KR100785655B1 - Self-assembled monolayers prepared by using a aminocalixarene derivatives, and protein chip using the self-assembled monolayers - Google Patents

Abstract

According to the present invention, a compound of the aminocalixarene derivative monomolecular layer prepared as a monomolecular layer on a solid substrate such as a glass or a gold substrate using the compound of Formula 1 or Formula 2, and the protein on the surface of the monomolecular layer of the aminocalixarene derivative using the same Protein chips prepared by immobilization by irreversible ion / molecular recognition of ionic or hydrogen-bonding functional groups external to the protein, and immobilized on the surface of the monomolecular layer directly in solution without denatured protein It is about.

[Formula 1]

Wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ' ₁ , R' ₂ , R ' ₃ , R' ₄ , R ' ₅ , R' ₆ , R ' ₇ and R' ₈ are independently of each other -H, -CH ₃ , -C ₂ H ₅ , -C ₃ H ₇ , -OCH ₃ , -Cl, -C ₆ H ₅ , -OH, -OCH ₂ CH ₃ , -Br, -CF ₃ , -OCH ₂ C ₆ H ₅ , -OC ₆ H ₅ , -OC ₆ H ₄ CH ₃ , -OC ₆ H ₄ C (CH ₃ ) ₃ , -OC ₆ H ₄ CF ₃ , -OC ₆ H ₄ Cl, -OCOCH ₃ , -NHCOCH ₃ , -CONHCH ₃ , -CN, COOH, and -COOR, wherein R in -COOR is -CH ₃ or -C ₂ And H ₅ , wherein Y ₁ , Y ₂ , Y _3, and Y ₄ are each independently a -H,-(CH ₂ ) _n -CH = O,-(CH ₂ ) _n -SH,-(CH ₂ CH ₂ O) _m -CH ₂ CH ₂ -CH = O,-(CH ₂ CH ₂ O) _m -CH ₂ CH ₂ -SH,-(CH ₂ ) _m -C ₆ H _4- (CH ₂ ) _c- Z and -CO- (CH ₂ ) _m-1 -C ₆ H _4- (CH ₂ ) _c -Z (n = 2-15, m = 1-10, c = 0-10, Z = -SH, -CHO, -COOH, -NH ₂ , and -C ₆ H ₄ -and C ₆ H ₅ are defined as phenyl groups).

[Formula 2]

Wherein R ₁ , R ' ₁ , R ₂ , R' ₂ , R ₃ , R ' ₃ , R ₄ , R' ₄ , R ₅ , R ' ₅ , R ₆ , R' ₆ , R ₇ , R ' ₇ , R ₈ and R' ₈ are independently of each other -H, -CH ₃ , -C ₂ H ₅ , -C ₃ H ₇ , -OCH ₃ , -Cl, -C ₆ H ₅ , -OH, -OCH ₂ CH ₃ , -Br, -CF ₃ , -OCH ₂ C ₆ H ₅ , -OC ₆ H ₅ , -OC ₆ H ₄ CH ₃ , -OC ₆ H ₄ C (CH ₃ ) ₃ , -OC ₆ H ₄ CF ₃ , -OC ₆ H ₄ Cl, -OCOCH ₃ , -NHCOCH ₃ , -CONHCH ₃ , -CN, COOH, and -COOR, wherein R in -COOR is -CH ₃ or -C ₂ And H ₅ , wherein Y ₁ , Y ₂ , Y _3, and Y ₄ are each independently a -H,-(CH ₂ ) _n -CH = O,-(CH ₂ ) _n -SH,-(CH ₂ CH ₂ O) _m -CH ₂ CH ₂ -CH = O,-(CH ₂ CH ₂ O) _m -CH ₂ CH ₂ -SH,-(CH ₂ ) _m -C ₆ H _4- (CH ₂ ) _c- Z and -CO- (CH ₂ ) _m-1 -C ₆ H _4- (CH ₂ ) _c -Z (n = 2-15, m = 1-10, c = 0-10, Z = -SH, -CHO, -COOH, -NH ₂ , and -C ₆ H ₄ -and C ₆ H ₅ are defined as phenyl groups).

Aminocalixarene derivatives, protein chips

Description

SELF-ASSEMBLED MONOLAYERS PREPARED BY USING A AMINOCALIXARENE DERIVATIVES, AND PROTEIN CHIP USING THE SELF-ASSEMBLED MONOLAYERS}

1 is a schematic diagram of preparing an amine slide glass according to the method published in the paper (Langmuir, 1996, Vol 12, pp 5338-5342).

FIG. 2 is a schematic diagram of an aminocalixarene derivative self-assembled monolayer prepared by chemical bonding between the prepared amine slide glass and an aminocalixarene derivative.

FIG. 3 is a schematic diagram illustrating a process for preparing a self-assembled monomolecular layer in which a thiol group attached derivative of a derivative of Formula 1 and Formula 2 is an aminocalixarene derivative.

Figure 4 is a schematic diagram showing the protein chip proposed in the present invention, when the amino acid solution is applied to a denatured protein solution without any chemical change on the surface of the solid substrate, such as gold or glass slide self-assembled into a monolayer, New technology to manufacture protein monolayer, that is, protein chip, in which protein is spontaneously immobilized on the surface of solid substrate by molecular recognition of hydrogen bondable functional groups such as ammonium ion recognition and guanidine group, so that protein is immobilized without vacancy at theoretical maximum density. The actual experimental results are as shown in FIG. 6.

Figure 5 shows the protein monomolecular layer by protein immobilization proposed in the present invention, that is, when the end group capable of hydrogen bonding such as an ammonium group or arginine of the protein in the aminocalixarene derivative during immobilization of the protein during protein chip production, the rest It is a schematic diagram which shows that immobilization of a protein advances by filling a vacancy with a cation, such as ammonium ion.

Figure 6 is an actual experimental result showing that the immobilization rate of the protein is affected by the ion concentration of NH ₄ ⁺ , when the antibody protein immobilization according to Example 3, the present invention is the highest density at 200 ~ 400 mM NH ₄ ⁺ ion The result is a new technology that can manufacture protein chips by immobilizing proteins at high speed in a short time of 10-30 minutes. The density of the immobilized protein was confirmed by binding the fluorinated antigen, and the result shows that the protein immobilization proceeds closer to the theoretical maximum density compared with the fluorescence sensitivity of the theoretical highest density shown in FIG. 7.

FIG. 7 is twice the amount combined with the protein of highest density in order to compare and analyze the result of FIG. 6, which is the result of spontaneous protein immobilization according to Example 3, with the result of immobilized protein theoretical highest density. , Fluorescence sensitivity measured by drying after 1x and 0.5x fluorinated antigen in the same space. In FIG. 6, the results measured at 1 hour using 400 mM of ammonium ion appeared almost the same as the fluorescence sensitivity when using 1-fold fluorescent antigen, indicating that protein immobilization proceeded at a very high speed and high density. Experimental results.

