Molecularly imprinted fluorescence sensor for detecting insulin autoantibody and preparation method thereof

Abstract

The invention provides a molecular imprinting fluorescence sensor for insulin autoantibody detection and a preparation method, and relates to the technical field of biological medicines.The molecular imprinting fluorescence sensor is characterized in that an aptamer capable of specifically recognizing IAA antibodies related to type 1 diabetes is used as a template, a template coordination fixing method is utilized, dopamine is used as a functional monomer, and the molecular imprinting fluorescence sensor is prepared through an auto-polymerization method. Fluorescent responsive imprinted nanoparticles based on fluorescent quantum dots are prepared, and PEG is grafted on the surface of the prepared fluorescent imprinted material by using a simple post-modification pegylation method. The molecular imprinting fluorescence sensor is used for quantitative detection of IAA < + > in a complex blood environment, especially under the individual condition of single autoantibody positive, not only can reduce the false positive rate and improve the disease predictability, but also provides a more accurate basis for risk prediction screening, typing, curative effect evaluation and guided treatment of early diabetes mellitus, and has good application prospects. And important support is provided for personalized diagnosis and treatment of diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a molecular imprinting fluorescent sensor for detecting insulin autoantibodies and a preparation method thereof. Background Art

[0002] Over the past 30 years, the prevalence of diabetes in my country has increased significantly. According to epidemiological data, the prevalence of diabetes was 0.67% in 1980, 9.7% in adults aged 20 and above in 2007-2008, and 11.2% in people aged 18 and above in 2015-2017. According to the new version of the International Diabetes Federation (IDF) map data, the number of diabetes patients in my country increased by 56% from 2011 to 2021. According to statistics, there were 116.4 million diabetes patients in my country in 2019, and it is expected to reach 145 million in 2045. Therefore, early diagnosis and treatment of diabetes patients and monitoring of the course of disease have become the goals pursued by the medical community. The "Guidelines for the Prevention and Treatment of Type 2 Diabetes in China (2020 Edition)" still uses the etiology classification system of WHO (1999 Edition) for the classification of diabetes, dividing diabetes into type 1 diabetes (T1DM), type 2 diabetes (T2DM), special types of diabetes and gestational diabetes. Among them, T1DM is mainly caused by immune-mediated destruction of pancreatic β cells, which leads to the release of multiple pancreatic autoantigens into the bloodstream and the production of different autoantibodies. Common islet-specific autoantibodies (ISAs) include: islet cell antibodies (ICA), insulin autoantibodies (IAA), glutamic acid decarboxylase antibodies (GADA), tyrosine phosphatase antibodies (IA-2A), and zinc transporter 8 antibodies (ZnT8A). These islet autoantibodies can be detected in the blood long before clinical symptoms appear. Since this pathology is characterized by an asymptomatic period that can last from several months to more than a decade, the detection and quantification of these autoantibodies can be used as a risk prediction for T1DM in children and adolescents. According to the recommendations of the 2022-ISPAD Guidelines: Staging of Type 1 Diabetes in Children and Adolescents, screening and testing of islet autoantibodies can identify individuals at risk for T1DM or with early T1DM, providing them with intervention measures to delay and ultimately prevent the disease. The sensitivity of one-time autoantibody screening in the 3-5 age group is about 35%, which can be increased to about 50% when repeated screening is performed at 2-3 years old and 5-7 years old. According to the "Guidelines for the Diagnosis and Treatment of Type 1 Diabetes in China", clinically diagnosed type 1 diabetes patients also need to undergo further testing for pancreatic autoantibodies to clarify the cause.

[0003] IAA is an autoantibody against endogenous insulin. It is the only type of specific antibody against pancreatic beta cells among diabetes-related antibodies. It is significantly associated with the occurrence of T1DM. In patients with type 1 diabetes, the older the age, the lower the titer of IAA will be. The positive rate in newly diagnosed type 1 diabetes is 30% to 40%, and it is very likely to appear before the onset of the disease. Many data studies have shown that IAA is crucial for predicting the onset of T1DM in children. It is an important criterion for screening high-risk children for immunotherapy, and the level of IAA is one of the main determinants for predicting the onset of T1DM. The higher the IAA level and the earlier the onset, the greater the predictive value. IAA can almost be detected in children under the age of 5 before the onset of the disease. If the detection rate of IAA can be further improved, the occurrence of T1DM can be predicted more accurately at an earlier stage. However, it should be noted that high-affinity IAA is associated with the appearance of multiple autoantibodies and the progression of clinical diseases. The affinity of IAA in the initial autoantibody sample is >10 9 L / mol, it can identify almost all children with multiple autoantibodies or clinical diabetes. 9 None of the children with a low affinity IAA level progressed to T1DM within 6 years. High affinity IAA is also associated with HLA-DRB1*04, a predictor of disease progression, but not with IAA titer. In addition, many studies have shown that autoantibody affinity seems to remain stable over time. Therefore, there is an urgent need to develop methods that can distinguish high-affinity and low-affinity IAA antibodies and perform quantitative detection, especially in the case of individuals with a single autoantibody positive. This can not only reduce the false positive rate and improve disease prediction, but also provide a more accurate basis for risk prediction screening, typing, efficacy evaluation and treatment guidance for early diabetes, and provide important support for personalized diagnosis and treatment of the disease.

