KR20090020384A - DNA chip detection method using an evanescent wave fluorescence microscope - Google Patents

Abstract

A method for detecting the DNA chip by using evanescent wave fluorescence microscopy is provided to excite the fluorescent material adhered to the target DNA by using the evanescent wave generated with SPR(surface Plasmon resonance), thereby confirming the position and number of base sequence in the low DNA concentration of about 1nM. The method for detecting the DNA chip by using evanescent wave fluorescence microscopy comprises the steps of: immobilizing the carrier on the chip; adhering the fluorescent material to the end of DNA; exciting the fluorescent material adhered to the DNA by using the evanescent wave generated with SPR; and inspecting the excited fluorescent material with the evanescent wave fluorescence microscopy.

Description

Detecting method of DNA chips using evanescent wave fluorescence microscopy

1 is a manufacturing process of the DNA chip.

Figure 2 is a photograph of the carrier after UV irradiation attached to the dicing tape and cut to a size of 400μm with a dicing machine.

3 is a process diagram of fixing a biotin-modified probe DNA to a carrier by a biotin-avidin binding method.

Figure 4 is a DNA chip microarray process diagram of the carrier fixed to the pattern chip.

5 is a photograph of a carrier fixed to a pattern chip.

Figure 6 is a photograph of the concentration of the fluorescent image according to the hybridization of the probe DNA (PB-1) and match DNA (CR-1).

Figure 7a is a graph measuring the hybridization according to the concentration of the probe DNA and match DNA in real time.

Figure 7b is a graph measuring the fluorescence concentration according to the concentration of the target DNA.

8 is a concentration-specific photograph of fluorescence images according to hybridization of probe DNA (PB-1) and mismatched DNA having mismatched nucleotide sequences in the central and terminal portions.

FIG. 9A is a ΔFI graph of real time measurements of matched probe DNA and hybridization of each mismatched DNA. FIG.

9B is a graph showing fluorescence intensity according to match with probe DNA and concentration of each mystery DNA.

The present invention uses an evanescent wave fluorescence microscope that can confirm the position and number of nucleotide sequences even at a low DNA concentration of about 1 nM by excitation of a fluorescent substance modified on the target DNA using the evanescent wave generated by SPR. It relates to a DNA chip detection method.

Recently, devices that integrate a variety of biomaterials, including DNA chips, into a single chip using microelectromechanical systems (MEMS) have attracted attention. Not only can a large number of analyzes be performed at the same time, it is expected to reduce the consumption of reagents and samples by micronization and to obtain complex information unknown from a single species or several kinds of recognition materials.

The DNA chip manufacturing method can be broadly divided into two types, a method using lithographic and a method using a stamp. The lithographic method uses a photoreaction of the deprotection group reaction at the extremity end in a high phase synthesis, and a random pattern can be formed as a fine spot by a combination of mask patterns that determine the irradiation position. Can be. However, there is a problem that the stretchable length is short, such as 20 to 30mer, and is not suitable for the construction of large molecules as a limitation of high synthesis. The method of using a stamp is to transfer various kinds of materials prepared in advance to the tip of a needle by a simple automated device and transfer them on a chip. This method has no material restrictions, but there is a problem that the total amount of transfer is not fixed or contamination with adjacent sites.

An object of the present invention devised to solve the above problems is to excite the fluorescent substance modified in the target DNA by using the evanescent wave generated by SPR to position and number of base sequences even at low DNA concentration of about 1 nM. The present invention provides a DNA chip detection method using an evanescent wave fluorescence microscope.

A method for detecting DNA of a biomaterial immobilized on a carrier, the method comprising: immobilizing a carrier on a chip; Modifying a fluorescent substance called Rhodamin at the 5 'end of the match DNA and mismatch DNA; Exciting the modified fluorescent material in DNA using an evanescent wave generated by SPR; And examining the excited fluorescent material with an evanescent wave fluorescence microscope.

Hereinafter, the DNA detection method of the biomaterial fixed to the carrier according to the present invention will be described in detail.

<Sample>

The match target DNA used in the present invention is the subunit A portion (gap), which is the base sequence of STL2, which occupies 90% or more of all the DNA sequences of verotoxin producing toxins in Escherichia coli O157: H7. 2 parts) was selected and used.

