Molecularly imprinted polymer for detection of pentraxin protein and method for preparing the same

Abstract

The present disclosure relates to a molecularly imprinted structure for detection of a pentraxin protein and a method for preparing the same by synthesizing a reactive group-pentraxin protein ligand complex specifically reacting with the pentraxin protein and being polymerizable with a crosslink agent to detect a pentraxin protein by using the complex. The present disclosure also provides a chip for detection of a C-reactive protein and a method for preparing the same, the chip including a molecularly imprinted layer having excellent sensitivity to a C-reactive protein and an improved binding force to a metal substrate by using click chemistry.

Claims

1. A chip for detection of a C-reactive protein, the chip comprising: a fixing layer formed on a metal substrate and having an azide or an acetylene end group R.sup.1; and a molecularly imprinted layer formed on the fixing layer and including R.sup.2X-phosphocholine (wherein R.sup.2 is an azide or an acetylene end group, and X is a spacer group having a length of about 0.1 nm to about 5 nm), wherein the phosphocholine group included in the molecularly imprinted layer is arranged to correspond to a phosphocholine binding site of each molecule of the C-reactive protein, and wherein the fixing layer and the molecularly imprinted layer may be bound through a click chemistry reaction between the end group R.sup.1 of the fixing layer and R.sup.2 included in R.sup.2X-phosphocholine of the molecularly imprinted layer.

2. The chip for detection of a C-reactive protein claimed in claim 1, wherein R.sup.2 of the molecularly imprinted layer is the acetylene group with the proviso that the end group R.sup.1 of the fixing layer is the azide group, and R.sup.2 of the molecularly imprinted layer is the azide group with the proviso that the end group R.sup.1 of the fixing layer is the acetylene group.

3. The chip for detection of a C-reactive protein claimed in claim 1, wherein the fixing layer is a polymer layer, a self assembled monolayer, or mica having the azide or the acetylene end group R.sup.1 on the surface thereof.

4. The chip for detection of a C-reactive protein claimed in claim 3, wherein the self assembled monolayer may be formed including HSYR.sup.1 (wherein R.sup.1 is the azide or the acetylene end group, and Y is a spacer group having a length of about 0.1 nm to about 5 nm).

5. The chip for detection of a C-reactive protein claimed in claim 1, wherein the molecularly imprinted layer is formed including acetylene-C.sub.1-30alkylene-phosphorylcholine, propargyloxy-C.sub.1-30alkylene-phosphorylcholine, or azido-C.sub.1-30alkylene-phosphorylcholine.

6. The chip for detection of a C-reactive protein claimed in claim 4, wherein the self assembled monolayer is formed including azido-C.sub.1-30alkylene-1-thiol, acetylene-C.sub.1-30alkylene-1-thiol, or propargyloxy-C.sub.1-30alkylene-1-thiol.

7. A method for preparing a chip for detection of a C-reactive protein, the method comprising: forming a fixing layer having an azide or an acetylene end group R.sup.1 on a metal substrate; mixing a C-reactive protein and a molecularly imprinted material containing R.sup.2X-phosphocholine (wherein R.sup.2 is an azide or an acetylene end group, and X is a spacer group having a length of about 0.1 nm to 5 nm) to obtain a mixture solution of the molecularly imprinted material/the C-reactive protein; immersing the metal substrate, on which the fixing layer is formed, in the mixture solution of the molecularly imprinted material/the C-reactive protein to obtain a complex layer of the metal substrate/the fixing layer/the molecularly imprinted material/the C-reactive protein through a click chemistry reaction between the end group R.sup.1 of the fixing layer and R.sup.2 included in R.sup.2X-phosphocholine of the molecularly imprinted material; and removing the C-reactive protein from the complex layer to obtain a molecularly imprinted layer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Non-limiting and non-exhaustive embodiments will be described in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments in accordance with the disclosure and are, therefore, not to be intended to limit its scope, the disclosure will be described with specificity and detail through use of the accompanying drawings, in which:

(2) FIG. 1 is a view schematically illustrating a process of binding between a pentraxin protein and a reactive ligand according to an illustrative embodiment;

(3) FIG. 2 is a view for explanation of a method for preparing a styrene-phosphocholine ligand by steps according to an illustrative embodiment;

(4) FIG. 3 is NMR analysis results for a styrene-phosphocholine ligand prepared according to an illustrative working example;

(5) FIG. 4 is a schematic view showing a method for preparing a molecularly imprinted polymer using a styrene-phosphocholine ligand according to an example;

(6) FIG. 5 is a schematic view showing a method for preparing a molecularly imprinted polystyrene film using a styrene-phosphocholine ligand according to an example;

(7) FIG. 6 is a schematic view showing a method for preparing a molecularly imprinted polymer using a styrene-phosphocholine ligand according to an example;

(8) FIG. 7 is a graph showing Langmuir plot to determine a binding constant for a MIP in an example;

(9) FIG. 8 shows analysis results for a selective binding force of a MIP using BSA and CA;

(10) FIG. 9 shows observation results for distribution of C-reactive proteins being bound to a NIP, which is a control group of a MIP, by means of AFM in an example;

(11) FIG. 10 is a microphotograph obtained by observing a polystyrene bead prepared according to an example by means of SEM;

(12) FIG. 11(a) is a chemical formula showing a method for preparing 11-azidoundecane-1-thiol (azido-thiol) and FIG. 11(b) is a chemical formula showing a method for preparing 6-propargylhexylphosphorylcholine (propargyl-PC) in an example;

(13) FIG. 12 is a process view showing a process for preparing a chip for detection of a C-reactive protein in an illustrative embodiment;

(14) FIG. 13 shows FT-IR spectrums for surface modification according to an example;

(15) FIG. 14 is a graph showing concentration-dependent binding of a C-reactive protein (CRP) to each of a control polymer (CM), and a molecularly non-imprinted monolayer, and a molecularly imprinted monolayer (examples);

(16) FIG. 15 is a graph for plotting binding of a C-reactive protein to each of a molecularly non-imprinted monolayer and a molecularly imprinted monolayer according to the Langmuir isotherm equation;

(17) FIG. 16 is a graph showing an effect of free phosphocholine in binding of a C-reactive protein to each of a molecularly non-imprinted monolayer and a molecularly imprinted monolayer (examples);

(18) FIG. 17 is a graph showing binding of a C-reactive protein in serums to each of a control polymer, a molecularly non-imprinted monolayer, and a molecularly imprinted monolayer (examples); and

(19) FIG. 18 is a graph showing an effect of free phosphocholine in binding of a C-reactive protein in a complex matrix in an example.