[0004] In view of the challenges of IAA detection, a large number of domestic and foreign institutions and scholars have initiated a series of research. At present, the methods for detecting IAA include immunoblotting, indirect immunofluorescence, radioimmunoassay, enzyme-linked immunosorbent assay, radioligand, and chemiluminescence, but these methods not only have more or less low sensitivity, poor specificity, weak anti-interference ability, and complex operation, but most of them are qualitative or semi-quantitative, only reporting positive and negative, lacking specific antibody concentrations, and unable to screen and detect IAA with high affinity. Therefore, it is of great scientific value and clinical significance to establish a rapid, sensitive, and efficient technology for screening and quantitative detection of high-affinity IAA. Summary of the invention

[0005] In view of this, one of the purposes of the present invention is to provide a molecular imprinting fluorescence sensor for the detection of insulin autoantibodies, so as to solve the technical problems of low sensitivity, poor specificity, weak anti-interference ability, complex operation and the like in the prior art methods for detecting IAA using immunoblotting, indirect immunofluorescence, radioimmunoassay, enzyme-linked immunosorbent assay, radioligand and chemiluminescence.

[0006] A second object of the present invention is to provide a method for preparing a molecular imprinting fluorescence sensor for detecting insulin autoantibodies.

[0007] In order to achieve one of the above-mentioned purposes, the present invention provides a molecular imprinting fluorescent sensor for detecting insulin autoantibodies, wherein the molecular imprinting fluorescent sensor is synthesized by using fluorescent nanoparticles with magnetically responsive carbon dots as carriers and by using an anti-fouling strategy of aptamer molecular imprinting and post-imprinting modified polyethylene glycol;

[0008] The fluorescent nanoparticles are carbon dots as light sources, and Zn 2+ Fluorescent nanoparticles with magnetically responsive carbon dots were obtained by using dopamine as a ligand for surface modification.

[0009] The aptamer molecular imprint is a single-stranded DNA sequence consisting of 15-30 nucleotides;

[0010] The polyethylene glycol is amino-functionalized polyethylene glycol of different molecular weights.

[0011] The molecular imprinting fluorescent sensor of the present invention can detect high-affinity insulin autoantibodies with high sensitivity, wherein the high-affinity insulin autoantibodies are insulin autoantibodies with an affinity of more than 10 9 L / mol, IAA affinity in initial autoantibody sample>10 9 L / mol, it can identify almost all children with multiple autoantibodies or clinical diabetes, while IAA affinity <10 9 None of the children with ≥60% leucopenia developed T1DM within 6 years.

[0012] According to an optional embodiment, the concentration range of the carbon dots is between 0.1-1 mg / mL, preferably between 0.3-0.7 mg / mL.

[0013] According to an optional embodiment, the thickness of the dopamine is 2-8 nm, preferably between 3-6 mm.

[0014] According to an optional embodiment, the particle size of the fluorescent nanoparticles is between 20-100 nm, preferably between 50-80 nm.

[0015] According to an optional embodiment, the single-stranded DNA sequence consisting of 15-30 nucleotides includes 5'-GGC CAGCTG TGC GGG TTT-3', 5'-GGC CAG CTG TGC GGG CAG-3', 5'-GGC CAG CTG TGC GGG TTTGAA-3', 5'-GGC CAG CTG TGC GGG TTT GAG-3', 5'-CCG GAC CTG AGA GAA GCC ATT GGG-3', 5'-CCG GAC CTG AGA GAA GCC ATT TCC-3', 5'-CCG GAC CTG AGA GAA GCC ATT GGGGGC-3' or 5'-CCG GAC CTG AGA GAA GCC ATT TCC GGC-3', but is not limited to these nucleotide combinations.

[0016] According to an optional embodiment, the aptamer molecular imprint is 5'-GGC CAG CTG TGC GGG TTTGAG-3', 5'-CCG GAC CTG AGA GAA GCC ATT TCC-3' or 5'-CCG GAC CTG AGA GAA GCC ATTTCC GGC-3', the amino group modified by the aptamer can be coupled with the fluorescent magnetic nanomaterial through a coordination bond, and the immobilization amount is preferably 2-3mmoL / g.

