KR100656564B1 - Biomolecule Detection Method Using Differential Microscopy and Microchip Used in Detection - Google Patents

Abstract

The present invention relates to a method of detecting the biomolecules using a differential microscopy microscope without staining a biomolecule such as DNA with a fluorescent dye or the like and a microchip used in the detection.

According to the detection method according to the present invention, since the DNA is not stained with a fluorescent dye, the movement of the DNA itself can be observed more accurately.

Description

A Method for Detecting Biomolecules Using Differential Interference Contrast Microscopy and a Microchip for the Same Method}

Figure 1a is a DIC microscope device used in the biomolecule detection method according to the present invention.

Figure 1b is a microchip of PDMS / glass material used in the biomolecule detection method according to the present invention.

1C and 1D are plan and perspective views of the microchip of FIG. 1B.

2 is a fluorescence image of λ-DNA stained with YOYO-1.

3 is an image of λ-DNA (A, C) and native λ-DNA (B, D) stained with YOYO-1, observed with a DIC microscope.

4 is a moving speed of λ-DNA and native λ-DNA stained with YOYO-1 according to the electric field in the microchannel.

Figure 5 shows the length of the line on the image according to the exposure time in the microchannel

6 shows the effect of pH on the length of native λ-DNA molecules at 10 ms exposure time (white circle) and 300 ms exposure time (black circle).

7 is a real-time DIC image showing the effect of PVP on the adsorption of [lambda] -DNA molecules in microchannels of PDMA / glass material.

FIG. 8 shows DIC images of (A) native 1-kb DNA molecules and (B) 500 nm carboxylated modified microspheres in a microchip made of PDMS / glass.

9 is a DIC image of (A) native 1-kb DNA and (B) native 48-kb DNA in a glass / glass microchip.

The present invention relates to a method of directly detecting a biomolecule in a microchannel using a differential microscopy without staining a biomolecule such as DNA or a protein with a fluorescent dye and the like and a microchip used in the detection. According to the present invention, each single-DNA molecule flowing through a microchip made of glass or a polymer or glass that can transmit light such as a microchip made of glass / glass or PDMS is subjected to a Normaski microdispersive microscope ( Differential Interference Contrast Microcopy (hereinafter referred to as DIC microscopy) can be clearly observed.

Single molecule detection technology (SMD) has recently attracted much attention in the life sciences. Observation and manipulation of single biomolecules can be attributed to biomolecules in molecular genetics, microchip assembly, biosensor design, DNA biophysics, and basic separation theories such as capillary electrophoresis (CE) and liquid chromatography (LC). It is to study the movement.

In general, fluorescence detection of molecules of interest is used for supersensory analysis and biophysical applications. For example, in order to observe DNA, a fluorescent dye such as YOYO-1 was combined with a DNA molecule to observe DNA, and the bound fluorescent substance-DNA molecule was observed.

However, dyes inserted or bound to DNA affect physical / chemical properties and the movement of each DNA molecule. In microchannels, native DNA molecules travel faster than DNA molecules labeled with YOYO-1. This is because YOYO-1 increases the molecular weight and the size of λ-DNA. Electric field strength and pH also affect the movement of single-DNA molecules. It can be seen that YOYO-labeled DNA is more stretched than native DNA.

As such, due to the influence of fluorescent dyes, results at the conventional monomolecular level are not obtained directly from the native molecule of interest, but indirectly from derivatives.

Therefore, in order to monitor in real time and to facilitate the study of the movement of each molecule, it is necessary to study a detection method for native biomolecules that are not labeled with fluorescent dyes in solution.

The basic principle of a DIC microscope developed by Nomaski et al. Is similar to a conventional interferometer in that the measurement and comparison beam systems interfere with each other. The difference is that the comparison beam also passes through the object. Allen, R. D .; David, G. B .; Nomarski, G. Z. Wiss. Mikr. 1969, 69, 193-221]. The distance between the two beams is very short, about 1 μm increments. Sometimes it is carefully chosen to be below the minimum resolution distance defined by the diffraction limit. In the DIC microscope, the object can be seen by the optical path difference. Although the observed height and depth only show local differences in the gradient of the optical path through the sample, the flexibility of the image obtained by the DIC is excellent.

However, despite the known properties, DICs are less well used as imaging methods relative to conventional fluorescence microscopy. DIC microscopy is only used to obtain images of fixed samples using conventional cover slips and slide glasses. However, techniques related to the detection and manipulation of single biomolecules in fluid flow using DIC microscopy are not known.