DETAILED DESCRIPTION OF THE INVENTION

(20) Hereinafter, illustrative embodiments will be described in more detail with reference to illustrative working examples. The illustrative working examples are merely exemplary, and the scope of the present disclosure should not be construed as being limited to the illustrative working examples.

Example 1

Preparation of a Styrene-Phosphocholine Ligand

(21) A process for preparing a styrene-phosphocholine ligand was described below with reference to FIG. 2. The products presented in FIG. 2 have been identified through NMR.

(22) 12-hydroxydecanoic acid and potassium carbonate were added into 100 ml of N,N-dimethylformamide and stirred at a room temperature for about 10 minutes. Thereafter, 4-vinylbenzyl chloride was added thereinto and stirred while being heated at less than about 80 C. After the reaction was finished, N,N-dimethylformamide was removed through distillation under reduced pressure. The reacting material was dissolved in dichloromethane and then washed with water. Dichloromethane layers were collected and dried with a magnesium sulfate. From the dried dichloromethane, the magnesium sulfate was removed by a depressurization filter, and dichloromethane was removed through distillation under reduced pressure. Thereafter, column chromatography purification using an eluent of hexane and ethyl acetate with a ratio of 3:1 was performed (yield=79%).

(23) .sub.1H NMR (CDCl.sub.3 400 MHz): (ppm) 7.41 (m, 4H), 6.75 (m, 1H), 5.78 (dd, J.sub.1=13.6, J.sub.2=4.0, .sub.1H), 5.29 (dd, J1=6.8, J2=4.0, 1H), 5.10 (d, J=7.2, 2H), 3.63 (t, J=4.4, 2H), 2..sub.37 (t, J=4.8, 2H), 1.64 (m, 4H), 1.27 (br s, 14H)

(24) .sub.13C NMR (CDCl3 100 MHz): (ppm) 173.74, 137.90, 137.52, 136.47, 136.40, 136.35, 135.62, 128.77, 128.47, 127.60, 126.36, 126.05, 126.01, 114.39, 114.30, 65.97, 65.83, 63.11, 34.35, 32.81, 29.56, 29.49, 29.41, 29.24, 29.12, 25.74, 24.96

(25) LRMS (ESI+) m/z: 355 (M+Na)

(26) FT-IR (neat) (cm.sub.1): 3328 (br w), 2914 (s), 2848 (s), 1728 (s), 1462 (m), 1386 (w), 1359 (w), 1332 (m), 1316 (w), 1256 (m), 1195 (s), 1107 (m), 1057 (m), 1042 (m), 1008 (m), 989 (m), 962 (m), 899 (m), 830 (w), 850 (w), 804 (m), 728 (m)

(27) 1.5 g of 4-Vinylbenzyl-12-hydroxyl dodecanoate (496 mmol) and 2.09 ml of triethylamine (14.9 mmol) were added into 20 ml of dichloromethane (amylene stabillized) and stirred while being cooled at 0 C. on an ice-bath. 0.684 ml of 2-chloro-2-oxo-1,3,2-dioxaphospholane (7.44 mmol) was added into the reacting material and stirred at 0 C. for about 1 hour. Thereafter, the ice-bath was removed, and the reacting material was reacted at a room temperature. After the reaction was finished, dichloromethane was removed through distillation under reduced pressure. The reacting material was dissolved in chloroform and undissolved solids were removed by means of a depressurization filter. Column chromatography purification using an eluent of hexane and ethyl acetate with a ratio of 3:1 was performed (since the intermediate compound was easily denatured, a next follow-up reaction was proceeded with after identification of .sub.1H NMR). 0.864 g of 12-(2-Oxo-25-[1,3,2]dioxaphospholan-2-yloxy)-dodecanoic acid 4-vinyl-benzyl ester (1.97 mmol) were added into a pressure bottle together with an acetonitrile anhydrous and cooled to 20 C. 0.55 ml of trimethylamine (5.92 mmol) was added into the cooled pressure bottle and heated at less than 60 C. with stirring.

(28) After the reaction was finished, acetonitrile was removed through distillation under reduced pressure. Column chromatography purification using an eluent of chloroform, methanol, and water with a ratio of 65:25:4 was performed (yield=24%).

(29) .sub.1H NMR (CDCl.sub.3 400 MHz): (ppm) 7.42 (m, 4H), 6.73 (m, 1H), 5.77 (dd, J.sub.1=13.6, J.sub.2=4.0, 1H), 5.27 (dd, J1=6.8, J2=4.0, 1H), 5.08 (d, J=7.2, 2H), 4.26 (br s, 2H), 3.84 (m, 4H), 3.41 (s, 9H), 2.36 (m, 2H), 1.62 (.sub.m, 4H), 1.24 (br s, 14H)

(30) .sub.13C NMR (CD.sub.Cl3 100 M.sub.Hz): (ppm): 173.68, 137.87, 137.50, 136.46, 136.39, 136.33, 135.61, 128.77, 128.44, 127.57, 126.35, 126.01, 125.99, 114.39, 114.30, 66.25, 65.95, 65.81, 65.66, 65.60, 59.20, 54.29, 34.32, 31.07, 31.00, 29.70, 29.64, 29.53, 29.49, 29.32, 29.16, 25.92, 24.96

(31) LRMS (ESI+) m/z: 520 (M+Na)

(32) FT-IR (neat) (cm.sub.1): 3367 (br w), 2923 (s), 2852 (m), 1732 (s), 1483 (w), 1380 (w), 1232 (s), 1164 (s), 1057 (s), 986 (s), 966 (s), 909 (s), 874 (w), 825 (s), 796 (s), 763 (s), 713 (s), 505 (s)

(33) The styrene-phosphocholine ligand (12-(4-vinylbenzyl)oxycarbonyl dodecyl phosphocholine) that has been prepared as described above was identified as a final product by means of NMR (Nuclear Magnetic Resonance; Avance 400; the Bruker company). FIG. 4 shows results of the NMR analysis.

Example 2

Preparation of a Polystyrene Film, in which a Styrene-Phophocholine Ligand is Molecularly Imprinted

(34) A method for preparing a molecularly imprinted polymer according to an illustrative embodiment is described below with reference to FIG. 5.

