JPH1151935A - Simultaneous detection method for plural kinds of target molecules - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simultaneous detection method, which is provided with high detection sensitivity and easy detection operability, for plural kinds of target molecules. SOLUTION: The subject method consists of four processes. In the first process, hapten-labeled molecules 21, 22 are prepared by labeling specific bonding molecules, which can specifically combine with plural kinds of target molecules 11, 12, with different kinds of haptens according to their kinds. In the second process, enzyme- labeled anti-hapten antibodies 31, 32, each of which consists of an anti-hapten antibody specifically coupled with a hapten and a different kind of enzyme according to the kind of the anti-hapten antibody, are prepared. In the third process, fluorescent substances 41, 42, which can react with the enzymes specifically so as to fluoresce in the different colors, are prepared. In the fourth process, the hapten-labeled molecules 21, 22 are combined with the target molecules 11, 12, the enzyme-labeled anti-hapten antibodies 31, 32 are coupled with the hapten-labeled molecules 21, 22, the fluorescent substrate are allowed to react with the enzymes, and the plural kinds of target molecules 11, 12 can be detected at the same time according to the different fluorescent colors.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for simultaneously detecting a plurality of types of target molecules using a fluorescent substrate.

[0002]

2. Description of the Related Art A multicolor detection kit for detecting nucleic acids having different base sequences using a plurality of types of chromogenic substrates of alkaline phosphatase (hereinafter also referred to as ALP) is commercially available from Boehringer Mannheim. A detection method using this kit will be described. First, as shown in FIG.
A911 to 913 are mixed, these are electrophoresed on the gel 92 (FIG. 13A), and then transferred to a nylon membrane 93 (FIG. 13B).

Next, digoxigenin (hereinafter referred to as DIG)
Also called. ) And probe D labeled with biotin (hereinafter also referred to as BIO).
An NA 922 and a probe DNA 923 labeled with fluorescein (hereinafter, also referred to as FLUOS) are prepared. Next, these probe DNAs 921 to 923 are mixed and the target DNA 911 on the membrane 93 is mixed.
To 913 (FIG. 13)
(C)).

[0004] Next, as shown in FIG.
ALP was applied to the probe DNA 921 labeled with IG.
The labeled anti-DIG antibody 931 is coupled. Next, a fluorescent substrate 941 obtained by combining a phosphoric acid form of naphthol AS-GR and Fast Blue B (diazonium salt) is reacted with ALP. Next, FIG.
As shown in (D2), the membrane is heated to inactivate ALP bound to the target DNA 911.

[0005] Next, as shown in FIG.
ALP is applied to the probe DNA 922 labeled with IO.
The labeled anti-BIO antibody 932 is coupled. Next, the phosphoric acid form of naphthol AS and Fa
A fluorescent substrate 942 combined with st Red TR (diazonium salt) is reacted. Next, FIG.
As shown in 2), the membrane is heated and the target D
ALP bound to NA912 is inactivated.

[0006] Next, as shown in FIG.
For the probe DNA 923 labeled with LUOS, A
The LP-labeled anti-FLUOS antibody 933 is coupled. Next, a fluorescent substrate 943 in which a phosphoric acid form of naphthol AS and Fast Blue B (diazonium salt) are combined is reacted with ALP. Then, FIG.
4 (F2), the membrane is heated to inactivate ALP bound to the target DNA 913.

[0007] Next, as shown in FIG. 14 (G), the nylon membrane 93 is irradiated with excitation light to emit a fluorescent material 941.
Excite the reaction product from ~ 943. This gives
The target DNA 911 is detected in green, the target DNA 912 is detected in red, and the target DNA 91 is detected in red.
3 is detected in blue.

[0008]

The conventional method for detecting DNA using the multicolor probe method has low sensitivity because the detection signal is color. In addition, any target DNA 91
Since 1 to 913 are detected by the reaction of alkaline phosphatase (ALP), a step of inactivating ALP must be performed every time the reaction of the enzyme bound to each target DNA is completed. Therefore, the detection operation is complicated.

[0009] When the inactivation of ALP is insufficient, in the enzymatic reaction of ALP bound to the target DNA, the AL bound to the target DNA last time is also used.
In some cases, P also reacts, making it impossible to distinguish the colors of the target DNAs of different types, making it impossible to use the multicolor probe method.

