Microalbuminuria detection method based on molecularly imprinted polymer and nano-enzyme

Abstract

The invention relates to the technical field of biomedical engineering, and discloses a urine microalbumin detection method based on a molecularly imprinted polymer and nano enzyme, which comprises the following steps: firstly, selecting C-terminal nonapeptide of ALB as an epitope, introducing lysine and combining with fructose to synthesize a saccharification epitope template; a microwell plate capable of specifically recognizing epitope imprinting of ALB is prepared through a boron affinity anchoring epitope controllable directional surface imprinting method, then boric acid modified ferroferric oxide nano-enzyme with high catalytic activity is synthesized and selectively combined with a sugar chain of ALB through the boron affinity effect, then a mobile phone detection technology is introduced, and the ALB epitope imprinting specific recognition microwell plate is obtained. And establishing a sandwich immune sandwich method based on the molecularly imprinted polymer and the nano-enzyme. According to the method, antibodies and biological enzymes are not needed, the detection cost is reduced, meanwhile, professionals and equipment are not needed, instant detection can be achieved in combination with a smart phone, the detection result is obtained rapidly, a rapid, specific, sensitive and convenient detection tool is provided for early diagnosis of CKD, and the method has important clinical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and in particular to a method for detecting urine microalbumin based on molecular imprinting polymer and nanozyme. Background Art

[0002] In recent years, the incidence of chronic kidney disease (CKD) has continued to rise, and 70% of kidney disease patients have entered the late stage when they first visit the doctor. CKD poses a great threat to the patient's cardiovascular, metabolic, endocrine, etc., and may even lead to death. Therefore, early diagnosis of kidney disease is particularly important. As an important means of disease diagnosis, the accuracy and efficiency of biomarker detection directly affect the treatment effect and survival rate of patients. Albumin (ALB) is a water-soluble globular glycoprotein that accounts for about half of the total serum protein. When the kidneys are healthy, ALB will remain in the blood, and when there is a problem with kidney function, it will leak into the urine through the kidney vascular membrane. Therefore, it is usually used as a biomarker to predict kidney damage. In normal individuals, the ALB content in 24h urine is <30mg, and when the level rises to 30-300mg, it may indicate the risk of renal failure. At present, ELISA, as the most commonly used detection method for biomarkers, has been widely used in clinical practice, in which antibodies and enzymes play a vital role. However, the preparation of antibodies is complex, the screening cycle is long, the price is high, and the storage is difficult. At the same time, the enzyme is unstable and easily inactivated, which leads to problems with the stability and repeatability of the detection method. In addition, the operation process is complicated and requires professionals and equipment. Therefore, it is urgent to establish an enzyme-free, antibody-free, efficient, accurate, and convenient ALB detection method, which is of great significance for the early diagnosis of CKD.

[0003] Molecularly imprinted polymers (MIPs) are chemically synthesized materials that simulate antibody recognition of antigens. Due to the presence of nano-imprinted cavities that are highly complementary to the shape, size and functional groups of the template molecules, they have similar binding properties to the template molecules as antibodies. Compared with antibodies, MIPs have the advantages of simple preparation, stable structure, low price, and tolerance to various harsh environments. MIPs have been widely used in immunoassays, proteomics, disease treatment and other fields. However, the traditional method of imprinting proteins is overall imprinting, which can easily cause the protein conformation to change and cannot be recognized. In addition, the template is huge and difficult to remove, and only a small amount of effective imprinting cavities can be formed. In the face of the above problems, Rachkov and Minoura proposed epitope imprinting, which uses characteristic short peptides instead of the entire protein as a template for de-imprinting. Boric acid can reversibly bind to cis-dihydroxy compounds (such as glycoproteins and carbohydrate compounds) by adjusting pH. It has the characteristics of binding under alkaline conditions and dissociation under acidic conditions. It has become an important functional group for selective recognition and imprinting of cis-dihydroxy compounds. In recent years, by combining boron affinity imprinting and epitope imprinting, Liu et al. proposed boron affinity anchored epitope controllable directional surface imprinting, which is a simple, universal and efficient protein imprinting method. The prepared MIPs have the advantages of high specificity, strong affinity and high imprinting efficiency.

[0004] Nanozymes are a type of nanomaterials with biocatalytic functions. They not only have the advantages of nanomaterials such as stable structure, adjustable activity, diverse functions, and simple preparation, but also exhibit enzyme-like activity. They are often used in biochemical detection, disease treatment and other fields. Nanozymes can be divided into three categories according to the type of material: metal-based nanozymes, metal oxides or metal sulfides, and carbon-based nanozymes. These nanozymes have oxidoreductase activity, hydrolase activity or lyase activity, and have developed into substitutes for natural enzymes. Compared with biological enzymes, nanozymes have the advantages of good stability, low cost and easy large-scale preparation. Common nanozymes include Fe 3 O 4 MNPs, MoS 2 NPs, MOF (Co / 2Fe), etc. In recent years, MIPs have been applied to the detection of biomarkers by combining them with nanozymes. For example, molecular imprinting slides combined with molecular imprinting Janus gold nanoparticle nanozymes were used to construct a double molecular imprinting immunosandwich method for α 2 -Colorimetric detection of macroglobulin; magnetic titanium dioxide molecular imprinted particles combined with boric acid-modified Janus gold nanoparticle nanozymes to establish an immunosandwich method for the visual detection of transferrin.