Recently, microchip technology, for example, a micro total analysis system (μ-TAS) or a lab-on-a-chip, has been developed in the microfluidic field. Microfluidics enable numerous studies in the chemical analysis of biomolecules, including high speed separation, high-throughput, parallel analysis, and microunit sample preparation.

The present invention discloses single molecule detection and manipulation using a DIC optical microscope without modifications such as fluorescent dye labels. The method according to the invention can be successfully applied to observe the real-time movement of biomolecules such as individual DNA or proteins in microchannels.

Accordingly, an object of the present invention is to provide a method for detecting the biomolecules using a differential microscopy without binding or staining a biomolecule such as DNA or protein with a fluorescent dye or the like.

The present invention also relates to a microchip used in the detection of the biomolecule.

The present invention relates to a method of detecting the biomolecules using a differential microscopy microscope and a microchip used in the detection without dyeing a biomolecule such as DNA or protein with a fluorescent dye or the like.

Biomolecule detection method according to the present invention,

Injecting a sample containing a biomolecule in a solution into a microchannel in the microchip;

Applying an electric field across the microchannel;

Enlarging the image in the microchannel with a differential interference microscope (DIC microscope); And

Photographing the enlarged image by an image capturing means such as a CCD camera to observe the biomolecule in the microchannel.

In the above method, the solution is preferably a buffer having a pH of 2 to 12. In addition, the thickness of the microchannel is preferably 1 ㎛ to 200 ㎛.

The strength of the applied electric field is preferably 0.1 to 1000 V / cm.

In the step of photographing the enlarged image with the CCD camera, the exposure time is preferably 5 ms to 300 ms.

According to the invention, the microchip used in the biomolecule observation method,

It includes an upper substrate and a lower substrate made of various kinds of polymer material through which light such as glass or PDMS is transmitted, wherein one of the upper substrate and the lower substrate has a negative microchannel. The height of the microchannel is preferably 1 μm to 200 μm and the width is 1 μm to 1000 μm.

Among the upper and lower substrates of the microchip, the thickness of the substrate on the side in contact with the objective lens of the DIC microscope when viewed with a DIC microscope is preferably 1 to 500 µm in consideration of the focal length of the objective lens. For example, when the objective lens of the DIC microscope is inspected in contact with the upper substrate of the microchip, the thickness of the upper substrate of the microchip is preferably 1 to 500 µm.

In the microchip according to the present invention, it is preferable that the upper portion of the upper substrate further includes a substrate or a tube having a reservoir having a hole formed therein. In the case where the thickness of the upper substrate is thin, the height of the reservoir can be easily adjusted by forming a substrate or a tube made of a light transmissive polymer such as glass or PDMS on the upper substrate.

On the other hand, when observing a biomolecule using a refractive differential interference microscope in the present invention, even if any one of the upper substrate or the lower substrate of the microchip used is an opaque substrate (for example, silicon substrate), Does not interfere with observing molecules.

Hereinafter, a biomolecule detection method and a microchip used according to the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited by the following examples.

Example 1 Experiment Preparation

end. Buffer Preparation

Gly-Gly, CHES, and sodium hydroxide were purchased from Sigma Chemical Co., St. Louis, Mo. Various buffer systems were prepared as follows: sodium acetate / acetic acid (pH 6.0-7.5), Gly-Gly / NaOH (pH 8.2), and CHES / NaOH (pH 9.0-10.0).

Kang, SH; Shortreed, MR; Yeung, ES Anal. Chem . Through a procedure as described in 2001 , 73 , 1091-1099, high concentrations of glacial acetic acid, sodium acetate, and sodium chloride (all purchased from Fisher Scientific, Fiar Lawn, NJ) were dissolved in very pure (18 MΩ) water. Acetic acid, sodium acetate, and sodium chloride 1 M solutions were used to prepare sodium acetate buffers of various pH. All solutions were filtered using a 0.2 μm filter before use.

I. DNA Sample Preparation

λ-DNA (48,502 bp) was purchased from Life Technologies, Grand Island, NY. DNA 1-kb fragment was purchased from Seegene Co., Seoul, Korea. All DNA samples were prepared in Gly-Gly buffer at 10 mM pH 8.2. DNA samples at 200 pM concentration were labeled with the insertion dye YOYO-1 (Molecular Probes, Eugene, OR). Labeled with one dye molecule ratio per 5 base pairs according to manufacturer's instructions. DNA samples labeled with YOYO-1 were incubated for 5 minutes prior to dilution and use.

