JP2005055285A - Dna chip and manufacturing method therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a DNA chip in which DNA fragments are bonded to a base surface by quick reactions of isocyanato groups and amino groups being introduced into ends of DNAs and which can keep the bonding more stably in comparison with conventional ionic bonds formed in reaction products, and to provide a method for manufacturing the DNA chip. <P>SOLUTION: At least a binder-containing layer which includes a polymer molecule with a structural unit expressed by formula, is formed on the base. In above-mentioned formula, roman letters R, R' represent principal chains of the polymer molecule composed of identical or different groups selected from aliphatic series and aromatic series. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a DNA chip useful for simultaneous analysis of gene expression, gene mutation, gene polymorphism and the like.

In the past, analysis of various gene expression in cells and tissues has been done by Northern plots in which RNA is prepared from cells and tissues, the RNA is immobilized on a membrane, and hybridization is performed using a specific probe of the gene to be analyzed. (Or dot plot) method, RT-PCR method using primers specific to the gene to be analyzed, and the like.
On the other hand, the number of genes that need to be analyzed has increased rapidly due to advances in gene research, and gene expression fluctuations have been rapidly and rapidly increased along with progress in genome projects and applications in the medical field. There is a growing need for analysis processing.

  Technological development for responding to such a request is progressing, and a DNA chip is used as an analysis means. The feature of the DNA chip is that thousands of different DNA fragments are aligned and fixed on a glass substrate at high density, and hybridization between the fixed DNA fragment and a very small amount of labeled target DNA fragment is performed. It is to detect a target DNA fragment with high sensitivity.

As a means for detecting the formed hybrid, a method using a fluorescent label or a radioactive label previously bound to a DNA fragment sample, a method using an intercalator having a fluorogenic group or a conductive group incorporated into the hybrid, etc. Are known.
By using such a DNA chip, it is possible to quickly analyze and analyze all genes of mammals such as humans and microorganisms having thousands of genes, and to analyze the expression level for all genes by labeled RNA. It can also be done. In addition, mutations such as gene defects can be analyzed by labeling genomic DNA.

As a method for producing the DNA chip, a method of directly synthesizing a DNA fragment on the surface of the substrate (on-chip method) and a method of fixing a DNA fragment prepared separately in advance on the surface of the substrate are known.
As an on-chip method, a combination of the use of a protective group that is selectively removed by light irradiation and the photolithography technology and solid-phase synthesis technology used in semiconductor manufacturing can be combined in a predetermined region of a minute matrix. A method of performing selective synthesis is typical.
Since the use of this method is limited to oligonucleotides, a plurality of oligonucleotide probes must be used to detect one mRNA.

  On the other hand, as a method for fixing a DNA fragment prepared in advance on the surface of a substrate, first, a microplate containing a plurality of types of probe DNAs is prepared. In addition, a glass plate is prepared, and the surface is coated with a DNA-glass binder such as poly-l-lysine. After this, the probe DNA contained in the microplate is attached to the pin, and the probe is attached to the pin on the glass substrate whose surface is coated with DNA-glass binder (poly-l-Lysine). Spot DNA in contact. This operation is repeated until all the probe DNAs contained in the microplate have been spotted to produce a DNA chip.

  Thus, conventionally, a DNA chip is manufactured by previously coating a glass substrate with a DNA / glass binder in advance and plotting the DNA on the glass substrate.

In the hybridization step of the DNA chip, the DNA chip in which the probe DNA is spotted on the glass substrate with the binder and the sample DNA labeled with the fluorescent substance are both put in the hybridization solution and hybridized. The hybridization solution is a mixed solution composed of formaldehyde, SSC (NaCl, trisodium citrate), SDS (sodium dodecyl sulfate), EDTA (ethylenediamidetetraacetic acid), distilled water, and the like, and the mixing ratio varies depending on the properties of the DNA used. At this time, if the sample DNA and the probe DNA on the DNA chip are complementary strand DNAs, the two take a double helix structure and bind to each other. On the other hand, if the two are not complementary strands, they will not bind, and the sample DNA labeled with a fluorescent substance will remain in the hybridization solution as it is, or a small part of it will bind to the binder coated on the glass substrate. In some cases, it remains unbound.
Thereafter, when the sample DNA labeled with the fluorescent substance remaining on the glass substrate is placed in a water tank or the like and washed, the sample DNA not bound to the probe DNA is excluded. After that, the fluorescent substance labeled on the sample DNA bound to the probe DNA is excited by light energy from a predetermined light source, and the light emitted by the fluorescent substance is detected by a photosensor such as a CCD. Detect hybridization with.

