CN109593113B

CN109593113B - Stepwise molecular imprinting method and molecularly imprinted material

Info

Publication number: CN109593113B
Application number: CN201811212895.XA
Authority: CN
Inventors: 赵美萍; 赵慕华; 黄山; 李梦圆
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2018-10-18
Filing date: 2018-10-18
Publication date: 2022-05-03
Anticipated expiration: 2038-10-18
Also published as: CN109593113A

Abstract

The invention relates to a step-by-step molecular imprinting method and a molecular imprinting material obtained by the method. The molecular imprinting process is divided into two steps, the functional monomer selected in the first step has two functions, one is certain interaction with template molecules, imprinting sites can be formed through the imprinting process, and the other is that after the polymerization reaction is finished, the functional monomer still contains active groups capable of further reaction, so that conditions are provided for the second step of imprinting. The functional monomer selected in the second step firstly needs to be capable of carrying out covalent reaction with the surface active group after the imprinting is finished once, and secondly can provide new interaction with the surface area of the template molecule. The invention introduces two types of functional monomers with different reaction conditions, thereby greatly improving the fine recognition capability of the template molecules and obviously enhancing the affinity.

Description

Stepwise molecular imprinting method and molecularly imprinted material

Technical Field

The invention belongs to the technical field of material preparation, relates to a step-by-step molecular imprinting method and a molecular imprinting material obtained by the method, and particularly relates to a magnetic nano-protein affinity material with high selectivity and high affinity, which is synthesized by performing molecular imprinting on dopamine and organic boric acid step by step.

Background

Molecular Imprinting Technique (MIT) is a Technique for preparing a polymer having a specific three-dimensional structural cavity that can specifically recognize and bind to a target compound. The Polymers they produce are known as Molecularly Imprinted Polymers (MIPs). MIPs have properties similar to natural antibodies and are considered as "plastic antibodies" with specific recognition capabilities for target molecules.

The molecularly imprinted polymer has important significance for recognizing protein molecules. On the one hand, the recognition effect is similar to the specificity of natural recognition molecules such as 'antigen-antibody'; on the other hand, MIP recognition of protein molecules has unique advantages, such as: compared with the preparation of antibodies, the imprinting material has the advantages of simple operation, low cost and short period, and certain antibodies and receptors which are difficult to obtain in nature can be obtained by a molecular imprinting technology. In addition, the MIP material has high stability, can resist harsh environments such as strong acid, strong alkali, organic reagents and the like, has long service life, can be generally reused and is convenient to store. Thus, MIPs have been widely used in the fields of protein isolation and purification, sensory detection, activity modulation, and drug delivery.

Since most proteins are water soluble, conventional molecular imprinting tends to be carried out in the organic phase, making the proteins susceptible to denaturation under such polymerization conditions. In addition, the molecular weight of the protein is large, and thus the template protein is difficult to remove from the polymeric layer. In response to these problems, various methods for imprinting protein molecules have been developed, such as surface imprinting, nano-imprinting, epitope imprinting, and post-imprinting modification. The surface imprinting is carried out by fixing the template molecules on the surface of the nano-particles, and the binding sites of the surface imprinting are mainly limited on the accessible surface, so that the surface imprinting is beneficial to the elution and recombination of the template molecules; the antigen epitope blot is a blot of a short peptide sequence in an exposed region on the surface of the protein, so that the cost and the time are saved; the post-blot modification is to introduce a new functional group, i.e., a new force, at the blotting site to increase the selectivity of the blotting material after the template molecule is eluted at the end of blotting (Takeda, K.; Kuwahara, A.; Ohmori, K.; Takeuchi, T.; molecular engineered binding sites on conjugated structural genes. journal of the American Chemical society.2009,131, 8833; Sunayama, H.; Ojoya, T.; Takeuchi, T.Fluorogenic protein reactivity in cellulose, fibrous membrane of reaction, coating of specific primer, coating of molecular primer, 14. modification, expression of the imprinted template molecule, 7. modification, 7. 7.A. In these methods, it is still difficult to avoid the conformational changes of the protein molecules during the imprinting reaction, so that the final imprinted material still has a large gap in affinity and selectivity compared to the biorecognition molecules.

Disclosure of Invention

In view of the above problems, the present invention provides a stepwise molecular imprinting method, and a molecularly imprinted material obtained by the method.

The stepwise molecular imprinting method comprises the following steps:

1. uniformly mixing a buffer solution containing nanoparticles, a target protein molecule and an aqueous solution of a first imprinting functional monomer, carrying out self-polymerization on the surface of the nanoparticles by the first imprinting functional monomer at room temperature, and carrying out a first-step imprinting reaction with the target protein molecule to obtain first molecularly imprinted nanoparticles;

2. separating the first molecular imprinting nano-particles, washing the first molecular imprinting nano-particles by using an original buffer solution, then adding a buffer solution containing a second imprinting functional monomer, standing the mixture at room temperature, and carrying out a second-step imprinting reaction on the second imprinting functional monomer and the first imprinting functional monomer and the target protein molecule;

3. and (3) removing target protein molecules by solvent washing to obtain the molecular imprinting material.

