Hybrid gold nanoparticles, their preparation method and applications

Abstract

This invention provides hybrid gold nanoparticles, their preparation method, and applications, relating to the field of new biomedical materials technology. The hybrid gold nanoparticles comprise a star-shaped polymer template and gold nanoparticles loaded onto the star-shaped polymer template (PAMAM-PLG-PEG). This invention uses the star-shaped polymer template (PAMAM-PLG-PEG) to complex with the precursor chloroauric acid, and under the action of a reducing agent, obtains water-soluble hybrid gold nanoparticles with controllable size, uniform morphology, and good stability; furthermore, these hybrid gold nanoparticles can be used in the detection of miRNAs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical new materials, and particularly relates to a hybrid gold nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Tumor is a malignant disease with high morbidity and mortality, which poses a great threat to human health. Early diagnosis is conducive to early intervention and treatment of tumor, reduces the morbidity and mortality, improves the survival rate, and improves the quality of life of patients. Liquid biopsy refers to a kind of in vitro diagnostic technology using human body fluid as a sample source for detection and analysis. Due to its low invasiveness, liquid biopsy technology is considered to be the first choice for early diagnosis of tumor. Among many liquid biopsy biomarkers, miRNA is widely distributed in tissues, blood, urine and other body fluids, so detecting circulating miRNA is an effective strategy for cancer diagnosis, prognosis and monitoring based on liquid biopsy.

[0003] In the existing research, gold nanoparticles are favored by researchers due to their excellent performance, but the gold nanoparticles prepared by the traditional method are prone to agglomeration, difficult to store for a long time, low in yield, and cannot be used in high concentration, and the post-modification means is single.

[0004] Therefore, it is urgent to develop a preparation method for improving the size, morphology and stability of gold nanoparticles, which is helpful to further promote the efficient detection of miRNAs.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The present application aims to provide a hybrid gold nanoparticle and a preparation method and application thereof. The hybrid gold nanoparticle is a kind of water-soluble gold nanoparticle with controllable size, uniform morphology and good stability, which can be used as a detection reagent for miRNA.

[0007] In order to achieve the above-mentioned purpose of the present application, the following technical solutions are adopted:

[0008] In a first aspect, the present application provides a hybrid gold nanoparticle, which comprises a star-shaped polymer template and gold nanoparticles loaded on the star-shaped polymer template.

[0009] The star-shaped polymer template has the following structure shown in formula I:

[0010]

[0011] In the formula, PAMAM represents dendrimer polyamide-amine, n is 20-100, m is 30-400, and X group represents a protecting group of carboxyl or amino group.

[0012] Preferably, the particle size of the hybrid gold nanoparticles is 20-40 nm.

[0013] Preferably, the protecting group of the amino group is selected from any one of Fmoc, Dmb or Bn, preferably Fmoc.

[0014] In a second aspect, the present application provides a preparation method of the hybrid gold nanoparticles according to the first aspect, comprising the following steps:

[0015] (1) glutamic acid 5-benzyl ester N-carboxylic cyclic anhydride is polymerized with dendritic macromolecule polyamide-amine to obtain polymer PAMAM-PBLG-NH2;

[0016] (2) the terminal amino group of the polymer PAMAM-PBLG-NH2 is modified for the first time by using a bromination reagent to obtain polymer PAMAM-PBLG-Br; and the terminal group of the polymer PAMAM-PBLG-Br is modified for the second time by using an azide reagent to obtain polymer PAMAM-PBLG-N3;

[0017] (3) polymer PAMAM-PBLG-N3 is subjected to click reaction with an end group functionalized alkyne polyethylene glycol to obtain polymer PAMAM-PBLG-PEG;

[0018] (4) under the joint action of trifluoroacetic acid and hydrobromic acid, polymer PAMAM-PBLG-PEG is subjected to reaction to obtain the star-shaped polymer template shown in formula I;

[0019] (5) the star-shaped polymer template shown in formula I is complexed with chloroauric acid and is reduced under the action of a reducing agent to obtain the hybrid gold nanoparticles.

[0020] Preferably, in step (1), the branching degree of the PAMAM ranges from 16 to 256 (G2-G6).

[0021] Preferably, in step (1), the molecular weight of the polymer PAMAM-PBLG-NH2 ranges from 80000 to 5120000 Dalton.

[0022] Preferably, in step (1), the molar ratio of glutamic acid 5-benzyl ester N-carboxylic cyclic anhydride to dendritic macromolecule polyamide-amine is (20-100):1.

[0023] Preferably, in step (1), the temperature of the polymerization reaction is 10-40℃, and the time of the polymerization reaction is 6-10h.

[0024] Preferably, in step (1), the polymerization reaction is carried out in a solvent, and the solvent comprises dichloromethane.

[0025] Preferably, in step (1), after the polymerization reaction, the following post-processing step is further included: the reaction solution obtained from the polymerization reaction is concentrated, and then precipitated using diethyl ether to obtain the polymer PAMAM-PBLG-NH2.

[0026] Preferably, in step (2), the brominating agent is 2-bromoisobutyryl bromide.