The present invention has been made in view of the above problems, and has as its object to provide a method for simultaneously detecting a plurality of types of target molecules, which has high detection sensitivity and is easy to perform a detection operation.

[0011]

According to the first aspect of the present invention, a specific binding molecule having a property of specifically binding to a plurality of types of target molecules to be detected is different for each type of the specific binding molecule. Preparing a hapten-labeled molecule obtained by labeling different types of haptens, an enzyme-labeled anti-hapten comprising an anti-hapten antibody that specifically couples with the hapten, and an enzyme of a different type for each type of the anti-hapten antibody A step of preparing an antibody; a step of preparing a fluorescent substance capable of reacting specifically with the enzyme to fluoresce in different colors; and a step of binding the hapten-labeled molecule to a plurality of types of target molecules to be detected. A step of coupling the enzyme-labeled anti-hapten antibody to the hapten-labeled molecule, and a step of reacting the enzyme with a fluorescent substrate. A simultaneous detection method of a plurality of types of target molecules, characterized in that it consists of a step of detecting several types of target molecules at the same time.

The most characteristic features of the present invention are that a hapten-labeled molecule obtained by labeling a specific binding molecule with a hapten, an enzyme-labeled anti-hapten antibody, and a fluorescent color correspond to the type of target molecule to be detected. Different fluorescent substrates were used.

That is, a hapten-labeled molecule is specifically bound to each of the target molecules, and an enzyme-labeled anti-hapten antibody labeled with a different enzyme is coupled to the target molecule using an antigen-antibody reaction. On the other hand, fluorescent substrates having different colors are reacted. Therefore, each target molecule can be detected with a different fluorescent color for each type.

Further, by performing each step of the present invention once, a plurality of types of target molecules can be simultaneously detected. It is not necessary to carry out an enzymatic reaction separately for each target molecule as in the prior art. Therefore, the operation is very simple as compared with the related art. In addition, detection can be performed in a short time regardless of the type of the target molecule.

Further, since different kinds of enzymes are reacted at the same time, there is no need to deactivate the enzymes after completion of each enzyme reaction as in the prior art, and the detection operation is simple.
In addition, there is no detection error due to defective enzyme inactivation, and the detection sensitivity is extremely high.

[0016] For example, as in the invention of claim 3, the target molecule is a nucleic acid, an antibody, an antigen, or a protein.

The specific binding molecule is not particularly limited as long as it has a property of specifically binding to the target molecule. For example, as in the invention of claim 4, the specific binding molecule is a nucleic acid probe or an antibody.

As in the invention of claim 5, the hapten is preferably gibberellin, biotin, or fluorescein. This improves the binding between the hapten and the anti-hapten antibody. As gibberellin, it is particularly preferable to use gibberellin A4 from the viewpoint of detection sensitivity. As the anti-hapten antibody, an anti-hapten antibody corresponding to such a hapten is used.

Different kinds of enzymes are selected for each anti-hapten antibody. As in the invention of claim 6, it is preferable that a plurality of kinds of the enzymes are selected from a group of hydrolytic enzymes such as alkaline phosphatase, acid phosphatase, peroxidase, esterase, and β-galactosidase. These are because the reaction with the fluorescent substrate is high, and the detection sensitivity is increased.

As in the invention of claim 7, the enzyme-labeled anti-hapten antibody is an alkaline phosphatase-labeled anti-gibberellin antibody, an acid phosphatase-labeled anti-gibberellin antibody, a peroxidase-labeled anti-gibberellin antibody, an esterase-labeled anti-gibberellin antibody, a β-galactosidase-labeled antibody. Anti-gibberellin antibody, alkaline phosphatase-labeled anti-biotin antibody, acid phosphatase-labeled anti-biotin antibody,
Peroxidase-labeled anti-biotin antibody, esterase-labeled anti-biotin antibody, β-galactosidase-labeled anti-biotin antibody, alkaline phosphatase-labeled anti-fluorescein antibody, acid phosphatase-labeled anti-fluorescein antibody, peroxidase-labeled anti-fluorescein antibody, esterase-labeled anti-fluorescein antibody, and β-galactosidase Preferably, a plurality of types are selected from the group of labeled anti-fluorescein antibodies. These have high binding to haptens and high reactivity with fluorescent substrates, so that detection sensitivity is further increased. When the hapten is gibberellin A4, an anti-gibberellin antibody is used.