The nanoparticles are SiO₂The magnetic nano-particles coated by the layer are ultrasonically dispersed in pure water, the final concentration is 0.1-0.2 mg/mL, and the ultrasonic time is more than 15 minutes;

the first imprinting functional monomer is dopamine, and the polymerization concentration is 0.1-0.2 mg/mL; the target protein molecule is DNase I, the concentration is 5-15 nM, the molecular imprinting time of the first step is 30-60 minutes, and the pH is 8.0-10.0; the second imprinting functional monomer is 3-aminophenylboronic acid with the concentration of 1-2 mM; the molecular imprinting time of the second step is not less than 10 minutes, and the pH value is 7.0-8.0. Or the target protein molecule is APE1, the concentration is 1-3 nM, the first-step imprinting time is 30-60 minutes, and the pH is 8.0-9.0; the second imprinting functional monomer is 2-naphthalene boric acid with the concentration of 1-2 mM, the second imprinting time is not less than 10 minutes, and the pH value is 8.0-10.0.

The method of the invention uses absolute ethyl alcohol to wash so as to remove the target protein molecules.

The invention separates out the molecular engram material by an external magnetic field, dries after washing, and preserves at 4 ℃ for further use. .

The molecular imprinting material comprises a nano particle, a first imprinting functional monomer polymerization layer and a second imprinting functional monomer modification layer, wherein the first imprinting functional monomer polymerization layer coats the nano particle; the first imprinting functional monomer can react with a target protein molecule to form a first molecular imprinting site and carry out covalent reaction with the second imprinting functional monomer; the second imprinting functional monomer can react with the target protein molecule to form a second molecular imprinting site.

The nanoparticles are inorganic or organic solid nanoparticles comprising SiO₂Various inorganic or organic solid particulate materials such as nanoparticles, gold nanoparticles, magnetic nanoparticles, and the like; magnetic nanoparticles are preferred in order to facilitate separation from the solution after the reaction;

the first print functional monomer comprises dopamine, or a derivative thereof, such as: catechol, dopa, noradrenaline, alpha-methyldopamine, gallic acid, tannic acid and other catechols. The polymeric layer is derived from dopamine self-polymerization, and also includes dopamine derivatives self-polymerization.

The second imprinting functional monomer comprises organic boric acid connected with different substituents, or an organic compound capable of reacting with catechol on the surface of a dopamine polymerization layer or a derivative self-polymerization layer thereof. The organic boric acid substituent is selected by considering the combination mode and the ability with the template protein, such as the template protein with surface charge under the imprinting condition, and electrostatic interaction can be selected; for template proteins rich in tyrosine, tryptophan and phenylalanine residues, pi-pi interactions and the like may be chosen. The boric acid modification layer can be further developed into a modification layer based on other groups capable of reacting with catechol on the surface of polydopamine, such as amino or sulfhydryl compounds.

The nanoparticles are preferably SiO comprising a magnetic core₂A nanoparticle; the first imprinting functional monomer is dopamine; the second imprinting functional monomer is 3-aminophenylboronic acid, and the target protein molecule is DNase I; or the first imprinting functional monomer is dopamine; the second imprinting functional monomer is 2-naphthalene boronic acid, and the target protein molecule is APE 1.

The molecularly imprinted material comprises a magnetic core, a silicon dioxide layer, a polydopamine layer and an organic boric acid modification layer from inside to outside in sequence; the diameter of the magnetic core is 5-10nm, the thickness of the silicon dioxide shell layer is 25-35nm, and the thicknesses of the polydopamine layer and the organic boric acid modification layer are 3-5 nm.

The molecular imprinting process is divided into two steps, the functional monomer selected in the first step has two functions, namely, the functional monomer has certain interaction with a template molecule (target protein molecule) and can form an imprinting site through the imprinting process, and the functional monomer still contains an active group capable of further reacting after the polymerization reaction is finished, so that conditions are provided for the imprinting in the second step. The functional monomer selected in the second step firstly needs to be capable of carrying out covalent reaction with the surface active group after the imprinting is finished once, and secondly can provide new interaction with the surface area of the template molecule. The step-by-step imprinting strategy can introduce two types of functional monomers with different reaction conditions, so that the fine recognition capability of the template molecules is greatly improved, and the affinity is obviously enhanced.

Dopamine is preferably used as a functional monomer for the first imprinting reaction. Dopamine has the characteristic of being capable of being polymerized into a film on the surface of almost all nano materials, and the formed Polydopamine (pDA) material has good biocompatibility (Lee, H.; Dellator, S.M.; Miller, W.M.; Messersmith, P.B. Mussel-induced surface chemistry for multifunctional coatings, science 2007,318, 426-430). Dopamine autopolymerization has been applied to surface blots of various protein molecules, such as hydrophobin type II (HBF II), Human Serum Albumin (HSA), Bovine Serum Albumin (BSA) (Riveros, D.; Cordova, G.; Michips, C.; Veracht, H.; Derdelinkx, H. multidopamine engineered semiconducting proteins as a method to purify and detect class II hydroxides from carbon sources of microorganisms, Talan 2016,160,761.Yin, Y.; Yan L.; Zhang, Z.; J.semiconducting immobilized polymeric nanoparticles on carbon nanoparticles for protein surface treatment, J.semiconducting nanoparticles, P.S. P.A. P.B.; nanoparticles, P.S. P.A. P.A.B.S.; moisture, P.S. P.A.S.; moisture, P.S. P.A.S. P.S. P.A.A.A.A. P.A. P.S. P.A. 1, P.S. P.A.S. P.A. P.S. P.A.P.S. P.S. P.P.S. P.S. P.P.S. P.A.P.P.P.P.P.P.S. P.S. P.P.P.P.P.P.P.P.S. P.S. P.P.S. P.S. P.P.S. P.P.P.P.P.P.S. P.P.S. P.P.P.P.P.P.P.P.P.S. P.P.P.P.P.P.S. P.P.P.P.P.P.P.P.S. P.P.P.P.P.P.P.S. P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.. However, due to the fact that the density of functional groups on the surface of the polydopamine is large and the types of the functional groups are single, the specificity and the affinity of the existing polydopamine molecularly imprinted material (pDA-MIP) to the target protein are not high enough.

