The present invention relates to novel tetraiminecalixarene derivatives, method of preparation thereof, self-assembled monolayer prepared by using them, unmodified protein fixation method using ionic recognition on the self-assembled monolayer, and protein chip preparation technology using it. More particularly, the present invention relates to tetraiminecalixarene derivatives capable of fixing protein such as antigen, antibody, etc., or compounds having an ammonium functional group, etc. on the surface of a solid substrate by molecular recognition, a method of preparation thereof, a method for preparing it in a monolayer on a solid substrate such as a glass substrate or a gold substrate, and a method for preparing a protein monolayer, i.e., protein chip using a self-assembled monolayer thereof.
Also, the present invention relates to a self-assembled monolayer prepared by using aminocalixarene derivatives, a method for fixing protein thereon in a monolayer by using ionic or molecular recognition, and a protein chip prepared by using the method. More particularly, the present invention relates to a aminocalixarene derivative monolayer prepared in a monolayer on a solid substrate such as a glass substrate or a gold substrate by using the compound of formula 1 or formula 2, a method for fixing protein such as antigen, antibody, etc., especially protein having the molecular weight of 10 kD or more, on the surface of the monolayer at high density by molecular recognition, and a protein chip prepared by said fixation of protein.
Development and differentiation of vital phenomenon is controlled by functional interaction such as protein-protein, protein-ligand(or agent), antigen-antibody, enzyme-substrate, etc. Thus, the research to discover the function or role of the biomolecule that uniquely interacts with a specific protein or ligand is becoming a main project in the fields of life science, health, medical treatment, etc. In order to fix such various proteins on a single solid substrate, i.e. a chip and measure interaction of protein-protein or protein-ligand, the protein chip wherein a number of proteins are fixed is being researched actively. Particularly, the method of fixing enzyme, antigen, antibody, etc. on a solid substrate is the most primary infratechnology in life science such as immunochemistry, enzymochemistry, etc. or in various fields wherein proteins are used, such as a diagnosis kit diagnosing diseases by using proteins. For example, the Enzyme-Linked Immunoassay, EIA, a method most commonly used for diagnosis, is a diagnosis method that antibody is physically absorbed in the bottom of 96-microtiter well made of plastics or antibody is fixed by a chemical bonding method that reacts various functional groups existing at the surface of the antibody to prepare a product, and it is reacted with the antigen of blood serum and developed in color using the secondary antibody-enzyme conjugate, and then the concentration of antigen is measured through fluorescence analysis in order to analyze a certain protein or a specific protein that becomes a disease cause. Various disease diagnosis kits prepared by this method are on the market.
Besides physical absorption or chemical bonding method, a protein fixation method that fixes protein such as antibody wherein biotin is attached by biotin-streptavidin molecular recognition after fixing a protein named streptavidin on a solid substrate wherein biotin is attached by hydrogen bonding is known.
The problems occurring in protein fixation technology such as conventional physical absorption method and chemical bonding method, and the method using biotin-streptavidin molecular recognition, which currently started being used, etc. are as follows.
1. Fixation Density: Although this is the field that more research has proceeded than any other among protein fixation technology, for various kinds of proteins currently being used for fixation, solid substrates capable of preparing a protein monolayer which completely covers the whole space upon fixation have rarely been published. Unless the density of protein fixed on the surface reaches the density that can make a perfect level of a monolayer (in case of antibody, it is 1.1-1.4 μg/cm2), the following problems can occur: that specific antigen is fixed not on the antibody but nonspecifically on the surface wherein solid substrate is directly exposed; that since the amount of fixed antibody is small, the amount of combined antigen becomes small, as a result causing difficulty in reading; and that the concentration of readable protein is not distinguished from conventional technology.
2. Reproducibility: It is the most important technology to put a product on the market in the protein fixation technology. When several to dozens of kinds of proteins are fixed, the precise diagnosis results can be obtained only when fixed protein achieves the same levels of fixation density and activity. Protein fixation technology currently used include the chemical bonding on a solid substrate, the biotin-streptavidin method, or a method using polymers that can make the maximum chemical bonding on a glass slide, such as polymers of the polylysine series. However, since most of them require reaction between protein and a specific functional group and fixation occurs after the reaction, the conventional protein fixation technology have many problems in maintaining the same results, i.e., reproducibility when several incomplete reactions are carried out.
3. Superhigh speed fixation: Upon fixation, the fixation occurs on the surface of a solid substrate in a solution phase, and if protein is dissolved in a solution phase, the activity of protein in a solution phase tends to decrease as time passes by. However, since the activity hardly decreases after fixation, a solid substrate capable of superhigh speed fixation and completing fixation within one hour needs to be developed in order to minimize the activity reduction upon fixation, but it has not been developed yet.
4. Unmodified protein Fixation: Even if the protein fixation density, i.e., the reproducibility where the number of proteins to be fixed are maintained at the same level within a certain range, is secured and if the same number of proteins is fixed, in order to carry out fixation maintaining the activity of protein at the same level within a certain range of error, the fixation should take place in a controllable solution phase and to achieve it, the development of technology that does not require a chemical bonding process which can affect the protein activity and fixes unmodified protein directly in a solution phase is necessary. Since, except the technology using crown compounds that the present researchers published several years ago, no unmodified protein fixation technology has been developed yet, unmodified protein fixation technology that does not involve attachment of a functional group and the development of the solid substrate wherein the technology can be applied is necessary.
5. Technology for an analysis of a trace protein
Since the concentration of antibody protein, etc. which can be analyzed by using conventional protein fixation technology remains at the level of several ng/ml, a diagnosis product capable of quantitative analysis of protein existing at a concentration lower than that has not been developed.
If a technology capable of quantitatively analyzing protein existing even at a concentration of several μg/ml or lower is developed, new cancer identification protein which couldn't be analyzed with conventional technology can be discovered, and at the same time, by making possible the quantitative analysis even when the concentration of cancer identification protein is very low, the development of new concept diagnosis protein that makes possible early diagnosis and disease prevention is expected to be possible; however, it has not been developed until now.
6. Long-Term Storage Technology
Protein loses its activity significantly when stored over a long period of about 3-6 months, and since the reduction ratio is not constant, it causes many problems with its commercialization. In order to obtain the same diagnosis results even upon such long storage, the Protein Chip Research Association, etc. has suggested the need for development of the technology minimizing the phenomenon that the protein fixed on a solid substrate by nonspecific attraction is attracted to the surface, which happens when protein is fixed at a low density or by physical absorption, by fixing protein at high density without empty space, that is, the technology to minimize the protein activity reduction caused by alteration of an active site, which happens when it is difficult to maintain the shape of protein, that is, the protein chip preparation technology capable of high density fixation without leaving empty space.
