elongation rate demonstrates the superiority of size analysis by relaxation time measurement over methods that identify size by measuring elongation length (Tegenfeldt et al., PNAS, 2004, 101, 30). Identifying size from elongation length requires the same external force for elongation. Achieving this for each molecule requires advanced molecular manipulation techniques such as optical tweezers, making it unsuitable for high-speed size analysis. In this embodiment, molecules are also elongated to observe the relaxation phenomenon; therefore, the peak resolution when performing size analysis using the elongation length was verified. Figure 19 shows the histograms of the elongation lengths of λDNA and T4 DNA measured in a nanoslit channel device. The black bar graphs represent λDNA, and the white bar graphs represent T4 DNA. The solid and dotted lines represent the results of fitting the respective histograms to a Gaussian distribution function. The average elongation lengths of each DNA were measured as l1=7.7 μm and l2=14.7 μm, and the full width at half maximum (FWHM) was measured as W1=3.6 μm and W2=7.3 μm. The resolution R, a quantitative evaluation index for peak discrimination, was evaluated using equation (6) and was found to be 1.27. Since the resolution of size analysis using relaxation time measurement was 2.9, it was found that discrimination could be performed with higher accuracy than size analysis using elongation length.

[0069] (Size analysis of mixed samples) DNA size separation and analysis techniques must separate and differentiate DNA samples containing various molecular weights. Therefore, to verify the effectiveness of the analytical method proposed in this study, we conducted size analysis experiments using relaxation time measurements with mixed samples of DNA of different sizes.

[0070] Two types of DNA samples were used: λDNA (48.5 kbp) and T4 DNA (166 kbp). After preparing each sample as described above, they were mixed in a 1:1 ratio. The device used in this embodiment was the tapered nanoslit channel shown in Figure 7(b). As described above, molecules were continuously relaxed within the nanoslit channel by applying a pulsed voltage. Since the relaxation phenomena of both λDNA and T4 DNA were observed with the pulsed voltage used there, the same pulsed voltage parameters were used in this experiment with the mixed sample (voltage value 15V, frequency 0.4Hz, duty cycle 0.5). Microscopic observation was performed at a fixed point above the center of the nanoslit. The observation conditions were the same as in Table 1. The number of frames captured was 1500, and the time required for capture was 60s.

[0071] Figure 20 shows the histogram of the measured relaxation times. There were a total of 60 data points (number of DNA molecules). Since two peaks were observed, a Gaussian distribution function was fitted as described above (Figure 22, dashed curve). The mean, standard deviation, and half-width of the left peak were τ'1=0.075s, σ'1=0.008s, and W'1=0.020s, respectively. The mean, standard deviation, and half-width of the right peak were τ'2=0.119s, σ'2=0.009s, and W'2=0.021s, respectively. The resolution R, a quantitative indicator of whether the two peaks are distinguishable, was measured at 2.2, exceeding the threshold of 1.5.

[0072] As mentioned above, the resolution was measured at 2.2, which is considered to be sufficient accuracy to distinguish between the sizes of λDNA (48.5 kbp) and T4 DNA (166 kbp). Furthermore, the size analysis time was approximately 60 seconds, which was the time required to capture the relaxation phenomenon, so it is considered that the desired high-speed single-molecule DNA size analysis was achieved.

[0073] The results of the above experiment are summarized below. Molecular elongation was achieved using nanoslit channels with a depth of 25 nm. The elongation length was measured from the brightness distribution of the elongated molecules and evaluated as the molecular elongation rate. By using tapered nanoslit channels that narrow in the direction of electrophoresis, effective molecular elongation was achieved through the action of Coulomb forces due to the electric field gradient in addition to viscous forces. By applying a pulsed voltage to DNA molecules within a nanoslit channel, we successfully observed continuous entropic relaxation. The time evolution of the elongation rate during the relaxation process was plotted, and the relaxation time for one DNA molecule was obtained as the time constant when fitted to an exponential function. The measured relaxation times for two DNA samples of different sizes, λDNA (48.5 kbp) and T4 DNA (166 kbp), were 0.075 s and 0.116 s, respectively. • Using a sample containing a mixture of two types of DNA of different sizes, size analysis was performed by measuring the relaxation time of a single DNA molecule in a nanoslit channel. Two distinguishable peaks were obtained from the histogram of the relaxation time measurements. The peak separation index R, calculated from the inter-peak distance and standard deviation, was 2.2, demonstrating sufficient accuracy for peak discrimination. The size analysis time in this study was approximately 60 seconds, which was required to observe the relaxation phenomenon. Therefore, we were able to obtain results that demonstrate the effectiveness of this new method for fast and highly accurate size analysis of a single DNA molecule.

[0074] The present disclosure has been described above based on embodiments. These embodiments are illustrative, and it will be understood by those skilled in the art that various modifications are possible in combinations of their components and processing processes, and that such modifications are also within the scope of the present disclosure. [Explanation of symbols]

[0075] 12 Substrate, 14 Nano-slit channel, 20 Cover member, 110, 112 Electrodes, 120 Power supply, 140 Detection unit.

Claims

1. A DNA molecule size measurement device comprising a substrate, a nanoslit channel formed on one surface of the substrate, and a cover member covering the nanoslit channel, wherein a single DNA molecule is stretched in the nanoslit channel by electrophoresis, and the time it takes for the stretched DNA molecule to relax is measured.

2. A DNA molecule size measuring device comprising a substrate, a nanoslit channel formed on one surface of the substrate, and a cover member covering the nanoslit channel, wherein a single DNA molecule is stretched in the nanoslit channel by electrophoresis, and the time it takes for the stretched DNA molecule to relax is measured.

3. The DNA molecule size measuring device according to claim 2, characterized in that the width of the entrance to the nanoslit channel into which one DNA molecule flows is 1 to 1000 μm, and the depth of the nanoslit channel is 1 to 1000 nm.

4. The DNA molecule size measuring device according to claim 2 or 3, characterized in that the nanoslit channel has a tapered shape.

5. A DNA molecule size measuring device according to any one of claims 2 to 4, At least a pair of electrodes are arranged so as to sandwich the nanoslit channel within the device, A power supply for applying voltage to the aforementioned electrode, Equipped with, A DNA molecule size measurement system that extends a single DNA molecule by electrophoresis within the nanoslit channel and measures the time it takes for the DNA molecule to relax.

6. The DNA molecule size measurement system according to claim 5, further comprising a detection unit for detecting the elongation of a single DNA molecule by electrophoresis and the relaxation caused by the cessation of electrophoresis.

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