Boric acid can specifically react reversibly covalently with vicinal diol structures, and this boron affinity (boron affinity) has been used in recent years in the design and development of molecularly imprinted polymers (Wang, H.; Xu, Q.; Shang, L.; Wang, J.; Rong, F.; Gu, Z.; Zhao, Y. boron affinity molecular implants for multiple layer-free biological assays.chemical Communication 2016,52,3296; Awino, J.; Gunasekara, R.Zhao, Y. Selective interaction of bonded-aldhexoses in water by bonded biological acid-functional, molecular immobilized-bonded, chemical interaction of moisture, III., Z.; tissue engineering, III., Z.; tissue, III., Z., S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.A.A.A.S.A.A.S.S.S.A.A.A.A. A. A.S.S.A. A. A.S.S.S.A. A. A.A.A. A. A.A. A. A.A. A. A.A.A. A.S.A. A. A.A. A. A.A.A. B.A. A. A.S.S.A. A. B.S.A.A. A. B.A. A. B.A. A. B.A. A. B.A. A. B.A. A. B.A. B.S. B.A. A. B.A. A. B.A. A. B.A. B.A.A.A. B.A.. However, the applications all depend on that the imprinted template molecule contains a vicinal diol structure, and the application range is narrow.

The invention preferentially selects organic boric acid molecules as functional monomers for the second imprinting. After the first step of dopamine self-polymerization reaction is completed, the organic boric acid molecules are continuously added by utilizing the rich vicinal diol structure on the surface of the dopamine self-polymerization reaction under the condition of not eluting the template molecules. The organic functional group in the organic boric acid molecule can provide interaction with the exposed region of the template protein, and the boric acid part can react with the vicinal diol structure on the surface of the polydopamine, so that secondary imprinting on the template molecule is realized. Through screening organic boric acid molecules with different substituent structures, the difference of the optimal secondary imprinting functional monomers for different template molecules is found, and the secondary imprinting reaction participates in further identification of the template molecules. Further performance characterization results show that the surface imprinted nano material obtained by the step imprinting method has significantly improved specificity and affinity to the target protein, and has wide application prospects in the field of affinity receptor materials for artificially synthesizing protein molecules.

The invention respectively prepares the molecular imprinting magnetic nanoparticles of deoxyribonuclease (DNase I) and abasic endonuclease 1(APE1) by utilizing the new method, is successfully used for directly separating and purifying target protein from serum, and well maintains the activity of the protein.

In the method, dopamine is added for polymerization in the presence of template protein, the residual dopamine solution is removed after the reaction is finished, and a proper organic boric acid functional monomer is continuously added for a second imprinting reaction under the condition of not eluting template molecules. The method integrates the recognition capability of two functional monomers, finely constructs binding sites in situ of template molecules, has better material selectivity and stronger affinity to the template molecules compared with the material obtained by the traditional one-time imprinting method. The method is simple and convenient to operate, good in controllability and free of initiators, cross-linking agents and the like. The obtained protein affinity adsorption material is convenient to use, can be directly used for separating and purifying target protein in complex biological samples such as serum, cell lysate and the like, can desorb and recover the target protein with high activity, and has wide application prospect.

Meanwhile, the method has better universality and can be popularized and applied to more target proteins.

Drawings

FIG. 1 is a schematic diagram of the principle of the stepwise molecular imprinting method of the present invention.

FIG. 2 is a structural formula of six kinds of organic boric acids containing different substituents. Wherein B1 (phenylboronic acid), B2 (3-carboxyphenylboronic acid), B3 (3-aminophenylboronic acid), B4 (3-hydroxyphenylboronic acid), B5 (2-naphthylboronic acid) and B6 (dodecylboronic acid) are provided.

FIG. 3 is a transmission electron micrograph (a) of a magnetic nanoimprint material (Fe)₃O₄@SiO₂(ii) a (b) Dopamine one-time blotting material (M1) and (c) dopamine-organoboronic acid stepwise blotting DNase I (M1-M2-B3).

FIG. 4 is a graph of imprinting of DNase I with dopamine as a function of polymerization time at different pH conditions.

FIG. 5 is a graph comparing the selectivity factors of imprinted polymers (MIPs) and non-imprinted polymers (NIPs) obtained by selecting different organoboronic acids during the second imprinting reaction. Template protein: DNase I; reference protein: RNase A.