8 shows amines present in the terminal groups such as lysine exposed to the outside of the protein, in which the protein immobilization in the aminocalixarene derivative self-assembled monolayer of the present invention, that is, the ammonium ion and the N-terminus of the protein, ie, the amine terminal In order to show that the attached ammonium ion is irreversibly ion-recognized and ultimately immobilizes the protein, a competition reaction between the amine compound to which the amine functional group is attached and the protein is performed, which impedes the immobilization of the protein according to the concentration of the amine compound. It is a schematic diagram showing.

9 is an experimental result according to Example 5, when the protein is immobilized in the aminocalixarene derivative self-assembled monolayer of the present invention, a compound with an ammonium group, such as lysine and benzylamine, is immobilized to the protein and ion recognition While competing to reduce the rate of immobilization of the actual protein, competition shows that aromatic amines such as aniline, which are present in the amine as non-ammonium ions in the buffer, are not recognized inside the aminocalixarene and do not undergo a competitive reaction. Reaction result. Even when this competition reaction is carried out using a guanidine functional group attached to an arginine terminal having good hydrogen bonding, the protein immobilization rate can be reduced. Therefore, the proposed protein monolayer layer production technique of ammonium ion, hydrogen bonding, etc. It is shown that ion recognition / molecular recognition of the C-D-H-M-C may work together to form a protein monolayer in which an immutable protein is immobilized on an aminocalixarene monolayer (protein chip substrate).

 10 is a schematic diagram of attaching a Cy-5 (Telechem, USA) fluorescent material to the antigenic protein bound to the antibody protein for fluorescence reading.

The present invention relates to a self-assembled monomolecular layer prepared using an aminocalixarene derivative, a method of immobilizing a protein into a monomolecular layer using ion recognition or molecular recognition thereto, and a protein chip prepared using the same. More specifically, the present invention provides an aminocalixarene derivative monomolecular layer prepared as a monomolecular layer on a solid substrate such as glass or a gold substrate using a compound of Formula 1 or Formula 2, and proteins such as antigens and antibodies on its surface, particularly 10 kD. The present invention relates to a method of immobilizing a protein having the above molecular weight on the surface of a single molecule layer by molecular recognition and a protein chip produced by immobilization of the protein.

The occurrence and differentiation of life phenomena is controlled by functional interactions of protein-proteins, protein-ligands (or drugs), antigen-antibodies, enzyme-substrates, and the like. Therefore, researches on the function or role of biomolecules that specifically interact with specific proteins or ligands have become a major challenge in the life sciences, health and medical fields. In order to fix these various proteins on one solid substrate, that is, a chip, and to measure protein-protein or protein-ligand interaction, many protein chips immobilized on the same chip have been studied.

In particular, the method of immobilizing enzymes, antigens, antibodies, etc. on a solid substrate is the most basic technology in the field of using a variety of proteins, such as diagnostic kits for determining diseases using proteins. For example, Enzyme-Linked Immunoassay (EIA), which is widely used in diagnosis, is a 96-microtiter well (96 microtiter) in which antibodies are made of plastic to analyze specific proteins or specific proteins that are pathogenic. The product is prepared by immobilizing the antibody by physisorption at the bottom of the well or by chemically bonding various functional groups present on the outside of the antibody with the functional groups on the surface of the plastic, and the specific antigen present in the serum After the reaction, as a method of diagnosing by measuring the concentration of the antigen by color development and fluorescence analysis using a secondary antibody-enzyme conjugate, various disease diagnosis kits using the above method are commercially available.

In addition to the physical adsorption or chemical binding method, a protein called streptoberdine is immobilized on the solid substrate to which biotin is chemically bonded by biotin and streptoberdine, and then biotin is bound to the biotin attachment site in streptoberdine. The method of immobilizing proteins, such as an antibody, by the binding force between biotin and streptoberdine is known.

Problems appearing in the protein immobilization technology, such as the conventional physical adsorption method, chemical bonding method, and the method using the biotin-streptoberdine binding force recently used as follows.

1. Immobilization Density: The area where most efforts are being made in protein immobilization technology, but various kinds of proteins used for immobilization are immobilized at such a high density that the entire space applied in solution is completely covered with protein without empty space. In other words, little technology has been published for the production of protein chips capable of immobilizing protein monolayers. If the density of the immobilized protein on the surface is not the density of the monolayer (1.1-1.4 ㎍ / cm ² in the case of antibodies) of the complete level, the various proteins are not covered with the protein on the surface of the solid substrate immobilized Nonspecific immobilization occurs on the surface of a solid substrate, that is, a fixed antigen is not immobilized on the antibody and the surface where the solid substrate is exposed as it is, or a small amount of immobilized antibody results in a small amount of bound antigen. The problem is that trace protein analysis or quantitative reading becomes difficult.

2. Reproducibility: The most important technology for immobilization of proteins to be used for diagnosis and research. When immobilized dozens of dozens of proteins, immobilized proteins must show the same level of immobilization density and activity for accurate diagnosis. Can be obtained. However, currently used protein immobilization techniques use physisorption, chemical bonding, or biotin-streptoberdine binding on solid substrates, most of which require reactions between proteins and specific functional groups, and several incomplete reactions. Proceed with the problem that the immobilized protein can not maintain the same activity at the same density, even if reacted with the same concentration of antigen protein does not show the same result. In other words, protein chips prepared by immobilizing proteins using existing techniques have many problems in ensuring reproducibility at a level that can be commercialized.

3. Ultra-fast immobilization: Immobilization proceeds from the solution phase to the surface of the solid substrate. When the protein is dissolved in the solution, the activity of the protein gradually decreases over time. However, since immobilization hardly decreases after immobilization, in order to minimize the decrease in activity during immobilization, it is necessary to develop a solid substrate capable of ultrafast immobilization which completely performs immobilization within 1 hour, but few examples have been developed so far.

4. Ultra trace protein analysis technology: By using the protein immobilization technology known to date, the concentration of the antigenic protein, etc., which can be analyzed remains at the level of several ng / ml, and a diagnostic product capable of quantitative analysis of the protein present at a lower concentration is available. It is not developed. When a technology that can quantitatively analyze proteins with concentrations of several ng / ml or less is developed, it is possible to discover new cancer marker proteins that cannot be analyzed using existing technologies. It is predicted that the development of a new concept of protein chips for quantitative progress and early diagnosis and disease prevention is possible, but has not been developed until now.