However, in this method, DNA such as poly-l-Lysine and glass have an ionic bond, so that the binding force is not sufficient. In some cases, the soybean sample was washed away. There is a problem that probe DNA and sample DNA are greatly lost due to such insufficient binding.
In addition, sample DNA that did not bind to the probe DNA is removed from the glass substrate by washing, even if the sample DNA is non-specifically bound to the binder part where the probe DNA is not spotted. Not. It appeared as noise at the time of detection, and the measurement accuracy was lowered.
For this reason, part of the sample DNA remains on the DNA chip in a state where it is not specifically bound to the probe DNA but is simply attached to the binding agent, and the fluorescent substance labeled on the sample DNA is also excited and emits light, resulting in noise. There was a problem of being detected as.

Therefore, an object of the present invention is to develop a material and a technique for stably fixing a DNA fragment in a sufficient amount on a substrate surface in order to solve the above-described problems.
In other words, DNA fragments can be bound to the substrate surface by a rapid reaction between an amino group and an isocyanate group introduced at the end of the DNA, and the reaction product can be compared with a conventional ionic bond. It is an object of the present invention to provide a DNA chip capable of maintaining stable binding and a method for producing the DNA chip.

〔上記式（化１）において、Ｒ，Ｒ’は、同一又は異なって、脂肪族、芳香族から選ばれる基で構成される高分子の主鎖を表す。〕
また、請求項２に記載のＤＮＡチップは、請求項１に記載のＤＮＡチップにおいて、前記結合剤含有層は、蒸着重合法により形成されたものであることを特徴とする。
また、請求項３に記載のＤＮＡチップは、請求項１又は２に記載のＤＮＡチップにおいて、マスキングにより前記結合剤含有層がパターン形成されたものであることを特徴とする。
また、請求項４に記載のＤＮＡチップは、請求項１又は２に記載のＤＮＡチップにおいて、マスキングを介して予め形成された前記結合剤含有層に紫外線を照射することにより、前記結合剤含有層がパターン形成されたものであることを特徴とする。
また、請求項５に記載のＤＮＡチップは、請求項１乃至４のいずれかに記載のＤＮＡチップにおいて、前記結合剤含有層に、プローブＤＮＡを結合したことを特徴とする。
本発明のＤＮＡチップの製造方法は、請求項６に記載の通り、請求項１に記載のＤＮＡチップの製造方法であって、下記構造式（化２、化３）で表される二種類以上の原料を蒸発させ、基材上に蒸着重合させて高分子の結合剤含有層を形成するようにしたことを特徴とする。

〔上記式（化２）において、Ｒは、脂肪族又は芳香族で構成されるモノマーの骨格を表す。〕

〔上記式（化３）において、Ｒ’は、脂肪族又は芳香族で構成されるモノマーの骨格を表す。〕 In order to solve the above problems, the present inventors have found the following solution as a result of intensive studies.
That is, in the DNA chip of the present invention, as described in claim 1, a binder-containing layer containing a polymer having a structural unit represented by the following formula (Chemical Formula 1) is formed on at least a substrate. It is characterized by that.

[In the above formula (Formula 1), R and R ′ are the same or different and each represents a polymer main chain composed of a group selected from aliphatic and aromatic. ]
The DNA chip according to claim 2 is characterized in that, in the DNA chip according to claim 1, the binder-containing layer is formed by a vapor deposition polymerization method.
The DNA chip according to claim 3 is the DNA chip according to claim 1 or 2, wherein the binder-containing layer is patterned by masking.
The DNA chip according to claim 4 is the DNA chip according to claim 1 or 2, wherein the binder-containing layer is formed by irradiating the binder-containing layer formed in advance through masking with ultraviolet rays. Is characterized in that it is patterned.
A DNA chip according to claim 5 is characterized in that in the DNA chip according to any one of claims 1 to 4, probe DNA is bound to the binder-containing layer.
The method for producing a DNA chip according to the present invention is the method for producing a DNA chip according to claim 1, as described in claim 6, wherein two or more types represented by the following structural formulas (Chemical Formula 2, Chemical Formula 3) are provided. The material is evaporated and vapor-deposited on the substrate to form a polymer binder-containing layer.