FIG. 6 shows the adsorption capacity of nanoparticles on template protein (DNase I) and reference protein (RNase A) obtained by a second blotting of DNase I with aminobenzeneboronic acid (B3) at different pH conditions.

FIG. 7 shows the adsorption capacity of the blots synthesized by the "one-pot" method for template protein (DNase I) and reference protein (RNase A) against different materials with or without template molecules in the second blotting step.

FIG. 8 is a Scatchard fit of M1-M2-B3 material adsorbed template protein DNase I.

FIG. 9 shows the efficiency of the desorption of DNase I bound to M1-M2-B3 using different desorbing solutions.

FIG. 10 shows the recovery of adsorption of DNase I by several materials followed by desorption with Tris buffer, 10 DNase I working buffer and 1mM sodium fluoride in sequence.

Figure 11 blotting factors for dopamine blotting APE1 under different pH and polymerization time conditions.

FIG. 12 selectivity factors for magnetic nano-imprinted particles (MIPs) and non-imprinted Nanoparticles (NIPs) when performing a second imprinting step of APE1 with different organic boronic acids. Template protein: APE 1; reference protein: DNase I.

Figure 13 APE1 was used to imprint the adsorption capacity of magnetic nanoparticles step by step at different pH conditions.

FIG. 14 shows the recovery rate of APE1 of APE1 step-imprinted magnetic nanomaterials under different desorption solutions.

FIG. 15APE1 step-blotting magnetic nanomaterials and control materials were used for separation and enrichment of APE1 in serum. The curves in the figure represent fluorescence plots of different materials desorbing recovered APE1 reacted with fluorescent probes.

Detailed Description

The invention is further described below by way of examples.

The first embodiment is as follows: magnetic nano affinity material for synthesizing DNase I by step-by-step imprinting method and performance characterization thereof

1. The synthesis step of the stepwise blotting method comprises:

as shown in fig. 1, the stepwise molecular imprinting method of this embodiment comprises:

(a) first-step blotting: the reaction was carried out in a 200. mu.L PCR tube containing 0.2mg/mL of Fe₃O₄@SiO₂(SiO₂Layer coated Fe₃O₄Magnetic nanoparticles, DNase I with the final concentration of 10nM (DNase I concentration can be within the range of 5-15 nM) and dopamine with the final concentration of 0.1mg/mL (dopamine concentration can be within the range of 0.1-0.2 mg/mL) are added into 10mM Tris buffer solution with the final concentration of 0.1-0.5 mg/mL and the ultrasonic time is more than 15 minutes) which is ultrasonically dispersed in pure water, and mixed blotting is carried out for 30-60 minutes at room temperature. After the blotting was completed, the dopamine on the surface which had not been polymerized was washed out with 10mM Tris buffer. The resulting material was designated M1.

When non-imprinted nanoparticles (N1) were synthesized as a control, the template molecule DNase I was not added and the other steps were the same.

(b) Second step blotting: after the magnetic separation of the primary blotting material M1 and the primary non-blotting material N1, the samples were blotted at room temperature for 15min without drying by directly adding 180. mu.L of 10mM Tris buffer containing 1mM 3-aminophenylboronic acid (B3) (the concentration of B3 may be in the range of 1-2 mM). The material was washed three times with absolute ethanol and dried.

Several control materials were prepared according to the same reaction conditions. The six kinds of boric acids containing different substituents are respectively numbered as B1 (phenylboronic acid), B2 (3-carboxyphenylboronic acid), B3 (3-aminophenylboronic acid), B4 (3-hydroxyphenylboronic acid), B5 (2-naphthoic acid) and B6 (dodecylboronic acid). The imprinting material obtained by the participation of template molecules in the two-step reaction is named as M1-M2-Bx (x is 1-6), wherein M1 represents a material obtained by primary imprinting of dopamine, and M2 represents a material obtained by secondary imprinting of boric acid derivatives and a polydopamine layer under the existence of a template through boron affinity; bx represents the type of boric acid derivative (x is 1-6, shown in figure 2); the first step of reaction is carried out by taking a template molecule into consideration, then the template molecule is eluted, and the second step of reaction is carried out by using a material named as M1-N2-Bx (x is 1-6); template molecules are not added in the first step of reaction, and a material added with the template in the second step of reaction is named as N1-M2-Bx (x is 1-6); the material without template molecules added in the two-step reaction is named as N1-N2-Bx (x is 1-6).

2. Morphology and surface charge of magnetic nanoimprint materials:

from FIG. 3, Fe₃O₄The magnetic core has a diameter of about 5-10nm and SiO₂The layer thickness was about 25-35nm and the thickness of polydopamine was about 3-5nm, indicating that one blot of dopamine self-polymerization successfully occurred. Table 1 shows the surface Zeta potentials of the magnetic nanoparticles obtained by the second imprinting step using B1-B6. As can be seen from the data in Table 1, the Zeta potential changes correspondingly after the polydopamine surface is combined with the boric acid group with different substituents. For B1, B2, B4, and B5, the negative values were significantly larger; for the material modified by B6, the Zeta potential becomes negative to a relatively small extent. This is consistent with the formation of negatively charged boron tetrahedra upon attachment of the boronic acid groups to the polydopamine surface. In contrast, the Zeta potential of M1-M2-B3 was less changed than that of M1 in the material after B3 modification because the amino group was positively charged under the measurement conditions (pH 7.3).