5. Protein immobilization without denaturation: protein immobilization density, ie, the reproducibility that the number of proteins to be immobilized remains the same within a certain value, and even if the number of identical proteins is immobilized at the same level within which the activity of the protein is within a certain error In order to proceed with immobilization, immobilization must be performed in a controllable solution. Therefore, it is essential to develop a technology in which a protein that does not need a chemical bonding step that may cause a problem of protein activity is directly immobilized in the solution without any chemical manipulation. However, in addition to the technology using the crown compound published several years ago by the researchers, no denatured protein immobilization technology has yet been developed. Therefore, it is necessary to develop a protein immobilization technology that does not require functional group attachment.

6. Long-term preservation technology: protein is significantly reduced in activity during long-term storage of about 3 to 6 months, the reduction is not constant, there are many problems in the commercialization. In order to obtain the same diagnosis result even in long-term preservation, the technique proposed by the recent protein chip research group mainly fixes the protein to high density without empty space, and thus is applied to the solid substrate that appears when the protein is fixed at low density or by physical adsorption. Development of technology that minimizes the phenomenon that the immobilized protein is attracted to the surface by non-specific attraction, that is, the deactivation caused by the degeneration of the protein active site due to difficulty in maintaining the shape of the protein, that is, the protein chip capable of high-density immobilization without empty space There is a need for development of manufacturing technology, and the research direction is in agreement.

An object of the present invention is to provide a high-density immobilization technology without empty space for ensuring the uniformity of protein immobilization density and maintaining the same activity in order to solve the problem of reproducibility of the product which makes it difficult to commercialize among the problems occurring in the existing various protein immobilization method It is. In addition, as the immobilization proceeds in solution, the activity of the protein decreases over time in the solution, thereby providing a technique for solving the problem of non-uniformity in readings, which occurs because the activity of the protein is not the same after immobilization. In addition, the active site showing the activity of the protein is mainly maintained by hydrogen bonding, which solves the problem of activity non-uniformity caused by the decrease in activity caused by the change in activity site caused by chemical bonding force with a solid substrate that is stronger than the hydrogen bonding force. It provides a new concept of denatured protein immobilization technology that enables the analysis of very small amount of protein quantitatively, and the aminocalaxarene derivative monolayer, which is essential for developing a reproducible protein chip that can be commercialized by combining the above technologies, is used. To provide a protein chip and related technology to be produced.

In addition, it is an object of the present invention by attaching the compound to glass substrates (amine slide glass), silicon wafers, fused silica or gold substrates with amine functional groups, and all kinds of proteins without any processing 10 ~ To provide a monomolecular layer of an aminocalixarene derivative which can be immobilized on a solid substrate at a high speed and high density in a short time of about 30 minutes to 1 hour on a solution containing a cation of 800 mM concentration to prepare a protein monolayer.

It is a further object of the present invention to provide a protein monolayer, ie, protein chip and its manufacturing technology, in which all kinds of proteins are densely fixed at high density and high speed without any denaturation.

The aminocalixarene derivative, which is essential for the preparation of self-assembled monolayers in which proteins are immobilized at high speed within 30 minutes to 1 hour without any processing, is a compound having the structure of Formula 1 or 2.

Wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ' ₁ , R' ₂ , R ' ₃ , R' ₄ , R ' ₅ , R' ₆ , R ' ₇ and R' ₈ are independently of each other -H, -CH ₃ , -C ₂ H ₅ , -C ₃ H ₇ , -OCH ₃ , -Cl, -C ₆ H ₅ , -OH, -OCH ₂ CH ₃ , -Br, -CF ₃ , -OCH ₂ C ₆ H ₅ , -OC ₆ H ₅ , -OC ₆ H ₄ CH ₃ , -OC ₆ H ₄ C (CH ₃ ) ₃ , -OC ₆ H ₄ CF ₃ , -OC ₆ H ₄ Cl, -OCOCH ₃ , -NHCOCH ₃ , -CONHCH ₃ , -CN, COOH, and -COOR, wherein R in -COOR is -CH ₃ or -C ₂ And H ₅ , wherein Y ₁ , Y ₂ , Y _3, and Y ₄ are each independently a -H,-(CH ₂ ) _n -CH = O,-(CH ₂ ) _n -SH,-(CH ₂ CH ₂ O) _m -CH ₂ CH ₂ -CH = O,-(CH ₂ CH ₂ O) _m -CH ₂ CH ₂ -SH,-(CH ₂ ) _m -C ₆ H _4- (CH ₂ ) _c- Z and -CO- (CH ₂ ) _m-1 -C ₆ H _4- (CH ₂ ) _c -Z (n = 2-15, m = 1-10, c = 0-10, Z = —SH, —CHO, —COOH, —NH ₂ , and —C ₆ H ₄ — and C ₆ H ₅ are defined as phenyl groups).

Wherein R ₁ , R ' ₁ , R ₂ , R' ₂ , R ₃ , R ' ₃ , R ₄ , R' ₄ , R ₅ , R ' ₅ , R ₆ , R' ₆ , R ₇ , R ' ₇ , R ₈ and R' ₈ are independently of each other -H, -CH ₃ , -C ₂ H ₅ , -C ₃ H ₇ , -OCH ₃ , -Cl, -C ₆ H ₅ , -OH, -OCH ₂ CH ₃ , -Br, -CF ₃ , -OCH ₂ C ₆ H ₅ , -OC ₆ H ₅ , -OC ₆ H ₄ CH ₃ , -OC ₆ H ₄ C (CH ₃ ) ₃ , -OC ₆ H ₄ CF ₃ , -OC ₆ H ₄ Cl, -OCOCH ₃ , -NHCOCH ₃ , -CONHCH ₃ , -CN, COOH, and -COOR, wherein R in -COOR is -CH ₃ or -C ₂ And H ₅ , wherein Y ₁ , Y ₂ , Y _3, and Y ₄ are each independently a -H,-(CH ₂ ) _n -CH = O,-(CH ₂ ) _n -SH,-(CH ₂ CH ₂ O) _m -CH ₂ CH ₂ -CH = O,-(CH ₂ CH ₂ O) _m -CH ₂ CH ₂ -SH,-(CH ₂ ) _m -C ₆ H _4- (CH ₂ ) _c- Z and -CO- (CH ₂ ) _m-1 -C ₆ H _4- (CH ₂ ) _c -Z (n = 2-15, m = 1-10, c = 0-10, Z = —SH, —CHO, —COOH, —NH ₂ , and —C ₆ H ₄ — and C ₆ H ₅ are defined as phenyl groups).

Compounds of formulas (1) and (2) of the present invention can be synthesized using starting materials of the following formula (3).