(35) Total 1 ml of styrene/DVB (divinylbenzene) as a crosslink agent, and DBK (dibenzylketone) as an initiator, were added to 10 ml of a standard phosphate buffer solution (pH 7.0) with a ratio of styrene:DVB:DBK=9:1:1. Thereafter, a C-reactive protein and a styrene-phosphocholine ligand, which have been reacted together in advance, were added thereto with a molar ratio of 1:5. Since the C-reactive protein and the styrene-phosphocholine ligand were strongly bound to each other via calcium ions, they have been reacted in advance in a binding buffer solution (0.1M Tris, 5 mM CaCl.sub.2, 150 mM NaCl, and pH 8.0) thereby inducing photo-polymerization thereafter. With respect to conditions for the polymerization reaction, photo-polymerization was performed at 37 C. for 2 hours such that a monomer (styrene-phophocholine ligand) is positioned on a surface of the crosslink agent layer in the state that the monomer is bound to the C-reactive protein (here, since the ligand has both a hydrophilic group and a hydrophobic group, the hydrophilic group is directed toward a water phase, and the hydrophobic group is directed toward a surface of styrene/DVB). Subsequently, photo-polymerization was performed at 90 C. for 2 hours such that a hard polystyrene polymer can be formed. After the polymerization reaction was finished, in order to remove the C-reactive protein, the reacting material was washed with an elution buffer solution (0.1 M Tris, 10 mM EDTA, 150 mM NaCl, pH 8.0) twice.

Experimental Example 1

Measurement of a Binding Force of a C-Reactive Protein to a Molecularly Imprinted Polymer

(36) A binding force of a C-reactive protein to a molecularly imprinted polymer was analyzed by detecting a C-reactive protein through the Bradford assay method and an immunoassay method. In this case, in addition to a molecularly imprinted polymer (MIP), a molecularly non-imprinted polymer (NIP) and a polystyrene polymer (control polymer; CP) were prepared to analyze a molecularly imprinting effect. The molecularly non-imprinted polymer was prepared by using a reactive ligand without molecularly imprinting of CRP. The polystyrene polymer was prepared without using a reactive ligand and CRP.

(37) (1) Bradford Assay Method

(38) 5 g of a CRP was added to the prepared molecularly imprinted polymer and reacted at 0 C. for 30 minutes. In order to remove a non-specifically bound protein, the reacting material was washed three times with PBS or repeatedly washed three times with a TBST buffer solution (0.1 M Tris, 150 mM NaCl, 0.1% Tween 20) containing 0.1% Tween 20. Thereafter, 2 ml of an elution buffer solution was added thereto, and the reacting material was kept at a room temperature for 15 minutes. In order to quantify an amount of the protein in the elution buffer solution, 1 ml of a Bradford reagent (the Sigma company) was added to 1 ml of an eluted protein solution, and the solution was kept at a room temperature for 10 minutes. Thereafter, absorbance was measured at 595 nm. In order to prepare a standard quantification curve, 0, 0.5, 1, 1.5, and 2 g/ml of BSA solutions were prepared. 1 ml of the Bradford reagent was added to 1 ml of each of the BSA solutions, and the solution was kept at a room temperature for 10 minutes. Thereafter, absorbance was measured at 595 nm (Varian-300 spectrophotometer).

(39) (2) Immunoassay Method

(40) 5 g of a CRP was added to the prepared molecularly imprinted polymer and reacted at 0 C. for 30 minutes. In order to remove a non-specifically bound protein, the reacting material was washed three times with PBS or repeatedly washed three times with a TBST buffer solution (0.1 M Tris, 150 mM NaCl, 0.1% Tween 20) containing 0.1% Tween 20. Thereafter, 10 g of anti CRP mouse monoclonal antibody (the Sigma company) was added thereto, and the reacting material was reacted at 0 C. for 30 minutes. In order to remove a non-specifically bound antibody, the reacting material was washed three times with PBS or repeatedly washed three times with a TBST buffer solution (0.1 M Tris, 150 mM NaCl, 0.1% Tween 20) containing 0.1% Tween 20. Thereafter, an anti mouse IgG (the Sigma company), to which alkaline phosphatase was bound, was diluted with a ratio of 1:30,000 and added thereto. Thereafter, the reacting material was reacted at 0 C. for 30 minutes. In order to remove a non-specifically bound antibody, the reacting material was washed three times with PBS or repeatedly washed three times with a TBST buffer solution (0.1 M Tris, 150 mM NaCl, 0.1% Tween 20) containing 0.1% Tween 20. Thereafter, 1 ml of an NPP solution (p-nitrophenolphosphate; the Sigma company), which is a substrate of alkali phosphatase, was added thereto, and the reacting material was kept at a room temperature for 10 minutes. Thereafter, absorbance was measured at 405 nm (Varian-300 spectrophotometer).

(41) TABLE-US-00001 TABLE 1 Effect of molecularly imprinting a C-reactive protein Assay method/Washing buffer Bradford assay (ug) Immunoassay (O.D) Sample PBS TBST PBS TBST CP 0.45 0.0073 0.13 0.021 NIP 1.4 0.051 0.27 0.041 0.48 0.061 0.024 0.0051 MIP 3.0 0.71 2.6 0.17 1.3 0.18 0.19 0.061 IF 2.1 9.8 2.6 8.1

(42) Table 1 shows comparison of molecularly imprinting factors (imprinting factor; IF) of C-reactive proteins of a MIP and a NIP. An amount of a C-reactive protein bound to the MIP was higher 8.1 or 9.8 times than an amount of a C-reactive protein bound to the NIP depending on a test method. It is understood that the C-reactive protein on the NIP was eluted during the washing process, whereas the C-reactive protein on the MIP was not eluted and remains.

Experimental Example 2

Determination of a Binding Constant to a MIP

(43) FIG. 7 is a graph using the Langmuir isotherm equation. Based on slope and a y-intercept value, a MIP-C-reactive protein binding constant K.sub.A is 3.010.sup.9/M. Below is the Langmuir isotherm equation:

(44) $\frac{C}{R}=\frac{C}{R_{\max }}+\frac{1}{R_{\max }}\frac{1}{K_A}$

(45) R.sub.max can be calculated from a reciprocal number of the slope, and K.sub.A can be calculated from the y-intercept.

(46) Following table 2 shows CRP binding constants that have been reported until now. The detection method using a molecularly imprinted polymer in an illustrative embodiment exhibits a binding constant value higher about three degrees than that in detection methods using a conventional anti C-reactive protein antibody.