TABLE 1 surface Zeta potential of step-by-step imprinted magnetic nanoparticles

3. And (3) selective test of the magnetic nano imprinting material for adsorbing protein:

and dispersing 40 mu g of the nanoparticle material obtained under different synthesis conditions in 100 mu l of a solution containing 20nM template protein (DNase I) and 20nM reference protein (RNase A) respectively, mixing for 15min, measuring the activity of the residual DNase I and RNase A in the supernatant, and calculating the adsorption capacity Q, the imprinting factor IF and the selectivity factor alpha of the DNase I and the RNase A adsorbed by the material. Wherein the print factor is calculated according to the following formula:

the selectivity factor is calculated according to the following formula:

Q_MIpand Q_NIpThe adsorption capacity (nmol/g) of the imprinted material and the non-imprinted material for DNase I; q_{DNase I}And Q_{RNase A}The adsorption capacities (nmol/g) of the blots for DNase I and RNase A, respectively.

The activities of DNase I and RNase a were measured using the following fluorescent probes, respectively:

DNase I probe:

RNase A probe:

wherein, the underlined italics shows a complementary sequence, the asterisk indicates that the phosphate framework at the position is subjected to sulfo-modification, TAMRA and FAM are fluorescent groups of tetramethylrhodamine and carboxyfluorescein respectively, and BHQ2 and BHQ1 are corresponding quenching groups respectively. Unless otherwise specified, all of the fluorescent probes mentioned hereinafter are the above sequences.

The pH condition and time of dopamine self-polymerization determine the density and thickness of a polydopamine layer, and fig. 4 shows that for template molecule DNase I, the optimal Imprinting Factor (IF) can be obtained by performing first-step imprinting reaction by using dopamine as a functional monomer, wherein the pH is preferably 8.0-10.0, and the imprinting is performed for 45min under the condition that the pH is 9.0, namely the constructed primary imprinting site is optimal.

In order to obtain the best second imprinting effect, the present example optimizes the type of organic boronic acid, the pH of the reaction and the reaction time in the boron affinity reaction, and as can be seen from fig. 5, the imprinting effect is best when 3-aminophenylboronic acid (B3) is used; the imprinting time is not less than 10 min; as can be seen from FIG. 6, the blotting pH is preferably 7.0-8.0. Among them, the optimum selectivity factor can be obtained under the condition that the pH value is 7.3.

To confirm the necessity of a two-step blotting procedure and to investigate the determinants of the resulting material selectivity, this example synthesized blotting material (OPM1-B3) directly added dopamine and 3-aminophenylboronic acid (B3) "one-pot" as control materials. The specific synthetic steps are as follows: fe dispersed in 10mM Tris (pH 9.0) buffer to a final concentration of 0.2mg/mL was added to each 200. mu.L PCR tube₃O₄@SiO₂DNase I at a final concentration of 10nM, dopamine at 0.1mg/mL and 3-aminophenylboronic acid at 1mM (B3), the reaction volume was 200. mu.L, and blots were mixed at room temperature for 45 min. After the blotting was completed, the material was magnetically separated and washed three times with anhydrous ethanol and then dried.

In this example, on the basis of the above "one-pot" reaction, after the above dopamine and 3-aminobenzeneboronic acid (B3) were mixed and blotted for 45min, 2. mu.L of 1. mu.M DNase I protein and 2. mu.L of 100mM 3-aminobenzeneboronic acid (B3) were added to the system, and blotting was continued at room temperature for 15 min. After completion of blotting, the material was magnetically separated and washed three times with anhydrous ethanol and dried to obtain a control material (OPM 1-M2-B3).

40. mu.g of each of the above-synthesized materials M1-M2-B3, M1-N2-B3, M1, N1-M2-B3, N1-N2-B3 and N1 and the "one pot" synthesized control materials OPM1-B3 and OPM1-M2-B3 were dispersed in 100. mu.L of Tris buffer containing 20nM template protein (DNase I) or 20nM reference protein (RNase A), allowed to stand for 15min, the activities of the remaining DNase I and RNase A in the supernatant were measured, and the adsorption capacity Q of each material for DNase I and RNase A was calculated.

As can be seen from FIG. 7, after the N1 material is further reacted, the binding capacity of the material to both proteins is slightly increased, which indicates that the aminobenzene boron structure randomly connected to the surface of the polydopamine has weak interaction with protein molecules, but the maximum adsorption capacity of the N1 series materials (N1-M2-B3, N1-N2-B3 and N1) to DNase I is only about 60% of that of M1 series (M1-M2-B3, M1-N2-B3 and M1) regardless of whether template molecules are added in the second step of reaction. The imprinting reaction in the first step is proved to form a large number of sites with strong DNase I binding effect on the polydopamine layer. In the M1 series material, if the second step reaction does not have the template molecule, the combination of DNase I is only slightly increased, and the combination of RNase A is basically unchanged, similar to the N1 series material, further explaining that the boron affinity reaction directly occurs on the polydopamine surface without the template molecule, and no new combination site is obviously increased, and the reaction is mainly on the original combination site. The above experimental results prove that the first imprinting process is the most critical step and is the basis for successful imprinting, and the second imprinting process is a modification process which further occurs on the basis of binding sites formed in the first step, and a new binding effect with a template molecule is introduced through a substituent of the organoboron compound. In the two-step imprinting process, the presence of the template molecule is a crucial factor.