In order to solve the problems of conventional protein fixation technology, the density of proteins fixed on the surface of a solid substrate must be high enough to form a perfect level of a monolayer, and the fixation density and the activity of the proteins must be maintained at the same level, and superhigh speed fixation must be possible to reduce activity reduction upon fixation.
An objective of the present invention is, in order to solve product reproducibility problems that have hindered protein chips from being put on the market among the above-mentioned problems of various conventional protein fixation methods, to provide high density fixation technology which do not leave empty space for obtaining homogeneous fixation density and maintaining the same level of activity. In addition, another objective of the present invention is to provide technology that solves the problems of the irregularity of reading results, which happens because the protein activity decreases in a solution phase as time passes by and thus the protein activity after fixation becomes irregular.
In addition, considering that the active site, which contributes to protein activity, is maintained mainly by hydrogen bonding, another objective of the present invention is also to provide iminecalixarene derivatives essential for developing reproducible protein chips by solving the problems of activity irregularity caused by the alteration of active sites that happens by the chemical bond stronger than hydrogen bond upon chemical bonding and the preparation method thereof.
In addition, another objective of the present invention is, by attaching said compounds to a glass substrate (amine slide glass) wherein amine functional groups are attached, siliconwafer, fused silica, or gold substrate, etc., to provide an iminecalixarene derivative monolayer or an aminocalixarene derivative monolayer wherein all kinds of proteins are fixed in a solution phase without any treatment at superhigh speed in a short time of 30 minutes to 1 hour to prepare a protein monolayer.
In addition, another objective of the present invention is to provide a technology for preparing a protein chip, i.e. a protein monolayer prepared using an iminecalixarene derivative monolayer or an aminocalixarene derivative monolayer wherein all kinds of proteins are fixed at high density and superhigh speed without any alteration.
Distinguished completely from the conventional method of preparing protein chip by fixing protein using conventional chemical bonding, physical absorption, or biotin-streptavidin, which is being generally used over the world, the present invention is a novel technology wherein once unmodified protein is coated on an imincalixarene derivative monolayer or an aminocalixarene derivative monolayer, protein is fixed on the surface of the monolayer irreversibly at superhigh speed through ionic/molecular recognition and at the same time with the maximum density fixation. Also, the present invention relates to a new protein chip preparation technology that not only keeps the amount of fixed proteins, i.e., the density, homogeneous by fixing proteins at the maximum level all the time, but also obtains high reproducibility by maintaining the same level of protein activity through superhigh speed fixation.
The protein monolayer prepared by using the novel iminecalixarene derivatives or the aminocalixarene derivatives developed according to the present invention is a novel technology that can solve the problems of heterogeneousness of fixation density and the activity reduction that occur in conventional protein fixation methods, and the problems of irregularity of an fixed protein activity, which is caused by the activity reduction of proteins left in a solution phase for a long time due to slow fixation technology. In addition, the active site that contributes to protein activity is mainly maintained by hydrogen bonds, and the present invention is a novel technology that, by using molecular recognition, can solve problems that the activity of fixed proteins is irregularly reduced since the alteration of the active site occurs by the chemical bond with a solid substrate stronger than the hydrogen bonds when chemical bonding is carried out using an aldehyde chip, etc.
In addition, the present invention provides a protein chip preparation technology that completely solves the reproducibility problem of the protein chips, which has been the biggest obstacle to putting protein chips on the market, by providing the preparation technology of an iminecalixarene derivative monolayer for protein fixation or an aminocalixarene derivative monolayer, novel iminecalixarene derivatives, and the preparation method thereof.
In addition, the present invention provides an iminecalixarene derivative monolayer or an aminocalixarene derivative monolayer that can make a protein monolayer by fixing all kinds of proteins, regardless of molecular weight, on a solid substrate in a solution phase in a short time of 30 minutes to 1 hour at superhigh speed without any treatment by attaching iminecalixarene derivatives of the formula 2 or 4 to glass substrate (amine slide glass) wherein amine functional groups are attached, siliconwafer, fused silica, etc., or attaching iminecalixarene thiol derivatives of the formula 2 or 4 to a gold substrate, or attaching aminocalixarene derivatives of the formula 6 or 7 to a glass substrate (amine slide glass) wherein amine, alcohol, or thiol functional groups are attached, siliconwafer, fused silica, or a gold substate.
In addition, the present invention provides protein chip preparation technology that can fix proteins of various sizes and shapes having a molecular weight of 1000 (10 kD) or more at an optimal condition with the maximum activity, by providing the world s first iminecalixarene derivative monolayer for protein fixation capable of controlling an fixation speed, increasing fixation speed through the raise of an ionic concentration as in the fixation according to an ion concentration presented in
In addition, the present invention provides protein chip preparation technology that can simplify protein chip preparation processes and prepare protein chips at a low cost since it can prepare protein chips at superhigh speed with only trace proteins, around 3 times the amount of protein needed to achieve the maximum density.
Particularly, according to the fluorescence analysis results of
The novel iminecalixarene derivative essential for preparation of a self-assembled monolayer wherein proteins are fixed in 30 minutes to 1 hour at a superhigh speed without having any treatment has a structure of the following formula 1, 2, 3, or 4.
(wherein, R1, R′1, R2, R′2, R3, R′3, R4, and R′4 are independently selected from the group consisting of —H, —CH3, —C2H5, —C3H7, —OCH3, —Cl, —C6H5, —OH, —OCH2CH3, —Br, —CF3, —OCH2C6H5, —OC6H5, —OC6H4CH3, —OC6H4C(CH3)3, —OC6H4CF3, —OC6H4Cl, —OCOCH3, —NHCOCH3, —CONHCH3, —CN, COOH, and —COOR, wherein R represents —CH3 or —C2H5).