[0027] Preferably, in step (2), the molar ratio of the brominating agent to the terminal amino group of the polymer PAMAM-PBLG-NH2 is (1-3):1.

[0028] Preferably, in step (2), in the process of the first modification, the reaction is first carried out at -5-5°C for 1-3h, and then carried out at 10-40°C for 20-24h.

[0029] Preferably, in step (2), in the process of the first modification, the reaction is carried out in the presence of a solvent, and the solvent comprises DMF.

[0030] Preferably, in step (2), after the first modification, the following post-processing step is further included: the reaction solution obtained from the first modification is precipitated using diethyl ether and water in sequence to obtain the polymer PAMAM-PBLG-Br.

[0031] Preferably, in step (2), the azide reagent is sodium azide.

[0032] Preferably, in step (2), the molar ratio of the azide reagent to the terminal bromine group of the polymer PAMAM-PBLG-Br is (18-22):1.

[0033] Preferably, in step (2), in the process of the second modification, the modification temperature is 10-40°C, and the modification time is 22-26h.

[0034] Preferably, in step (2), in the process of the second modification, the reaction is carried out in the presence of a solvent, and the solvent comprises DMF.

[0035] Preferably, in step (2), after the second modification, the following post-processing step is further included: the reaction solution obtained from the second modification is dissolved in dichloromethane, unreacted azide reagent is washed away with water, the organic phase is concentrated, and then precipitated using diethyl ether to obtain the polymer PAMAM-PBLG-N3.

[0036] Preferably, in step (3), the molecular weight of the end group functionalized alkynyl polyethylene glycol is 2000-20000 Dalton.

[0037] Preferably, in step (3), the click reaction is carried out in the presence of a catalyst; wherein the catalyst is ascorbic acid.

[0038] Preferably, in step (3), the click reaction is carried out in the presence of a promoter; wherein the promoter is copper sulfate.

[0039] Preferably, in step (3), the click reaction is carried out in the presence of a ligand; wherein the ligand is pentamethyldiethylenetriamine.

[0040] Preferably, in step (3), the molar ratio of the end group functionalized alkynyl polyethylene glycol, the polymer PAMAM-PBLG-N3, the catalyst, the promoter and the ligand is (1-2):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5).

[0041] Preferably, in step (3), the temperature of the click reaction is 30-50℃, and the time of the click reaction is 22-26h.

[0042] Preferably, in step (3), the click reaction is carried out in the presence of a solvent, and the solvent includes DMF.

[0043] Preferably, in step (3), after the click reaction, the following post-processing step is further included: dialysis and lyophilization of the reaction solution obtained by the click reaction to obtain the polymer PAMAM-PBLG-PEG.

[0044] Preferably, in step (3), the molecular weight of the dialysis is 500-10000 Dalton.

[0045] Preferably, in step (3), the time of the dialysis is 48-96h.

[0046] Preferably, in step (4), the volume ratio of trifluoroacetic acid to hydrobromic acid is (2-4):1.

[0047] Preferably, in step (4), the concentration of trifluoroacetic acid is 90-150mg / mL.

[0048] Preferably, in step (4), the concentration of hydrobromic acid is 30-50mg / mL.

[0049] Preferably, in step (4), the temperature of the reaction is 10-40℃, and the time of the reaction is 2-6h.

[0050] Preferably, in step (4), after the reaction, the following post-processing steps are included: dialysis of the reaction solution, freeze-drying, to obtain the star-shaped polymer template of formula I.

[0051] Preferably, in step (4), the molecular weight of the dialysis is 500-10000 Dalton.

[0052] Preferably, in step (4), the dialysis time is 48-96h.

[0053] Preferably, in step (5), the reducing agent is borane-tert-butylamine complex.

[0054] Preferably, in step (5), the molar ratio of chloroauric acid, reducing agent and carboxyl in the star-shaped polymer template of formula I is (4-6):(8-12):1.

[0055] Preferably, in step (5), the complexing temperature is 10-40℃, and the complexing time is 42-54h.

[0056] Preferably, in step (5), the reduction temperature is 50-70℃, and the reduction time is 1-3h.

[0057] In a third aspect, the application provides a hybrid gold nanoparticle as described in the first aspect for use as a detection reagent for miRNA.

[0058] Compared with the prior art, the application has the following beneficial effects:

[0059] (1) The preparation method described in the application uses a star-shaped polymer template of formula I to complex with the precursor chloroauric acid, and under the action of a reducing agent, water-soluble gold nanoparticles with controllable size, uniform morphology and good stability are obtained.

[0060] (2) The preparation of the star-shaped polymer template of formula I described in the application has simple steps, strong repeatability, and can be produced on a large scale, providing a beneficial reference for the preparation of various inorganic nanoparticles that are difficult to synthesize.

[0061] (3) The hybrid gold nanoparticles described in the application can be applied to the detection of miRNA, coupled with double-stranded DNA, and after the target miRNA appears, one of the double-stranded DNAs will be replaced, releasing the reporter strand with a FAM group, and detecting the fluorescence signal. BRIEF DESCRIPTION OF DRAWINGS

[0062] In order to make the technical solutions in the specific embodiments or prior art of the present application more clearly understood, the drawings needed in the description of the specific embodiments or prior art will be briefly introduced as follows. Obviously, the drawings described below are some embodiments of the present application, and for those skilled in the art, other drawings can be obtained without creative labor on the basis of these drawings.