As can be seen from FIG. 7, the OPM1-B3 material obtained by adding dopamine and organic boric acid into the imprinting system in a one-pot manner and the OPM1-M2-B3 material supplemented with template molecules and organic boric acid after the one-pot manner have a reduced adsorption capacity on reference protein (RNase A) compared with the one-pot imprinting material M1, but have relatively low binding capacity on target protein (DNase I) and no obvious improvement on selectivity. This is probably because the organoboronic acid directly undergoes a boron affinity reaction with the dopamine monomer, the generated dopamine borate is difficult to undergo a self-polymerization reaction, and in addition, the organoboronic acid may also affect the self-assembly of dopamine and DNase I, reducing the imprinting efficiency. The experimental results prove that the imprinting reaction is carried out by adding dopamine and boric acid in two steps, and the dopamine and boric acid are respectively used for completing the construction and fine processing of imprinting sites, so that the material is ensured to have better selectivity.

4. Affinity of magnetic nano-imprinting material to target protein DNase I:

the adsorption capacities of materials such as M1-M2-B3, N1-M2-B3, M1-N2-B3, M1 and N1 in DNase I solutions with different concentrations were respectively measured. The data obtained were fitted by the Scatchard method (FIG. 9).

Wherein Q and Q_maxRespectively the adsorption amount of the unit mass of the blotting material to the DNase I protein and the maximum protein adsorption amount (mu mol/g) of the unit mass of the blotting material, c is the concentration (nmol/L) of the DNase I protein in the supernatant when the adsorption reaches the equilibrium, and K is_dIs the dissociation equilibrium constant (μmol/L) of the material and DNase I protein, K_aIs the binding constant (L/mol) of the material to the DNase I protein.

K for several materials compared from Table 2_aAnd Q_maxIt can be seen that the affinity of the step imprinting material M1-M2-B3 is improved by 2 orders of magnitude compared with that of the one-time imprinting material M1; compared with the material which is not imprinted in the first step and is only imprinted in the second step, the affinity is improved by 4 times, which indicates that the two-step imprinting method increases new acting force at the original imprinting site and improves the binding capacity of the material to the template protein.

TABLE 2 apparent affinity constants and maximum adsorption capacities for DNase I for different materials

5. Desorption performance of DNase I magnetic nano imprinting affinity material (M1-M2-B3)

(a) Comparison of desorption efficiency when different desorption solutions were used alone: mu.L of 1 XDNase I buffer (DNase I buffer composition: 2.5mM MgCl. sub.2.5 mM) was added to each 200. mu.L PCR tube containing 40. mu.g of step-blot magnetic nanoparticles M1-M2-B3 having adsorbed DNase I₂，10mM Tris-HCl，0.5mM CaCl₂pH 7.6@25 ℃ C.), 10 XDnase I buffer, 1mM NaF, 1mM KHF, 1mM KBF)₄10 XDase I buffer containing 1mM NaF, 10 XDase I buffer containing 1mM KHF, and 10 XDase I buffer containing 1mM KBF₄Desorbing the buffer solution for 10min at room temperature, performing magnetic separation, and directly measuring the DNase I activity of the supernatant.

The data in FIG. 10 show that a 10-fold increase in DNase I buffer concentration increases the desorption efficiency of the template protein, but the recovery rate is still less than 50%. The recovery rate of 60% can be achieved by directly desorbing with 1mM NaF, and the recovery rate can be improved to 96% by using 10 XDnase I buffer containing 1mM NaF. These data indicate that there are at least two different types of forces between the template molecules and the imprinted sites in the MIP layer.

(b) Comparison of desorption intensities for different desorption solutions in continuous use: in a 200. mu.L PCR tube containing 40. mu.g of the above DNase I-adsorbed step-wise imprinted magnetic nanoparticle M1-M2-B3, desorption was carried out successively with 100. mu.L of 10mM Tris buffer, 10 XDnase I buffer and 1mM NaF, and after mixing at room temperature for 10min, magnetic separation was carried out to measure the DNase I activity of the supernatant.

Several other control materials were tested under the same conditions and the results are shown in figure 11. According to the previous studies, the direct elution with 10mM Tris buffer is a weak adsorption on the material surface; the interaction between polydopamine and DNase I, namely the acting force generated by the first-step blotting, can be destroyed by using 10 times DNase I buffer solution; further elution with fluoride ions disrupts the binding of the organoboron compound to the protein, i.e., the force generated by the secondary blot. As can be seen from FIG. 11, in the non-imprinted material N1 of the first step, proteins were mainly bound to the surface of the material by very weak action, and were directly eluted with only 10mM Tris buffer, and the force was not changed much after the surface was bound to B3. And for the material M1 with template molecules participating in imprinting in the first step, the proportion of weak adsorption is obviously reduced. The second reaction, with or without template molecules, provides substantially stable binding sites for polydopamine. From the comparison of the data of M1-N2-B3 and M1, it can be seen that the weak binding effect of the second reaction, which can be destroyed directly with 10mM Tris buffer, is not changed much if no template molecule exists, i.e. the surface-linked aminobenzeneboronic acid does not provide significantly more strong binding force, while the weak binding effect of the material M1-M2-B3 obtained by the step-by-step blotting method is obviously reduced compared with that of M1, and has been converted into strong binding effect. Therefore, compared with the material obtained by one-step imprinting, the two-step imprinting method not only improves the selectivity of the material, but also enhances the affinity of the material to the target protein.