(wherein, R1, R′1, R2, R′2, R3, R′3, R4, and R′4 are independently selected from the group consisting of —H, —CH3, —C2H5, —C3H7, —OCH3, —Cl, —C6H5, —OH, —OCH2CH3, —Br, —CF3, —OCH2C6H5, —OC6H5, —OC6H4CH3, —OC6H4C(CH3)3, —OC6H4CF3, —OC6H4Cl, —OCOCH3, —NHCOCH3, —CONHCH3, —CN, COOH, and —COOR, wherein R represents —CH3 or —C2H5. Also, said Y1, Y2, Y3 and Y4 are independently selected from the group consisting of —H, —(CH2)n—CH═O, —(CH2)n—SH, —(CH2CH2O)m—CH2CH2—CH═O, —(CH2CH2O)m—CH2CH2—SH, —(CH2)m—C6H4—(CH2)c-Z and —CO—(CH2)m-1—C6H4—(CH2)c-Z (wherein, n=2˜15, m=1˜10, c=0˜10, Z=—SH, —CHO, —COOH or —NH2, and —C6H4 and —C6H5 are defined as phenyl group.).
(wherein, R1, R′1, R2, R′2, R3, R′3, R4, and R′4 are independently selected from the group consisting of —H, —CH3, —C2H5, —C3H7, —OCH3, —Cl, —C6H5, —OH, —OCH2CH3, —Br, —CF3, —OCH2C6H5, —OC6H5, —OC6H4CH3, —OC6H4C(CH3)3, —OC6H4CF3, —OC6H4Cl, —OCOCH3, —NHCOCH3, —CONHCH3, —CN, COOH, and —COOR, wherein R represents —CH3 or —C2H5)
(wherein, R1, R′1, R2, R′2, R3, R′3, R4, and R′4 are independently selected from the group consisting of —H, —CH3, —C2H5, —C3H7, —OCH3, —Cl, —C6H5, —OH, —OCH2CH3, —Br, —CF3, —OCH2C6H5, —OC6H5, —OC6H4CH3, —OC6H4C(CH3)3, —OC6H4CF3, —OC6H4Cl, —OCOCH3, —NHCOCH3, —CONHCH3, —CN, COOH, and —COOR, wherein R represents —CH3 or —C2H5. Also, said Y1, Y2, Y3 and Y4 are independently selected from the group consisting of —H, —(CH2)n—CH═O, —(CH2)n—SH, —(CH2CH2O)m—CH2CH2—CH═O, —(CH2CH2O)m—CH2CH2—SH, —(CH2)m—C6H4—(CH2)c-Z and —CO—(CH2)m-1—C6H4—(CH2)c-Z (wherein, n=2˜15, m=1˜10, c=0˜10, Z=—SH, —CHO, —COOH or —NH2, and —C6H4 and —C6H5 are defined as phenyl group.).
(wherein, R1, R2, R3, R4, R5, R6, R7, R8, R1, R2, R3, R4, R5, R6, R7 and R8 are independently selected from the group consisting of —H, —CH3, —C2H5, —C3H7, —OCH3, —Cl, —C6H5, —OH, —OCH2CH3, —Br, —CF3, —OCH2C6H5, —OC6H5, —OC6H4CH3, —OC6H4C(CH3)3, —OC6H4CF3, —OC6H4Cl, —OCOCH3, —NHCOCH3, —CONHCH3, —CN, COOH, and —COOR, wherein R represents —CH3 or —C2H5. Also, said Y1, Y2, Y3 and Y4 are independently selected from the group consisting of —H, —(CH2)n—CH═O, —(CH2)n—SH, —(CH2CH2O)m—CH2CH2—CH═O, —(CH2CH2O)m—CH2CH2—SH, —(CH2)m—C6H4—(CH2)c-Z and —CO—(CH2)m-1—C6H4—(CH2)c-Z (wherein, n=2˜15, m=1˜10, c=0˜10, Z=—SH, —CHO, —COOH or —NH2, and —C6H4 and —C6H5 are defined as phenyl group.).
(wherein, R1, R2, R3, R4, R5, R6, R7, R8, R1, R2, R3, R4, R5, R6, R7 and R8 are independently selected from the group consisting of —H, —CH3, —C2H5, —C3H7, —OCH3, —Cl, —C6H5, —OH, —OCH2CH3, —Br, —CF3, —OCH2C6H5, —OC6H5, —OC6H4CH3, —OC6H4C(CH3)3, —OC6H4CF3, —OC6H4Cl, —OCOCH3, —NHCOCH3, —CONHCH3, —CN, COOH, and —COOR, wherein R represents —CH3 or —C2H5. Also, said Y1, Y2, Y3 and Y4 are independently selected from the group consisting of —H, —(CH2)n—CH═O, —(CH2)n—SH, —(CH2CH2O)m—CH2CH2—CH═O, —(CH2CH2O)m—CH2CH2—SH, —(CH2)m—C6H4—(CH2)c-Z and —CO—(CH2)m-1—C6H4—(CH2)c-Z (wherein, n=2˜15, m=1˜10, c=0˜10, Z=—SH, —CHO, —COOH or —NH2, and —C6H4 and —C6H5 are defined as phenyl group.).
The compounds of the formula 6 and 7 of the present invention can be synthesized using the compound of the formula 5 as a starting material.
The iminecalixarene derivative of the formula 1 is synthesized by using a compound of the formula 5 synthesized according to the example 2 as a starting material and reacting the amine functional group of the formula 5 with a suitable aromatic aldehyde.
The iminecalixarene derivative of the formula 2 is synthesized by reacting a compound having an aldehye, thiol, or carboxyl functional group, etc. at the terminal with —OH, i.e. alcohol functional groups, which is attached to the calixarene of the compound of the formula 1, through ether or ester bond, while alkyl groups having aldehyde or thiol at the terminal are attached to each —OH independently.
The iminecalixarene derivative of the formula 3 is synthesized by using a compound of the formula 5 synthesized according to the example 6 as a starting material and reacting the amine functional group of the formula 5 with a suitable aromatic aldehyde.
The iminecalixarene derivative of the formula 4 is synthesized by reacting a compound having an aldehye, thiol, or carboxyl functional group, etc. at the terminal with —OH, i.e. alcohol functional groups, which is attached to the calixarene of the compound of the formula 3, through ether or ester bond, while alkyl groups having aldehyde or thiol at the terminal are attached to each —OH independently.
In addition, the present invention provides a technology of preparing a oligo-DNA chip, i.e. an oligo-DNA monolayer wherein all kinds of oligo-DNAs are fixed at a high density by attaching said compound of the formula 2 to gold substrate or glass substrate wherein amine groups are attached.
In addition, the present invention provides a technology of preparing an aminocalixarene derivative monolayer by attaching the compound of said formula 6 or 7 to a gold substrate or a solid substrate such as a glass, siliconwafer, fused silica, wherein functional groups that can form chemical bonds are attached, and a protein chip, i.e. a protein monolayer wherein all kinds of proteins are fixed at a high density.