However, DNA samples used for DIC detection were prepared in only 10 mM Gly-Gly buffer without insert dye or binding dye. For single molecule imaging experiments, these DNA samples were further diluted to 10-20 pM immediately before starting the experiment in a suitable buffer. It was confirmed that the addition of Gly-Gly buffer did not change the pH of the final sample solution.

All. DIC Microscope and CCD Camera

To investigate, an upright Olympus BX51 microscope (Olympus Optical Co., Ltd., Shinjuku-ku, Tokyo, Japan) equipped with a DIC slider (U-DICT, Olympus) was used. 1A shows the DIC microscope device. A 20-fold objective lens (Olympus UPLFL20 × / 0.5N.A., W.D. 1.6) was used. CCD (Cool SNAP fx, Photometrics, Tucson, AZ) cameras were mounted on top of the microscope with halogen lamps and Hg lamps. CCD exposure time is 5-500 ms at a 20 MHz digitization rate. Electrophoresis and single DNA molecular manipulations were performed in the 0-5 kV range using a high voltage power supply (DVHV-100, Digital Bio Technology Co, Ltd., Seoul, South Korea). The electric field was applied at 0.41-81.6 V / cm to the DNA sample. Then, 0.2 and 0.5 μm microbeads (FluoSpheres carboxylate-modified microsphere, Molecular Probes) were used to calibrate the size determination of each single DNA molecule in the microchannel. MetaVue software (Version 5.0, Downingtown, PA) was used for DNA image acquisition and data processing.

la. Microchip Manufacturing for DIC

PDMS / glass microchips for DIC are shown in FIG. 1B. 1C and 1D are plan and perspective views of the microchip of FIG. 1B. The microchip was manufactured in a micro fabrication facility of Digital Biotechnology.

4.9 cm long straight channels were made with polydimethylsiloxane (PDMS) made from Sylgard 184A and B (Dow Corning, Corning, NY). Masters were fabricated using embossed channels on a Corning silicon wafer using standard micro photolithography techniques.

PDMS was prepared, degassed in vacuo for 30 min and poured onto the channel master. Curing at 80 ° C. for 3 hours, followed by cooling at room temperature for 3 hours. PDMS was separated from the silicone template and formed a pattern of negative channels. The blades were then cut to a suitable size and 2 mm diameter holes were punched into the cured polymer using a punching machine (Bollmanncrip) to form a reservoir. PDC-32G-2 Basic Plasma Cleaner (Harric Scientific Co., Broadway Ossing, NY) using a glass substrate (No. 1 Corning cover glass; 60 mm long × 2 mm wide, 0.16 mm thick) and The PDMS of the embossed channel was bonded to surround the microchannel and to increase the stability of the flexible PDMS chip. A small PDMS plate with holes of 2 mm diameter was left on the PDMS / glass microchip and the reservoir volume of the reservoir was increased.

The microchip consists of an upper substrate 200 made of PDMS and a lower substrate 100 made of glass (No. 1 Corning cover glass). The upper substrate 200 has a thickness of 140 μm, and the lower substrate has a thickness of 160 μm. The dimensions of the channel formed on the upper substrate 200 are 49 mm long, 50 μm wide, and 50 μm thick. The reservoirs 2, 4 are 2.0 mm in diameter. In addition, holes (1, 3) for applying an electric field are formed at both ends of the channel formed on the upper substrate (200). Preferably, the upper portion of the upper substrate 200 is further bonded with a substrate 300 (1.5 mm thick) in which a hole for increasing the depth of the reservoir is formed. When the substrate 300 is further provided, the total thickness of the chip is 1.8 mm.

Glass / glass microchips were fabricated by using a 500 μm-thick glass substrate to form a channel on one substrate and then joining the two substrates together. The size of the channel is 50 mm long, 70 μm wide, 10 μm thick. Others are the same as for PDMS / Glass microchips.

hemp. result

Kang, SH; Shortreed, MR; Yeung, ES Anal. Chem . 2001 , 73 , 1091-1099, each λ-DNA molecule at the fused silica / water interface forms an arbitrary coil and their conformation fluctuates rapidly above pH 5.5. As the pH is further reduced to 4.5, DNA molecules are gradually attracted onto the fused silica surface, similar to the molecular combing process.