The tetraaminocalix [4] arene of Formula 3 is converted to tetranitrocalix [4] arene according to the method of Reference 1 below by reducing the calligraphy [4] arene (calix [4] arene) [Reference 1: Journal of Organic Chemistry, 1990 Vol 55, pp 5639-5643; Journal of Organic Chemistry, 1979 Vol 44, pp 1233-1238].

The present invention is an aminocalixarene derivative monomolecular layer prepared by attaching a compound of Formula 1 and Formula 2 to a solid substrate such as glass, silicon wafer, molten quartz, etc., with a functional group capable of chemical bonding such as a gold substrate or an amine functional group, And to provide a protein monolayer, that is, protein chip manufacturing technology to immobilize all kinds of proteins at high density.

1 shows a method of introducing various functional groups into a solid substrate in order to attach an aminocalixarene derivative to a solid substrate, and shows a method of introducing an amine functional group into a glass slide glass. same.

Glass substrates with amine functionality (glass slide glass) were prepared using silanol (-Si) according to the method published in Langmuir (1997 Vol 13, pp 4305-4308; Langmuir, 1996 Vol 12, pp 5338-5342). -OH) functional group was prepared in the form of a glass substrate (amine slide glass, or amine chip) having an amine end group through a chemical reaction on the surface of the glass substrate rich in the surface.

FIG. 2 shows a method for attaching a compound having an aldehyde functional group in a derivative of Formula 1 or 2 to an amine slide glass, which is one of the solid substrates used to prepare a protein monolayer by immobilizing a protein proposed in the present invention. . A glass substrate having an amine functional group, that is, an amine slide glass, is placed in a mixed solution of chloroform and THF (CHCl ₃ : THF = 9: 1) in which the compounds of Formula 1 and Formula 2 are dissolved at a concentration of 0.1 to 5 mM. After 1 to 5 hours, washed with chloroform, acetone, and ethanol, and then dried, and the aminocalixarene derivative monolayer as shown in FIG. 2 is completed. The formation of monolayers is confirmed using surface reflection infrared spectroscopy. Glass substrates used here are generally all kinds of slide glass or glass commercially available. Most biomaterials, such as proteins, are prepared by dissolving pH in buffers between 7 and 8, so it is safe to use aminocalixarene derivatives imine-bonded with amine slide glass when used for this purpose. It was confirmed. When developing products that require long-term use, the derivatives are immobilized to solid substrates for 1-5 minutes in THH or methanol solvents with 1-3% of BH ₃ / THF or NaBH ₄ , which is a well known reduction reaction. A monomolecular layer of aminocalixarene derivatives amine-bonded with a solid substrate is prepared by using a reaction of reducing guarine imine with an amine functional group.

3 schematically shows a process in which the aminocalixarene derivative is prepared as a self-assembled monolayer on a gold substrate. A specific method for preparing a self-assembled monolayer of aminocalixarene derivatives on a gold substrate is as follows.

A solution obtained by dissolving a compound attached with a thiol in a compound of Formula 1 or 2 in an organic solvent such as chloroform (CHCl ₃ ) at a concentration of 0.1 to 5 mM is prepared. Gold substrate was immersed in the prepared solution for 1 to 5 hours and then taken out, washed with chloroform, acetone and water, respectively, and dried to complete the self-assembled monolayer of aminocalixarene derivatives as shown in FIG. 3. The gold substrate used here may be any kind of gold thin film, but generally, vacuum deposition of 2 to 10 nm of chromium (Cr) or titanium (Ti) on glass, fused silica, silicon wafers, plastic substrates, etc. After the deposition, the substrate was vacuum deposited to a thickness of 50 to 200 nm. The prepared substrate is gold piranha solution immediately prior to use _{(Piranha solution: conc.H 2 SO 2} : 30% H 2 O 2 = 3: 1), soak for 1 minute, wash in purified water, and dry with blowing with nitrogen. The formation of monolayers is confirmed using surface reflection infrared spectroscopy.

In addition, the present invention provides a method for immobilizing denatured protein by recognition of multiple ions / molecules by recognizing one or more of ammonium group of protein or guanidine group of arginine in ion self-assembled monolayer of aminocalixarene derivative will be.

In addition, the immobilization method of the present invention manufactures all kinds of diagnostic biochips, research protein chips, etc. using protein immobilization, that is, a technology based on the production of products using all kinds of protein immobilization. Even results are inventions that have never been published.

4 is a Cy-5-antigen conjugate (conjuate) in which an antibody protein is immobilized by spontaneous ion / molecule recognition on an aminocalixarene derivative monomolecular layer developed in the present invention, and then a Cy-5 fluorescent substance is attached to the immobilized antibody. It is a schematic diagram confirming the density and activity of the immobilized antibody protein by binding to the antigen-antibody reaction.

Figure 5 is when the immobilization of the protein proceeds in the schematic diagram of Figure 4, when the end group capable of hydrogen bonding, such as the ammonium group or arginine of the protein inside the aminocalix arene derivative, the remaining empty space filled with cations such as ammonium ion It is a schematic diagram which shows that fixation of a protein advances.

As shown in the schematic diagram of FIG. 4, the immobilization was carried out on the aminocalixarene derivative monolayer using the antigen (Ag) and the antibody (Ab) of C-reactive protein (CRP). One real study is the result. This result shows the immobilization rate and the immobilization density of the protein in the aminocalixarene derivative monolayer, and the activity of the immobilized protein. High purification of the protein was carried out using NH ₄ ⁺ ions and the same concentration of antibody protein (3 times the theoretical density to be immobilized on the surface (1.4 μg / cm ² ) and 3 μl each of 45 μg / ml concentration). It was. The immobilization time was set to 10 minutes, 30 minutes, and 1 hour, and the antibody protein was immobilized during this time. Density analysis of the immobilized antibody was performed by allowing a sufficient amount of the fluorinated antigen protein and antibody protein to bind with the immobilized antibody protein in a 1: 1 manner by an antigen-antibody reaction. Fluorescence sensitivity analysis confirmed the results using a scanner (GSI lite, USA). According to the analysis results, when immobilized using NH ₄ ⁺ ions 0-100 mM, it was confirmed that the antibody showed about 40% of the theoretical maximum fluorescence when the antibody protein immobilization proceeded for about 1 hour. On the other hand, in the experimental results of immobilizing the antibody at 400 mM concentration of NH ₄ ⁺ ion, it can be seen that it shows almost the same level as the theoretical maximum fluorescence shown in FIG. In particular, at 400 mM concentration, about 65% of the theoretical maximum fluorescence was observed even after 30 minutes of immobilization, which indicates that the protein was immobilized to a high level of protein monolayer in a short period of 30 minutes to 1 hour. to be. In addition, the results show that the higher the concentration of ions filling the remaining space after the functional groups of the protein is recognized, the faster the immobilization, the actual study confirming the effect of the ion concentration in the schematic diagram shown in FIG. The result is However, the result of the 800 mM concentration is lower than 400 mM, in connection with this, it is well known that high ion concentrations often cause degeneration at the active site of the antibody protein and thus lose activity. The results of 800 mM show that immobilization proceeded rapidly and at high density, but the activity of immobilized proteins was significantly reduced. In the present experiment, the antibody protein was used as it is without any degeneration.