(47) TABLE-US-00002 TABLE 2 Comparison of binding constants in the detection method of an illustrative embodiment and a conventional detection method Binding Detection constant Receptor method (K.sub.A, M.sup.1) Reference Anti-CRP antibody SPR 1.4 10.sup.6 1 (immobilized) Anti-CRP antibody fiber-optic 3.8 10.sup.8 2 (immobilized) detection Exposed PC on SPR 9.9 10.sup.6 3 supported lipid monolayers.sup.a PC-appended ELISA 7.1 10.sup.6 4 supramolecular assembly The MIP in this MSIA 3.0 10.sup.9 study 1. M. H. F. Meyer, M. Hartmann and M. Keusgen, Biosens. Bioelectron., 2006, 21, 1987 2. C. Chou, H. Y. Hsu, H. T. Wu, K. Y. Tseng, A. Chiou, C. J. Yu, Z. Y. Lee and T. S. Chan, J. Biomed. Opt., 2007, 12, 024025 3. S. F. Sui, Y. T. Sun and L. Z. Mi, Biophys. J., 1999, 76, 333; 4. B. A. Rosenzweig, N. T. Ross, D. M. Tagore, J. Jayawickramarajah, I. Saraogi and A. D. Hamilton, J. Am. Chem. Soc., 2009, 131, 5020.

Experimental Example 3

Analysis of a Selective Binding Force of a MIP

(48) In order to identify a specific binding force of a MIP to a CRP, 5 g of bovine serum albumin (BSA) and 5 g of carbonic anhydrase (CA) were added into each of the MIP and the NIP. The binding force was analyzed in the same manner as described in Experimental Example 1.

(49) FIG. 8 shows binding forces of the MIP and the NIP to BSA and CA. As shown in FIG. 8, the PS and the NIP exhibit a low binding force of less than 0.5 g to the CRP, BSA, and CA. However, the MIP exhibits a higher binding force of more than 2.5 g only to the C-reactive protein. It shows that the MIP was specifically bound to the CRP.

Experimental Example 4

AFM Analysis

(50) FIG. 9 shows observance results for distribution of C-reactive proteins being bound to a MIP and a NIP by means of an atomic force microscope (AFM; the Vecco company; model code: D-V). Upon observation, the height of a side surface of the protein on the MIP is relatively regular, while the protein on the NIP is partially agglutinated or bound with its side surface having irregular height. In case of the molecularly imprinted protein, pentamers are simultaneously bound thereby exhibiting a plane and regular height of the side surface. However, on the surface of the molecularly non-imprinted polymer, binding through only one ligand is possible. Accordingly, the protein binding shape and the protein distribution were irregular, which were different from those in the MIP.

Example 3

Preparation of a Polystyrene Bead, in which a Functional Monomer is Molecularly Imprinted

(51) A process for preparing a molecularly imprinted polystyrene bead was the same as Example 1. However, in order to disperse styrene/DVB on an aqueous solution, 0.1% Brij 78 was added as a dispersant, and then, photo-polymerization was performed. Under the same conditions as those in Example 2, the 0.1% Brij 78 dispersant was added, such that styrene/DVB was dispersed in a bead form through vigorous stirring. During the photo-polymerization, styrene/DVB was continuously dispersed through stirring.

(52) FIG. 10 shows results of observation of the prepared polystyrene bead by means of an SEM. The size of the bead is in a range of 1.2 m to 1.8 m. A molecularly imprinting factor is 3.6. A detection limit, which can be identified by the Bradford assay method, is 0.30.1 g/ml. Accordingly, CRP detection is possible at a concentration below a blood concentration range. Upon measurement of surface conductivity, the surface conductivity of the polystyrene bead, to which no ligand is bound, is 0.0767 mS/cm. The surface conductivity of the bead, in which a ligand is molecularly imprinted, is 9.98 mS/cm. Since one end of the reactive ligand has a choline group and a phosphate group, they exhibit + and charges. For this reason, the surface conductivity of the bead in which a ligand is molecularly imprinted appears to have increased, compared to that of the polystyrene bead to which no ligand is bound.

Example 4

Synthesis of 11-azidoundecane-1-thiol

(53) All reagents for chemical synthesis were purchased from Sigma-Aldrich Chemical and used without additional purification. Human plasma and all buffer components were also purchased from Sigma-Aldrich Chemical. For a CRP-negative human serum (N-serum), components of the CRPA Latex Test Set purchased from Cenogenics (Morganville, USA) were used. Phosphate buffered saline (PBS) and HyClone(R) were purchased from Thermo Scientific (Rochester, USA). 1H (400 MHz) and 13C (100 MHz) NMR spectrums were measured by using Bruker Spectrospin 400.

(54) 11-bromo-1-undecanol (1 g, 3.98 mmol), sodium azide (285 mg, 4.38 mmol), and potassium iodide were dissolved in ethanol and refluxed for 20 hours. In the solution, a solvent was removed under reduced pressure, and a residue was dissolved in diethylether. The residue mixture dissolved in the ether was washed with water and dried on an anhydrous magnesium sulfate. The solvent was removed under reduced pressure. A non-purified product produced by the removal of the solvent was purified on silica gel (R.sub.f=0.3, hexane:EtOAc=5:1) through column chromatography to obtain a compound 1 (868 mg, 102.2%).

(55) IR: 3332, 2924, 2853, 2091

(56) .sup.1H NMR (400 MHz, CDCl.sub.3) 3.57 (t, 2H, OCH.sub.2), 3.32 (s, 1H, OH), 3.25 (t, 2H, N.sub.3CH.sub.2), 1.57 (m, 4H, HOCH.sub.2CH.sub.2 (CH.sub.2).sub.7CH.sub.2), 1.28 (m, 14H, HOCH.sub.2CH.sub.2(CH.sub.2).sub.7)

(57) .sup.13C NMR (100 MHz, CDCl.sub.3) 62.4, 51.4, 32.6, 29.5, 29.4, 29.1, 28.8, 26.7, 25.7

(58) Exact mass calcd for C.sub.11H.sub.23N.sub.3O: 213.18. found: 236 [M+Na].sup.+.