[0005] However, these current detection methods still face many problems. For example, MIPs use the entire protein as a template for imprinting, which results in limited sites on the protein surface that can be used for enzyme labeling. More importantly, current detection methods require professional equipment, making it difficult to achieve instant detection. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for detecting urinary microalbumin based on molecular imprinting polymers and nanozymes, which utilizes the high specific recognition ability of molecular imprinting polymers and the catalytic activity of nanozymes to achieve efficient detection of albumin. By selecting the C-terminal nonapeptide of ALB as the epitope, a saccharification epitope template is prepared through saccharification treatment, and a molecular imprinting polymer with high affinity is prepared by a boron affinity anchored epitope controllable directional surface imprinting method. At the same time, a boric acid-modified ferroferric oxide nanozyme with high catalytic activity is synthesized, which selectively binds to the sugar chain of ALB through boron affinity, thereby achieving quantitative detection of urinary microalbumin.

[0007] The method for detecting urinary microalbumin based on molecular imprinting polymer and nanozyme provided by the present invention comprises the following steps:

[0008] S1. Preparation of molecular imprinted polymer: amino modification, boric acid modification, and epitope imprinting are performed on the microplate in sequence;

[0009] S2, preparing boronic acid modified nanozymes;

[0010] S3. Detection operation:

[0011] S3.1, mixing the pretreated urine sample with the prepared molecularly imprinted polymer to allow albumin to specifically bind to the molecularly imprinted polymer;

[0012] S3.2, adding boric acid-modified nanozymes to label the captured albumin;

[0013] S3.3, triggering the catalytic activity of the nanozyme by adding a substrate, so that the nanozyme catalyzes the substrate to produce a detectable signal (such as a color change), and the signal intensity is proportional to the albumin content in the sample;

[0014] S3.4. Use smartphone software to analyze the intensity of the color change and calculate the concentration of urine microalbumin using a preset algorithm;

[0015] S3.5. Based on the test results and combined with clinical guidelines, provide a basis for early diagnosis of chronic kidney disease;

[0016] S4. Verify specificity and stability.

[0017] Preferably, in S1, the specific steps of preparing the molecularly imprinted polymer include:

[0018] Preparation of amino-modified microplate: Concentrated nitric acid and concentrated sulfuric acid (v / v, 1:3) were mixed and added to the microplate, and then placed at room temperature and then washed with water until neutral, then dried, and then aired to room temperature, followed by adding 10% APTES aqueous solution, shaking at room temperature and then heating to solidify the amino group, and finally washed with distilled water and anhydrous ethanol three times each, and dried to obtain the amino-modified microplate;

[0019] Preparation of boric acid-modified microplate: Add 150 μL of 10 mg / mL FPBA and 150 μL of 10 mg / mL NaBH to the amino-modified microplate. 3 CN methanol solution, sealed and shaken at room temperature, washed with water and anhydrous ethanol three times each to obtain a boric acid-modified microplate;

[0020] Preparation of epitope-imprinted microplate: The above-mentioned boronic acid-modified microplate is subjected to operations of fixing glycosylated epitopes, directional imprinting, and clearing glycosylated epitopes to obtain an epitope-imprinted microplate.

[0021] Preferably, the synthesis steps of the nanozyme include preparing amino-modified ferrosoferric oxide nanoparticles by a solvothermal method and then modifying the boronic acid group.

[0022] Preferably, in S4, the specific test method is: using albumin as the target protein and other proteins as competitive proteins, detecting each protein separately, and verifying the specific recognition ability of the detection method for urinary microalbumin.

[0023] Preferably, in S4, the method of stability testing is: conducting a long-term stability test on the prepared imprinted polymer and nanozyme at room temperature to ensure long-term maintenance of the detection performance.

[0024] Preferably, the detection method is suitable for early diagnosis of chronic kidney disease.

[0025] Compared with the related art, the method for detecting urine microalbumin based on molecular imprinting polymer and nanozyme provided by the present invention has the following beneficial effects:

[0026] The present invention abandons the limitation of the traditional ELISA detection method that relies on expensive antibodies and unstable enzymes, and significantly reduces the detection cost and improves the convenience of detection through the dual recognition of molecular imprinting polymers and nanozymes;

[0027] The molecularly imprinted polymer prepared by boron affinity anchor epitope controllable directional surface imprinting method has a highly specific recognition ability for the C-terminal nonapeptide of urinary microalbumin. Combined with the boric acid-modified nanozyme with high catalytic activity, it achieves high-sensitivity detection of albumin with a wide linear detection range and a minimum detection limit as low as 0.3μg / mL.