In addition, the present invention provides a protein chip prepared by producing a protein monolayer (i.e., a protein chip) on an aminocalixarene derivative monolayer by using an invariant protein, at an ultrafast, high reproducibility, and at the highest density level. It's about technology.

7 is 1: 1 binding of the immobilized antibody (1.4 μg / cm ² ) to the maximum and the antigen (35 ng / 0.0314 cm ² , dry spot 2 mm in diameter, binding density 1.1 μg / cm ² ) After fluorescence, the same amount of fluorinated antigen was applied to a solid substrate and dried, followed by fluorescence measurement. When 1 and 2 times the amount of the above test results were applied, the brightness of the fluorinated antigen was almost the same as that of the fluorinated antigen with respect to the antibody immobilized for 1 hour using NH ₄ ⁺ 400 mM in FIG. 6. On the other hand, the application of 1/2 fold shows that the fluorescence is reduced by half.

8 is a schematic diagram of a competition reaction showing the process of analyzing the effect on the immobilization density by adding compounds having an ammonium function in the immobilization step to confirm whether immobilization of the antibody protein actually occurs through ion recognition. At pH 7, the amine compound is present in the form of 100% ammonium ions, and it is a schematic showing that immobilization of the protein will be hindered by adding an appropriate amount of such amine compound.

FIG. 9 shows the results of the research actually conducted according to the schematic diagram of FIG. 8. Benzylamine with ammonium functional group, lysine with ammonium end group, and aniline with amine functional group but not changed to ammonium ion in pH 7 buffer This is the actual study result of antibody protein immobilization reaction by adding 10 mM aniline). In order to compare with the result of the competition reaction was also carried out with the immobilization without adding any amine compound (control). The density of the immobilized antibody protein was quantitatively analyzed by binding the immobilized antibody protein to the fluorescent protein at a 1: 1 level. In the case of immobilization with 10 mM lysine and benzylamine, the immobilization of the protein was only 1/4 of the antibody protein immobilization compared to the control immobilization, indicating that the immobilization of the protein was severely hampered by a compound having an ammonium functional group. Giving. Addition of arginine with guanidine end groups also resulted in impeding immobilization. In contrast, when aniline having an amine functional group that does not change to ammonium was added under immobilization conditions, the immobilization density was similar to that of no addition, and thus, aniline did not interfere with the immobilization process. These results are the actual experimental results showing that the immobilization proceeds by the ion / molecule recognition inside the aminocalixarene derivatives when the antibody protein is actually immobilized outside the protein.

10 is a schematic diagram showing the attachment of Cy-5 (Telichem, USA) fluorescent material to the antigenic protein bound to the antibody protein for fluorescence reading.

In addition, the present invention provides an ultrafast fixation technique capable of high-density immobilization of proteins within a short time within 1 hour, an aminocalixarene derivative monolayer which enables immobilized protein immobilization without any manipulation to the protein itself, Techniques for immobilizing proteins on surfaces, and protein chips prepared using the same are provided.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited thereto.

EXAMPLE

Example 1

Preparation of aminocalix arene derivative monolayer of Figure 2

A solution in which an aldehyde end group is attached to an organic solvent such as CHCl ₃ in an aminocalixarene derivative of Formula 1 or Formula 2 is dissolved at a concentration of 0.1-5 mM. After immersing the slide glass (amine chip) attached to the amine functional group prepared as shown in Figure 1 for 1-24 hours, and washed with chloroform, acetone, and water, and then dried, it appears in Figure 2 An aminocalixarene derivative monolayer is prepared.

Example 2

Preparation of Amino-Calixarene Derivative Monolayer on Gold Substrate of FIG.

A solution obtained by dissolving a derivative having a thiol end group in an aminocalixarene derivative of Formula 1 or Formula 2 in an organic solvent such as CHCl ₃ at a concentration of 0.1 to 5 mM is prepared. As shown in Figure 3, the gold substrate is immersed in the solution prepared for 1 to 24 hours and then taken out and washed with chloroform, acetone, and water, respectively, and dried to prepare the aminocalixarene derivative monolayer shown in Figure 3. The gold substrate used herein may be used in various forms, but generally, chromium (Cr) or titanium (Ti), such as glass, fused silica, silicon wafers, plastics, etc., is vacuum-deposited at 5 to 20 nm. The substrate is then vacuum deposited to 100 to 300 nm thick gold. The gold substrate thus prepared is dipped in a piranha solution (concentrated sulfuric acid: mixed solution of 30% hydrogen peroxide = 3: 1) for about 10 seconds to 1 minute, washed with water, and dried under nitrogen flow. Use immediately. The generation of monolayers is analyzed using surface reflection infrared spectroscopy (FTIR-ERS).

Example 3

Effect of Ammonium Ion Concentration on Immutable Protein Immobilization by Molecular Recognition of FIG.

Immobilization of Antibody Proteins Containing Ammonium Ions by Concentration

The concentration, composition, etc. of the washing solution, blocking solution used to immobilize the protein on the aminocalixarene derivative monomolecular layer prepared in Example 2, ie, the aminocalixarene derivative chip, are shown in Table 1. As the immobilization solution used for immobilization of the antibody protein, the immobilization solution containing ammonium ions by concentration and the reaction solution used when reacting the fluorescently attached antigen are shown in Table 2. First, a well plastic punched into a 2 mm diameter was attached to the chip to fix the protein. Immobilization solutions 1, 2, 3, 4, 5, 6 and 7 of Table 2 containing the same concentration (three times 135 ng / 0.0314 cm ² of theoretical maximum immobilization) and different concentrations of ammonium ions Each 3 microliter was applied to the chip by using a micropipette, and then immobilized at room temperature for 10 minutes, 30 minutes and 1 hour in a thermostat maintained humidity. The density of the protein to be immobilized at the immobilization of 1 hour or more did not show a significant difference from the result at 1 hour. The antibody protein was applied and immersed in the blocking solution of Table 1 to block portions other than the immobilized spots. The well plastic attached to the chip was detached and soaked at room temperature for 30 minutes. After blocking, the chip was immersed in the washing solution 1 of Table 1 for 2 minutes, and then immersed in the washing solution 2 of Table 1 for 2 minutes, then taken out and dried.