[0021] As the fluorescent substrate, a plurality of types of fluorescent substrates that fluoresce in different colors are selected. As in the invention of claim 8, the fluorescent substrate of the alkaline phosphatase or the acid phosphatase has any one of the skeletons shown in FIG. 1, FIG. 2, and FIG. In FIG. 2, R1,
R2 is one or more selected from the group consisting of an alkyl group, an aromatic group, an alkoxyl group, and a halogen group. In FIG. 3, R3 is an organic substance having an aromatic group or a condensed aromatic group; n1 is preferably 1 to 10. This increases the fluorescence intensity and improves the detection sensitivity. The fluorescent substrate having the skeleton shown in FIG. 1 to FIG. 3 reacts with alkaline phosphatase or acid phosphatase and is excited, whereby the one shown in FIG.
2 fluoresces green, and FIG. 3 fluoresces yellow-green.

According to the ninth aspect of the present invention, the fluorescent substrate for peroxidase has a skeleton shown in FIG. 4, in which R4 is hydrogen, an alkyl group, an aromatic group, an alkoxyl group and 1 selected from the group of halogen groups
It is preferable to use at least two species. This increases the fluorescence intensity and improves the detection sensitivity. The fluorescent substrate having the skeleton shown in FIG. 4 emits orange when excited by reacting with peroxidase.

As in the tenth aspect, the fluorescent substrate of the esterase has any one of the skeletons shown in FIGS. 5, 6, and 7, and in FIG. Is an organic substance having an aromatic group or a condensed aromatic group, n2 is 1 to 10, and in FIG. 6 and FIG.
R7 is preferably one or more selected from the group consisting of an alkyl group, an aromatic group, an alkoxyl group and a halogen group. This increases the fluorescence intensity,
The detection sensitivity is improved. Each of the fluorescent substrates having the skeleton shown in FIGS. 5 to 7 emits yellow-green fluorescence by reacting with the esterase.

Next, the invention according to claim 2 is characterized in that specific binding molecules having the property of specifically binding to a plurality of types of target molecules to be detected are different for each type of the specific binding molecules. Preparing a hapten-labeled molecule obtained by labeling different types of haptens, an enzyme-labeled anti-hapten comprising an anti-hapten antibody that specifically couples with the hapten, and an enzyme of a different type for each type of the anti-hapten antibody A step of preparing an antibody, a step of adding the hapten-labeled molecule to a sample to be determined for the presence or absence of a target molecule to be detected, a step of adding the enzyme-labeled anti-hapten antibody to the sample, Adding the above-mentioned fluorescent substrate to the sample, and examining the presence / absence of fluorescence emitted from the sample and the color of the fluorescent light to determine whether a plurality of types of target molecules exist in the sample. A simultaneous detection method of a plurality of types of target molecules, characterized in that, characterized in that comprising the step of examining whether Luke.

The present invention utilizes the invention of claim 1 to simultaneously detect whether or not a plurality of types of target molecules are present in a subject. If the target molecule to be detected does not exist in the sample, the sample does not show any fluorescence, but if there is at least one type of target molecule, the target molecule in the sample must be Fluorescent to different colors. When fluorescence from a fluorescent substrate is detected, which fluorescent color corresponds to which target molecule is predicted by the type of fluorescent substance that reacts with the enzyme bound to the target molecule by an antigen-antibody reaction. From the target molecule.

It is preferable that the constituent elements in the second aspect of the invention use those of the third to tenth aspects. The above-mentioned inventions of claims 1 to 10 can be used for luminescence, clinical examination, immunology, genetic diagnosis, particularly for examining the presence or absence of metastasis of an oncogene.

[0027]

BEST MODE FOR CARRYING OUT THE INVENTION

Embodiment 1 A method for simultaneously detecting a plurality of types of target molecules according to an embodiment of the present invention will be described with reference to FIGS. To explain the outline of this example, first, FIG.
As shown in (B), DNA fragments as target molecules 11 and 12 were electrophoresed in agarose gel 2,
Next, these are transferred to the membrane 3.