[0017] According to an optional embodiment, the amino-functionalized polyethylene glycols of different molecular weights include H2N-PEG-NHS-1000, H2N-PEG-NHS-2000, H2N-PEG-NHS-3400, H2N-PEG-NHS-5000 or H2N-PEG-NHS-10000, but are not limited to polyethylene glycols of these molecular weights.

[0018] According to an optional embodiment, the polyethylene glycol is H2N-PEG-NHS-1000, H2N-PEG-NHS-3400 or H2N-PEG-NHS-10000, and the grafting amount is preferably 4.8-5.5 mmoL / g.

[0019] In order to achieve the second objective above, the present invention provides a method for preparing a molecular imprinted fluorescent sensor as described above, comprising the following steps:

[0020] Step 1: Fluorescent nanoparticles of magnetically responsive carbon dots (Fe3O4@CDs@PDA-Zn 2+ ) synthesis;

[0021] Step 1.1: Synthesize magnetic Fe3O4 nanoparticles of different particle sizes using a hydrothermal method.

[0022] Ferric chloride hexahydrate and anhydrous sodium ethanol are dispersed in ethylene glycol respectively, mixed evenly after being completely dissolved, and transferred to a hydrothermal reactor for hydrothermal heating. The reaction system is subjected to magnetic separation and washing to obtain Fe3O4 magnetic nanoparticles.

[0023] Step 1.2: Magnetic Fe3O4 nanoparticles are loaded with fluorescent quantum dots.

[0024] Carbon dots CDs with fluorescent light source were added into the magnetic microsphere solution and mechanically stirred for 2 hours. After the reaction, the solution was washed with pure water twice to obtain Fe3O4@CDs nanoparticles.

[0025] Step 1.3: Surface modification with polydopamine layer

[0026] The Fe3O4@CD nanoparticles were redispersed in 20 mL of NaOH aqueous solution, and 5 mL of dopamine solution was added under mechanical stirring conditions. The reaction was allowed to react overnight at room temperature, and a thin layer of polydopamine was coated on the surface of Fe3O4@CDs. After magnetic separation, the nanoparticles were washed with ethanol and pure water until neutral. The prepared nanoparticles were labeled as Fe3O4@CDs@PDA.

[0027] Step 1.4, Fe3O4@CDs@PDA-Zn 2+ Preparation

[0028] The rich functional groups contained in polydopamine can form coordination bonds with metal ions. The above Fe3O4@CDs@PDA nanoparticles were ultrasonically dispersed in 10mL Zn(NO3)2 solution and reacted for 10h at room temperature. After the reaction, magnetic separation was performed to obtain Fe3O4@CDs@PDA-Zn 2+ .

[0029] Step 2: Preparation of aptamer molecularly imprinted magnetic nanoparticles

[0030] Weigh the modified Zn 2+ Nanoparticles Fe3O4@CDs@PDA-Zn 2+ , and the amino-modified aptamer were added to PBS (10 mM, pH 7.0, containing 0.5 M NaCl) and reacted at room temperature for 2 h. After the reaction, the aptamer was washed twice with pure water to remove the unfixed template aptamer and obtain Fe3O4@CDs@Apt.

[0031] Disperse the above Fe3O4@CDs@Apt (about 500 mg) in 1 g 10 mL IAA +After mechanical stirring for 1 h in a PBS solution without DNA hydrolase, the mixture was washed twice with pure water and dispersed in 20 mL of Tris buffer. Dopamine (40 mg) was then added and the reaction was continued at room temperature for 3 h. After the reaction was completed, 0.1 M EDTA-2Na + The solution was eluted twice to allow the template molecules to be effectively eluted. The prepared imprinted nanoparticles were labeled as Fe3O4@CDs@Apt@MIP.

[0032] Step 3. PEGylation of Imprinted Polymer

[0033] The imprinted nanoparticles were dispersed in 1.5 mL of PEG-NHS, and then 40 μL of 11 μL / mL triethylamine solution (dissolved in ethanol) was added to the reaction system. The reaction was stirred at room temperature for 10 h to obtain high affinity insulin autoantibodies (IAA + ) Molecular imprinting fluorescence sensor with high sensitivity detection.

[0034] The molecular imprinting fluorescent sensor for detecting insulin autoantibodies and the preparation method provided by the present invention have the following technical effects:

[0035] High affinity IAA + It is associated with the appearance of multiple autoantibodies and the progression of clinical disease, while low-affinity IAA - None of the children developed T1DM within 6 years. However, most of the current methods for detecting IAA are qualitative or semi-quantitative, lacking specific antibody concentrations and unable to accurately predict the severity of IAA. + 、IAA - Combining quantum dots with aptamer molecular imprinting technology (MIP) to prepare fluorescence-responsive molecular imprinting polymers, and using a simple post-modification PEGylation method to increase anti-fouling ability, it can simultaneously have the high sensitivity of quantum dot fluorescence detection and the high specificity of molecular imprinting polymers, which is a promising method for developing high-sensitivity IAA + Testing provides a new approach.