(59) The compound 1 (868 mg, 4.09 mmol), methanesulfonyl chloride (1.26 g, 11.0 mmol), and triethylamine (2.44 g, 24.1 mmol) were dissolved in THF. The reaction mixture was stirred at a room temperature for 2 hours. After addition of ice water, an organic phase in the mixture was separated from an aqueous phase in the mixture. The aqueous phase was extracted twice by diethylether. The organic phase was washed with 1 M HCl, deionized water, and saturated sodium bicarbonate. After the organic phase was dried on an anhydrous magnesium sulfate, a solvent in the organic phase was removed under reduced pressure. A resultant non-purified product was purified on silica gel (R.sub.f=0.4, hexane:EtOAc=5:1) through column chromatography to obtain a compound 2 (1.17 g, 98.2%).

(60) IR: 2925, 2854, 2092, 1180

(61) .sup.1H NMR (400 MHz, CDCl.sub.3) 4.21 (t, 2H, OCH.sub.2), 3.26 (t. CH.sub.2N.sub.3), 3.00 (s, 3H, CH.sub.3S), 1.74 (m, 2H, OCH.sub.2CH.sub.2), 1.59 (m, 2H, CH.sub.2CH.sub.2N.sub.3) 1.39-1.18 (m, 14H, OCH.sub.2CH.sub.2(CH.sub.2).sub.7)

(62) .sup.13C NMR (100 MHz, CDCl.sub.3) 70.4, 51.4, 37.1, 29.4, 29.3, 29.11, 29.10, 29.0, 28.8, 26.7, 25.4

(63) Exact mass calcd for C.sub.12H.sub.25N.sub.3O.sub.3S: 291.16. found: 314 [M+Na].sup.+.

(64) The compound 2 (1.17 mg, 4.01 mmol) and potassium thioacetate (917 mg, 8.03 mmol) were dissolved in 90 mL of DMF. The reaction mixture was stirred at a room temperature for 1 hour. In the reaction mixture, a solvent was removed under reduced pressure, and a residue was dissolved in diethyl ether. An organic phase formed of the residue was washed with water and dried on an anhydrous magnesium sulfate. The solvent was removed under reduced pressure. A resultant non-purified product was purified on silica gel (R.sub.f=0.7, hexane:EtOAc=9:1) through column chromatography to obtain a compound 3 (831 mg, 76.4%).

(65) IR: 2924, 2853, 2092, 1690

(66) .sup.1H NMR (400 MHz, CDCl.sub.3) 3.25 (t, 2H, CH.sub.2N.sub.3), 2.85 (t, 2H, SCH.sub.2), 2.31 (s, 3H, CH.sub.2CO), 1.57 (m, 4H, SCH.sub.2CH.sub.2 (CH.sub.2).sub.7CH.sub.2), 1.35-1.27 (m, 14H, SCH.sub.2CH.sub.2(CH.sub.2).sub.7)

(67) .sup.13C NMR (100 MHz, CDCl.sub.3) 195.8, 51.4, 30.6, 29.5, 29.4, 29.15, 29.11, 28.86, 28.81, 26.7

(68) Exact mass calcd for C.sub.13H.sub.25N.sub.3OS: 271.17. found: 294 [M+Na].sup.+.

(69) The compound 3 (588 mg, 2.17 mmol) was dissolved in 40 ml of methanol and 2 ml of concentrated HCl. The reaction mixture was stirred for 3 hours and quenched with water. In the reaction mixture, an aqueous phase was extracted twice by diethyl ether. In the reaction mixture, an organic phase was washed with water and dried on an anhydrous magnesium sulfate. A solvent of the organic phase was removed under reduced pressure. A resultant non-purified product was purified on silica gel (R.sub.f=0.8, hexane:EtOAc=9:1) through column chromatography to obtain a compound 4 (471 mg, 94.6%).

(70) IR: 2923, 2852, 2090

(71) .sup.1H NMR (400 MHz, CDCl.sub.3) 3.25 (t, 2H, CH.sub.2N.sub.3), 2.51 (q, 2H, SCH.sub.2), 1.60 (m, 4H, SCH.sub.2CH.sub.2(CH.sub.2).sub.7CH.sub.2), 1.35-1.28 (m, 15H, HSCH.sub.2CH.sub.2 (CH.sub.2).sub.7)

(72) .sup.13C NMR (100 MHz, CDCl.sub.3) 51.4, 34.1, 29.5, 29.1, 29.0, 29.8, 28.4, 26.7, 24.6.

Example 5

Synthesis of 6-propagylhexylphosphorylcholine (propagyl-phosphorylcholine)

(73) A solution of 1,6-hexandiol (3 g, 25.4 mmol) in DMF (20 ml) is dropwised into a suspension of sodium hydride (1.52 g, 38.1 mmol) in the DMF (20 ml) on an ice bath, and stirred for 30 minutes under the ice bath condition. Propagyl bromide (5.7 g, 38.1 mmol) in the DMF (20 ml) was added to the mixture, and then, stirred at a room temperature for 20 hours. The solvent was removed under reduced pressure. A resultant non-purified product was dissolved in diethylether. The mixture was washed with water, dried on an anhydrous magnesium sulfate, and purified on silica gel (R.sub.f=0.5, hexane/EtOAc=1:1) through column chromatography to obtain a compound 5 (1.80 g, 45.4%).

(74) IR: 3373, 3292, 2933, 2858, 1093

(75) .sup.1H NMR (400 MHz, CDCl.sub.3) 4.13 (s, 2H, HCCCH2O), 3.59 (t, 2H, CH.sub.2OH), 3.52 (t, 2H, CH.sub.2OCH.sub.2), 3.03 (s, 1H, CH.sub.2OH), 2.48 (s, 1H, HCCCH.sub.2O), 1.58 (m, 4H, OCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2), 1.38 (m, 4H, OCH.sub.2CH.sub.2CH.sub.2CH.sub.2)

(76) .sup.13C NMR (100 MHz, CDCl.sub.3) 79.8, 74.3, 70.0, 62.3, 57.9, 32.5, 29.3, 25.8, 25.5

(77) Exact mass calcd for C.sub.9H.sub.16O.sub.2: 156.12. found: 179 [M+Na].sup.+.

(78) The compound 5 (600 mg, 3.84 mmol), 2-chloro-1,3,2-dioxaphosphoran2-oxide (1.09 g, 7.68 mmol), and triethylamine (777 mg, 7.68 mmol) were dissolved in 30 ml of DCM. The reaction mixture was stirred at a room temperature for 72 hours under a darkroom condition. The solvent was removed under reduced pressure. A resultant non-purified product was purified on silica gel (R.sub.f=0.3, hexane/EtOAc=1:4) through column chromatography to obtain a compound 6 (700 mg, 69.5%).