[0063] Figure 1 SEC characterization chart of the molecular weight of the polymer PAMAM-PBLG-NH2 provided for Example 1.

[0064] Figure 2 Infrared characterization chart of the polymer PAMAM-PBLG-N3 provided for Example 1.

[0065] Figure 3 NMR characterization chart of the polymer PAMAM-PBLG-PEG provided for Example 1.

[0066] Figure 4 TEM characterization chart of the hybrid gold nanoparticles provided for Example 1.

[0067] Figure 5 Fluorescence chart of the hybrid gold nanoparticles provided for Example 1 for detecting miRNA. DETAILED DESCRIPTION

[0068] Unless otherwise defined, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. In this application, the use of "or" means "and / or" unless specifically stated otherwise, for example, "comprises" or "comprising" means "includes or is inclusive of, and "consists of" or "consisting of" means "includes and is inclusive of, and not limited to, except as otherwise noted or required by context.

[0069] It should be noted that specific details are set forth in the following description in order to provide a thorough understanding of the application. However, the application can be practiced without many of the details set forth in this description, many of which are well known in the art. Therefore, the present application is not limited to the embodiments described herein but can be practiced with the under standing of the claims. It should be noted that where costs are discussed, the costs are relative to the costs of the prior art.

[0070] The technical solutions of the present application will be described clearly and completely in combination with the embodiments. Obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0071] In a first aspect, the present application provides a hybrid gold nanoparticle, which comprises a star-shaped polymer template and gold nanoparticles loaded on the star-shaped polymer template.

[0072] The star-shaped polymer template has the following structure shown in Formula I:

[0073]

[0074] In the formula, PAMAM represents dendrimer polyamide-amine, n is 20-100, m is 30-400, and X represents a protecting group of carboxyl or amino.

[0075] In the present application, the star-shaped polymer template (which can be referred to as PAMAM-PLG-PEG) shown in Formula I is used to complex with a precursor chloroauric acid, and under the action of a reducing agent, water-soluble gold nanoparticles with controllable size, uniform morphology and good stability can be obtained. The PAMAM-PLG-PEG provided by the present application can effectively solve the problems of easy agglomeration, poor stability, difficult long-term preservation, low yield, inability to use at high concentration, and single post-modification means of gold nanoparticles in the preparation process.

[0076] As an optional embodiment, n is 20-100, for example, it can be 20 / 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, etc.

[0077] As an optional embodiment, m is 30-400, for example, it can be 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, etc. As an optional embodiment, the particle size of the hybrid gold nanoparticle is 20-40 nm, for example, it can be 20 nm, 22 nm, 24 nm, 26 nm, 28 nm, 30 nm, 32 nm, 34 nm, 36 nm, 38 nm, 40 nm, etc.

[0078] As an optional embodiment, the protecting group of the amino group is selected from any one of Fmoc, Dmb or Bn, and is preferably Fmoc.

[0079] In a second aspect, the present application provides a preparation method of the hybrid gold nanoparticle according to the first aspect, which comprises the following steps:

[0080] (1) glutamic acid 5-benzyl ester N-carboxyl cyclic anhydride is subjected to a polymerization reaction with dendrimer polyamide-amine to obtain a polymer PAMAM-PBLG-NH2; the reaction formula is as shown below:

[0081]

[0082] (2) the terminal amino group of the polymer PAMAM-PBLG-NH2 is modified for the first time by using a bromination reagent to obtain a polymer PAMAM-PBLG-Br; and the terminal group of the polymer PAMAM-PBLG-Br is modified for the second time by using an azide reagent to obtain a polymer PAMAM-PBLG-N3; the reaction formula is as shown in the following:

[0083]

[0084] (3) the polymer PAMAM-PBLG-N3 is subjected to a click reaction with an end group functionalized alkynyl polyethylene glycol to obtain a polymer PAMAM-PBLG-PEG; the reaction formula is as shown in the following:

[0085]

[0086] (4) the polymer PAMAM-PBLG-PEG is subjected to a reaction under the joint action of trifluoroacetic acid and hydrobromic acid to obtain the star-shaped polymer template shown in formula I; the reaction formula is as shown in the following:

[0087]

[0088] (5) the star-shaped polymer template shown in formula I is complexed with chloroauric acid and is reduced under the action of a reducing agent to obtain the hybrid gold nanoparticles.

[0089] In the application, dendritic macromolecules polyamide-amine (PAMAM) with different branching degrees are used as initiators to initiate a polymerization reaction of 5-benzyl glutamate N-carboxylic acid anhydride (BLG-NCA), and end group modification and click reaction are performed to graft end group functionalized alkynyl polyethylene glycol (ALK-PEG-X), and finally, under the joint action of trifluoroacetic acid and hydrobromic acid, deprotection is performed to obtain a water-soluble star-shaped polymer template (PAMAM-PLG-PEG). Then, a precursor chloroauric acid is added to complex the PLG segment in PAMAM-PLG-PEG, and under the action of a reducing agent, water-soluble gold nanoparticles with controllable size, uniform morphology and good stability are obtained, and the gold nanoparticles are applied in the detection of miRNA. The polymer template prepared in the application has the advantages of simple steps, strong repeatability and large-scale production, and provides a beneficial reference for the preparation of various inorganic nanoparticles that are difficult to synthesize.