[0036] The molecular imprinted fluorescent sensor and preparation method, with the help of the strategy of directional aptamer imprinting and post-imprinting modified polyethylene glycol, takes the aptamer that can specifically recognize the IAA antibody related to type 1 diabetes (T1DM) as a template, uses the method of coordination fixed template, takes dopamine as the functional monomer, and prepares the fluorescent responsive imprinted nanoparticles based on fluorescent quantum dots by self-polymerization method, and grafts PEG on the surface of the prepared fluorescent imprinted material by a simple post-modification polyethylene glycolization method, thereby achieving IAA + The molecular imprinting fluorescence sensor is used to detect IAA in a complex blood environment.+ Quantitative detection, especially in the case of individuals with a single autoantibody positive, can not only reduce the false positive rate and improve disease prediction, but also provide a more accurate basis for risk prediction screening, classification, efficacy evaluation and treatment guidance for early diabetes, and provide important support for personalized diagnosis and treatment of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 This is the schematic diagram of the preparation of molecular imprinted fluorescence sensor;

[0039] Figure 2 yes Figure 1 Fluorescence spectrum of molecular imprinted fluorescent sensor;

[0040] Figure 3 yes Figure 1 Molecularly imprinted fluorescent sensor for detecting different concentrations of IAA in serum + Calibration curve of fluorescence response. DETAILED DESCRIPTION

[0041] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0042] The molecular imprinted fluorescence sensor and the preparation method of the present invention are described in detail below with reference to specific Examples 1-6.

[0043] Embodiment 1:

[0044] (1) Synthesis of Fe3O4

[0045] Disperse 1.3g of ferric chloride hexahydrate and 5g of anhydrous sodium ethanol in 40mL of ethylene glycol, mix them evenly after they are completely dissolved, continue stirring for 1h, transfer them to a hydrothermal reactor, heat at 200℃ for 8h, and obtain Fe3O4 magnetic nanoparticles after magnetic separation and washing of the reaction system. The average particle size is 50nm.

[0046] (2) Preparation of green fluorescent carbon dots CDs

[0047] Weigh 5.26 g of sodium citrate into a 100 mL beaker, add 50 mL of ultrapure water and sonicate until completely dissolved, transfer it to a high-pressure reactor after cooling, add 1.68 mL of ethylenediamine, react at 150 ° C for 5 h, and after the reaction is completed, cool it to room temperature and store it at 4 ° C.

[0048] (3) Synthesis of Fe3O4@CDs

[0049] 0.8 g Fe3O4 was added into 20 mL 0.3 mg / mL carbon dot CDs solution with fluorescent light source, and mechanically stirred for 2 h. After the reaction, the solution was washed with pure water twice to obtain Fe3O4@CDs nanoparticles.

[0050] (4) Preparation of Fe3O4@CDs@PDA

[0051] 0.5g Fe3O4@CD nanoparticles were redispersed in 20mL NaOH aqueous solution, and 5mL dopamine solution was added under mechanical stirring. The reaction was allowed to proceed overnight at room temperature. A thin layer of polydopamine was coated on the surface of Fe3O4@CDs. After magnetic separation, the nanoparticles were washed with ethanol and pure water until neutral. The prepared nanoparticles were labeled as Fe3O4@CDs@PDA. The thickness of dopamine was 4.5nm.

[0052] (5)Fe3O4@CDs@PDA-Zn 2+ Preparation

[0053] The Fe3O4@CDs@PDA nanoparticles were ultrasonically dispersed in 10 mL of 0.1 mmol / L Zn(NO3)2 solution and reacted for 10 h at room temperature. After the reaction, magnetic separation was performed to obtain Fe3O4@CDs@PDA-Zn 2+ .

[0054] (6) Preparation of Fe3O4@CDs@Apt

[0055] 0.3 mmol of amino-modified aptamer 5'-CCG GAC CTG AGA GAA GCC ATT TCC-3' was dissolved in 20 mL of PBS solution without DNA hydrolase, and 0.1 g of Fe3O4@CDs@PDA-Zn 2+ , mechanically stirred at room temperature for 2 h. After the reaction, the mixture was washed twice with pure water to remove the unfixed template aptamers.