Reaction with Fluorescent Antigen Proteins

The antibody protein immobilized on the aminocalixarene derivative monolayer is reacted with the fluorescent CRP antigen protein bound to the antibody of the monolayer. The protein was immobilized in 15 mL of the fluorescently attached antigen solution (antigen concentration 1.4 µg / ml) described in Table 2, and the aminocalixarene derivative chip immobilized was maintained at 37 ^° C. for 1 hour in a humidified thermostat. The coupling reaction was carried out. After the antigen-antibody reaction was finished, the chip was soaked in Wash Solution 1 of Table 1 for 2 minutes, then dipped in Wash Solution 2 for 2 minutes, dried and analyzed using a fluorescent scanner (GSI Lumonics, USA). The result is shown in FIG. According to the results of FIG. 6, in the immobilization solution containing ammonium ion concentration (NH ₄ ⁺ ) at a low concentration level of about 0 to 100 mM, when immobilized for 1 hour, the protein immobilization density was about 40% of the theoretical maximum 1 culture. As a level, a weak immobilization density is shown. In contrast, when the ammonium ion concentration (NH ₄ ⁺ ) was about 400 mM, the immobilization density was shown to be 90% of the theoretical maximum, and the immobilization time was also very high even at a short time of 30 minutes to 1 hour. do. When the ammonium ion concentration is 800 mM, the ion concentration is too high, and as is well known, the active site of the antibody protein is denatured, resulting in a decrease in activity. Therefore, the immobilization of the protein proceeds at the highest density as in the case of 400 mM. However, due to the reduced activity of immobilized antibody, the binding reaction with the fluorinated antigen is lower than when using 400 mM concentration, so that the actually attached fluorinated antigen appears to be 50% of the theoretical maximum. .

Cleaning solution and blocking solution 1x PBS buffer (NaCl 8g, KCl 0.2g, Na ₂ HPO ₄ 1.44g, KH ₂ PO ₄ 0.24g) / DW 1000ml Blocking solution Skim milk powder 2.5g + 1xPBS 50ml = 50ml Cleaning solution 1 Tween20 300µl + 1xPBS 1000ml = 1000ml Cleaning solution 2 (NaCl 8g, KCl 0.2g, Na ₂ HPO ₄ 1.44g, KH ₂ PO ₄ 0.24g) / DW 1000ml

Immobilized Solution and Fluorescently Attached Antigen Solution NH ₄ ⁺ concentration and amount of NH ₄ ⁺ Cl added (90%) glycerol 300 μg / ml CRP Ab Distilled Water (DW) Total volume Immobilization Solution 1 0 mM 0 μl 14 μl 7.5 μl 28.5 μl 50 μl Immobilization Solution 2 25 mM (0.5 M) 2.5 μl 14 μl 7.5 μl 26 μl 50 μl Immobilization Solution 3 50 mM (0.5 M) 5 μl 14 μl 7.5 μl 23.5 μl 50 μl Immobilization Solution 4 100 mM (0.5 M) 10 μl 14 μl 7.5 μl 18.5 μl 50 μl Immobilization Solution 5 200 mM (0.5 M) 20 μl 14 μl 7.5 μl 8.5 μl 50 μl Immobilization Solution 6 400 mM (4 M) 5 μl 14 μl 7.5 μl 23.5 μl 50 μl Immobilization Solution 7 800 mM (4 M) 10 μl 14 μl 7.5 μl 18.5 μl 50 μl Fluorescent Antigen Solution Antigen concentration 1.4 µg / ml = (300 µl 5% milk + 27 µl Cy5-CRP antigen (818 µg / ml) + 14.7 ml = 1xPBS = 15 ml)

Example 4

Fluorescence Analysis Technology for Maximum Density Analysis of Protein Immobilization

The experimental results of FIG. 7 were measured by applying two times, one time, or 0.5 times the amount of fluorescent antigen protein required to bind 1: 1 with CRP antibody protein immobilized at the theoretical highest density onto glass slides of the same area, followed by drying. One is fluorescence sensitivity. This assay was conducted to obtain comparative data to analyze how the immobilization density of the antibody protein proceeded in Example 3 approached the theoretical maximum and what level of activity of the immobilized antibody protein is.

It is used to bind 1: 1 with the 1.4 μg / cm ² antibody (CRP Ab 160 kD / mol) required to cover 1 times the theoretical immobilization maximum as a CRP antibody protein at an area of 1 cm ² of the surface of the aminocalixarene derivative monolayer. Apply 0.5 times, 1 times, and 2 times the amount of the required fluorinated antigen (CRP Ag 115 kD / mol) (1.1 μg / cm ² ) and dry them to fluoresce directly with the fluorescence scanner (GSI Lumonics, USA). Analyzed. Application of 1 μl of fluorescently-labeled antigen results in a circular spot with a diameter of 2 mm and its area is 0.0314 cm ^2. Therefore, when 1 μl of 35 μg / ml of fluorescently-labeled antigen is applied, a surface density of 1.1 μg / cm ² is achieved. As a result, a measuring point having a diameter of 2 mm, in which the amount of the antigen is present, results in the same number of fluorescences as a result of binding a 1-fold amount of the fluorinated antigen to the antibody protein immobilized on the aminocalixarene derivative monolayer in a 1: 1 ratio. The result of FIG. 7 is a result of measuring a fluorescence sensitivity using a scanner by generating a spot in which the antigen is physically adsorbed, and comparing the result with the result obtained through the antigen-antibody reaction obtained in FIG. It becomes possible. 2 times and 0.5 times were obtained by applying the fluorinated antigens at twice the concentration and 0.5 times the concentration, respectively, in the same manner. In the case of the theoretical maximum value of 1 times, the antibody protein immobilization density was shown to be almost the same as the fluorescence sensitivity obtained by immobilizing with 400 mM ammonium ion for 1 hour and reacting with the fluorinated antigen. The result shows that the monomolecular layer is composed at a level similar to that of, and the activity is high enough to perform 1: 1 binding to the antigen.