6. DNase I magnetic nano-imprinting affinity material (M1-M2-B3) is used for separating and enriching DNase I in serum

mu.L of human serum sample (from Beijing university Hospital) was diluted into 50. mu.L of DNase I buffer containing 0.2mg/mL of the material to be tested, subjected to ultrasonic dispersion, and then allowed to stand for 10 min. The concentration of DNase I in the supernatant was determined by magnetic separation. The material was washed with 50. mu.L of Tris buffer. After discarding the supernatant, 10 XDNase I working buffer containing 1mM NaF was added, and the mixture was allowed to stand for 10min by ultrasonic dispersion, and the DNase I concentration in the supernatant was determined by magnetic separation. Two control groups were simultaneously prepared, and DNase I activity in the supernatant was determined by performing adsorption-desorption experiments on DNase I in 10-fold diluted serum samples using 40. mu. g N1-N2-B3 and M1 materials, respectively.

Table 3 adsorption-desorption data of three different nanomaterials for DNase I in serum diluted samples

The results in Table 3 show that the M1-M2-B3 material can effectively bind DNase I protein in serum diluted samples, and can further desorb the protein for recovery. Whereas the control materials N1-N2-B3 and M1 only partially bound DNase I in the serum diluted samples.

Example two: magnetic nano affinity material synthesized by step imprinting method and APE1 and performance characterization thereof

1. APE1 magnetic nano affinity material prepared by stepwise imprinting method

(a) First-step blotting: the reaction was carried out in a 200. mu.L PCR tube containing 0.2mg/mL of Fe₃O₄@SiO₂(SiO₂Layer coated Fe₃O₄The final concentration of the magnetic nanoparticles dispersed in pure water by ultrasonic is 0.1-0.5 mg/mL, in this example, 0.2mg/mL and the ultrasonic time is more than 15 minutes) of 10mM Tris buffer solution is added with APE1 (the concentration of APE1 can be both in the range of 1-3 nM) and 0.1mg/mL dopamine (the concentration of dopamine can be both in the range of 0.1-0.2 mg/mL) with the final concentration of 1.5nM, and mixed blotting is carried out for 45min at room temperature, the pH is 8.0-9.0, and the optimal pH is 9.0. Is printed completelyAfter that, the dopamine whose surface has not been polymerized is washed away with 10mM Tris buffer. The resulting material was designated M1. Non-imprinted control material N1 was synthesized under the same conditions without the addition of template molecules.

(b) Second step blotting: after the magnetic separation of the primary blotting material M1 and the primary non-blotting material N1, drying was not necessary, and 180. mu.L of 10mM Tris buffer containing 1mM 2-naphthalene boronic acid (B5) (the concentration of B5 may be in the range of 1 to 2 mM) was directly added thereto, and blotting was carried out at room temperature for 15min (preferably less than 10 min) at a pH of 8.0 to 10.0, more preferably at a pH of 9.0. The material was washed three times with absolute ethanol and dried. Other control materials were prepared according to the same reaction conditions. The nomenclature is the same as in embodiment one.

Mu.g of the synthesized APE1 magnetic nanomaterial was dispersed in 100. mu.l of a solution containing 3nM template protein (APE1) or reference protein (DNase I) and mixed for 15min, and the remaining APE1 and DNase I activities in the supernatant were measured. The adsorption capacity Q, the imprinting factor IF and the selectivity factor α of the material for adsorption of APE1 and DNase I were calculated.

The DNA probe sequence for detecting the APE1 activity is as follows:

APE1 probe: 5' -T C TC*(dT-ROX)AGAGXCGT(dTBHQ2)C*A*C*T*G*

T*AGTTTATA*C*A*G*T*GAATCTCTCTAG*T*C*T-3’

Wherein underlined italics indicates the complementary sequence, asterisks indicate the thio-modified base, and ROX and BHQ2 are 5(6) -carboxy-X-rhodamine hydrochloride and its corresponding quenching group, respectively.

The results in FIG. 12 show that the optimum pH of APE1 first-step blotting carried out by dopamine polymerization is 9.0, and better blotting effect can be achieved after 60min of blotting. The comparative experiment of fig. 13 and fig. 14 shows that B5 (2-naphthalene boronic acid) is added to perform the second imprinting under the condition of pH 9.0, and the imprinting time is 15min, so that the nonspecific adsorption of the material to DNase I can be effectively reduced, and higher selectivity can be obtained. The results further show the advantages of the stepwise imprinting method, and the selectivity of the imprinting sites can be effectively regulated and controlled by changing the substituent of the organic boronic acid.