The glass substrate wherein amine functional group is attached (glass slide glass) is prepared in the form of a glass substrate having an amine terminal group (amine slide glass or amine chip) through a chemical reaction on the surface of a glass substrate having sufficient silanol (—Si—OH) functional groups according to the method described in the paper (Langmuir, 1997, Vol. 13, pp 4305-4308; Langmuir, 1996, Vol. 12, pp 5338-5342).
The detailed method for preparing a self-assembled iminecalixarene derivative monolayer on a glass substrate wherein amine groups are attached is as follows:
First, the glass substrate wherein said amine functional group is attached (glass slide glass) is prepared in the form of a glass substrate having an amine terminal group (amine slide glass or amine chip) through a chemical reaction on the surface of a glass substrate having sufficient silanol (—Si—OH) functional groups according to the method described in the paper (Langmuir, 1997, Vol. 13, pp 4305-4308; Langmuir, 1996, Vol. 12, pp 5338-5342). The thus-prepared glass substrate wherein the amine functional group is attached and the compound of formula 2 are added to a mixed solution of chloroform and THF dissolved in a concentration of 0.1-5 mM (CHCl3:THF=9:1). After 1˜5 hours, it is washed with chloroform, acetone and ethanol in that order and dried, and then an iminecalixarene self-assembled monolayer as
A solution is prepared by dissolving a compound, wherein thiol is attached among the compounds of formula 2 or 4, in an organic solvent such as chloroform (CHCl3), etc. in a concentration of 0.1-5 mM. After a gold substrate is put into the thus-prepared solution and left for 1˜5 hours, it is taken out and washed with chloroform, acetone and ethanol in that order and dried, and then a self-assembled monolayer of iminecalixarene derivative as
In addition, the present invention provides a method of fixing an unmodified protein by multiionic/multimolecular-recognizing the ammonium group of the protein or one or more of the guanidine groups in arginine, using four nitrogens of the iminecalixarene derivative.
In addition, the fixation method of the present invention is an original, fundamental technology for preparing all types of products using protein fixation such as diagnosis biochips, protein chips for research use, etc., and no other research results, not even similar ones, have been published in this regard all over the world until now.
A solution is prepared by dissolving a compound, wherein thiol is attached among the compounds of formula 6 or 7, in an organic solvent such as chloroform (CHCl3), etc. in a concentration of 0.1-5 mM. After a gold substrate is put into the thus-prepared solution and left for 1˜5 hours, it is taken out and washed with chloroform, acetone and ethanol in that order and dried, and then a self-assembled monolayer of aminocalixarene derivative as
In addition, the present invention provides a method of fixing an unmodified protein by multiionic/multimolecular-recognizing the ammonium group of the protein or one or more of the guanidine groups in arginine.
In addition, the fixation method of the present invention is an original, fundamental technology for preparing all types of products that use protein fixation such as diagnosis biochips, protein chips for research use, and no other research results, not even similar ones, have been published in this regard all over the world until now.
According to the analysis result, it is confirmed that in case of fixation using NH4+ ion in a concentration of 0-100 mM, about 40% of the maximum fluorescence is achieved after about 1 hour of protein fixation. On the other hand, in case of fixation using NH4+ ion in a concentration of 400 mM, the fluorescence is almost the same in 1 hour as the theoretically maximum fluorescence shown in the
In addition, the present invention relates to a protein chip prepared by forming a protein monolayer (i.e., protein chip) on an aminocalixarene derivative monolayer at superhigh speed, high density, and high reproducibility using an unmodified protein, and the preparation method thereof.
Such results show that the protein fixation proceeds actually by ion recognition, and especially if there is a p-p stacking interaction between the iminecalixarene ring and the benzene ring of amine compounds, amine compounds are more easily recognized by the iminecalixarene derivative, and thus inhibit the protein fixation faster, which shows that on the iminecalixarene derivative monolayer of the present invention, proteins are mostly fixed by the recognition of amine groups at the outside of the protein.
From said results, it can be found that in the present invention four amine compounds, i.e. ammonium functional groups are irreversibly recognized on the surface of an iminecalixarene derivative monolayer and that the protein is fixed at a high density with ammonium functional groups outside the protein being recognized irreversibly on the surface of an iminecalixarene derivative monolayer.
In addition, the present invention provides a superhigh speed fixation technology, novel iminecalixarene derivatives or aminocalixarene derivatives that make possible a fixation of proteins that have undergone no treatment, i.e. unmodified proteins, and monolayers thereof, and the protein chip using said monolayers.
Hereinafter, the present invention is described in more detail by the following examples. However, the present invention is not restricted thereto.
A light-yellow solid product, 5,11,17,23-tetraaminocalix[4]arene (TACA) is obtained in the yield of 75% by having 5,11,17,23-tetranitrocalix[4]arene (TNCA) as a starting material and synthesizing it according to the synthesizing method presented in the following cited reference.
[Cited Reference: Van Wagenigen, A. M. A.; Snip, E.; Verboom. W.; Reinhoudt, D. N.; Boerrigter, H.; Liebigs Ann/Recueil 1997. pp 2235-2245].
Experiment is prepared by putting TACA (100 mg, 0.2 mmol) and magnetic bar in a dried round-bottom flask. After adding 15 ml of acetonitrile in the reaction vessel is added, it is mixed. After 2,4-dimethoxybenzaldehyde (330 mg, 2.4 mmol) is put, it is mixed for two hours under nitrogen exchange at room temperature. After the reaction, the solvent is removed under reduced pressure by decompressed filtering; the reaction product is dissolved in 3 ml of CH2Cl2; and then 15 ml of n-hexane is added to obtain a light-brown solid product. The product is dissolved in 3 ml of CH2Cl2 once again, and 15 ml of n-hexane is slowly added to obtain 196 mg (yield 88%) of a light-brown solid product, TMBICA (5,11,17,23-tetra-2,4-dimethoxybenzyliminecalix[4]arene).