[In the above formula (Formula 2), R represents a skeleton of a monomer composed of aliphatic or aromatic. ]

[In the above formula (Formula 3), R ′ represents a skeleton of a monomer composed of an aliphatic group or an aromatic group. ]

As described above, according to the present invention, DNA can be efficiently and stably immobilized by covalently bonding the reactive group on the surface of the substrate and the reactive group introduced at the end of the DNA.
In addition, according to the present invention, the sample hybridized with the probe DNA is firmly bound by binding the probe DNA to the reactive group portion on the surface of the base material, thereby enabling highly accurate measurement. . Furthermore, since necessary probe DNA and a sample are not lost in the measurement, the cost for the measurement can be suppressed.
Further, according to the present invention, the isocyanate group can be uniformly distributed on the surface of the substrate by forming the binder-containing layer on the surface of the substrate by vapor deposition polymerization.
In addition, according to the present invention, it is possible to form a pattern by irradiation with ultraviolet rays, so that it is possible to specify a location where DNA is fixed.

As described above, the DNA chip of the present invention is one in which a binder-containing layer containing a polymer having a structural unit represented by the following formula (Formula 1) is formed on at least a substrate.
Thereby, a base material and probe DNA can be combined more reliably, and probe DNA and sample DNA can be used effectively without a probe DNA and sample DNA flowing down from a base material in a washing process etc. It is what I did.

  In the above formula (Formula 1), R and R ′ are the same or different and are not particularly limited as long as they represent a main chain of a polymer composed of a group selected from aliphatic and aromatic. However, those having no reactive group in the aliphatic and aromatic groups are preferable.

  The material of the substrate is preferably transparent glass, silicon or polyethylene terephthalate, polycarbonate such as cellulose acetate or bisphenol A, polymer such as polystyrene or polymethyl methacrylate. Among these, glass or silicon is particularly preferable. This is due to the ease of surface treatment and the ease of analysis using a fluorescence scanning device. Glass having a silica surface layer is also preferably used. The thickness of the substrate is preferably in the range of 100 to 2000 μm.

In addition, the binder-containing layer of the present invention is preferably made of a material such as glass by evaporating and decomposing the raw material by a vapor deposition polymerization method, particularly by a CVD method, and polymerizing and depositing the material on a substrate. Adheres well to the material, and the amino group (NH ₂ ) present in the structural formula binds to the phosphate group (PO ₄ ) of the DNA fragment that serves as the probe, so that the probe DNA is well immobilized on the substrate. be able to.
Further, mask deposition may be performed using a mask having a predetermined pattern during film formation. Thus, by performing mask vapor deposition, a binder-containing layer pattern can be formed with high accuracy, DNA adheres to unnecessary portions and remains as garbage, and S / N deterioration can be prevented. .
The pattern is preferably formed by irradiating with ultraviolet rays after the film formation. By irradiating with ultraviolet rays, the isocyanate group is cross-linked and does not exist, so that DNA is not fixed in the ultraviolet irradiation portion. Since ultraviolet rays are used, a pattern can be formed with higher accuracy than mask vapor deposition in which molecules may wrap around the mask.

  The binder-containing layer of the present invention can be produced by the following method.

  In the vacuum chamber, the following two types of materials of Chemical Formula 2 and Chemical Formula 3 are heated and evaporated at the same time, and a polymer film is obtained by a polymerization reaction on the substrate.

  At that time, if each heating temperature is set so that the vapor pressures of Chemical Formula 2 and Chemical Formula 3 are the same, a polymer film having a monomer composition ratio of 1: 1 can be obtained. By increasing the heating temperature of Chemical Formula 2 above the above temperature, Chemical Formula 2 becomes excessive, and Chemical Formula 1 in which the terminal is -N = C = O can be obtained.

  The formed polymer film may have a film thickness of one molecule, but is usually about 0.3 to 10 μm.