All of the DNA used in the present invention was used by purifying 0.2μM double-stranded DNA in Nisshinbo, Japan by synthetic synthesis and HPLC (High Performance Liquid Chromatography). The base sequence of each DNA is shown in Table 1. Biotin was modified at the 5 terminus to fix the probe DNA on the carrier by biotin-avidin binding. For evanescent wave fluorescence microscopy, a fluorescent substance called Rhodamin was modified at the five ends of the match and each mismatched DNA. This biodine-avidin binding method can utilize the principle that avidin, a glycoprotein, has a very affinity for biotin, a low molecular weight vitamin. For reference, avidin is a glycoprotein present in egg white and has a three-dimensional structure that can bind to biotin.

3,3'-dithiodipropionic acid, a reagent used for immobilization of DNA, was used as the product of PFALTZ & BAUER. In addition, NHS (N-hydroxy succinimide, C ₄ H ₅ NO ₃ ), avidin (Mw = 67,000), Tris [2-Amino-2-hydroxymethy1-1,3-propanediol], H ₂ NC (CH ₂ OH) ₃ , Sodium chloride (NaC 1), 2-aminoethanol (H ₂ NCH ₂ CH ₂ OH) were used as silver from Wako Pure Chemical Industries, Ltd. EDC (1-Ethy1-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, C ₈ H ₁₇ N _{3 ·} HCI) was used as a product of TOKYO Kasei Organic Chemistry.

Kind of DNA base sequence probe (PB-1) match (CR-1) Mismatch 1 (MR-1) Mismatch 2 (MR-2) Mismatch 3 (MR-3) Mismatch 22 (MR-22) Mismatch 33 (MR-33) 5 'TGCAGAGTGGTATAACTG 3' 5 'CAGTTATACCACTCTGAA 3' 5 'GGTTTCCATGACAACGGA 3' 5 'CAGTTATGCCACTCTGCA 3' 5 'CAGTTATGCCACTCTGCG 3' 5 'CAGTTATAGGACTCTGCA 3' 5 'CAGTTATACCACTCTGGG 3'

Wherein the match DNA (CR-1) and mismatch DNA (MR-1) are part of the DNA nucleotide sequence of the subunit A portion of Escherichia coli O157: H7. The underlined portion of the base sequence represents the mismatched base sequence. Here, MR-2 and MR-3 of mismatched DNA have one mismatched nucleotide sequence at the center and the end, and two MR-22 and MR-33 at the middle and the end of the mismatched DNA. Had mismatched nucleotide sequences. This mass-matched DNA was used to determine how the location and number of mismatched nucleotide sequences affect DNA hybridization.

All DNA was provided in Powerer format and used by dissolving at a concentration of 100 μM in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.4). In addition, the concentration of all DNA was adjusted from 01.nM to 10μM by aqueous buffer solution (10mM Tris-HCI, 0.2M NaC1, pH7.9) and stored at -20 ℃ when not used.

In addition, the indicator used in the present invention (indicator) is Methylen blue (MB) and Mitoxantrone (MXT). For reference, MB is a crystal that has no dark green blue odor. It is well dissolved in water, ethanol and chloroform to become a blue solution, but insoluble in ether and weak in sunlight. There are a method of synthesizing from p-aminodimethylanilan made from dimethylaniline as a raw material, and a method obtained by reacting dimethylamine after brominating phenothiadine. Colorless by reducing with Ti (III) salt, V (II) salt, CR (II) salt, Sn (II) salt, Cu (I) salt, Mo (III) salt, W (V) salt, etc. in acidic solution Since it becomes a leuco compound, it is used for these detection and measurement. In addition, it is used for redox indicators, cell nuclear stains, blood cell stains, fungicides, analgesics and the like. In addition, MXT is an anthracenedion drug and is widely used as a secondary treatment for breast cancer, acute leukemia, and non-Hodgkin's lymphoma by interacting with DNA. MXT has a two-dimensional heterocyclic ring structure that is positively charged to the extramolecular nitrogen chain, so it is inserted and stabilized between the bases of negatively charged bibasic DNA. Therefore, MXT is used in DNA biosensor research by electrochemical measurement because it reacts between hydrogen bonds of dibasic DNA, but the exact mechanism is not yet known.