[0056] (7) Preparation of Fe3O4@CDs@Apt@MIP

[0057] Disperse the above Fe3O4@CDs@Apt (about 500 mg) in 1 g 10 mL IAA + After mechanical stirring for 1 h in a PBS solution without DNA hydrolase, the mixture was washed twice with pure water and dispersed in 20 mL of Tris buffer. Dopamine (40 mg) was then added and the reaction was continued at room temperature for 3 h. After the reaction was completed, 0.1 M EDTA-2Na + The solution was eluted twice to allow the template molecules to be effectively eluted. The prepared imprinted nanoparticles were labeled as Fe3O4@CDs@Apt@MIP.

[0058] (8) PEGylation of Imprinted Polymers

[0059] 100 mg of imprinted nanoparticles were dispersed in 1.5 mL of 0.2 g of PEG-NHS-1000, and then 40 μL of 11 μL / mL triethylamine solution (dissolved in ethanol) was added to the reaction system. The mixture was stirred for reaction at room temperature for 10 h, washed twice with pure water, and dried under vacuum at 60 °C for 12 h to obtain PEGylated imprinted nanoparticles, which were labeled as Fe3O4@CDs@MIP-PEG-1.

[0060] Embodiment 2:

[0061] (1) Synthesis of Fe3O4

[0062] Disperse 2.6g of ferric chloride hexahydrate and 10g of anhydrous sodium ethanol in 40mL of ethylene glycol, mix them evenly after they are completely dissolved, continue stirring for 1h, transfer them to a hydrothermal reactor, heat at 200℃ for 8h, and wash the reaction system by magnetic separation to obtain Fe3O4 magnetic nanoparticles with an average particle size of 30nm.

[0063] (2) Preparation of green fluorescent carbon dots CDs

[0064] Weigh 5.26 g of sodium citrate into a 100 mL beaker, add 50 mL of ultrapure water and sonicate until completely dissolved, transfer it to a high-pressure reactor after cooling, add 1.68 mL of ethylenediamine, react at 150 ° C for 5 h, and after the reaction is completed, cool it to room temperature and store it at 4 ° C.

[0065] (3) Synthesis of Fe3O4@CDs

[0066] 0.8 g of Fe3O4 was added into 20 mL of 0.65 mg / mL carbon dot CDs solution with fluorescent light source, and mechanically stirred for 2 h. After the reaction, the solution was washed with pure water twice to obtain Fe3O4@CDs nanoparticles.

[0067] (4) Preparation of Fe3O4@CDs@PDA

[0068] 0.5g Fe3O4@CD nanoparticles were redispersed in 20mL NaOH aqueous solution, 3.5mL dopamine solution was added under mechanical stirring, and the reaction was allowed to proceed overnight at room temperature. A thin layer of polydopamine was coated on the surface of Fe3O4@CDs, and the nanoparticles were separated by magnetic separation and washed with ethanol and pure water to neutrality. The prepared nanoparticles were labeled as Fe3O4@CDs@PDA. The thickness of dopamine was 3nm.

[0069] (5)Fe3O4@CDs@PDA-Zn 2+ Preparation

[0070] The Fe3O4@CDs@PDA nanoparticles were ultrasonically dispersed in 10 mL of 0.1 mmol / L Zn(NO3)2 solution and reacted for 10 h at room temperature. After the reaction, magnetic separation was performed to obtain Fe3O4@CDs@PDA-Zn 2+ .

[0071] (6) Preparation of Fe3O4@CDs@Apt

[0072] 0.3 mmol of amino-modified aptamer 5'-GGC CAG CTG TGC GGG TTT GAG-3' was dissolved in 20 mL of PBS solution without DNA hydrolase, and 0.1 g of Fe3O4@CDs@PDA-Zn 2+ , mechanically stirred at room temperature for 2 h. After the reaction, the mixture was washed twice with pure water to remove the unfixed template aptamers.

[0073] (7) Preparation of Fe3O4@CDs@Apt@MIP

[0074] Disperse the above Fe3O4@CDs@Apt (about 500 mg) in 1 g 10 mL IAA + After mechanical stirring for 1 h in a PBS solution without DNA hydrolase, the mixture was washed twice with pure water and dispersed in 20 mL of Tris buffer. Dopamine (40 mg) was then added and the reaction was continued at room temperature for 3 h. After the reaction was completed, 0.1 M EDTA-2Na + The solution was eluted twice to allow the template molecules to be effectively eluted. The prepared imprinted nanoparticles were labeled as Fe3O4@CDs@Apt@MIP.

[0075] (8) PEGylation of Imprinted Polymers

[0076] 100 mg of imprinted nanoparticles were dispersed in 1.5 mL of 0.2 g of PEG-NHS-10000, and then 40 μL of 11 μL / mL triethylamine solution (dissolved in ethanol) was added to the reaction system. The mixture was stirred for 10 h at room temperature, washed twice with pure water, and dried under vacuum at 60 °C for 12 h to obtain PEGylated imprinted nanoparticles, which were labeled as Fe3O4@CDs@MIP-PEG-2.