[0090] As an optional implementation, in step (1), the branching degree of the PAMAM ranges from 16 to 256 (G2-G6).

[0091] As an optional implementation, in step (1), the molecular weight of the polymer PAMAM-PBLG-NH2 is in the range of 80000-5120000 Dalton, for example, it can be 80000 Dalton, 100000 Dalton, 150000 Dalton, 200000 Dalton, 250000 Dalton, 300000 Dalton, 350000 Dalton, 400000 Dalton, 450000 Dalton, 500000 Dalton, 600000 Dalton, 800000 Dalton, 1000000 Dalton, 2000000 Dalton, 3000000 Dalton, 4000000 Dalton, 5000000 Dalton, 5120000 Dalton, etc.

[0092] As an optional implementation, in step (1), the molar ratio of glutamic acid 5-benzyl ester N-carboxyl cyclic anhydride to dendrimeric polyamide-amine is (20-100):1, for example, it can be 20:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, etc.

[0093] As an optional implementation, in step (1), the temperature of the polymerization reaction is 10-40℃, for example, it can be 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc., and the time of the polymerization reaction is 6-10h, for example, it can be 6h, 6.5h, 7h, 7.5h, 8h, 8.5h, 9h, 9.5h, 10h, etc.

[0094] As an optional implementation, in step (1), the polymerization reaction is carried out in a solvent, and the solvent includes dichloromethane.

[0095] As an optional implementation, in step (1), after the polymerization reaction, the following post-processing step is further included: the reaction solution obtained by the polymerization reaction is concentrated, and then precipitated using diethyl ether to obtain the polymer PAMAM-PBLG-NH2.

[0096] As an optional implementation, in step (2), the bromination reagent is 2-bromoisobutyryl bromide.

[0097] As an optional implementation, in step (2), the molar ratio of the bromination reagent to the terminal amino group of the polymer PAMAM-PBLG-NH2 is (1-3):1, for example, it can be 1:1, 1.2:1, 1.4:1, 1.5:1, 1.6:1, 1.8:1, 2:1, 2.2:1, 2.4:1, 2.5:1, 2.6:1, 2.8:1, 3:1, etc.

[0098] As an optional implementation, in step (2), in the process of the first modification, first, react at -5-5℃ (for example, it can be -5℃, -4℃, -3℃, -2℃, -1℃, 0℃, 1℃, 2℃, 3℃, 4℃, 5℃, etc.) for 1-3h (for example, it can be 1h, 1.2h, 1.4h, 1.5h, 1.6h, 1.8h, 2h, 2.2h, 2.4h, 2.5h, 2.6h, 2.8h, 3h, etc.), and then react at 10-40℃ (for example, it can be 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc.) for 20-24h (for example, it can be 20h, 20.5h, 21h, 21.5h, 22h, 22.5h, 23h, 23.5h, 24h, etc.).

[0099] As an optional implementation, in step (2), in the process of the first modification, it is carried out in the presence of a solvent, and the solvent includes DMF.

[0100] As an optional implementation, in step (2), after the first modification, the following post-processing step is further included: the reaction solution obtained by the first modification is sequentially settled with diethyl ether and water to obtain the polymer PAMAM-PBLG-Br.

[0101] As an optional implementation, in step (2), the azide reagent is sodium azide.

[0102] As an optional implementation, in step (2), the molar ratio of the azide reagent to the terminal bromine group of the polymer PAMAM-PBLG-Br is (18-22):1, for example, it can be 18:1, 18.5:1, 19:1, 19.5:1, 20:1, 20.5:1, 21:1, 21.5:1, 22:1, etc.

[0103] As an optional implementation, in step (2), in the process of the second modification, the modification temperature is 10-40℃, for example, it can be 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc., and the modification time is 22-26h, for example, it can be 22h, 22.5h, 23h, 23.5h, 24h, 24.5h, 25h, 25.5h, 26h, etc.

[0104] As an optional implementation, in step (2), in the process of the second modification, it is carried out in the presence of a solvent, and the solvent includes DMF.

[0105] As an optional implementation, in step (2), after the second modification, the following post-processing step is further included: the reaction solution obtained by the second modification is dissolved with dichloromethane, unreacted azide reagent is washed away with water, the organic phase is concentrated, and then ether is used for precipitation to obtain the polymer PAMAM-PBLG-N3.

[0106] As an optional implementation, in step (3), the molecular weight of the terminal group functionalized alkynyl polyethylene glycol is 2000-20000 Dalton, for example, it can be 2000 Dalton, 3000 Dalton, 4000 Dalton, 5000 Dalton, 6000 Dalton, 7000 Dalton, 8000 Dalton, 9000 Dalton, 10000 Dalton, 11000 Dalton, 12000 Dalton, 13000 Dalton, 14000 Dalton, 15000 Dalton, 16000 Dalton, 17000 Dalton, 18000 Dalton, 19000 Dalton, 20000 Dalton, etc.