<Production of carrier>

The carrier manufacturing process of the cover glass is shown in FIG. First, the cover glass substrate (0.13∼0.16mm, 18mm ㅧ 18mm, Takahashi Giken, Glass Co.) was cleaned for 30 minutes in the order of pure water, acetone, and pure water using an ultrasonic cleaner (W-222, Honda). . And, one surface of the cover glass has a spin coater (1H-D3, MIKASA) while being rotated by 10 seconds at 500rpm, 20 seconds at 1,000~4,000rpm (C ₄ F _{9) 3} N by using a 0.45~9.0wt solvent solution CPFP diluted to a concentration of% was applied to a thickness of 0.5-2.0 μm by dropping 100 μL with a pipette to make hydrophobic. Subsequently, it put in 115 degreeC constant temperature oven (DS64, YAMATO SCIENFITIC), and hard-baked for 4 hours.

Then, on the opposite side, about 200 크롬 of chromium was deposited by using a resistance heating type small vacuum deposition apparatus (SVC-700TURBO-TM, SANYU) using a oxton boat, and then gold was maintained while maintaining vacuum (2Φ 500L). Was deposited at about 2,000 cc. The film thickness was measured using a film thickness monitor (TM-200R, Maxtek) using a crystal oscillator (6 MHz, PKG 10, LEYBOLD INFICON), and the deposition rate was 0.5 to 1.0 Å / s for chrome and 5.0 for gold. It adjusted so that it might become a range of -10.0 mW / s. The degree of vacuum was measured with an ion vacuum gauge (ULVAC GI-TL3) and kept below 10 ^-6 Torr. After vapor deposition, no heat treatment such as annealing was performed. Subsequently, attach the cover glass to the adhesive dicing tape (Adwill D-210, LINTEC), and then use a dicing machine (A-WD-10A, Tokyo Seimitsu) to use a diamond cutter (52D-0.1T-40H, Asahi). Diamond) was cut to a size of 400μm while spraying pure water at a rate of 0.5mm / s to prepare a carrier. Afterwards, the dicing tape was irradiated with UV for 5 minutes, and the adhesiveness was weakened from 19,600mN / 25mm (catalog value) before UV irradiation to 250mN / 25mm after UV irradiation, so that the carrier easily fell off the adhesive tape. By this process, about 1,000 to 8,000 carriers could be produced, and photographed with a digital camera as shown in FIG.

 In FIG. 2, the gold part is a manufactured carrier, and the white part is a dicing tape. Since the carrier on the dicing tape can be easily peeled off with tweezers or the like, it can be used as a DNA chip.

<Fixing of Probe DNA>

The process of immobilizing the biotin-modified probe DNA to the carrier by the biotin-avidin binding method is shown in FIG. 3.

First, the carrier was immersed in 3 ml of 3,3'-dithiodipropionic acid aqueous solution at 1 mM concentration for 20 minutes at room temperature, and then reacted with carboxylic acid for 1 hour with NHS and EDC made of 100 mg / ml concentration as a mixed solution and dried. . In addition, avidin was immersed in a 1 ml solution prepared in an aqueous buffer solution of 0.2 mg / ml for 30 minutes. Next, the carrier was immersed in 1 ml of 1 M aqueous ethanolamine solution for 30 minutes to inactivate carboxylation. Finally, the carrier modified with avidin was immersed in a 1 ml solution of biotinylated probe DNA in an aqueous buffer solution at 25 ° C. for 1 hour. Here, the fixed amount of biotinylated probe DNA was controlled by the time to soak in the DNA solution.

Through this process, the probe DNA was fixed by binding to avidin on the surface of the DNA chip microarray.