However, in PDMS / glass microchips, neither λ-DNA (YOYO-DNA) nor native λ-DNA molecules stained with insertion dye YOYO-1 were eluted below pH 6.5. This is due to the adsorption of microchannels on the PDMS surface.

For 300 ms exposure time above pH 7.0, fluorescence images of YOYO-DNA show linearity (see FIG. 2). This is due to the movement of the DNA by prolonged exposure, resulting in an integrated orbit in the form of streaks. Depending on the position on the molecule's perpendicular to the focal plane, the lines exhibit different thicknesses. FIG. 2 is a fluorescence image of λ-DNA stained with embedded YOYO-1 obtained from an epi-fluorescence microscope in PDMS / glass microchannel. The electric field is 20.4 V / cm, and the objective lens is UPLFL20 × / 0.5W.D. 1.6 was used, the CCD exposure time was 300 ms, and the sample was 10 pM YOYO-1 labeled λ-DNA.

The motion of each native [lambda] -DNA in the microchannels can be easily monitored in real time without YOYO-1 labeling by a Normasky DIC microscope. The trajectory of native λ-DNA molecules is similar to the worm (earthworm) shape at 300 ms exposure time (see FIGS. 3 (A) and 3 (B)). However, dye insertion changes the motion of the λ-DNA molecules. The fact that the length of the line is mainly the result of DNA motion (electrophoresis) can be seen from C and D of FIG. 3. Thus, short exposure times essentially eliminate the contribution to the line length of the movement.

In FIG. 3, (A) shows a λ-DNA molecule labeled with YOYO-1 under a DIC microscope at 300 ms exposure, and (B) shows a native λ-DNA under a DIC microscope at 300 ms exposure. (C) is a DIC microscope photograph of λ-DNA molecules labeled with YOYO-1 at 10 ms exposure, and (D) is a DIC microscope image of native λ-DNA at 10 ms exposure. The electric field is 10.2 V / cm and the sample is 20 pM λ-DNA.

The rate of travel of λ-DNA as a function of electric field in the microchannels is shown in FIG. 4. The rate of the λ-DNA molecules increases with the applied electric field. Native DNA molecules (black circles in FIG. 4) move faster than YOYO-DNA molecules (white triangles in FIG. 4). At pH 8.2, both native λ-DNA and YOYO-DNA are purely negatively charged. However, YOYO-1 inserts one of five DNA bases. YOYO-1 (C ₄₉ H ₅₈ I ₄ N ₆ O ₂ ; MW 1270.65) increases the molecular weight and size of DNA. In addition, since YOYO-1 is a positive charge, similar to ethidium bromide, the ionic charge is reduced to reduce the electrophoretic motion of the DNA-YOYO complex. Thus, YOYO-DNA is slower than native DNA molecules.

In FIG. 4 the rate of movement of each λ-DNA molecule as a function of applied electric field in PDMS / glass microchannels was monitored by DIC microscopy. The CCD exposure time is 10 ms, the sample is 20 pM λ-DNA molecules, the black circles represent native λ-DNA and the white triangles represent λ-DNA labeled YOYO-1. Error bars are the standard deviation for five measurements.

In general, because the molecule moves relative to the camera during the exposure time, the trajectory appears as a line on the image. Horteed, MR; Li, H .; Huang, W.-H .; Yeung, ES Anal. Chem . 2000 , 72 , 2879-2885, line lengths were used to determine electrophoretic motion in molecular identification. In other words, fast molecules have long lines, and slower molecules have shorter lines. In this specification, the measured lengths of the two types of λ-DNA molecules vary linearly with exposure time (see FIG. 5). Native DNA molecules are spherical at the shortest exposure time of 10 ms. They are not distorted by the flow. However, Figure 5 shows that YOYO labeled DNA molecules extend longer than native DNA molecules. This can be explained by the fact that dye insertion extends the length of the chain and makes the molecule more linear.

5 shows the length of the line of each DNA molecule under an electric field of 10.2 V / cm. The sample is a 20 pM λ-DNA molecule, the black circle is a native λ-DNA molecule, and the white triangle is a λ-DNA molecule labeled YOYO-1. Other conditions are the same as those in FIG.