(79) The compound 6 (700 mg, 2.66 mmol) and trimethylamine (1.57 g, 26.6 mmol) were dissolved in 8 ml of acetonitrile. The reaction mixture was stirred at 60 C. for 20 hours. The solvent was removed under reduced pressure. A resultant non-purified product was purified on silica gel (chloroform:methanol is 2:1; and chloroform:methanol:water is 50:50:4; R.sub.f=0.2) through column chromatography. The solvent was removed under reduced pressure. The residue was dissolved in anhydrous chloroform and filtered to obtain a compound 7 (411 mg, 48.1%).

(80) IR: 3350, 3296, 2936, 2860, 1086, 1059

(81) .sup.1H NMR (400 MHz, CD.sub.3OD) 4.28 (m, 2H, POCH.sub.2CH.sub.2N), 4.15 (d, 2H, HCCCH.sub.2O), 3.90 (q, 2H, CH.sub.2CH.sub.2CH.sub.2OP), 3.69 (m, 2H, POCH.sub.2CH.sub.2N+), 3.54 (t, 2H, HCCCH.sub.2OCH.sub.2), 3.27 (s, 9H, N.sup.+(CH.sub.3).sub.3), 2.85 (t, 1H, HCCCH2O), 1.64 (m, 4H, OCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2), 1.44 (m, 4H, OCH.sub.2CH.sub.2CH.sub.2CH.sub.2)

(82) .sup.13C NMR (100 MHz, CDCl.sub.3) 79.7, 74.8, 69.6, 65.5, 59.0, 57.4, 53.5, 30.4, 29.2, 25.6, 25.3

(83) Exact mass calcd for C.sub.14H.sub.28NO.sub.5P: 321.17. found: 322 [M+H]).

Example 6

Surface Modification of an SPR Chip

(84) FIG. 12 shows a whole scheme for surface modification of an SPR chip. In order to prepare a self assembled monolayer (SAM) having an azide(N.sub.3)-terminated compound, an SPR chip (15 mm15 mm10 mm) was immersed in 2 mM (20 ml) of an ethanol solution of 11-azidoundecane-1-thiol at a room temperature for 12 hours (FIG. 12). The substrate was thoroughly washed with ethanol several times, and then, dried under N.sub.2 gas. For pre-test for a click reaction, a gold substrate (5 mm5 mm1 mm) was coated with 11-azidoundecane-1-thiol in the same manner as described above, and placed in a reaction mixture containing 1 mol of propagyl-phosphorylcholine in 1 ml of PBS (FIG. 12), 0.5 mol of copper sulfate (II).pentahydrate, and 1 mol of a sodium ascorbic acid at 4 C. for 16 hours.

(85) In order to prepare a molecularly imprinted monolayer (MIM), 3.32 nmol of propagyl phosphorylcholine (PC) was pre-cultured on an ice bath for 30 minutes, with 0.66 nmol of a C-reactive protein (CRP) (molar ratio of 5:1 for propagyl phosphorylcholine: CRP) in 1 ml of a binding buffer (0.1 M Tris/HCl, 150 mM NaCl, 5 mM CaCl.sub.2, pH 8.0). A 1,3-dipolar cycloaddition reaction, which is one of click chemistry reactions, was performed by placing the SPR chip coated with the self assembled monolayer of 11-azidoundecane-1-thiol in 20 ml of a PBS buffer solution containing 1.66 nmol of copper sulfate (II).hydrate, 3.32 nmol of a sodium ascorbate, and the propagyl phosphorylcholine-CRP complex pre-cultured at 4 C. for 16 hours. The molar ratio of propagyl phosphorylcholine:copper sulfate:a sodium ascorbate was 1:0.5:1.

(86) In order to prepare a molecularly non-imprinted monolayer, all steps were carried out as those for the molecularly imprinted monolayer, except that no CRP was added to the reaction mixture. After the reaction was finished, the surface was thoroughly washed with cool PBS and dried under N.sub.2 gas.

(87) In order to block an azide end that has not been involved in the click reaction, propagyl alcohol for another click reaction was added to the substrate. The substrate was immersed in 20 ml of PBS containing 20 mol of propagyl alcohol, 10 mol of copper sulfate (II).hexahydrate, and 20 mol of a sodium ascorbate at a room temperature for 5 hours.

(88) In order to prepare a control polymer (CM), the SPR chip coated with the N.sub.3-terminated self assembled monolayer was directly modified by propagyl alcohol through a click reaction. A surface, which was not coated with propagyl phosphorylcholine, was obtained.

(89) In order to remove the CRP from the molecularly imprinted monolayer, the surface-modified SPR chip and the gold substrate were immersed in an elution buffer (0.1 M Tris/HCl, 10 mM EDTA, 150 mM NaCl, pH 8.0) at a room temperature for 30 minutes. Finally, the substrate was thoroughly washed with deionized water, acetone, and ethanol, and dried under N.sub.2 gas.

(90) Analysis of Surface Characteristics of an SPR Chip by Means of FT-IR

(91) An FT-IR/ATR spectrum was acquired in a single reflection mode by using the Thermo Nicolet iS10 FT-IR spectrometer equipped with a smart aperture gazing angle (SAGA). IR spectrums of a gold surface, which has been functionalized in each of the above-described reaction steps, were obtained in a range of 650 cm.sup.1-4000 cm.sup.1. All the spectra were equalized through 512 scan and reported in a penetration mode to a clean gold surface.

(92) Analysis of CRP Binding Through a Surface Plasmon Resonance (SPR) Research

(93) CRP binding to each substrate was measured by SPR LAB (the K-MAC company). Dilution and elution of the sample were carried out with a flow rate of 20 ml/min at 25 C. by means of a binding buffer. Binding of the CRP onto the SPR chip was carried out by one dose of injection of 300 of the protein solution of 100 pM to 400 nM in the binding buffer. A N-serum was diluted to be a 2% solution in a binding buffer. BSA was prepared to be a 1% solution in PBS. In the binding buffer, 10 mM of free phosphocholine (f-phosphocholine) was used to identify phosphorylcholine specific binding of the CRP. The protein binding was recorded with a resonance angle and reported with incidence angle shift ().