[0077] Example 3

[0078] (1) Synthesis of Fe3O4

[0079] Disperse 0.65g of ferric chloride hexahydrate and 2.5g of anhydrous sodium ethanol in 40mL of ethylene glycol, mix them evenly after they are completely dissolved, continue stirring for 1h, transfer them to a hydrothermal reactor, heat at 200℃ for 8h, and obtain Fe3O4 magnetic nanoparticles after magnetic separation and washing of the reaction system. The average particle size is 80nm.

[0080] (2) Preparation of green fluorescent carbon dots CDs

[0081] Weigh 5.26 g of sodium citrate into a 100 mL beaker, add 50 mL of ultrapure water and sonicate until completely dissolved, transfer it to a high-pressure reactor after cooling, add 1.68 mL of ethylenediamine, react at 150 ° C for 5 h, and after the reaction is completed, cool it to room temperature and store it at 4 ° C.

[0082] (3) Synthesis of Fe3O4@CDs

[0083] 0.8 g Fe3O4 was added into 20 mL 0.5 mg / mL carbon dot CDs solution with fluorescent light source, and mechanically stirred for 2 h. After the reaction, the solution was washed with pure water twice to obtain Fe3O4@CDs nanoparticles.

[0084] (4) Preparation of Fe3O4@CDs@PDA

[0085] 0.5g Fe3O4@CD nanoparticles were redispersed in 20mL NaOH aqueous solution, and 6.5mL dopamine solution was added under mechanical stirring conditions. The reaction was allowed to proceed overnight at room temperature. A thin layer of polydopamine was coated on the surface of Fe3O4@CDs. After magnetic separation, the nanoparticles were washed with ethanol and pure water to neutrality. The prepared nanoparticles were labeled as Fe3O4@CDs@PDA. The thickness of dopamine was 8nm.

[0086] (5)Fe3O4@CDs@PDA-Zn 2+ Preparation

[0087] The Fe3O4@CDs@PDA nanoparticles were ultrasonically dispersed in 10 mL of 0.1 mmol / L Zn(NO3)2 solution and reacted for 10 h at room temperature. After the reaction, magnetic separation was performed to obtain Fe3O4@CDs@PDA-Zn 2+ .

[0088] (6) Preparation of Fe3O4@CDs@Apt

[0089] 0.25 mmol of amino-modified aptamer 5'-CCG GAC CTG AGA GAA GCC ATT TCC GGC-3' was dissolved in 20 mL of PBS solution without DNA hydrolase, and 0.1 g of Fe3O4@CDs@PDA-Zn 2+ , mechanically stirred at room temperature for 2 h. After the reaction, the mixture was washed twice with pure water to remove the unfixed template aptamers.

[0090] (7) Preparation of Fe3O4@CDs@Apt@MIP

[0091] Disperse the above Fe3O4@CDs@Apt (about 500 mg) in 1 g 10 mL IAA + After mechanical stirring for 1 h in a PBS solution without DNA hydrolase, the mixture was washed twice with pure water and dispersed in 20 mL of Tris buffer. Dopamine (40 mg) was then added and the reaction was continued at room temperature for 3 h. After the reaction was completed, 0.1 M EDTA-2Na + The solution was eluted twice to allow the template molecules to be effectively eluted. The prepared imprinted nanoparticles were labeled as Fe3O4@CDs@Apt@MIP.

[0092] (8) PEGylation of Imprinted Polymers

[0093] 100 mg of imprinted nanoparticles were dispersed in 1.5 mL of 0.2 g of PEG-NHS-3400, and then 40 μL of 11 μL / mL triethylamine solution (dissolved in ethanol) was added to the reaction system. The mixture was stirred for 10 h at room temperature, washed twice with pure water, and dried under vacuum at 60 °C for 12 h to obtain PEGylated imprinted nanoparticles, which were labeled as Fe3O4@CDs@MIP-PEG-3.

[0094] Example 4

[0095] (1) Synthesis of Fe3O4

[0096] Disperse 1.3g of ferric chloride hexahydrate and 5g of anhydrous sodium ethanol in 40mL of ethylene glycol, mix them evenly after they are completely dissolved, continue stirring for 1h, transfer them to a hydrothermal reactor, heat at 200℃ for 12h, and wash the reaction system by magnetic separation to obtain Fe3O4 magnetic nanoparticles with an average particle size of 60nm.