It is preferable to bind probe DNA to the binder-containing layer.
Thus, the raw material can be used effectively without the probe DNA being peeled off even in the water washing step or the like.
In addition, the DNA fragment used as a probe can be divided into two types according to the purpose. In order to examine the expression of a gene, a polynucleotide such as cDNA, a part of cDNA, or EST can be used. The functions of these polynucleotides may be unknown, but are generally amplified by PCR using a cDNA library, genomic library, or whole genome as a template based on sequences registered in a database. (Hereinafter referred to as “PCR product”). Alternatively, those that are not amplified by the PCR method can also be used. In order to examine gene mutations and polymorphisms, it is preferable to synthesize various oligonucleotides corresponding to mutations and polymorphisms based on known standard sequences and use them. Furthermore, in the case of base sequence analysis, it is preferable to synthesize and use 4n (n is the length of the base) type oligonucleotide. The base sequence of the DNA fragment is preferably known.

  In the spotting of DNA fragments, an aqueous solution in which a DNA fragment is dissolved or dispersed in an aqueous medium is dispensed into a 96-well or 384-well plastic plate, and the dispensed aqueous solution is applied onto a substrate using a spotter device. It is performed by dripping.

The DNA fragments to be spotted are preferably in the range of 10 ² to 10 ⁵ types / cm ² with respect to the surface of the base material. The amount of the DNA fragment is in the range of 1 to 10 ^-15 mol, and the weight is preferably several ng or less. The aqueous solution of DNA fragments is fixed in the form of dots on the substrate surface by spotting, but the distance between the dots is preferably in the range of 0 to 1.5 mm, particularly in the range of 100 to 300 μm. It is preferable that it exists in. The size of one dot is preferably in the range of 50 to 300 μm in diameter. The amount to be spotted is preferably in the range of 100 pl to 1 μl, particularly preferably in the range of 1 to 100 nl.

  In addition, after the spotting, it is preferable to perform incubation as necessary. After incubation, it is preferred to wash away the DNA fragments that have not been spotted.

  The shape of the dots on the substrate surface is almost circular. The absence of variation in shape is important for quantitative analysis of gene expression and single nucleotide mutation analysis.

  The lifetime of the DNA chip produced as described above is several weeks for the cDNA chip to which the cDNA is immobilized, and is longer for the oligo DNA chip to which the oligo DNA is immobilized. These DNA chips are used for gene expression monitoring, nucleotide sequence determination, mutation analysis, polymorphism analysis, and the like. The detection principle is hybridization with a labeled target nucleic acid.

  As a target nucleic acid that is a sample, a DNA fragment sample or an RNA fragment sample whose sequence or function is unknown is used.

  For the purpose of examining gene expression, the target nucleic acid is preferably isolated from a eukaryotic cell or tissue sample. If the target is a genome, it is preferably isolated from any tissue sample except red blood cells. Arbitrary tissues other than red blood cells are preferably peripheral blood lymphocytes, skin, hair, semen and the like. If the target is mRNA, it is preferably extracted from a tissue sample in which the mRNA is expressed. mRNA is labeled by incorporating labeled dNTP ("dNTP" means deoxyribonucleotide whose base is adenine (A), cytosine (C), guanine (G) or thymine (T)) by reverse transcription reaction. It is preferable to use cDNA. As dNTP, it is preferable to use dCTP because of chemical stability. The amount of mRNA required for one hybridization depends on the amount of liquid and the labeling method, but is preferably several μg or less. When the DNA fragment on the DNA chip is an oligo DNA, it is desirable to make the target nucleic acid low in molecular weight. In prokaryotic cells, since selective extraction of mRNA is difficult, it is preferable to label total RNA.

  For the purpose of examining gene mutation and polymorphism, the target nucleic acid is preferably obtained by PCR of the target region in a reaction system containing a labeled primer or labeled dNTP.

  The labeling method includes an RI method and a non-RI method, but it is preferable to use a non-RI method. Examples of the non-RI method include a fluorescence labeling method, a biotin labeling method, a chemiluminescence method, and the like, but it is preferable to use a fluorescence labeling method. As the fluorescent substance, any substance can be used as long as it can bind to the base portion of the nucleic acid, but cyanine dyes (for example, CyDye ™ series Cy3, Cy5, etc.), rhodamine 6G reagent, N-acetoxy-N2-acetylamino It is preferable to use fluorene (AAF), AAIF (iodine derivative of AAF) or the like.