(94) Analysis of Surface Characteristics of an SPR Sensor

(95) In order to introduce an azide group onto a surface of the gold substrate for continuous click chemistry, the gold substrate was immersed in 2 mM of an ethanol solution of 11-azidoundecane-1-thiol, which was azide-containing thiol, at a room temperature for 12 hours. Characteristics of a self assembled monolayer obtained from the immersion were analyzed by means of FT-IR/ATS. In the IR spectrums, characteristic peaks of the self assembled monolayer were observed at 2850 cm.sup.1 (symmetric methyl CH stretching), 2930 cm.sup.1 (non-symmetric methyl CH stretching), and 2090 cm.sup.1 (non-symmetric N.sub.3 stretching mode) (FIG. 13b).

(96) In order to identify that the click chemistry is caused by propagyl phosphorylcholine, an excess molar concentration of propagyl phosphorylcholine (1 mM) was added to the reaction mixture containing the gold substrate coated with 11-azidoundecane-1-thiol. FIG. 13c shows an IR spectrum of the gold substrate, in which propagyl phosphorylcholine at a 4-site of triazole was substituted with an excess amount of propagyl phosphorylcholine. In the spectrum, the non-symmetric N.sub.3 stretching peak has disappeared at 2090 cm.sup.1. This shows that mounting of propagyl phosphorylcholine onto the surface was accomplished by successful coupling between an azide group and an acetylene group.

(97) In synthesis of a molecularly non-imprinted monolayer (Click-NIM) and a molecularly imprinted monolayer (Click-MIM), a molar concentration of propagyl phosphorylcholine was 166 nM. In the IR spectrum, the non-symmetric N.sub.3 stretching peak has not disappeared at 2090 cm.sup.1 (FIG. 13d and FIG. 13e). This shows that the free N.sub.3 group remains on the surface.

(98) In order to quench an activity surface of the free N.sub.3 group, propagyl alcohol, which is another agent for blocking click reaction, is added. After the blocking reaction, IR spectrums (FIGS. 13f, 13g, and 13h) for the control polymer (Blocking-CM), the molecularly non-imprinted monolayer (Blocking-NIMM), and the molecularly imprinted monolayer (Blocking-MIM) show that the N.sub.3 group is completely protected by propagyl alcohol. It is identified that the non-symmetric N.sub.3 stretching peak has disappeared completely at 2090 CM.sup.1. The peaks at 2850 CM.sup.1 (symmetric methyl CH stretching) and 2930 CM.sup.1 (non-symmetric methyl CH stretching) were preserved even after the click reaction.

(99) Analysis of Binding Characteristics of a CRP to a Molecularly Imprinted Monolayer

(100) The SPR technology relates to an optical method to measure a refractive index of a very thin layer of a material absorbed to a metal. Degree of binding between a target molecule in a solution and a molecular receptor fixed onto the chip surface was easily observed and quantified by monitoring reflectivity variation. One of advantages of the SPR technology relates to exhibiting high sensitivity to the target molecule or the receptor molecule without using labeling. The SPR was used to research binding of the CRP onto the modified gold surface.

(101) In the system used in an illustrative embodiment, i.e., SPR LAB (the K-MAC company), reflectivity was measured with a function for an incidence angle of a light beam. Angle variation, to a coated gold sensor surface, was measured with about 1 milli degree of resolution corresponding to 10 g/mm.sup.2 of a protein bound onto the coated surface. Analysis for the SPR system was measured by variation of a resonance angle, which was related to an amount of an analyte bound onto the sensor surface. A difference in a normal-state SPR signal is defined by incidence angle shift () and proportional to a mass of an absorbed protein (0.11 ng/mm.sup.2). The incidence angle shift was calculated by an incidence angle after washing with a buffer for 5 minutes and an incidence angle prior to injection of a protein.

(102) Through an X-ray crystallography research, it was identified that a diameter of the CRP was 12 nm, and a binding region of the SPR chip was 28.9 mm. Accordingly, based on 115 kDa of the molecular weight of the CRP, the whole binding ability of the SPR chip calculated to 49 ng (=1.7 ng/mm.sup.2).

(103) FIG. 14 shows an SPR signal by binding of a C-reactive protein (CRP) to a gold substrate, a control polymer (CM), a molecularly non-imprinted monolayer (NIM), and a molecularly imprinted monolayer (MIM), respectively. In case of the control polymer, no CRP binding was observed. In case of the molecularly imprinted monolayer (MIM) with 10 nM of the CRP concentration, binding of the CRP was almost half in comparison with that in the saturated state. Binding in case of 100 nM of the CRP concentration is almost same with that in the saturated state. In case of the molecularly non-imprinted monolayer (NIM), no CRP binding was observed up to 100 nM of the CRP concentration. CRP binding rapidly increases from 200 nM to 400 nM. The concentration-dependent binding of the CRP to each of the molecularly non-imprinted monolayer and the molecularly imprinted monolayer shows that phosphorylcholine has been successfully introduced onto the gold surface through click chemistry, and provides a binding ability of the CRP to each of the molecularly non-imprinted monolayer and the molecularly imprinted monolayer. If there is no non-specific binding in binding between the molecularly non-imprinted monolayer and the CRP, it is likely that a total CRP binding ability (the number of binding sites) in the molecularly non-imprinted monolayer is greater than that in the molecularly imprinted monolayer.

(104) A binding constant and a maximum binding ability (R.sub.max) between the CRP and each of the molecularly non-imprinted monolayer and the molecularly imprinted monolayer were calculated by using a Langmuir absorption model. The correlation between CRP concentration and angle shift as shown FIG. 14 is expressed with a following reaction formula, i.e., a modified Langmuir equation model:

(105) $\begin{array}{cc}\frac{C}{R}=\frac{C}{R_{\max }}+\frac{1}{R_{\max }.Math.K_A}& \left(1\right)\end{array}$

(106) Here, R is incidence angle shift caused by CRP binding in an equilibriuma state, C is CRP concentration, R.sub.max is angle shift when C is infinite, and K.sub.A is an apparent binding constant.

(107) As a result of plotting according to the above equation (1), a graph in a straight line was obtained, and a proportion of slope to the intercept was K.sub.A (FIG. 15). A K.sub.A value for the molecularly imprinted monolayer and the CRP was determined to be 1.3310.sup.8 M.sup.1. However, binding between the molecularly non-imprinted monolayer and the CRP was not plotted by the Langmuir equation, and C and C/R are in the in inverse proportion to each other. Although an exact cause for the relation of inverse proportion is not provided herein, it is assumed that the relation of inverse proportion can be explained in non-specific binding of the CRP to the molecularly non-imprinted monolayer.