[0097] (2) Preparation of green fluorescent carbon dots CDs

[0098] Weigh 5.26 g of sodium citrate into a 100 mL beaker, add 50 mL of ultrapure water and sonicate until completely dissolved, transfer it to a high-pressure reactor after cooling, add 1.68 mL of ethylenediamine, react at 150 ° C for 5 h, and after the reaction is completed, cool it to room temperature and store it at 4 ° C.

[0099] (3) Synthesis of Fe3O4@CDs

[0100] 0.8 g Fe3O4 was added into 20 mL 0.5 mg / mL carbon dot CDs solution with fluorescent light source, and mechanically stirred for 2 h. After the reaction, the solution was washed with pure water twice to obtain Fe3O4@CDs nanoparticles.

[0101] (4) Preparation of Fe3O4@CDs@PDA

[0102] 0.5g Fe3O4@CD nanoparticles were redispersed in 20mL NaOH aqueous solution, and 6.5mL dopamine solution was added under mechanical stirring conditions. The reaction was allowed to proceed overnight at room temperature. A thin layer of polydopamine was coated on the surface of Fe3O4@CDs. After magnetic separation, the nanoparticles were washed with ethanol and pure water to neutrality. The prepared nanoparticles were labeled as Fe3O4@CDs@PDA. The thickness of dopamine was 8nm.

[0103] (5)Fe3O4@CDs@PDA-Zn 2+ Preparation

[0104] The Fe3O4@CDs@PDA nanoparticles were ultrasonically dispersed in 10 mL of 0.1 mmol / L Zn(NO3)2 solution and reacted for 10 h at room temperature. After the reaction, magnetic separation was performed to obtain Fe3O4@CDs@PDA-Zn 2+ .

[0105] (6) Synthesis of Fe3O4@CDs@Apt

[0106] 0.3 mmol of amino-modified aptamer 5'-CCG GAC CTG AGA GAA GCC ATT TCC GGC-3' was dissolved in 20 mL of PBS solution without DNA hydrolase, and 0.1 g of Fe3O4@CDs@PDA-Zn 2+ , mechanically stirred at room temperature for 2 h. After the reaction, the mixture was washed twice with pure water to remove the unfixed aptamer, and Fe3O4@CDs@Apt was obtained.

[0107] (7) Preparation of Fe3O4@CDs@Apt@MIP

[0108] Disperse the above Fe3O4@CDs@Apt (about 500 mg) in 1 g 10 mL IAA + After mechanical stirring for 1 h in a PBS solution without DNA hydrolase, the mixture was washed twice with pure water and dispersed in 20 mL of Tris buffer. Dopamine (40 mg) was then added and the reaction was continued at room temperature for 3 h. After the reaction was completed, 0.1 M EDTA-2Na + The solution was eluted twice to allow the template molecules to be effectively eluted. The prepared imprinted nanoparticles were labeled as Fe3O4@CDs@Apt@MIP.

[0109] (8) PEGylation of Imprinted Polymers

[0110] 100 mg of imprinted nanoparticles were dispersed in 1.5 mL of 0.2 g of PEG-NHS-1000, and then 40 μL of 11 μL / mL triethylamine solution (dissolved in ethanol) was added to the reaction system. The mixture was stirred for 10 h at room temperature, washed twice with pure water, and dried under vacuum at 60 °C for 12 h to obtain PEGylated imprinted nanoparticles, which were labeled as Fe3O4@CDs@MIP-PEG-4.

[0111] Example 5

[0112] like Figure 1 and Figure 2 As shown, the fluorescence sensor is used to detect different concentrations of IAA in serum. + The fluorescence response and the corresponding calibration curve.

[0113] Disperse 20 mg Fe3O4@CDs@MIP-PEG-1 evenly in 100 mL of saline, take 1 mL of Fe3O4@CDs@MIP-PEG-1 dispersion and add it to 1 mL of IAA +In the standard solution, mix well. Select 2, 4, 8, 16, 32, 64, and 128nM for linear experiments. After the mixed solution is incubated at 25°C for 10 minutes, the fluorescence intensity is measured on a fluorescence spectrophotometer. The slit widths of the fluorescence test excitation and emission are 5nm and 10nm respectively. The excitation wavelength is 350nm, and the detection range of the emission spectrum is 360-600nm.

[0114] like Figure 1 As shown in Figure 2, the fluorescence intensity of Fe3O4@CDs@MIP-PEG increases with the increase of IAA + decreases with increasing concentration, such as Figure 2 As shown, F0 / F = 0.0065C + 1.0357 (R 2 =0.9939), and the detection limit (LOD) value was 0.25 nM.