1H NMR (300 MHz, CDCl3): δ 10.0(s, 4H, OH), 8.66(s, 4H N═CH), 7.96(d, 4H,ArH) 6.98(s, 8H, ArH), 6.56˜6.37(m, 8H, ArH) 4.3(d, 4H, ArCH2Ar J=13 Hz), 3.81(s, 12H, OCH3), 3.73(s, 12H, OCH3), 3.58(d, 4H, ArCH2Ar J=13 Hz)
After a magnetic bar, TMBICA (100 mg, 0.1 mmol), K2CO3 (145 mg, 1.1 mmol), and sodium iodide (142 mg, 0.9 mmol) are put in that order in a dried round-bottom flask, it is dried under decompressed pressure, and 15 mg of anhydrous acetonitrile is added to the reaction vessel under nitrogen exchange, and then it is mixed on a heater. 6-bromohexanal (113 mg, 0.6 mmol) is added, and the temperature of the reaction vessel is raised to 80° C. Then, it is mixed for 24 hours. After the reaction, the reaction product is cooled down to room temperature, and the solvent is removed under decompressed pressure. To remove the additional insoluble solid product obtained during the reaction, the reaction product is dissolved in 10 ml of CH2Cl2, and the insoluble solid product is removed by decompressed filtering. The filtered solution is decompressed to remove the solvent.
After the reaction product is dissolved in 2 ml of CH2Cl2, 15 ml of n-hexane is slowly added, and then the reaction product is extracted. The extracted product is decompressed and filtered to obtain a yellow solid product. After this product is dried, it is dissolved in 2 ml of CH2Cl2 for refining. Then, the reaction product is slowly extracted by slowly adding 20 ml of n-hexane slowly to obtain 100 mg (yield 85%) of a light-yellow solid product, TMBICADA (5,11,17,23-tetra-2,4-dimethoxybenzyliminecalix[4]arene-1,3-dihexanal).
1H NMR (300 MHz CDCl3): δ 9.76(s, 2H, CHO), 8.79(s, 2H, n=CH) 8.58(s, 2H, N═CH) 8.04(d, 2H, ArH) 7.88(d, 2H, ArH), 7.25(s, 4H, ArH), 6.84(s, 4H, ArH) 6.55˜6.35(m, 8H, ArH), 4.32(d, 4H, ArCH2Ar, J=13 HZ), 4.02(t, 4H, OCH2), 3.90˜3.17(m, 24H, OCH3), 3.46(d, 4H ArCH2Ar J=13 Hz), 2.56(t, 4H, CH2CHO) 2.16˜1.6(m 12H, CH2CH2)
After TMBICA (500 mg, 0.46 mmol), anhydrous K2CO3 (636 mg, 4.6 mmol) and sodium iodide (620 mg, 4.14 mmol) are put in a dried round-bottom flask and then 160 ml of anhydrous acetonitrile is added, it is mixed for 15 minutes at room temperature. 1,4-dibromobutane (1.0 g, 0.55 ml, 4.6 mmol) is added to the reaction vessel and the temperature of the reaction vessel is raised to 80° C. Then, it is reacted for 24 hours. After the reaction vessel is cooled down to room temperature, the solvent is decompressed and removed, and the remaining product is dissolved with 150 ml of CH2Cl2. Solid precipitates obtained during the reaction, such as KBr, KI, K2CO3, are removed by being decompressed and filtered and the filtered solution is decompressed to remove the solvent. After being extracted with ethyl acetate/n-hexane, it is decompressed and filtered to obtain a light-brown solid product, 5,11,17,23-tetra-2,4-methoxybenzyliminecalix[4]arene-1,3-butylbromine (TMBICAB, 5,11,17,23-tetra-2,4-methoxybenzyliminecalix[4]arene-1,3-butylbromide). This solid is recrystallized with CHCl3/n-hexane to obtain a light-yellow TMBCIAB (490 mg, 79%).
1H NMR (300 MHz CDCl3): δ 8.79(s, 2H, N═CH), 8.55(s, 2H, N═CH), 8.06(d, 2H, ArH) 7.90(d, 2H, ArH), 7.26(s, 4H, ArH), 6.85(s, 4H, ArH) 6.61˜6.46(m, 8H, ArH), 4.33(d, 4H, ArCH2Ar, J=13 HZ), 4.03(t, 4H, OCH2), 3.94˜3.21(m, 24H, OCH3), 3.45(d, 4H ArCH3Ar J=13 Hz), 3.32(t, 4H, CH2Br) 2.41˜2.34(m, 4H, OCH2CH2), 2.24-2.18(m, 4H, CH2CH2Br)
After TMBICAB (500 mg, 0.37 mmol) and potassium thioacetate (170 mg, 1.48 mmol) are put in a dried round-bottom flask, and dissolved in 50 ml of acetone, it is sonic reacted for 90 minutes at room temperature under nitrogen exchange. After the reaction, the solvent is removed under decompressed pressure, and the remaining product is dissolved with 30 ml of CH2Cl2. Then, the precipitate that is not dissolved is decompressed and filtered, and then the filtered solution is cleaned with water twice and the organic layer is separated and dried with MgSO4. The solid obtained by depressing and filtering the organic layer and decompressing and drying the filtered solution is recrystallized using ethyl acetate/n-hexane, and decompressed and filtered to obtain a light-brown solid crystal. The thus-obtained solid crystal is put in the round-bottom flask, and dissolved in a mixed solution in a ratio of CH2Cl2:methanol=5:1, and sonic reacted at room temperature under nitrogen exchange. After 1 minute, 1.0 M KOH (1.5 ml, 1.5 mmol) is added, and it is sonic reacted for 30 minutes. After the reaction, the solvent is removed under decompressed pressure, and then it is dissolved in 4 ml of CH2Cl2 and washed with 0.1 M HCl solution once. After the organic layer is separated and dried with MgSO4, it is decompressed and filtered. The solvent of the filtered solution is removed under decompressed pressure and 25 ml of n-hexane is slowly added to obtain a light-yellow 5,11,17,23-tetra-2,4-methoxybenzyliminecalix[4]arene-1,3-bytylmercaptane (TMBICAT). The thus-obtained TMBICAT is recrystallized with ethyl acetate/n-hexane to obtain a white solid crystal, TMBICAT (370 mg, yield 79%).