Example 5

Measurement of immobilization rate decrease by competition with ammonium functional compound in protein immobilization

Immobilization of the competitive protein with the amine functional compound on the aminocalixarene chip shown in the schematic diagram of FIG. 8 was performed for 1 hour using 400 mM ammonium ion as in the antibody protein immobilization method including the ammonium ion of Example 3 Proceed in a manner that, the concentration, composition, etc. of the sample solution containing the amine functional compound used are shown in Table 3. 3 μl of competing solutions 1, 2, 3 and 4, each containing no amine compound (control) and 10 mM aniline, benzylamine, lysine, etc. After the application by immobilization for 1 hour, the antibody was immobilized in the same manner as in Example 3 after the immobilization of the antibody, and the aminocalaxarene derivative monolayer was washed and blocked. The antibody-antigen reaction was carried out in the same manner as the reaction with the fluorescent antigen of Example 3, and the same treatment was performed using a fluorescence scanner (GSI Lumonics, USA). The results are shown in FIG. In the case of using aniline in which the amine functional group is not changed to the ammonium functional group under the antibody protein immobilization conditions containing 10 mM each of the amine compounds, the aniline protein immobilization density is almost the same as that in the absence of the amine compound. Indicated. On the other hand, when using benzylamine or lysine in which the amine functional group is present in the ammonium group under the experimental conditions, a sharp decrease in fluorescence occurs, which results in the amine compounds being ion / molecularly recognized inside the aminocalixarene derivatives competitively with the antibody protein. This impedes the immobilization of the antibody protein, resulting in a significantly lower density. In addition, this is a result demonstrating that the immobilization of the protein in the aminocalixarene derivative monolayer is achieved by immobilization of an amine functional group (ammonium group in solution) by irreversible ion / molecular recognition.

Immobilization Solution Containing 10 mM Amine Functional Compound Contains 10 mM amine compound Amount of amine compound (90%) glycerol 300 μg / ml CRP Ab Distilled Water (DW) (4 M) NH ₄ ⁺ Cl Total volume Competitive Solution 1 contrast 0 μl 28 μl 15 μl 47 μl 10 μl 100 μl Competitive Solution 2 aniline 10 μl (100 mM) 28 μl 15 μl 37 μl 10 μl 100 μl Competitive Solution 3 Benzylamine 10 μl (100 mM) 28 μl 15 μl 37 μl 10 μl 100 μl Competitive Solution 4 Lysine 10 μl (100 mM) 28 μl 15 μl 37 μl 10 μl 100 μl

In addition, the immobilization of the protein in the aminocalixarene derivative monolayer proposed in the present invention is theoretically high density, i.e., ultrafast / high density, in a short time of about 30 minutes to 1 hour without denaturation of the protein by molecular / ion recognition. As a result of immobilization on the surface of the monomolecular layer, it is a denatured protein immobilization technology that can produce protein chips.

Example 6

The binding reaction between Cy5 (fluorescent material) and antigen for fluorescence adhesion to the antigenic protein of FIG. 10

Fluorescent antigen protein was prepared by reacting Cy-5, a fluorescent substance provided by Telechem Co., Ltd., according to the binding reaction condition with the provided protein as follows.

325 μl of Cy-5 fluorescent material diluted in carbonate buffer was prepared and placed in a tube. 175 μl of CRP antigen protein (2.86 mg / ml) diluted in 1 × PBS buffer was added to the tube, followed by reaction at room temperature for 30 minutes. After the reaction, the reactants were purified by column (Column Purification, SephadexTM G-50), and the ratio of the amide functional groups of the protein and the aromatic absorption band of the fluorescent material was analyzed according to the analysis method provided by Telechem. After measurement, it was confirmed that one level of fluorescence was attached to each protein, and then used for binding reaction with the antibody protein. The amount of the fluorinated antigen protein bound to the immobilized antibody protein was measured using a fluorescence scanner (GSI Lumonics, USA), and then the density of the immobilized protein was confirmed by fluorescence analysis.

The present invention is completely different from the method of manufacturing protein chips by immobilizing proteins using chemical bonds, physisorption, biotin-streptoberdine, etc., which are generally used worldwide, and have a concentration of 10-800 mM. By applying the denatured protein to the aminocalixarene derivative monolayer in an aqueous solution containing a cation of, it is not only fast but also irreversibly immobilized on the surface of the monomolecular layer through ion / molecule recognition, and at the same time, it can be immobilized at the highest density. It is a new technology. In addition, the present invention relates to a new protein chip manufacturing technology in which the amount of the immobilized protein, that is, the density is uniform, and the immobilization at high speed is maintained uniformly so that the protein activity is uniformly maintained, thus ensuring high reproducibility. .

The protein monolayer, i.e., the protein chip, prepared using the aminocalixarene derivative monolayer proposed in the present invention has a problem of non-uniformity and deactivation of immobilization density occurring in various conventional protein immobilization methods, and solution phase when immobilized at a slow rate. It is a new technology that can solve all the problems of non-uniformity of immobilized protein activity by reducing the activity of protein that has existed for a long time. In addition, the active site showing the activity of the protein is mainly maintained by hydrogen bonds, and the modification of the active site by the chemical bonds with the solid substrate stronger than the hydrogen bond forces during the chemical bonding proceeds using an aldehyde chip, etc. There is a problem that the activity of the protein is irregularly reduced, the present invention is a novel technology that can solve the problem by using a molecule / ion recognition for immobilization, a force similar to the hydrogen bonding force to maintain the active position.

In addition, the present invention provides a protein chip manufacturing technology that completely solves the problem of reproducibility of protein chips, which has been the most problematic in the conventional protein chip production by providing a protein immobilized aminocalixarene derivative monolayer, and a protein chip using the same. To provide.

In addition, the present invention, the aminocalixarene derivative of the formula (1) or (2) through various chemical bonds are attached to functional groups such as amines, alcohols, thiols, glass substrates (amine slide glass), silicon wafers, fused silica (fused silica) ), And by attaching to the gold substrate to provide a variety of solid substrates attached with a monolayer of aminocalixarene derivatives, all kinds of proteins regardless of molecular weight in a short time of 30 minutes to 1 hour in solution without any processing The present invention provides a variety of solid substrates that are immobilized at high speed and a protein monolayer and a protein monolayer manufactured by using the same.

In addition, the present invention, by using the immobilization according to the ion concentration of Figure 6 or the competition reaction of Figure 9, etc., by increasing the ion concentration to increase the immobilization rate further, or by including a part of the amine compound to adjust the immobilization rate to the optimal state Providing the world's first single layer of aminocalixarene derivatives for protein immobilization with immobilization rate control function, it is manufactured by immobilizing proteins of various sizes and shapes of more than 10000 (10kD) while maintaining the highest activity under optimal conditions. Provided is a protein chip and its manufacturing technology.

In addition, the present invention enables the production of protein chips at a very high speed with only a small amount of protein that is three times the amount of protein required to achieve the highest immobilization density, which simplifies the protein chip manufacturing process and makes it possible to manufacture protein chips at low cost. Provide protein chip manufacturing technology.

In particular, according to the embodiment, the immobilized protein maintains the theoretical high density and maintains high activity of binding to the antigen, thereby providing a technology for manufacturing a diagnostic protein chip having the highest sensitivity. This technology will be applied to development of various kinds of protein chips, diagnostic antibody chips, and biochip manufacturing new technologies.