FIG. 6 shows the effect of pH on the length of native λ-DNA molecules at 10 ms exposure time (white circle) and 300 ms exposure time (black circle). Above pH 7.0, the glass surface has a negatively charged silanoate (SiO ⁻ ) group. Electrostatic repulsive forces overwhelm the interaction between the surface of the microchannel and the DNA molecules. DNA molecules can move freely on the surface and form linear for long exposure times.

Below pH 6.9, hydrophobic interactions between microchannels and DNA molecules are stronger than electrostatic forces. The PDMS surface has a different chemical composition than the glass surface (SiO-) (Si (CH ₃ ) ₂ -O-Si (CH ₃ ) ₂ ) -O-Si (CH ₃ ) ₂ ) -O-Si (CH ₃ ) ₂ PH) has previously been described by Kang, SH; Shortreed, MR; Yeung, ES Anal. Chem . 2001 , 73 , 1091-1099. Thus, DNA molecules are adsorbed onto the PDMS surface of the microchannels by hydrophobic interactions.

As the pH decreases to 6.5, λ-DNA molecules are immobilized on the PDMS surface. Thus, the length of the lines associated with the motion of the DNA molecules decreases even with long exposure times (black circles in FIG. 6).

Below pH 6.5, DNA molecules are strongly adsorbed on the PDMS surface and DNA molecules are not eluted through the PDMS / free microchannels. These results indicate that even though both electrostatic and hydrophobic interactions are related to adsorption, hydrophobic interactions rather than electrostatic interactions at the liquid-solid interface of the microchip are the main driving force of DNA adsorption.

6 shows the line lengths of native λ-DNA molecules (10 pM) at different pHs. The electric field is 10.2 V / cm, the running buffer is 2 mM sodium acetate / acetic acid (pH 6.5-7.5), 10 mM Gly-Gly / 0.1M sodium hydroxide (pH 8.2), 25 mM CHES / 0.1M sodium hydroxide (pH 9.0) -10.0), the white circle is 10 ms exposure time and the black circle is 300 ms exposure time.

Each λ-DNA molecule does not exhibit the same movement and shape in the fluid flow direction at pH 8.2. Solutions containing λ-DNA molecules migrate electrokinically, while some DNA molecules begin to adsorb to the surface. Adsorption of the ends of DNA molecules under bulk flow has been reported to extend the length of DNA molecules [Kang, SH; Shortreed, MR; Yeung, ES Anal. Chem . 2001 , 73 , 1091-1099]. However, no permanent fixation was observed at the channel surface. This is because the adsorption / desorption behavior of DNA molecules at basic pH can be caused by strong hydrophobic interactions and weak electrostatic repulsion between the PDMS surface and the DNA molecules.

It was confirmed that λ-DNA molecules in 25 mM sodium acetate buffer (pH 6.0) strongly adsorbed to the PDMS surface, but did not elute through the microchannel. Therefore, DNA molecules could not be observed at the ends of the microchannels (see FIG. 7A). This means that DNA molecules can be observed, but no other types of molecules are observed.

However, in the same pH buffer containing 0.3% polyvinylpyrrolidone (PVP, Mr 1,000,000), the polymer dynamically coats the surface of the microchannels and each DNA molecule is not electrokinetic (FIG. 7). B). These results are consistent with the fact that PVP inhibits electroosmotic flow and PVP prevents DNA from adsorbing to the fused silica surface and liquid-solid interface in PDMS as well as capillary electrophoresis.

7 is a real-time DIC image showing the effect of PVP on the adsorption of λ-DNA molecules in PDMA / free microchannels. The electric field is 10.2 V / cm, the CCD exposure time is 10 ms, and the sample is 10 pM native λ-DNA molecules. The running buffer is (A) 0% PVP and (B) 0.3% PVP (Mr 1,000,000) in 25 mM sodium acetate / acetic acid buffer (pH 6.0). Due to the adsorption, the molecule appears to move along the channel in (B), but not in (A).

The minimum size detectable in PDMS / glass microchips by DIC microscopy according to the invention is 1-kb DNA molecules (about 85 nm, see A in FIG. 8). However, for linear DNA, detection of 50bp DNA molecules is also possible. In addition, 50 nm microspheres can be observed without any pretreatment. 8B is an observation of 500 nm microspheres.

FIG. 8 is a DIC image of (A) native 1-kb DNA molecule and (B) 500 nm carboxylated modified microspheres in PDMS / glass microchip. The electric field is 10.2 V / cm, the CCD exposure time is 10 ms, and the running buffer is 10 mM Gly-Gly buffer (pH 8.2 with 0.1 M NaOH). Arrows in FIG. 8 indicate 1-kb DNA molecules intact.