Next, as shown in FIG. 8C, haptens (DIG, Flous) are labeled on a probe DNA having a property of specifically binding to a plurality of types of target molecules 11 and 12 to be detected. A plurality of types of hapten-labeled molecules 21 and 22 are prepared. Next, the hapten-labeled molecules 21 and 22 are bound to the target molecules 11 and 12, respectively.

Next, as shown in FIG. 9 (D), the hapten-labeled molecules 21 and 22 are coupled to enzyme-labeled anti-hapten antibodies 31 and 32 labeled with a plurality of different enzymes. Next, fluorescent substrates 41 and 42 are reacted with each enzyme. Next, as shown in FIG. 9 (E), by irradiating the membrane 3 with excitation light, the plurality of target molecules 11 and 12 are simultaneously detected with different fluorescent colors.

As the two types of target molecules 11 and 12, λ DNA and Co digested with a restriction enzyme (EcoRI) were used.
Use 1E1. The hapten-labeled molecules 21 and 22 include DIG-labeled DNA probes complementary to λDNA / EcoRI and Flous-labeled λDNA.
Use a DNA probe complementary to / EcoRI.

The enzyme-labeled anti-hapten antibodies 31 and 32 include a POD-labeled anti-DIG antibody and an ALP-labeled anti-Flou antibody.
s antibody. As the fluorescent substance 41 for POD, 4-benzoylamino-2-methoxy
-5-methylbenzenamine (FIG. 1
0), and HN is used as the fluorescent substance 42 for ALP.
PP (own product) (FIG. 11) is used.

Hereinafter, this will be described in detail. EcoR1
Λ DNA and ColEI digested in the above were mixed at 5 ng and 1 ng per electrophoresis lane, respectively, and electrophoresed using a 1% agarose gel. Power supply voltage is 50V
The electrophoresis buffer used was TAE (Tris acetate E).
DTA) was used. After electrophoresis, the agarose gel was immersed in 0.25N hydrochloric acid for 5 minutes, and further immersed in 0.5N sodium chloride for 10 minutes.

Next, the DNA in the agarose gel was transferred to a nylon membrane by vacuum blotting. This was added to double concentration SSC (1 × SSC; 15 mM N
The operation of washing with aCl, 15 mM citric acid (pH 7.5) for 5 minutes was repeated twice. Then, by UV irradiation, DN
A was fixed to the membrane.

Next, for blocking, a commercially available hybridization solution (DIG Easy Hyb. Boehringer Mannheim) was used and immersed in the hybridization solution at 37 ° C. for 30 minutes. Next, DIG
DNA probe (100 ng) complementary to λ / EcoRI labeled with, and ColEI labeled with Flous
/ EcoRI and a DNA probe (50 ng) complementary to each other were mixed in 1 ml of DIG EasyHyb (Boehringer Mannheim hybridization solution),
Put this on a membrane as a probe solution,
Hybridization was performed for 0 hours.

Double concentration SSC, concentration 0.1% SDS
(Sodium dodecyl sulfate) twice for 5 minutes. Further, it was washed twice at 55 ° C. for 15 minutes using 1/10 concentration SSC and 0.1% SDS. Subsequently, it was immersed in a blocking buffer (commercially available) for 20 minutes.

POD-labeled anti-DIG antibody and ALP-labeled anti-F
mixed solution with each other (concentration 1/1000, respectively)
(Concentration 1/2000) was placed on the membrane and allowed to stand for 30 minutes. Then, add 3 minutes for 10 minutes with blocking buffer.
Washed twice.

Next, 2 mM 4-benzoylamimi was used.
no-2-methyoxy-5-methylbenz
eneamine, 80 μM HNPP, 0.1 M T
ris (pH 7.5), 10 mM magnesium chloride,
Fluorescent substrate solution consisting of 0.003% hydrogen peroxide
The enzyme reaction was carried out at room temperature for 3 hours on a membrane. After the reaction, the membrane was irradiated with UV excitation light, and a fluorescence signal was observed.

As shown in FIG. 9 (E), λ / EcoRI as the target molecule 11 emits orange light, ColEI / EcoRI as the target molecule 12 becomes blue, and two color fluorescent signals are detected on the membrane 3. Was done.

Embodiment 2 In this embodiment, three types of target molecules were simultaneously detected. As the three kinds of target molecules, λDNA, ColE1, and pBR322 cut with EcoRI were used.