2. Desorption performance of APE1 magnetic nano imprinting affinity material:

in a containerTo a 200. mu.L PCR tube containing 40. mu.g of the stepwise-imprinted magnetic nanoparticles having APE1 adsorbed thereon, 100. mu.L of 10 XBuffer 1.1 buffer (1 XBuffer 1.1 buffer composed of 10mM Bis Tris Propane-HCl and 10mM MgCl) was added₂100. mu.g/ml BSA, pH 7.0@25 ℃), 5mM NaF solution and 10 XBuffer 1.1 buffer containing 5mM NaF, were mixed and left at room temperature for 10min, and the activity of APE1 enzyme in the supernatant was directly measured after magnetic separation. And calculating the recovery rate of APE1 protein by different desorption solutions.

FIG. 15 shows that about 70% of APE1 can be recovered by using 10 XBuffer 1.1 buffer solution, while the recovery rate of the desorption solution containing only NaF solution is only about 20%, which indicates that the template molecules in the desorbed MIP layer are mainly assisted by metal ions and salts, and at the same time, about 20% of the proteins are tightly bound with the organoboron compound and are not easily released. The forces generated by both blotting reactions were overcome simultaneously with 10 XBuffer 1.1 buffer containing 5mM NaF, with 95% recovery of APE1 protein.

3. Enrichment effect of APE1 magnetic nano imprinting affinity material on APE1 in serum:

400 μ g of APE1 magnetic nano-blot material and 200 μ l of serum were incubated at 37 ℃ for 30min, magnetic separated for 15min, and the supernatant was discarded. The activity of APE1 was determined with a DNA probe by desorbing for 15min with 50. mu.l of a 10 XBuffer 1.1 mixture containing 5mM NaF, taking 2. mu.l of the supernatant after magnetic separation and adding 50. mu.l of Buffer 4(Buffer 4 Buffer composition 50mM potassium acetate,20mM Tris-acetate,10mM magnesium acetate,1mM DTT, pH 7.9@25 ℃) of Triton X-100 to a final concentration of 0.06%. At the same time, 3 control groups were set, one time blotting material MIP, one time non-blotting polymer NIP, M1-N2-B5 (no template molecule was present in the second reaction step), and the above experiment was repeated under the same conditions. FIG. 15 shows the result of APE1 separation and enrichment of APE1 step-imprinted magnetic nanomaterials and control materials in serum. The curves in the figure represent fluorescence plots of different materials desorbing recovered APE1 reacted with fluorescent probes. Compared with the data of the untreated serum sample shown in the figure, only the M1-M2-B5 material in the four materials realizes effective enrichment of APE1 in serum, and the actual enrichment efficiency reaches 87% +/-4% calculated according to 4 times of theoretical enrichment factor.

It will be understood by those skilled in the art that the above embodiments should not be construed as limiting the present invention and that various modifications and changes may be made within the spirit and scope of the present invention. For example, the nanoparticles of the embodiments can be various inorganic or organic solid nanoparticles; dopamine in the examples may be replaced with derivatives of dopamine; the organic boronic acid may be replaced by an organic compound that reacts with catechol on the surface of the dopamine polymer layer or its derivative homopolymer. The scope of the invention should be determined from the following claims.

Claims

1. A stepwise molecular imprinting method comprises the following steps:

1) uniformly mixing a buffer solution containing nanoparticles, a target protein molecule and an aqueous solution of a first imprinting functional monomer, carrying out self-polymerization on the surface of the nanoparticles at room temperature by the first imprinting functional monomer, and carrying out a first-step imprinting reaction with the target protein molecule to obtain first molecularly imprinted nanoparticles, wherein the first imprinting functional monomer is dopamine, and the polymerization concentration is 0.1-0.2 mg/mL;

2) separating the first molecular imprinting nano-particles, washing the first molecular imprinting nano-particles by using an original buffer solution, adding a buffer solution containing a second imprinting functional monomer, standing the mixture at room temperature, and carrying out covalent reaction on the second imprinting functional monomer and the first imprinting functional monomer and carrying out a second-step imprinting reaction on the second imprinting functional monomer and a target protein molecule, wherein the second imprinting functional monomer is 3-aminobenzene boric acid or 2-naphthalene boric acid, and the concentration of the second imprinting functional monomer is 1-2 mM;

3) and (3) removing target protein molecules by solvent washing to obtain the molecular imprinting material.

2. The method of claim 1, wherein the nanoparticles are SiO₂The magnetic nano-particles coated by the layer are ultrasonically dispersed in pure water, the final concentration is 0.1-0.5 mg/mL, and the ultrasonic time is more than 15 minutes;

the target protein molecule is DNase I, the concentration is 5-15 nM, the molecular imprinting time of the first step is 30-60 minutes, and the pH is 8.0-10.0; the molecular imprinting time of the second step is not less than 10 minutes, and the pH value is 7.0-8.0.

3. The method of claim 1, wherein the nanoparticles are SiO₂The magnetic nano-particles coated by the layer are ultrasonically dispersed in pure water, the final concentration is 0.1-0.5 mg/mL, and the ultrasonic time is more than 15 minutes;

the target protein molecule is APE1, the concentration is 1.0-3.0 nM, the first-step imprinting time is 30-60 minutes, and the pH is 8.0-9.0; the second imprinting time is not less than 10 minutes, and the pH value is 8.0-10.0.

4. The method according to claim 1, wherein the target protein molecule is removed by washing with absolute ethanol.

5. The method of claim 2 or 3, wherein the molecularly imprinted material is separated by an applied magnetic field.