  Hybridization is performed by spotting an aqueous solution prepared by dissolving or dispersing a labeled target nucleic acid, which has been dispensed in a 96- or 384-well plastic plate, onto the DNA chip prepared above. It is preferable. The amount of spotting is preferably in the range of 1 to 100 nl. Hybridization is preferably carried out in the temperature range of room temperature to 70 ° C. and in the range of 6 to 20 hours. After completion of hybridization, washing with a mixed solution of a surfactant and a buffer is preferably performed to remove unreacted target nucleic acid. As the surfactant, sodium dodecyl sulfate (SDS) is preferably used. As the buffer solution, a citrate buffer solution, a phosphate buffer solution, a borate buffer solution, a Tris buffer solution, a Good buffer solution, or the like can be used, and a citrate buffer solution is preferably used.

  A feature of hybridization using a DNA chip is that the amount of labeled nucleic acid used is very small. Therefore, it is necessary to set optimum conditions for hybridization according to the chain length of the DNA fragment immobilized on the substrate and the type of the labeled target nucleic acid. For gene expression analysis, it is preferable to perform long-term hybridization with low stringency so that low-expressing genes can be sufficiently detected. For detection of single nucleotide mutations, it is preferable to perform hybridization with high stringency for a short time. In addition, two types of target nucleic acids labeled with different fluorescent substances are prepared, and these are simultaneously used for hybridization, so that the expression level can be compared and quantified on the same DNA chip.

An embodiment of the present invention will be described below with reference to the drawings.
(Formation of binder-containing layer)

FIG. 1 shows a configuration diagram of an apparatus 10 used in the method for producing a DNA chip of the present invention.
As shown in FIG. 1, a base material 3 for forming a binder-containing layer is held downward by a base material holder 4 in a vacuum chamber 2 connected to an evacuation system 1. In addition, an evaporation source ejection port 5 for evaporating the raw material monomer is provided immediately below the center point of the substrate 3 at the lower part of the vacuum chamber 2, and another evaporation source ejection port 6 is provided beside it. The heater 8 (not shown) and a thermocouple (not shown) 8 are controlled to a predetermined temperature at which the evaporation amounts of the raw material monomers a and b are constant. A shutter 9 is formed between the evaporation source ejection ports 5 and 6 and the substrate 3, and the thickness of the binder-containing layer is adjusted by the shutter 9.

Next, a method for producing the DNA chip of the present invention using the apparatus 10 will be described.
A 4 cm methylene-dianiline (hereinafter abbreviated as MDA) having a vapor pressure at 40 ° C. of 1 × 10 ⁻⁵ Pa (hereinafter abbreviated as MDA) (Chemical Formula 4) is used in the evaporation source container 7 with a 10 cm square glass plate as the base material 3. The evaporation source container 8 is filled with 4,4 ′ diphenyl methane diisocyanate (hereinafter abbreviated as MDI) (chemical formula 5) having a vapor pressure of 5 × 10 ⁻³ Pa at 40 ° C., and a shutter 9 The vacuum exhaust system 1 evacuates until the total pressure of the atmospheric gas in the vacuum chamber 2 becomes 5 × 10 ⁻⁵ Torr or less in a closed state. The inner diameter of the evaporation source ejection ports 5 and 6 from the center point of the substrate 3 was set to 2 cm, and the distance from the evaporation source ejection ports 5 and 6 was set to 8 cm. Subsequently, the heaters of the evaporation source containers 7 and 8 were controlled to heat the MDA to 77 ° C. and the MDI to 82 ° C. At this time, the total pressure of the atmospheric gas in the vacuum chamber 2 was 4 × 10 ⁻⁵ Torr. In this state, the shutter 9 was opened for 30 minutes and vapors of both monomers were directed onto the substrate 3 to obtain a binder-containing layer having a thickness of about 1 μm.

  The introduced source gas was polymerized at the glass substrate interface, and a polymer film represented by the following structural formula 6 was formed. An infrared absorption spectrum of the binder-containing layer of the obtained DNA chip is shown in FIG.

As can be seen from FIG. 2, the absorption of isocyanate (frequency: 2270 cm ⁻¹ ) was large, and it was found that the isocyanate group was excessively present on the surface.