[0107] As an optional implementation, in step (3), the click reaction is carried out in the presence of a catalyst; wherein the catalyst is ascorbic acid.

[0108] As an optional implementation, in step (3), the click reaction is carried out in the presence of a promoter; wherein the promoter is copper sulfate.

[0109] As an optional implementation, in step (3), the click reaction is carried out in the presence of a ligand; wherein the ligand is pentamethyl diethylene triamine.

[0110] As an optional implementation, in step (3), the molar ratio of the terminal group functionalized alkynyl polyethylene glycol, the polymer PAMAM-PBLG-N3, the catalyst, the promoter and the ligand is (1-2):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5), for example, it can be 1:1:1:1:1, 1.1:1:1:1:1, 1.2:1:1:1:1, 1.3:1:1:1:1, 1.4:1:1:1:1, 1.5:1:1:1:1, etc.

[0111] As an optional implementation, in step (3), the temperature of the click reaction is 30-50°C, for example, it can be 30°C, 32°C, 34°C, 35°C, 36°C, 38°C, 40°C, 42°C, 44°C, 45°C, 46°C, 48°C, 50°C, etc., and the time of the click reaction is 22-26h, for example, it can be 22h, 22.5h, 23h, 23.5h, 24h, 24.5h, 25h, 25.5h, 26h, etc.

[0112] As an optional implementation, in step (3), the click reaction is carried out in the presence of a solvent, and the solvent includes DMF.

[0113] As an optional implementation, in step (3), after the click reaction, the following post-processing step is further included: dialysis and freeze-drying of the reaction solution obtained by the click reaction to obtain the polymer PAMAM-PBLG-PEG.

[0114] As an optional implementation, in step (3), the molecular weight of the dialysis is 500-10000 Dalton, for example, it can be 5000 Dalton, 10000 Dalton, 15000 Dalton, 20000 Dalton, 25000 Dalton, 30000 Dalton, 40000 Dalton, 50000 Dalton, 60000 Dalton, 70000 Dalton, 80000 Dalton, 90000 Dalton, 100000 Dalton, etc.

[0115] As an optional implementation, in step (3), the time of the dialysis is 48-96h, for example, it can be 48h, 54h, 60h, 66h, 72h, 78h, 84h, 90h, 96h, etc.

[0116] As an optional implementation, in step (4), the volume ratio of trifluoroacetic acid to hydrobromic acid is (2-4):1, for example, it can be 2:1, 2.2:1, 2.4:1, 2.6:1, 2.8:1, 3:1, 3.2:1, 3.4:1, 3.6:1, 3.8:1, 4:1, etc.

[0117] As an optional implementation, in step (4), the concentration of trifluoroacetic acid is 90-150mg / mL, for example, it can be 90mg / mL, 95mg / mL, 100mg / mL, 105mg / mL, 110mg / mL, 115mg / mL, 120mg / mL, 125mg / mL, 130mg / mL, 135mg / mL, 140mg / mL, 145mg / mL, 150mg / mL, etc.

[0118] As an optional embodiment, in step (4), the concentration of the hydrobromic acid is 30-50 mg / mL, for example, it can be 30 mg / mL, 32 mg / mL, 34 mg / mL, 36 mg / mL, 38 mg / mL, 40 mg / mL, 42 mg / mL, 44 mg / mL, 46 mg / mL, 48 mg / mL, 50 mg / mL, etc.

[0119] As an optional embodiment, in step (4), the temperature of the reaction is 10-40℃, for example, it can be 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc., and the time of the reaction is 2-6 h, for example, it can be 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h, etc.

[0120] As an optional embodiment, in step (4), after the reaction, the following post-treatment step is further included: dialysis and freeze-drying of the reaction solution obtained by the reaction to obtain the star-shaped polymer template represented by formula I.

[0121] As an optional embodiment, in step (4), the molecular weight of the dialysis is 500-10,000 Dalton, for example, it can be 5,000 Dalton, 10,000 Dalton, 15,000 Dalton, 20,000 Dalton, 25,000 Dalton, 30,000 Dalton, 40,000 Dalton, 50,000 Dalton, 60,000 Dalton, 70,000 Dalton, 80,000 Dalton, 90,000 Dalton, 100,000 Dalton, etc.

[0122] As an optional embodiment, in step (4), the time of the dialysis is 48-96 h, for example, it can be 48 h, 54 h, 60 h, 66 h, 72 h, 78 h, 84 h, 90 h, 96 h, etc.

[0123] As an optional embodiment, in step (5), the reducing agent is borane tert-butylamine complex.

[0124] As an optional embodiment, in step (5), the molar ratio of the chloroauric acid, the reducing agent, and the carboxyl group in the star-shaped polymer template represented by formula I is (4-6):(8-12):1, for example, it can be 4:8:1, 4.2:8.5:1, 4.5:9:1, 5:10:1, 5.5:11:1, 6:12:1, etc.