<Fixing of carrier to pattern chip>

In order to fix the carrier (400 μm) on the pattern chip (500 μm), the pattern chip was fixed to the shale as shown in FIG. 4, and a suspension of ethanol 90% and 10% pure water was added thereto. Then, when about 1500 ~ 4000 carriers are added to the suspension and shaken, the carriers sink by gravity, and the hydrophobic portions of the carriers on which the pattern chip and the biotinylated DNA are modified by hydrophobic interaction are randomly bound at numerous sites. The carrier was immobilized. The carrier photograph fixed to the pattern chip by hydrophobic interaction is shown in FIG. 5. As shown in Fig. 5, most carriers are in contact with the pattern chip and the hydrophobic portions.

<Evancent wave fluorescence microscope experiment>

By using a mixture of carriers immobilized with biomaterials having different chemical and biochemical properties in suspension, multi-item measurement and highly integrated microarray DNA chips obtained by immobilizing various biomaterials at high density were obtained. The interaction between DNAs was monitored in real time using an evanescent wave fluorescence microscope (Y-FL, Nikon) by exciting the modified DNA on the target DNA using the evanescent wave generated by SPR. In addition to DNA and PNA, various materials examined in the field of biosensors, such as proteins such as enzymes, antibodies, receptors, peptides, living cells of microorganisms and higher organisms, and hybrid materials thereof may be mentioned.

That is, in order to prevent light scattering of the DNA chip microarray in which the probe DNA is immobilized, it is fixed on the prism in which oil (n _d = 1.516, 23 ° C) is injected. After injecting Rhodamine-modified match and 400 μl of each mismatched DNA, adjusted from 0.1nM to 10μM, onto the DNA chip, the presence or absence of fluorescent material by hybridization of the probe DNA and the target DNA was imaged with a CCD camera. The change value of the fluorescence intensity obtained by the fluorescence image was recorded by the Histogram program. The measurement time was 90 minutes, and the measurement temperature was performed under the conditions of room temperature (23 ° C). Accordingly, it was possible to observe how the position and number of mismatched nucleotide sequences affect DNA hybridization from the change in fluorescence intensity according to the match and hybridization of each mismatched DNA.

<Reaction Characteristics of Matched DNA According to Concentration>

Fluorescence images of the hybridization of the probe DNA (PB-1) and the match DNA (CR-1) in the probe DNA among the DNA shown in Table 1 were measured at various concentrations, and the results are shown in the photograph of FIG. 6.

As shown in the photograph of FIG. 6, the fluorescence intensity obtained by the evanescent wave fluorescence microscope increases in proportion to the concentration of the matched DNA, and the fluorescence image is confirmed up to a concentration of about 1 nM. Accordingly, the measurable concentration of the evanescent wave fluorescence microscope used in the present invention was about 1 nM or more, and the result could be obtained even at a concentration about 1000 times lower than that of the BIACORE apparatus.

In addition, the results of measuring the hybridization according to the concentration of the probe DNA and the match DNA in real time is shown in Figure 7a, the fluorescence concentration according to the concentration of the target DNA is shown in Figure 7b. Here, ΔF.I. represents a change in fluorescence intensity, which is a value obtained by subtracting the fluorescence intensity before injection from the fluorescence intensity after injection of the target DNA. The concentration of probe DNA was adjusted to 1 μM and used.

As shown, the fluorescence intensity of the fluorescent material modified in the target DNA according to the evanescent wave generated by SPR depends on the refractive index of the medium proximate the DNA chip microarray surface. In other words, immobilizing the probe DNA on the surface of the DNA chip microarray formed with the gold thin film and injecting a sample containing the target DNA increases the mass of DNA on the surface of the DNA chip microarray by DNA hybridization. The refractive index of the microarray surface is increased. The evanescent wave generation intensity I _Z increases with this change in refractive index.

The evanescent wave generation intensity and the fluorescence intensity are proportional to each other. As shown in FIG. 7A by comparing the change in fluorescence intensity as an image, as the time increases, the change value ΔF.I. It shows saturation in about 45 minutes. This increases linearly as the refractive index of the DNA chip microarray surface increases due to the hybridization of probe DNA and match DNA with time, and after about 45 minutes, the change of the refractive index of the DNA chip microarray surface due to hybridization of DNA It is thought that ΔF.I. is saturated because there is no.