[0115] Example 6 Recovery rate of fluorescence sensor in actual samples

[0116] Take 20 mg of each of the fluorescent sensors Fe3O4@CDs@MIP-PEG-1, Fe3O4@CDs@MIP-PEG-2, Fe3O4@CDs@MIP-PEG-3 and Fe3O4@CDs@MIP-PEG-4 in Examples 1, 2, 3 and 4, and evenly disperse them in 100 mL of normal saline. Then take 1 mL of the dispersion and add 1 mL of IAA with different calibration concentrations. + Human serum solution (IAA + The calibration concentrations were (0 nM, 10 nM, 25 nM and 50 nM), and after incubation at 25 °C for 10 min, the fluorescence intensity was measured on a fluorescence spectrophotometer. Figure 2 Calculate IAA by fitting the standard curve + The recovery rate of fluorescent nanoparticles in actual samples was evaluated.

[0117] Table 1

[0118]

[0119] From Table 1, we can see that the fluorescence sensor is sensitive to IAA at different concentrations. + The recovery rate is 94.2%-101%, which can be applied to the detection in actual biological samples.

[0120] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A molecular imprinting fluorescence sensor for detecting insulin autoantibodies, characterized in that: The molecularly imprinted fluorescent sensor is synthesized by using fluorescent nanoparticles with magnetically responsive carbon dots as carriers and by using an anti-fouling strategy of aptamer molecular imprinting and post-imprinting modified polyethylene glycol; The fluorescent nanoparticles are carbon dots as light sources, and Zn 2+ Fluorescent nanoparticles with magnetically responsive carbon dots were obtained by using dopamine as a ligand for surface modification. The aptamer molecular imprint is a single-stranded DNA sequence consisting of 15-30 nucleotides; The polyethylene glycol is amino-functionalized polyethylene glycol of different molecular weights.

2. The molecular imprinting fluorescent sensor for detecting insulin autoantibodies according to claim 1, characterized in that: The concentration of the carbon dots is in the range of 0.1-1 mg / mL.

3. The molecular imprinting fluorescent sensor for detecting insulin autoantibodies according to claim 1, characterized in that: The thickness of the dopamine is 2-8 nm.

4. The molecular imprinting fluorescent sensor for detecting insulin autoantibodies according to claim 1, characterized in that: The particle size of the fluorescent nanoparticles is between 20-100 nm.

5. The molecular imprinting fluorescent sensor for detecting insulin autoantibodies according to claim 1, characterized in that: The single-stranded DNA sequence consisting of 15-30 nucleotides includes 5'-GGC CAG CTG TGC GGG TTT-3', 5'-GGCCAG CTG TGC GGG CAG-3', 5'-GGC CAG CTG TGC GGG TTT GAA-3', 5'-GGC CAG CTG TGCGGG TTT GAG-3', 5'-CCG GAC CTG AGA GAA GCC ATT GGG-3', 5'-CCG GAC CTG AGA GAAGCC ATT TCC-3', 5'-CCG GAC CTG AGA GAA GCC ATT GGG GGC-3' or 5'-CCG GAC CTG AGAGAA GCC ATT TCC GGC-3', but is not limited to these nucleotide combinations.

6. The molecular imprinting fluorescent sensor for detecting insulin autoantibodies according to claim 5, characterized in that: The aptamer molecular imprint is 5'-GGC CAG CTG TGC GGG TTT GAG-3', 5'-CCG GAC CTG AGAGAA GCC ATT TCC-3' or 5'-CCG GAC CTG AGA GAA GCC ATT TCC GGC-3'.

7. The molecular imprinting fluorescent sensor for detecting insulin autoantibodies according to claim 1, characterized in that: The amino-functionalized polyethylene glycols of different molecular weights include H2N-PEG-NHS-1000, H2N-PEG-NHS-2000, H2N-PEG-NHS-3400, H2N-PEG-NHS-5000 or H2N-PEG-NHS-10000, but are not limited to polyethylene glycols of these molecular weights.

8. The molecular imprinting fluorescent sensor for detecting insulin autoantibodies according to claim 7, characterized in that: The polyethylene glycol is H2N-PEG-NHS-1000, H2N-PEG-NHS-3400 or H2N-PEG-NHS-10000.

9. A method for preparing a molecularly imprinted fluorescent sensor as claimed in any one of claim 18, characterized in that: The following steps are involved: Synthesis of fluorescent nanoparticles of magnetically responsive carbon dots; Preparation of aptamer molecularly imprinted magnetic nanoparticles; PEGylation of imprinted polymers.

10. The method for preparing a molecularly imprinted fluorescent sensor according to claim 9, characterized in that: The synthesis of the fluorescent nanoparticles of magnetically responsive carbon dots comprises: Magnetic Fe3O4 nanoparticles of different sizes were synthesized by hydrothermal method; Magnetic Fe3O4 nanoparticles loaded with fluorescent quantum dots; Surface modification with a polydopamine layer; Fe3O4@CDs@PDA-Zn 2+ Preparation.

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