[0125] As an optional embodiment, in step (5), the temperature of the complexing is 10-40℃, for example, it can be 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc., and the time of the complexing is 42-54h, for example, it can be 42h, 44h, 46h, 48h, 50h, 52h, 54h, etc.

[0126] As an optional embodiment, in step (5), the temperature of the reducing is 50-70℃, for example, it can be 50℃, 52℃, 54℃, 56℃, 58℃, 60℃, 62℃, 64℃, 66℃, 68℃, 70℃, etc., and the time of the reducing is 1-3h, for example, it can be 1h, 1.2h, 1.4h, 1.5h, 1.6h, 1.8h, 2h, 2.2h, 2.4h, 2.5h, 2.6h, 2.8h, 3h, etc.

[0127] In a third aspect, the present application provides a use of the hybrid gold nanoparticle as described in the first aspect as a detection reagent of miRNA.

[0128] The present application is further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to the existing methods or directly purchased from the market.

[0129] Example 1

[0130] The present example provides a hybrid gold nanoparticle, which is prepared by the following steps:

[0131] S1, Preparation of polymer PAMAM-PBLG-NH2

[0132] The dendritic macromolecule polyamide-amine (PAMAM) (G2-G6) with different branching degrees is dissolved in methanol, and then rapidly added to a monomer glutamic acid 5-benzyl ester N-carboxyl cyclic anhydride (BLG-NCA) dichloromethane solution, and stirred vigorously. After the reaction is completed at room temperature for 8h, the reaction solution is concentrated by rotation, and then precipitated in diethyl ether to obtain the polymer PAMAM-PBLG-NH2.

[0133] The molecular weight characterization of the polymer is shown in Figure 1 The molar ratio of the raw materials, the molecular weight, and the corresponding obtained polymer PAMAM-PBLG-NH2 are shown in Table 1 below:

[0134] Table 1

[0135]

[0136]

[0137] S2, Preparation of PAMAM-PBLG-N3:

[0138] The PAMAM-PBLG-NH2 with molecular weight of 320000 Dalton in Table 1 above was dissolved in super dry DMF, and 2-bromoisobutyryl bromide (BIBB) was gradually added dropwise under ice bath condition, and reacted at 0℃ for 2h, and continuously reacted at room temperature for 22h. After reaction, the reaction solution was diluted with dichloromethane, and after water washing for 3 times, the product was precipitated with ether to obtain the polymer PAMAM-PBLG-Br;

[0139] The polymer PAMAM-PBLG-Br was dissolved in super dry DMF, and sodium azide was added, and reacted at room temperature for 24h, and then the reaction solution was diluted with dichloromethane, and the excess unreacted sodium azide was washed with water, and finally the product was precipitated with ether to obtain the polymer PAMAM-PBLG-N3.

[0140] The polymer PAMAM-PBLG-N3 prepared by using the PAMAM-PBLG-N3 with molecular weight of 320000 Dalton in Table 1 was taken as an example to perform infrared characterization of the terminal azide modification, as shown in Figure 1, the azide characteristic peak appeared at 2200cm-1. Figure 2 -1

[0141] S3, polymer PAMAM-PBLG-PEG:

[0142] The product polymer PAMAM-PBLG-N3 of S2, alkyne-polyethylene glycol-Fmoc (the molecular weight of polyethylene glycol is 5000 Dalton), anhydrous copper sulfate, ligand PMDETA were dissolved in super dry DMF, and the oxygen in the solution was removed after blowing with argon for 30min, and ascorbic acid dissolved in DMF was added (the molar ratio of alkyne-polyethylene glycol-Fmoc, PAMAM-PBLG-N3, anhydrous copper sulfate, ascorbic acid, PMDETA is 1.5:1:1:1:1), and the oxygen was removed again after blowing for 30min, and the reaction was carried out at 40℃ for 24h; the reaction solution was dialyzed (the molecular weight of dialysis is 50000 Dalton, and the dialysis time is 48h), and freeze-dried to obtain the polymer PAMAM-PBLG-PEG.

[0143] The polymer PAMAM-PBLG-PEG was taken as an example to perform nuclear magnetic resonance characterization, as shown in Figure 2, a is the hydrogen at the polyethylene glycol position, b is the hydrogen at the benzyl position, and c, d, e are the hydrogens at the benzene ring position in -Fmoc, respectively. Figure 3

[0144] S4, preparation of the star-shaped polymer template (PAMAM-PLG-PEG) represented by formula I:

[0145] ​​​The polymer PAMAM-PBLG-PEG obtained in S3 is dissolved using trifluoroacetic acid and hydrobromic acid is added (wherein the volume ratio of trifluoroacetic acid (100 mg / mL) and hydrobromic acid (33 mg / mL) is 3:1; the mass ratio of polymer PAMAM-PBLG-PEG and the total volume of acid is 75 mg: 1 mL), and deprotection is carried out at room temperature for 4 h; the reaction solution is dialyzed (the molecular weight of dialysis is 10000 Dalton, and the dialysis time is 48 h), and freeze-drying is performed to obtain the water-soluble star-shaped polymer template PAMAM-PLG-PEG.