The hapten-labeled molecules include DIG-labeled DNA probe complementary to λDNA / EcoRI, Flous-labeled DNA probe complementary to ColE1 / EcoRI, and BIO-labeled pBR.
A DNA probe complementary to H.322 / EcoRI was used.

POD is used as an enzyme-labeled anti-hapten antibody.
A labeled anti-DIG antibody, an ALP-labeled anti-Flous antibody,
An esterase-labeled anti-BIO antibody was used. As a fluorescent substance for POD, 4-benzoylamino-
2-methyoxy-5-methylbenzene
Amine (FIG. 10), HNPP (FIG. 11) is used as a fluorescent substance for ALP, and BNF acetate (our product) is used as a fluorescent substance for esterase.
(FIG. 12) was used.

Using these, the above-mentioned target molecules were simultaneously detected in the same manner as in Embodiment 1. As a result, λ / EcoRI fluoresces orange and ColEI
/ EcoRI fluoresces blue and pBR322 / EcoR
I fluoresced yellow-green. As described above, fluorescent signals of three colors indicating three types of target DNAs were detected.

[0043]

According to the present invention, it is possible to provide a method for simultaneously detecting a plurality of types of target molecules, which has high detection sensitivity and easy detection operation.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing a chemical structural formula of a fluorescent substrate capable of reacting with alkaline phosphatase or acid phosphatase in the present invention.

FIG. 2 is an explanatory diagram showing a chemical structural formula of a fluorescent substrate capable of reacting with alkaline phosphatase or acid phosphatase in the present invention.

FIG. 3 is an explanatory diagram showing a chemical structural formula of a fluorescent substrate capable of reacting with alkaline phosphatase or acid phosphatase in the present invention.

FIG. 4 is an explanatory view showing a chemical structural formula of a fluorescent substrate capable of reacting with peroxidase in the present invention.

FIG. 5 is an explanatory diagram showing a chemical structural formula of a fluorescent substrate capable of reacting with an esterase in the present invention.

FIG. 6 is an explanatory view showing a chemical structural formula of a fluorescent substrate capable of reacting with an esterase in the present invention.

FIG. 7 is an explanatory view showing a chemical structural formula of a fluorescent substrate capable of reacting with an esterase in the present invention.

FIG. 8 is an explanatory diagram showing a method for simultaneously detecting two types of target molecules in the first embodiment.

FIG. 9 is an explanatory view following FIG. 8 showing a method for simultaneously detecting two types of target molecules.

FIG. 10 is a diagram illustrating a 4-benzo according to the first and second embodiments.
ylamino-2-methyoxy-5-methy
BRIEF DESCRIPTION OF THE DRAWINGS FIG.

FIG. 11 is an explanatory view showing a chemical structural formula of HNPP in the first and second embodiments.

FIG. 12 is an explanatory diagram showing a chemical structural formula of BNF acetate in Embodiment Example 2.

FIG. 13 is an explanatory view showing a conventional multicolor probe method.

FIG. 14 is an explanatory view following FIG. 13 showing a conventional multicolor probe method.

[Explanation of symbols]

 11,12. . . Target molecule, 21, 22,. . . Hapten-labeled molecules, 31, 32. . . Enzyme-labeled anti-hapten antibody, 41, 42. . . Fluorescent substrate,

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/543 501 G01N 33/543 501D 33/566 33/566 // C12Q 1/68 C12Q 1/68 A (72) Inventor Atsushi Hattori Aichi 5-50, Hachiken-cho, Kariya-shi, Aisin Co., Ltd. Aisin Cosmos Research Institute (72) Inventor Paedi Iera Lady 5-50, Hachigen-cho, Kariya, Aichi Prefecture Aisin Cosmos Research Co., Ltd. Ken Takeshi 2-18-19 Mitake, Togo-cho, Aichi-gun, Aichi Prefecture

Claims (10)