(108) Upon calculation, a maximum coverage of the CRP on the SPR chip was 49 ng. However, when 100 nM of the CRP was injected into a sample tube (buffer volume of 100 nM=12.0 ng/ml and chip surface=51.5 ml), total 618 ng of the CRP was injected onto the chip surface during driving of the sample. Since CRP binding to the NIM started in concentration between 100 nM and 200 nM of the CRP, the surface can be covered completely by the CRP at an initial stage of the CRP binding. Thereafter, protein-protein attraction causes sudden non-specific binding of the CRP to the NIM in addition to PC-specific absorption.

(109) With the proviso that 1 ng0.1, R.sub.max of the MIM is 0.014, which corresponds to 4 ng/mm.sup.2 of the CRP binding and 8% of a calculated maximum coverage of the SPR chip surface.

(110) Effect of Free Phosphocholine

(111) In order to study whether specific binding of a CRP to a molecular receptor occurs, an effect of free phosphocholine (fPC) had been researched. For the molecularly imprinted monolayer, two sets of 300 L of a binding buffer sample containing 200 nM of the CRP and 10 mM of the fPC were prepared. One sample was directly injected without undergoing pre-culture. The other sample was injected after undergoing pre-culture at a room temperature for 15 minutes. FIG. 16 shows results for an effect of fPC in CRP binding to the molecularly imprinted monolayer and the molecularly non-imprinted monolayer. In the injection of the sample, the rapid decrease of the incidence angle was merely a result of an effect of the fPC (data therefor are not illustrated) by deprivation of calcium ions from the surface in the form of a calcium-fPC ionic compound. If there is no pre-culturing of the CRP and the fPC, the incidence angle shift (0.007) was observed in the molecularly imprinted monolayer. An amount of CRP binding corresponded to injection of 8.2 nM of the CRP according to a standard curve (FIG. 14, y=0.0016 ln(x)+0.0368) for a binding ability of the molecularly imprinted monolayer, and was almost half of R.sub.max (0.014) of the molecularly imprinted monolayer. In the case where the sample has been pre-cultured, no binding was observed on the molecularly imprinted monolayer. This means that the fPC formed a complex with the CRP during the pre-culturing of the sample. The fPC-CRP complex cannot be bound to the molecular receptor on the molecularly imprinted monolayer. However, if there is no pre-culturing of fPC-CRP, the CRP can be partially bound to a PC receptor site. This shows that CRP binding to the molecularly imprinted monolayer is very specific to the molecular receptor synthesized through molecularly imprinting and click chemistry. When a CRP-fPC solution that had been pre-cultured for 15 minutes was injected into the molecularly non-imprinted monolayer, the binding was observed with 0.004 of angle shift. The result was partially due to that the CRP binding to the molecularly non-imprinted monolayer according to the relation shown in FIG. 14 is non-specific binding. This is probably because when the binding site of the CRP to phosphorylcholine is blocked by the fPC in the sample, absorption onto the surface of the molecularly non-imprinted monolayer occurs.

(112) In the molecularly non-imprinted monolayer, considering results of the Langmuir isotherm research presented in FIG. 15, which is not applied to the Langmuir isotherm model, it is believed that binding of the CRP to the molecularly non-imprinted monolayer involved not only the phosphorylcholine receptor-adjusted specific binding but also non-specific binding which is not bound via the phosphorylcholine receptor.

(113) Selectivity of the SPR Chip

(114) Binding selectivity of a molecular receptor (molecularly imprinted structure) synthesized in an illustrative embodiment has been researched. A 2% N-serum is injected. After the surface is stabilized, 200 nM of a CRP is injected. In the binding of the CRP to the CM and the molecularly non-imprinted monolayer, the binding of the CRP was not occurred after the 2% N-serum covers the surface. However, in the molecularly imprinted monolayer, incidence angle shift by 200 nM (0.020) and 400 nM (0.032) of the CRP was observed even after the 2% N-serum covers the surface (FIG. 17). This means that the molecular receptor on the molecularly imprinted monolayer surface was not substituted with other serum proteins. In FIG. 14, a concentration-dependent CRP binding curve shows that incidence angle shift is 0.013 in case of 200 nM of the CRP and 0.014 in case of 400 nM of the CRP. The difference in angle shift resulted from the following different measurement conditions: (1) the 2% N-serum improved stability of the binding state of the CRP to the molecular receptor through protein-protein interaction, and reduced removal of the bound CRP from the surface through washing; (2) instrumental deviation caused by injection of a large amount of protein, which corresponds to 420 g of a serum protein in 300 l of the 2% N-serum (based on 70 mg/ml of original serum protein concentration). In order to identify phosphorylcholine-specific binding of the CRP to the SPR chip under the presence of an N-serum protein, 10 mM of the fPC was treated in the sample solution (2% N-serum+200 nM of the CRP) and pre-cultured for 15 minutes. Prior to injection of the sample, 1% BSA was injected on the surface for protection from non-specific binding by a large amount of proteins in the serum. FIG. 18 shows that even if serum proteins are simultaneously treated in the molecular receptor, the fPC completely inhibited the CRP binding to the molecularly imprinted monolayer. This means that the incidence angle shift by the CRP and the N-serum mixture, as observed in the molecularly imprinted monolayer, was caused from the CRP-receptor binding, and not non-specific binding of other proteins. When the 2% serum and 200 nM of the CRP were simultaneously treated, 0.026 of angle shift was observed (FIG. 18). As a result, the CRP in serum proteins was specifically bound to the receptor and thus can be applied to detection of CRPs in a human serum.

INDUSTRIAL APPLICABILITY

(115) As described, the present disclosure relates to a method for preparing a molecularly imprinted structure, providing an improved effect in detection of pentraxin proteins such as a C-reactive protein, a serum amyloid P component, and PTX-3. The present disclosure may be applied to various types of molecularly imprinted polymers, using a complex of a reactive group-pentraxin protein ligand capable of polymerizing a pentraxin protein and a reactive ligand directly on a reactive surface. Also, it is possible to prepare a film and a bead for detection of a pentraxin protein by using the method. Accordingly, it is possible to more easily detect and purify a pentraxin protein by using a complex of a molecularly imprinted reactive group-pentraxin protein ligand having a stronger binding force than that of a ligand of a single pentraxin protein.