[0146] S5, Preparation of hybrid gold nanoparticles:

[0147] The polymer template PAMAM-PLG-PEG is dissolved in DMF, and the precursor chloroauric acid trihydrate is added, and stirring is carried out at room temperature under N2 atmosphere for 48 h, so that the chloroauric acid is fully complexed with the -COOH of the polymer PLG segment, and then the borane tert-butylamine complex TBAB is added (wherein the molar ratio of chloroauric acid trihydrate, TBAB, and carboxyl in the polymer template PAMAM-PLG-PEG is 5:10:1), and reaction is carried out at 60°C for 2 h to obtain the hybrid gold nanoparticles.

[0148] Comparative Example 1

[0149] This comparative example provides a gold nanoparticle, which is prepared by the following steps:

[0150] First, 18 mg of gold chloride is mixed with 100 mL of boiling deionized water, and then 3 mL of 1% (w / v) trisodium citrate is added, and stirring is carried out for 30 min. The change in the color of the solution indicates that the size of the particles is reduced. Then the solution is cooled to room temperature, and finally filtered through a 0.2 mm membrane.

[0151] 50 mL of streptavidin (1 mg / mL) and 1 mL of Au NPs (pH = 6.5) are mixed, and incubation is carried out at 37°C for 30 min. Then, BSA (final concentration of 1%) is added, and incubation is carried out at 37°C for another 5 min, and after centrifugation at 12500 rpm, the obtained streptavidin-modified Au NPs are re-dispersed in 250 mL of PBS (0.1M, pH = 7.4), and stored at 4°C until use.

[0152] Test Example 1

[0153] Gold nanoparticle morphology test

[0154] Test sample: the hybrid gold nanoparticles provided in Example 1.

[0155] Test method: transmission electron microscopy (TEM) characterization.

[0156] Figure 4 TEM characterization figure of the hybrid gold nanoparticles provided in Example 1. As shown in the figure, the hybrid gold nanoparticles of the present application are spherical particles with uniform size, uniformly dispersed without agglomeration, and the particle size is 20-40 nm. Figure 4

[0157] Test Example 2

[0158] Water-solubility and stability test

[0159] Test sample: the hybrid gold nanoparticles provided in Example 1.

[0160] Test method:

[0161] (1) Solubility of gold nanoparticles in water: saturated state at 25°C under stirring at a speed of 800 rpm for 10 min;

[0162] (2) Storage time of gold nanoparticles in water: no precipitation phenomenon was observed after storage at 25°C under a humidity of 50-90% for more than 1 year without light protection.

[0163] Test result:

[0164] The solubility of the hybrid gold nanoparticles provided in Example 1 in water is 10 mg / mL; and the hybrid gold nanoparticles provided in Example 1 can be stored at 25°C for more than 365 days without precipitation or deterioration. Thus, the hybrid gold nanoparticles prepared by the preparation method of the present application not only have the advantages of controllable size and uniform morphology, but also have certain water-solubility and good stability.

[0165] Test Example 3

[0166] Detection of miRNA

[0167] Test sample: the hybrid gold nanoparticles provided in Example 1.

[0168] Test method: the prepared gold nanoparticles were coupled with double-stranded DNA, and after the target miRNA appeared, one of the DNAs in the double-stranded DNA was replaced to release the reporter strand with FAM group, and whether the fluorescence signal appeared was detected.

[0169] Test result:

[0170] As shown in the figure, after the addition of miRNA, the fluorescence signal was restored, and the fluorescence signal increased with the increase of the concentration. Figure 5

[0171] ​​It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A hybrid gold nanoparticle, characterized in that, The hybrid gold nanoparticles comprise a star polymer template and gold nanoparticles loaded on the star polymer template; The star polymer template has a structure shown in the following formula I: Formula I; In the formula, PAMAM represents a dendrimer polyamide-amine, n is 20-100, m is 30-400, and X represents a protecting group of carboxyl or amino.

2. The hybrid gold nanoparticle of claim 1, wherein, The hybrid gold nanoparticles have a particle size of 20-40 nm.

3. The hybrid gold nanoparticle of claim 1, wherein, The protecting group of amino is selected from any one of Fmoc, Dmb and Bn.

4. A method of preparing the hybrid gold nanoparticle according to claim 1 or 2, characterized in that, The preparation method comprises the following steps: (1) glutamic acid 5-benzyl ester N-carboxyl cyclic anhydride is subjected to a polymerization reaction with a dendrimer polyamide-amine to obtain a polymer PAMAM-PBLG-NH2; (2) a bromination reagent is used to modify terminal amino groups of the polymer PAMAM-PBLG-NH2 for the first time to obtain a polymer PAMAM-PBLG-Br; and an azide reagent is used to modify terminal groups of the polymer PAMAM-PBLG-Br for the second time to obtain a polymer PAMAM-PBLG-N3; (3) the polymer PAMAM-PBLG-N3 is subjected to a click reaction with an alkyne polyethylene glycol functionalized at a terminal group to obtain a polymer PAMAM-PBLG-PEG; (4) under the joint action of trifluoroacetic acid and hydrobromic acid, the polymer PAMAM-PBLG-PEG is subjected to a reaction to obtain the star polymer template shown in the formula I; (5) the star polymer template shown in the formula I is complexed with chloroauric acid and is reduced under the action of a reducing agent to obtain the hybrid gold nanoparticles.