[Claims] 1. A specific binding molecule having a property of specifically binding to a plurality of types of target molecules to be detected is labeled with a different type of hapten for each type of the specific binding molecule. A step of preparing a hapten-labeled molecule; a step of preparing an enzyme-labeled anti-hapten antibody comprising an anti-hapten antibody that specifically couples with the hapten and an enzyme of a different type for each type of the anti-hapten antibody; A step of preparing a fluorescent substance capable of reacting specifically with an enzyme to fluoresce in a different color; a step of binding the hapten-labeled molecule to a plurality of types of target molecules to be detected; A step of coupling a labeled anti-hapten antibody, a step of reacting the enzyme with a fluorescent substrate, and a step of coupling the plurality of target molecules with different fluorescent colors. Simultaneously detecting a plurality of target molecules. 2. A specific binding molecule having a property of specifically binding to a plurality of types of target molecules to be detected, wherein different types of haptens are labeled for each type of the specific binding molecule. A step of preparing a hapten-labeled molecule, a step of preparing an enzyme-labeled anti-hapten antibody comprising an anti-hapten antibody that specifically couples with the hapten, and a different type of enzyme for each type of the anti-hapten antibody; Adding the hapten-labeled molecule to the subject to be determined whether there is a target molecule to be determined, adding the enzyme-labeled anti-hapten antibody to the subject, and adding the fluorescent substrate to the subject Inspecting whether or not a plurality of types of target molecules are present in the subject by examining the presence or absence of fluorescence emitted from the subject and the fluorescent color. A method for simultaneous detection of a plurality of types of target molecules, characterized by comprising: 3. The method according to claim 1, wherein the target molecule is a nucleic acid, an antibody, an antigen, or a protein. 4. The method according to claim 1, wherein:
The method for simultaneously detecting a plurality of types of target molecules, wherein the specific binding molecule is a nucleic acid probe or an antibody. 5. The method according to claim 1, wherein:
The method for simultaneously detecting a plurality of types of target molecules, wherein the hapten is gibberellin, biotin, or fluorescein. 6. The method according to claim 1, wherein:
A method for simultaneously detecting a plurality of types of target molecules, wherein a plurality of types of the above enzymes are selected from a group of hydrolytic enzymes such as alkaline phosphatase, acid phosphatase, peroxidase, esterase, and β-galactosidase. 7. The method according to claim 1, wherein:
The above enzyme-labeled anti-hapten antibodies include alkaline phosphatase-labeled anti-gibberellin antibody, acid phosphatase-labeled anti-gibberellin antibody, peroxidase-labeled anti-gibberellin antibody, esterase-labeled anti-gibberellin antibody, β-galactosidase-labeled anti-gibberellin antibody, alkaline phosphatase-labeled anti-biotin antibody, acidic Phosphatase-labeled anti-biotin antibody, peroxidase-labeled anti-biotin antibody, esterase-labeled anti-biotin antibody, β-galactosidase-labeled anti-biotin antibody, alkaline phosphatase-labeled anti-fluorescein antibody, acid phosphatase-labeled anti-fluorescein antibody, peroxidase-labeled anti-fluorescein antibody, esterase-labeled anti-fluorescein Antibody, and β
-A method for simultaneously detecting a plurality of types of target molecules, wherein a plurality of types are selected from a group of galactosidase-labeled anti-fluorescein antibodies. 8. The method according to claim 6, wherein the fluorescent substrate of the alkaline phosphatase or the acid phosphatase has any one of the skeletons shown in FIG. 1, FIG. 2, and FIG. And FIG. 2, R1 and R2 are one or more selected from the group consisting of an alkyl group, an aromatic group, an alkoxyl group and a halogen group. In FIG. 3, R3 is an aromatic group or A method for simultaneously detecting a plurality of types of target molecules, wherein the method is an organic substance having a condensed aromatic group, and n1 is 1 to 10. 9. The method according to claim 6, wherein the fluorescent substrate of peroxidase has a skeleton shown in FIG.
In FIG. 4, R4 is one or more selected from the group consisting of hydrogen, an alkyl group, an aromatic group, an alkoxyl group, and a halogen group. A method for simultaneously detecting a plurality of types of target molecules. 10. The fluorescent substrate according to claim 6, wherein the fluorescent substrate of the esterase has one of the skeletons shown in FIG. 5, FIG. 6, and FIG. Is an organic substance having an aromatic group or a condensed aromatic group, and n2
Are 1 to 10, and in FIG. 6 and FIG.
6, wherein R7 is one or more selected from the group consisting of an alkyl group, an aromatic group, an alkoxyl group, and a halogen group;