1H NMR (300 MHz CDCl3): δ 3.78(s, 2H, N═CH), 8.54(s, 2H, N═CH), 8.03(d, 2H, ArH) 7.87(d, 2H, ArH), 7.24(m, 4H, ArH), 6.83(s, 4H, ArH) 6.59˜6.43(m, 8H, ArH), 4.33(d, 4H, ArCH2Ar, J=13 HZ), 4.03(t, 4H, OCH2), 3.93˜3.21(m, 24H, OCH3), 3.45(d, 4H ArCH2Ar J=13 Hz), 3.04(t, 4H, CH2SH) 2.39(m, 4H, OCH2CH2), 2.21-2.14(m, 4H, CH2CH2)
TACA (500 mg, 1.0 mmol) and magnetic bar are put in a dried round-bottom flask. After 60 ml of acetonitrile is added to the reaction vessel, it is mixed. After 2,5-dimethoxybenzaldehyde (1.65 g, 12 mmol) is put, it is mixed for two hours under nitrogen exchange at room temperature. After the reaction, the solvent is removed under reduced pressure by decompressed filtering; the reaction product is dissolved in 5 ml of CH2Cl2; and then 30 ml of n-hexane is added to obtain a light-brown solid product. The product is dissolved in 5 ml of CH2Cl2 once again, and 30 ml of n-hexane is slowly added to obtain 920 mg (yield 85%) of a light-brown solid product, 5,11,17,23-tetra(2,5-dimethoxy)benzyliminecalix[4]arene (2,5-TMBICA, 5,11,17,23-tetra-2,5-dimethoxybenzyliminecalix[4]arene).
1H NMR (300 MHz, CDCl3): δ 10.5(s, 4H, OH), 8.75(s, 4H N═CH), 7.57(d, 4H, ArH) 7.02(s, 8H, ArH), 6.86(m, 8H, ArH) 4.28(d, 4H, ArCH2Ar J=13 Hz), 3.79(s, 12H, OCH3), 3.72(s, 12H, OCH3) 3.58(d,4H,ArCH2Ar J=13 Hz)
After a magnetic bar, 2,5-TMBICA (500 mg, 0.46 mmol), K2CO3 (625 mg, 4.6 mmol), and sodium iodide (630 mg, 4.2 mmol) are put in that order in a dried round-bottom flask, it is dried under decompressed pressure, and 130 ml of anhydrous acetonitrile is added to the reaction vessel under nitrogen exchange, and then it is mixed on a heater. 6-bromohexanal (520 mg, 2.76 mmol) is added, and the temperature of the reaction vessel is raised to 80° C. Then, it is mixed for 24 hours. After the reaction, the reaction vessel is cooled down to room temperature, and the solvent is removed under decompressed pressure. To remove the additional insoluble solid product obtained during the reaction, the reaction product is dissolved in 120 ml of CH2Cl2, and the insoluble solid product is removed by decompressed filtering. The filtered solution is decompressed to remove the solvent. After the reaction product is dissolved in 5 ml of CH2Cl2, 30 ml of n-hexane is slowly added to extract the reaction product. The extracted product is decompressed and filtered to obtain a yellow solid product. After this product is dried, it is dissolved in 5 ml of CH2Cl2 for refining. Then, the reaction product is slowly extracted by slowly adding 30 ml of n-hexane to obtain 510 mg (yield 87%) of a light-yellow solid product, 5,11,17,23-tetra-2,5-dimethoxybenzyliminecalix[4]arene-1,3-dihexanal (2,5-TMBICADA).
1H NMR (300 MHz CDCl3): δ 9.75(s,2H,CHO), 8.86(s, 2H, N═CH) 8.66(s, 2H, N═CH) 7.55(d, 2H, ArH), 7.45(d, 2H, ArH), 7.09(s, 4H, ArH), 6.95(s, 4H, ArH), 6.90˜6.72(m, 8H, ArH), 4.32(d, 4H, ArCH2Ar, J=13 Hz), 4.03(t, 4H, OCH2), 3.87˜3.65(m, 24H, OCH3), 3.45(d, 4H ArCH2Ar J=13 Hz), 2.55(t, 4H, CH2CHO), 2.10˜1.5(m 12H, CH2CH2)
After 2,5-TMBICA (300 mg, 0.28 mmol), anhydrous K2CO3 (387 mg, 2.8 mmol) and sodium iodide (378 mg, 2.52 mmol) are put in a dried round-bottom flask, and 150 ml of anhydrous acetonitrile is added, it is mixed for 10 minutes at room temperature. 1,4-dibromobutane (600 mg, 0.34 ml, 2.8 mmol) is added to the reaction vessel and the temperature of the reaction vessel is raised to 80° C. Then, it is reacted for 24 hours. After the reaction, the reaction vessel is cooled down to room temperature; the solvent is decompressed and removed; and the remaining product is dissolved with 140 ml of CH2Cl2. Solid precipitates obtained during the reaction, such as KBr, KI, K2CO3, are removed by being decompressed and filtered and the filtered solution is decompressed to remove the solvent. After being extracted with ethyl acetate/n-hexane, it is decompressed and filtered to obtain a light-brown solid product, 5,11,17,23-tetra-2,5-dimethoxybenzyliminecalix[4]arene (2,5-TMBICA, 5,11,17,23-tetra-2,5-dimethoxybenzyliminecalix[4]arene-1,3-butylbromide). This solid is recrystallized with CHCl3/n-hexane to obtain a light-yellow 2,5-TMBICA (300 mg, 79%).
1H NMR (300 MHz, CDCl3): δ 8.87(s, 2H, N═CH) 8.67(s, 2H, N═CH) 7.65(d, 2H, ArH), 7.49(d, 2H, ArH), 7.09(s, 4H, ArH), 6.95(s, 4H, ArH), 6.89˜6.71(m, 8H, ArH), 4.32(d, 4H, ArCH2Ar, J=13 Hz), 4.03(t, 4H, OCH2), 3.87˜3.65(m, 24H, OCH3), 3.45(d, 4H ArCH2Ar J=13 Hz), 3.42.(t, 4H, CH2Br), 2.41˜2.33(m, 4H, OCH2CH2), 2.23-2.17(m, 4H, CH2CH2Br)
After 2,5-TMBICAB (500 mg, 0.37 mmol) and potassium thioacetate (200 mg, 1.75 mmol) are put in a dried round-bottom flask, and dissolved in 60 ml of anhydrous acetone, it is sonic reacted for 90 minutes at room temperature under argon exchange. After the reaction, the solvent is removed under decompressed pressure, and the remaining product is dissolved with 30 ml of CH2Cl2. Then, the precipitate that is not dissolved is decompressed and filtered, and then the filtered solution is cleaned with water twice and the organic layer is separated and dried with MgSO4. Thereafter, the solid obtained by decompressing and filtering the organic layer and removing the filtered solution under decompressed pressure, and then drying the remaining product is recrystallized using ethyl acetate/n-hexane, and decompressed and filtered to obtain a light-brown solid crystal. The thus-obtained solid crystal is put in the round-bottom flask, and dissolved in a mixed solution in a ratio of CH2Cl2:methanol=5:1, and sonic reacted at room temperature under argon exchange. After 1 minute, 1.0 M KOH (1.5 ml, 1.5 mmol) is added, and it is sonic reacted for 30 minutes. After the reaction, the solvent is removed under decompressed pressure, and then it is dissolved in 5 ml of CH2Cl2 and washed once with 0.1 M HCl solution and water each. After the organic layer is separated and dred with MgSO4, it is decompressed and filtered, and then the solvent of the filtered solution is removed under decompressed pressure. By slowly adding 30 ml of n-hexane, a solid product is extracted to obtain a light-yellow 5,11,17,23-tetra-2,5-methoxybenzyliminecalix[4]arene-1,3-bytylmercaptane (2,5-TMBICAT). The thus-obtained 2,5-TMBICAT is recrystallized with ethyl acetate/n-hexane to obtain a white solid crystal, 2,5-TMBICAT (385 mg, yield 82%).