And, as shown in Figure 7b, the fluorescence intensity obtained by the evanescent wave fluorescence microscope increases in proportion to the concentration of DNA, it can be seen that there is a concentration dependency up to a concentration of about 1 nM.

Response characteristics of mismatched DNA under various conditions

Fluorescence images of hybridization of probe DNA (PB-1) and mismatched DNAs (MR-2 to MR-33) having mismatched nucleotide sequences in the central and terminal portions of each DNA shown in Table 1 were obtained at a concentration of 1 μM. The measurement results are shown in FIG. 8.

As shown in Fig. 8, the match obtained by the evanescent wave fluorescence microscope and the fluorescence image of each mismatched DNA show a clear difference. Therefore, the evanescent wave fluorescence microscope used in the present invention can be used to compare the fluorescence intensities according to the match and hybridization of each mismatched DNA to observe how the position and number of mismatched nucleotide sequences affect DNA hybridization. have.

On the other hand, the results of measuring the hybridization of the probe DNA and the match and each mismatched DNA in real time is shown in Figure 9a, the fluorescence intensity according to the match of the probe DNA and the concentration of each mystery DNA is shown in Figure 9b.

Comparing the results of FIG. 9A, it can be seen that match DNA has the largest change value FI of fluorescence intensity compared to each mismatched DNA, and DNA hybridization is most likely to occur. In contrast, MR-1 having a 12mer mismatched DNA nucleotide sequence shows the smallest change in fluorescence intensity FI and the most difficult DNA hybridization.

Comparing the change value FI of the fluorescence intensity with respect to the position of the mismatched nucleotide sequence using MR-2 and MR-3 in mismatched DNA, the case where the mismatched nucleotide sequence is located at the terminal is larger. Conversely, the more mismatched nucleotide sequences are located in the center, the more difficult DNA hybridization is.

Next, comparing the change in fluorescence intensity FI with respect to the number of mismatched base sequences using MR-2 and MR-22 or MR-3 and MR-33, the number of mismatched base sequences is small. The larger the change value is, the more difficult the DNA hybridization is.

In addition, as shown in FIG. 5B, the fluorescence intensity obtained by the evanescent wave fluorescence microscope increases almost linearly with the concentration, and even for each mismatched DNA, the concentration is up to the concentration of about 1 nM like the match DNA. You can see that there is a dependency.

As described above, according to the DNA chip detection method using the evanescent wave fluorescence microscope of the present invention, since the fluorescent substance modified in the target DNA is excited using the evanescent wave generated by SPR, about 1 nM Even at low DNA concentrations, there is an advantage in confirming the location and number of nucleotide sequences.

Claims (5)

In the DNA detection method of a biomaterial fixed to a carrier, Immobilizing the carrier on a chip; Modifying a fluorescent material at the ends of the DNA; Exciting the modified fluorescent material in DNA using an evanescent wave generated by SPR; A method for detecting a DNA chip using an evanescent wave fluorescence microscope, comprising: inspecting the excited fluorescent substance with an evanescent wave fluorescence microscope. The method of claim 1, wherein the fluorescent material is a rhodamine (Rhodamin) DNA detection method using an evanescent wave fluorescence microscope. The biomaterial according to claim 1, wherein the biomaterial is any one of biosensor materials such as DNA, RNA, enzymes, antibodies, proteins such as receptors, peptides, living cells of microorganisms and higher organisms, and hybrid materials thereof. DNA chip detection method using cent wave fluorescence microscope. The method of claim 1, wherein the DNA is matched or mismatched DNA chip detection method using an evanescent wave fluorescence microscope. The method of claim 1, wherein the biomaterial is immobilized on the carriers. Fixing a biotin-modified DNA at the 5 ′ end via a thiol derivative and avidin; Dipping the carrier in an aqueous solution of 3,3′-dithiodipropionic acid; Reacting with carboxylic acid with NHS and EDC as a mixed solution in the aqueous solution and then drying; Dipping avidin in a solution prepared with buffer; Dipping the carrier in an aqueous ethanolamine solution and inactivating the carboxyl group; Dipping a biotinylated probe DNA into a buffer of a carrier modified with avidin in a solution; and a DNA chip detection method using an evanescent wave fluorescence microscope.

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