Thereafter, the probe DNA contained in the microplate was attached to the pin, and the probe DNA attached to the pin was contacted and spotted on the glass plate on which the polymer film was formed. This operation was repeated until all the probe DNA contained in the microplate had been spotted to produce a DNA chip.
In this way, the amino group introduced at the end of the probe DNA is covalently bonded to the isocyanate group in the binder-containing layer containing the polymer of the DNA chip of this example.

  In the hybridization step of the DNA chip, the DNA chip on which the probe DNA was bound and spotted on a glass plate and the sample DNA labeled with a fluorescent substance were both put in a hybridization solution and hybridized. The hybridization solution is a mixed solution composed of formaldehyde, SSC (NaCl, trisodium citrate), SDS (sodium dodecylsulfate), EDTA (ethylenediamidetetraacetic acid), distilled water, and the like, and the mixing ratio varies depending on the properties of the DNA used.

  Thereafter, the sample DNA labeled with the fluorescent substance remaining on the glass plate was placed in a water bath or the like and washed away, and the sample DNA not bound to the probe DNA was discharged.

  At this time, it was confirmed that the probe DNA bound on the substrate remained almost without being peeled off, and the DNA was not peeled off in the water washing step.

  After that, the fluorescent substance labeled on the sample DNA bound to the probe DNA is excited by light energy from a predetermined light source, and the light emitted by the fluorescent substance is detected by a photosensor such as a CCD. Hybridization was detected at.

  As a result, it was confirmed that the intended hybridization was performed accurately and the S / N ratio was good.

In Example 1, after the binder-containing layer was formed, the binder-containing layer was covered with a pattern in which an opening was provided at a position to be a spot portion, and an ultraviolet ray was irradiated for 10 minutes to prepare a DNA chip.
FIG. 3 shows an infrared absorption spectrum of the ultraviolet irradiation portion of the DNA chip. FIG. 3 shows that there is no isocyanate absorption (2270 cm ⁻¹ ) compared to FIG. 2, and there is no isocyanate group in the binder-containing layer.
From the above results, it became possible to produce a DNA immobilization part and a non-immobilization part by patterning the ultraviolet irradiation part and the non-irradiation part by masking.

  Thereafter, the probe DNA in the microplate was applied without spotting, rinsed with distilled water, and dried to prepare a DNA chip. Otherwise, a DNA chip was produced in the same manner as in Example 1.

  When the obtained DNA chip was evaluated in the same manner as in Example 1, it was found that the S / N was improved without garbage.

Configuration diagram of an apparatus used in a method of manufacturing a DNA chip according to an embodiment of the present invention The graph which shows the infrared absorption spectrum of one Example of this invention The graph which shows the infrared absorption spectrum of the other Example of this invention

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Vacuum exhaust system 2 Vacuum tank 3 Base material 4 Base material holder 5 Evaporation source ejection port 6 Evaporation source ejection port 7 Evaporation source container 8 Evaporation source container 9 Shutter 10 Apparatus used for manufacturing method of DNA chip

Claims (6)

A DNA chip comprising a binder-containing layer containing a polymer having a structural unit represented by the following formula (Chemical Formula 1) on at least a substrate.

[In the above formula (Formula 1), R and R ′ are the same or different and each represents a polymer main chain composed of a group selected from aliphatic and aromatic. ]   The DNA chip according to claim 1, wherein the binder-containing layer is formed by vapor deposition polymerization.   The DNA chip according to claim 1 or 2, wherein the binder-containing layer is patterned by masking.   The DNA chip according to claim 1 or 2, wherein the binder-containing layer is patterned by irradiating the binder-containing layer previously formed through masking with ultraviolet rays.   5. The DNA chip according to claim 1, wherein probe DNA is bound to the binder-containing layer. The method for producing a DNA chip according to claim 1, wherein two or more kinds of raw materials represented by the following structural formulas (Chemical Formula 2 and Chemical Formula 3) are evaporated and polymerized by vapor deposition on a substrate. A method for producing a DNA chip, wherein an agent-containing layer is formed.

[In the above formula (Formula 2), R represents a skeleton of a monomer composed of aliphatic or aromatic. ]

[In the above formula (Formula 3), R ′ represents a skeleton of a monomer composed of an aliphatic group or an aromatic group. ]

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