1H NMR (300 MHz CDCl3): δ 8.88(s, 2H, N═CH) 8.67(s, 2H, N═CH) 7.67(d, 2H, ArH), 7.51(d, 2H, ArH), 7.10(s, 4H, ArH), 6.96(s, 4H, ArH), 6.89˜6.71(m, 8H, ArH), 4.33(d, 4H, ArCH2Ar, J=13 Hz), 4.03(t, 4H, OCH2), 3.87˜3.65(m, 24H, OCH3), 3.46(d, 4H ArCH2Ar J=13 Hz), 3.09.(t, 4H, CH2SH), 2.38˜2.34(m, 4H, OCH2CH2), 2.19-2.12(m, 4H, CH2CH2SH)
Preparation of Iminecalixarene Derivative Monolayer as Shown in
A solution, wherein TMBICADA synthesized in example 3 is dissolved in an organic solvent such as CHCl3, etc. in a concentration of 0.1˜5 mM, is prepared. As shown in
Preparation of an Iminecalixarene Derivative Monolayer on a Gold Substrate as Shown in
A solution, wherein TMBICAT synthesized in example 5 is dissolved in an organic solvent such as CHCl3, etc. in a concentration of 0.1-5 mM, is prepared. As shown in
The monolayer of other iminecalixarene derivatives as shown in example 9 is also prepared by the same method.
Preparation of the Aminocalixarene Derivative Monolayer of
A solution, wherein a derivative having an aldehyde terminal among aminocalixarene derivatives of formula 6 or formula 7 is dissolved in an organic solvent such as CHCl3, etc. in a concentration of 0.1˜5 mM, is prepared. A slide glass (amine chip) wherein an amine functional group is attached, prepared as shown in
General Unmodified Protein Fixation Method by Molecular Recognition Shown in
Fixation of Antibody Protein
The sample solutions used to immobilize protein on the iminecalixarene derivative monolayer prepared in example 1, concentrations, compositions, etc. are as shown table 1. First, for the fixation of protein, a well plastic having a hole with a diameter of 2 mm is attached onto the chip. On the chip wherein the well plastic is attached, a protein sample solution prepared as shown in table 1 is coated in 3 ul each using a micropipet, and it is fixed in a plastic chamber for 1 hour at room temperature. At this time, the amount of coated antibody is three times as much as can cover the surface once. After fixation, in order to block the positions other than the spots wherein protein is fixed, it is put in a blocking solution. After the well plastic is detached from the chip, the chip is left in the blocking solution for 30 minutes at room temperature. After blocking, in order to clean the chip, it is put in a cleaning solution 1 for 2 minutes, and a cleaning solution 2 for 2 minutes, and then taken out and dried.
Reaction with Antibody Protein
After being dried, the chip wherein antibody is fixed is put in 15 ml of the prepared antigen solution in order to react with fluorescent attached antigen, and incubated in a chamber for 1 hour at 37° C. After the antigen-antibody reaction is completed, the chip is put in cleaning solution 1 for 2 minutes, and in cleaning solution 2 for 2 minutes. Then it is dried and analyzed using a fluorescence scanner (GSI Lumonics, U.S.A.).
Preparation of the Aminocalixarene Derivative Monolayer on a Gold Substrate of
A solution, wherein a derivative having a thiol terminal among aminocalixarene derivatives of formula 6 or formula 7 is dissolved in an organic solvent such as CHCl3, etc. in a concentration of 0.1-5 mM, is prepared. As shown in
Ion Concentration Effect Upon Protein Fixation
The sample solutions used for the protein fixation involving ions on the iminecalixarene derivative monolayer, their concentration and composition, etc. are as shown in Table 2 or 4. First, for the fixation of protein, well plastic with a hole having a diameter of 2 mm is attached onto the chip. On the chip wherein well plastic is attached, a protein sample solution prepared as shown in table 2 or 4 is coated using a micropipet in 3 ul for each ion and concentration. Then, it is fixed in a plastic chamber for 10 minutes, 30 minutes, and 1 hour for each ion. After fixation, in order to block the positions other than the spots wherein protein is fixed, the chip is put in a blocking solution of table 1, and the well plastic is detached from the chip, and then the chip is left in the blocking solution for 30 minutes at room temperature. After blocking, the chip is put in cleaning solution 1 of table 1 for 2 minutes, and cleaning solution 2 of table 1 for 2 minutes for cleaning, and then taken out and dried.
Reaction of the Fixed Antibody and Antigen
After being dried, the chip wherein antibody is fixed as a monolayer is put in 15 my of fluorescent attached protein (antibody) solution 2 of table 1, and incubated in a chamber for 1 hour at 37° C. in order to react with fluorescent attached antigen. After antigen-antibody reaction is completed, the chip is put in cleaning solution 1 of table 1 for 2 minutes, and cleaning solution 2 of table 1 for 2 minutes, and then dried. The analysis result using a fluorescence scanner (GSI Lumonics, U.S.A.) is shown in
The Effect of an Ammonium Ion Concentration Upon the Fixation of Unmodified Protein by Molecular Recognition of
The Fixation of Antibody Proteins Containing Ammonium Ions in Different Concentrations
The concentrations, compositions, etc. of the cleaning solutions and the blocking solution used to immobilize proteins on the aminocalixarene derivative monolayer prepared in example 12, i.e. aminocalixarene derivative chip is as shown in Table 5. The fixation solutions containing ammonium ions in different concentrations as an fixation solution used for fixation of antibody proteins and the reaction solution used for the reaction of fluorescent antigen are as shown in
The Reaction with Fluorescent Attached Protein
The method for reacting the fluorescent CRP antigen protein to be combined with the antibody of the fixed antibody protein monolayer on the aminocalixarene derivative monolayer is as follows. After the aminocalixarene derivative chip wherein protein is fixed in 15 ml of fluorescent antigen solution (antigen concentration 1.4 g/ml) presented in Table 6, combination reaction was carried out maintaining 37° C. for 1 hour. After antigen-antibody reaction is completed, the chip is put in cleaning solution 1 of Table 5 for 2 minutes, and then cleaning solution 2 for 2 minutes, and dried. Then, it is analyzed using fluorescence scanner (GSI Lumonics, U.S.A.). The result is as shown in
Fluorescence Analysis Technology for the Analysis of Protein Fixation Maximum Density
The result shown in
Competitive Fixation with an Amine Functional Group Upon Protein Fixation
Competitive protein fixation with an amine functional group compound on the iminecalixarene chip shown in
In addition, this is the result showing that on the iminecalixarene derivative monolayer developed in the present invention the protein fixation proceeds at a superhigh speed by ionic/molecular recognition.