5. The method of claim 4, wherein the gold nanoparticles are prepared by the method comprising the steps of: In step (1), the branching degree of the PAMAM ranges from G2 to G6; and the molecular weight of the polymer PAMAM-PBLG-NH2 ranges from 80000 to 5120000 Dalton.

6. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (1), the molar ratio of the glutamic acid 5-benzyl ester N-carboxyl cyclic anhydride to the dendrimer polyamide-amine is (20-100):

1.

7. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (1), the temperature of the polymerization reaction is 10-40℃, and the time of the polymerization reaction is 6-10 h.

8. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (1), the polymerization reaction is performed in a solvent, and the solvent comprises dichloromethane.

9. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (1), after the polymerization reaction, the following post-processing step is further included: The reaction solution obtained in the polymerization reaction is concentrated, and then is settled using diethyl ether to obtain the polymer PAMAM-PBLG-NH2.

10. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (2), the bromination reagent is 2-bromoisobutyryl bromide.

11. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (2), the molar ratio of the bromination reagent to the terminal amino groups of the polymer PAMAM-PBLG-NH2 is (1-3):

1.

12. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (2), in the process of the first modification, first, a reaction is performed at -5-5℃ for 1-3 h, and then a reaction is performed at 10-40℃ for 20-24 h.

13. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (2), in the process of the first modification, the reaction is performed in the presence of a solvent, and the solvent comprises DMF.

14. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (2), after the first modification, the following post-processing step is further included: The reaction solution obtained in the first modification is precipitated with ether and water in sequence to obtain the polymer PAMAM-PBLG-Br.

15. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (2), the azide reagent is sodium azide.

16. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (2), the molar ratio of the azide reagent to the terminal bromine group of the polymer PAMAM-PBLG-Br is (18-22):

1.

17. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (2), during the second modification, the modification temperature is 10-40℃, and the modification time is 22-26 h.

18. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (2), during the second modification, the modification is carried out in the presence of a solvent, and the solvent includes DMF.

19. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (2), after the second modification, the following post-processing steps are further included: The reaction solution obtained in the second modification is dissolved in dichloromethane, and unreacted azide reagent is removed by washing with water. The organic phase is concentrated, and then precipitated with ether to obtain the polymer PAMAM-PBLG-N3.

20. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (3), the molecular weight of the terminal group functionalized alkyne polyethylene glycol is 2000-20000 Dalton.

21. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (3), the click reaction is carried out in the presence of a catalyst; wherein the catalyst is ascorbic acid; In step (3), the click reaction is carried out in the presence of a promoter; wherein the promoter is copper sulfate; In step (3), the click reaction is carried out in the presence of a ligand; wherein the ligand is pentamethyl diethylene triamine; In step (3), the molar ratio of the terminal group functionalized alkyne polyethylene glycol, the polymer PAMAM-PBLG-N3, the catalyst, the promoter and the ligand is (1-2):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5).

22. The method of claim 4, wherein the hybrid gold nanoparticles are prepared by the method comprising the steps of: In step (3), the temperature of the click reaction is 30-50℃, and the time of the click reaction is 22-26 h.

23. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (3), the click reaction is carried out in the presence of a solvent, and the solvent includes DMF.

24. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (3), after the click reaction, the following post-processing steps are further included: The reaction solution obtained in the click reaction is dialyzed and freeze-dried to obtain the star-shaped polymer template represented by formula I.

25. The method for preparing hybrid gold nanoparticles according to claim 24, characterized in that, In step (3), the dialysis has a molecular weight of 500-10000 Dalton; and the dialysis time is 48-96 h.

26. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (4), the volume ratio of trifluoroacetic acid to hydrobromic acid is (2-4):

1.

27. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (4), the concentration of trifluoroacetic acid is 90-150 mg / mL; and the concentration of hydrobromic acid is 30-50 mg / mL.

28. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (4), the temperature of the reaction is 10-40℃, and the time of the reaction is 2-6 h.

29. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (4), after the reaction, the following post-processing steps are further included: The reaction solution obtained in the reaction is dialyzed and freeze-dried to obtain the star-shaped polymer template represented by formula I.

30. The method for preparing hybrid gold nanoparticles according to claim 29, characterized in that, In step (4), the dialysis has a molecular weight of 500-10000 Dalton; and the dialysis time is 48-96 h.

31. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (5), the reducing agent is borane tert-butylamine complex.

32. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (5), the molar ratio of the chloroauric acid, the reducing agent and the carboxyl group in the star-shaped polymer template of formula I is (4-6):(8-12):

1.

33. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (5), the temperature of the complexing is 10-40℃, and the time of the complexing is 42-54 h.

34. The method for preparing hybrid gold nanoparticles according to claim 4, characterized in that, In step (5), the temperature of the reducing is 50-70℃, and the time of the reducing is 1-3 h.

35. Use of the hybrid gold nanoparticle according to any one of claims 1-3 as a detection reagent for miRNA.

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