Claims (10)

The aminocalixarene derivative monomolecular layer prepared by attaching the aminocalixarene derivative of Formula 1 or Formula 2 to a solid substrate: [Formula 1]

Wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ' ₁ , R' ₂ , R ' ₃ , R' ₄ , R ' ₅ , R' ₆ , R ' ₇ and R' ₈ are independently of each other -H, -CH ₃ , -C ₂ H ₅ , -C ₃ H ₇ , -OCH ₃ , -Cl, -C ₆ H ₅ , -OH, -OCH ₂ CH ₃ , -Br, -CF ₃ , -OCH ₂ C ₆ H ₅ , -OC ₆ H ₅ , -OC ₆ H ₄ CH ₃ , -OC ₆ H ₄ C (CH ₃ ) ₃ , -OC ₆ H ₄ CF ₃ , -OC ₆ H ₄ Cl, -OCOCH ₃ , -NHCOCH ₃ , -CONHCH ₃ , -CN, COOH, and -COOR, wherein R in -COOR is -CH ₃ or -C ₂ And H ₅ , wherein Y ₁ , Y ₂ , Y _3, and Y ₄ are each independently a -H,-(CH ₂ ) _n -CH = O,-(CH ₂ ) _n -SH,-(CH ₂ CH ₂ O) _m -CH ₂ CH ₂ -CH = O,-(CH ₂ CH ₂ O) _m -CH ₂ CH ₂ -SH,-(CH ₂ ) _m -C ₆ H _4- (CH ₂ ) _c- Z and -CO- (CH ₂ ) _m-1 -C ₆ H _4- (CH ₂ ) _c -Z (n = 2-15, m = 1-10, c = 0-10, Z = -SH, -CHO, -COOH, -NH ₂ , and -C ₆ H ₄ -and C ₆ H ₅ are defined as phenyl groups, provided that (i) R ₁ to R ₈ , R ' ₁ to R ' ₈ is H at the same time and Y ₁ = Y ₃ = H, Y ₂ = Y ₄ =-(CH ₂ ) _n -CH = O, and (ii) R ₁ to R ₈ , R ' ₁ to R' ₈ are simultaneously H and Y ₁ = Y ₃ = H, Y ₂ = Y ₄ = -(CH ₂ ) _n -SH is excluded); [Formula 2]

Wherein R ₁ , R ' ₁ , R ₂ , R' ₂ , R ₃ , R ' ₃ , R ₄ , R' ₄ , R ₅ , R ' ₅ , R ₆ , R' ₆ , R ₇ , R ' ₇ , R ₈ and R' ₈ are independently of each other -H, -CH ₃ , -C ₂ H ₅ , -C ₃ H ₇ , -OCH ₃ , -Cl, -C ₆ H ₅ , -OH, -OCH ₂ CH ₃ , -Br, -CF ₃ , -OCH ₂ C ₆ H ₅ , -OC ₆ H ₅ , -OC ₆ H ₄ CH ₃ , -OC ₆ H ₄ C (CH ₃ ) ₃ , -OC ₆ H ₄ CF ₃ , -OC ₆ H ₄ Cl, -OCOCH ₃ , -NHCOCH ₃ , -CONHCH ₃ , -CN, COOH, and -COOR, wherein R in -COOR is -CH ₃ or -C ₂ And H ₅ , wherein Y ₁ , Y ₂ , Y _3, and Y ₄ are each independently a -H,-(CH ₂ ) _n -CH = O,-(CH ₂ ) _n -SH,-(CH ₂ CH ₂ O) _m -CH ₂ CH ₂ -CH = O,-(CH ₂ CH ₂ O) _m -CH ₂ CH ₂ -SH,-(CH ₂ ) _m -C ₆ H _4- (CH ₂ ) _c- Z and -CO- (CH ₂ ) _m-1 -C ₆ H _4- (CH ₂ ) _c -Z (n = 2-15, m = 1-10, c = 0-10, Z = -SH, -CHO, -COOH, -NH ₂ , and -C ₆ H ₄ -and C ₆ H ₅ are defined as phenyl groups, provided that (i) R ₁ to R ₈ , R ' ₁ to R ' ₈ is H at the same time and Y ₁ = Y ₃ = H, Y ₂ = Y ₄ =-(CH ₂ ) _n -CH = O, and (ii) R ₁ to R ₈ , R ' ₁ to R' ₈ are simultaneously H and Y ₁ = Y ₃ = H, Y ₂ = Y ₄ = -(CH ₂ ) _n -SH is excluded). delete A compound according to claim 1, wherein in formula (1) or formula (2), Y ₁ , Y ₂ , Y ₃ and Y ₄ are independently of each other-(CH ₂ ) _n -CH = O,-(CH ₂ CH ₂ O) _m − CH ₂ CH ₂ -CH = O,-(CH ₂ ) _m -C ₆ H _4- (CH ₂ ) _c -CH = O and -CO- (CH ₂ ) _m-1 -C ₆ H _4- (CH ₂ ) _c -CH = O (n = 2-15, m = 1-10, c = 0-10, and -C ₆ H ₄ -and C ₆ H ₅ are defined as phenyl groups) amino An aminocalixarene derivative monolayer prepared by attaching a callix arene derivative to a solid substrate selected from the group consisting of glass, silicon wafer, and molten quartz to which an amine functional group is attached, through an imine bond and a reduced amine bond. The compound of claim 1, wherein in formula (1) or (2), Y ₁ , Y ₂ , Y _3, and Y ₄ are independently of each other,-(CH ₂ ) _n -SH,-(CH ₂ CH ₂ O) _m -CH ₂ CH ₂ -SH,-(CH ₂ ) _m -C ₆ H _4- (CH ₂ ) _c -SH and -CO- (CH ₂ ) _m-1 -C ₆ H _4- (CH ₂ ) _c -SH Aminocalixarene derivatives selected from the group (n = 2-15, m = 1-10, c = 0-10, and -C ₆ H ₄ -and C ₆ H ₅ are defined by phenyl groups) An aminocalixarene derivative monomolecular layer prepared by adhesion. A protein chip produced by immobilizing a protein on a monomolecular layer of the aminocalixarene derivative according to any one of claims 1, 3 and 4. 6. The protein chip of claim 5, wherein the protein is an antibody. 6. The protein chip according to claim 5, prepared by spontaneous protein immobilization by irreversible molecule / ion recognition. The protein chip according to claim 5, wherein the protein to be immobilized has a molecular weight of 10 kD or more. The protein chip of claim 5, wherein the protein chip is prepared at a concentration of 10 mM to 800 mM of cation. The protein chip of claim 9, wherein the cation is NH ₄ ⁺ .

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