9 is a DIC image of 10 pM of (A) Nateb 1-kb DNA molecule and (B) Nateb 48-kb DNA molecule in glass / glass microchip. The electric field is 20 V / cm, the CCD exposure time is 10 ms, and the running buffer is 0.3% PVP and 10 mM Gly-Gly buffer (pH 8.2).

 Without labeling with fluorescent dyes, the use of DIC microscopy allows direct observation of the movement of native single-DNA molecules in microchannels in solution in real time. The basic principle of the DIC is due to the local difference in the optical thickness of the sample. Thus, the slope of the optical path difference of the single-DNA molecules makes it visible in the channel. However, images of λ-DNA molecules can only be seen within microchannels with a thickness of 2 mm or less (see B and C in FIG. 1). This is due to the distance of the DIC object and the working distance (W.D.) of the DIC lens. The real-time motion of the λ-DNA molecules on the PDMS / glass surface is affected by the adsorption / desorption balance between the DNA molecules and the microchannel surface and the shift due to the applied electric field. The insertion dye YOYO-1 affects native single DNA molecules, confirming that λ-DNA molecules always move faster than YOYO-DNA molecules.

According to the detection method according to the present invention, since the DNA is not stained with a fluorescent dye, the movement of the DNA itself can be observed more accurately. Therefore, the single molecule detection technique (SMD) using the DIC microscope according to the present invention can be applied to a variety of microchip studies and DNA biophysics studies in liquid at room temperature.

Claims (15)

Injecting a sample containing a biomolecule in a solution into a microchannel in the microchip; Applying an electric field across the microchannel; Enlarging the image in the microchannel with a differential interference microscope (DIC microscope); And And capturing the enlarged image by an image photographing means to observe biomolecules in the microchannels. The method of claim 1, wherein the biomolecule is DNA, characterized in that 50 bp or more in size. The method of claim 1, wherein the biomolecule is a protein and is at least 50 nm in size. The method of claim 1 wherein the solution is a buffer having a pH of 2 to 12. The method of claim 1, wherein the microchannels have a thickness of 1 μm to 200 μm. 6. The method of claim 5 wherein the intensity of the applied electric field is between 0.1 and 1000 V / cm. 7. The method according to claim 6, wherein the image capturing means is a CCD camera. 8. The method of claim 7, wherein the exposure time in the step of photographing the enlarged image with the CCD camera is 1 ms to 500 ms. The method of claim 1, The biomolecule is DNA or protein, The solution is a buffer of pH 2 to 12, The thickness of the microchannel is 1 to 200㎛, The intensity of the applied electric field is 1 to 1000 V / cm, The image photographing means is a CCD camera, The exposure time in the step of capturing the magnified image with the CCD camera, characterized in that 1 to 500 ms. The method according to claim 1 or 9, The microchip includes an upper substrate and a lower substrate of glass or a transparent polymer material, One of the upper substrate and the lower substrate is formed with a microchannel intaglio, The substrate thickness of the upper substrate and the lower substrate in contact with the objective lens of the DIC microscope is 1 to 500㎛. delete A microchip used in the biomolecule observation method according to claim 1, Including an upper substrate and a lower substrate of glass or a transparent polymer material, One of the upper substrate and the lower substrate is formed with a microchannel intaglio, The substrate thickness of the upper substrate and the lower substrate in contact with the objective lens of the DIC microscope is 1 to 500㎛, The microchannel has a height of 1 μm to 200 μm and a width of 1 μm to 1000 μm. delete The microchip of claim 12, further comprising a substrate or a tube made of glass or a light-transmitting polymer having a reservoir formed with a hole in an upper portion of the upper substrate. Injecting a sample containing a biomolecule in a solution into a microchannel in the microchip; Applying an electric field across the microchannel; Enlarging the image in the microchannel with a refractive differential microscope (refractive DIC microscope); And Photographing the enlarged image by an image photographing means, and observing a biomolecule in the microchannel, The microchip includes an upper substrate and a lower substrate, One of the upper substrate and the lower substrate is made of glass or a transparent polymer material, the other substrate is made of silicon, The method of observing biomolecules using a refraction DIC microscope, characterized in that the microchannel is intaglio formed on any one of the upper substrate and the lower substrate.

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