Measurement of Fixation Speed Reduction by a Competitive Reaction with an Ammonium Functional Group Upon Protein Fixation
Competitive protein fixation with an amine functional group compound on the aminocalixarene chip shown in
In addition, the protein fixation on the aminocalixarene derivative presented in the present invention is a technology of fixing unmodified proteins, which prepares a protein chip by performing fixation on the surface of a monolayer in a short time of about 30 minutes to 1 hour at a theoretically maximum density, that is, at a superhigh speed and a high density by molecular/ionic recognition.
Conjugation Reaction of Cy5 (Fluorescent Material) and Antigen of
Fluorescent attached antigen protein is prepared by reacting Cy-5, a fluorescent material provided by Telechem (U.S.A), as follows according to the provided condition for conjugation reaction with the protein.
325 μl of Cy-5 fluorescent material diluted in a carbonate buffer is put in a tube. After 175 μl of CRP antibody protein (2.86 mg/ml) diluted in a 1×PBS solution is put in said tube, it is reacted for 30 minutes at room temperature. After the reaction, the reaction product is column-refined (Sephadex™ G-50), and by analyzing the absorption band ratio of the amide functional group of protein and the aromatic group of the fluorescent material according to the analysis method provided by Telechem, the number of fluorescence attached to each protein is measured. After it is confirmed that about 1 fluorescence is attached to each protein, it is used for the combination reaction with antibody protein. After the amount of antigen protein combined with the fixed antibody protein is measured using a fluorescence scanner (GSI Lumonics, U.S.A.), density of the fixed protein is confirmed through fluorescence analysis.
Analysis of Iminecalixarene Derivative, TBICAP (5,11,17,23-tetrabenzyliminecalix[4]arene-1,3-dipropyl) Used for NMR Analysis of the Iminecalixarene Derivative and the Amine Compound in
After TBICA (500 mg, 0.59 mmol), anhydrous K2CO3 (826 mg, 5.9 mmol) and sodium iodide (1.3 g, 8.9 mmol) are put in a dried round-bottom flask, it is dried for 30 minutes under decompressed pressure. Then, after 120 ml of anhydrous acetonitrile is added under nitrogen exchange, it is mixed on a heater. After 10 minutes, 1-bromobutane (0.51 ml, 5.9 mmol) is added to the reaction vessel, and it is mixed for 24 hours with raising the temperature of the reaction vessel to 80° C. gradually. After the reaction, the solvent is decompressed and removed, and the remaining product is dissolved with CH2Cl2. Solid precipitates obtained during the reaction, such as KBr, KI, K2CO3 are removed by being decompressed and filtered and the filtered solution is decompressed to remove the solvent. The liquid product remaining in the flask is dissolved in 2 ml of CH2Cl2, and 13 ml of n-hexane is added slowly to extract the product. The extracted solid product is dissolved in 2 ml of CH2Cl2 once again, and 13 ml of n-hexane is added slowly to obtain 495 mg (yield 90%) of a yellow solid product, TBICAP (5,11,17,23-tetrabenzyliminecalix[4]arene-1,3-dipropyl)
1H NMR (300 MHz CDCl3): 8.47(s, 2H, N═CH), 8.27(s, 2H, N═CH), 7.86-7.72(m, 8H, ArH), 7.42-7.33(m, 12H, ArH), 7.09(s, 4H, ArH), 6.86(s, 4H, ArH), 4.37(d, 4H, ArCH2Ar), 4.02(t, 4H, OCH2), 3.46(d, 4H, ArCH2Ar), 2.10(q, 4H, OCH2CH2), 1.35(t, 6H, OCH2CH2CH3)
The Research on Molecular Recognition Between Allylamine and TBICAP by Using NMR
13.8 mg (920 g/mol, 0.015 mol) of TBICAP synthesized in example 21 is put in a vial, and dissolved in 5 ml of DMSO to make 3 mM. Then, after 500 μl of this TBICAP solution is put in an NMR tube, 2.5 μl of trimethylamine is added to the tube to make the solution under the basic condition. Allylamine is dissolved in D2O2 to a concentration of 500 mM, and 50 μl is taken and put in the prepared NMR tube, and then NMR is measured with molecular recognition proceeding, the result of which is shown in
The Research on Molecular Recognition of TBICAP with Alanine, Phenylalanine, Phenethylamine, etc. Using NMR
TBICAP material (13.8 mg, 0.015 mmol) synthesized in example 21 is measured and put in a vial, and dissolved in 5 ml of DMSO to make 3 mM. Then, after each amine compound is prepared in the same way as in example 21, it is added to NMR tube in the ratio of 1:16 as in example 22. By obtaining NMR spectrum by the hour, time needed for molecular recognition to proceed completely is grasped, leading to the analysis of a compound that allows an easy, or strong, molecular recognition. According to the result shown in
Distinguished completely from the conventional method of preparing protein chip by fixing protein using conventional chemical bonding, physical absorption, or biotin-streptavidin, which is being generally used over the world, the present invention is a novel technology wherein once unmodified protein is coated on an imincalixarene derivative monolayer or an aminocalixarene derivative monolayer, protein is fixed on the surface of the monolayer irreversibly at superhigh speed through ionic/molecular recognition and at the same time with the maximum density fixation.
Number | Date | Country | Kind |
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10-2005-0103857 | Nov 2005 | KR | national |
10-2005-0110824 | Nov 2005 | KR | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/KR2006/002258 | 6/14/2006 | WO | 00 | 11/28/2007 |