[0028] The detection method of the present invention can maintain detection performance above 89.4% after being placed at room temperature for 6 months, which is significantly better than the traditional method, is easy to transport and store, and is suitable for long-term on-site detection and home detection;

[0029] Combined with smartphone software to analyze the intensity of color changes, rapid and instant test results can be obtained without the need for professional equipment and personnel, greatly simplifying the operation process and improving test efficiency;

[0030] The detection method of the present invention does not require the use of expensive antibodies and enzymes, has low material preparation costs, good stability, and significantly reduces transportation and storage costs;

[0031] In addition to being applicable to the early diagnosis of chronic kidney disease, the method of the present invention can also be extended to the detection of other glycoprotein biomarkers and has broad clinical application prospects;

[0032] The detection method of the present invention has a simple preparation process, a wide range of material sources, and is easy to achieve standardization and large-scale production, thus providing convenience for large-scale screening and disease management. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The schematic diagram of the preparation of the immunosandwich method based on MIPs and nanozymes and the instant detection of urinary microalbumin is shown in the present invention;

[0034] Figure 2 : SEM images of untreated microplate (a), epitope-imprinted microplate (b) and non-imprinted microplate (c);

[0035] Fe 3 O 4 MNPs@NH 2 (d) and Fe 3 O 4 TEM image of MNPs@BA (e);

[0036] Fe 3 O 4 MNPs@NH 2 and Fe 3 O 4 IR spectrum of MNPs@BA (f);

[0037] UV absorption spectra of different systems (g): (1) TMB+H 2 O 2 +Fe 3 O 4 MNPs@BA, (2)TMB+Fe 3 O 4 MNPs@BA, (3)TMB+H2 O 2 , (4) TMB;

[0038] Figure 3 H 2 O 2 (a) and Michaelis-Menten curves of TMB (b) and the corresponding H 2 O 2 (c) and Lineweaver-Burk double reciprocal plots of TMB (d);

[0039] Figure 4 Fe at different pH 3 O 4 Relative activity of MNPs@BA (a), Fe 3 O 4 (b) Relationship between the catalytic activity of MNPs@BA and time;

[0040] Figure 5 Fe in the pH range of 2 to 12 3 O 4 Relative activity of MNPs@BA (a), Fe in the range of 4 to 75 °C 3 O 4 Relative activity graph of MNPs@BA;

[0041] Figure 6 Comparison of the binding capacity of HRP to microplates with different modifications (a), Fe 3 O 4 Comparison of the binding ability of MNPs@BA to ALB and BSA;

[0042] Figure 7 The capture capacity of epitope-imprinted microplates prepared at different molar ratios of APTES / UPTES / IBTES / TEOS (a) and different imprinting times (b) for ALB and the corresponding IF values;

[0043] Figure 8 The extraction time of epitope-imprinted microplate was investigated within 10 to 60 min (a); Fe 3 O 4 The labeling time of MNPs@BA was investigated within 20 to 70 min (b);

[0044] Fig. 9 To investigate the specificity of five proteins based on the immunosandwich method of MIPs and nanozymes;

[0045] Fig.10 To investigate the stability of the immunosandwich method based on MIPs and nanozymes within 6 months;

[0046] Fig.11 Linear relationship diagram for smartphone-assisted MIPs- and nanozyme-based immunosandwich assay. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with the accompanying drawings and implementation modes.

[0048] In this paper, we prepared epitope-imprinted microplates and boric acid-modified Fe 3 O 4 MNPs(Fe 3 O 4 MNPs@BA) was used to construct an immunosandwich method based on MIPs and nanozymes for the instant detection of urinary microalbumin. Figure 1 As shown, the microplate was first modified with amino and boronic acid groups in sequence, and then the boron affinity anchored epitope controllable directional surface imprinting method was used to select the C-terminal nonapeptide of ALB as the epitope. A lysine was introduced into its C-terminus and combined with fructose to synthesize the glycosylated epitope template, which was then fixed on the boronic acid-modified microplate. Aminopropyltriethoxysilane (APTES), 3-ureidopropyltriethylthiosilane (UPTES) and isobutyltriethoxysilane (IBTES) were selected as functional monomers, and tetraethyl orthosilicate (TEOS) was used as a crosslinker for directional imprinting. The prepared epitope-imprinted microplate was used to specifically identify ALB. The Fe 3 O 4 MNPs@NH 2 , and then modified with boronic acid groups to obtain Fe 3 O 4 MNPs@BA was used to label the captured ALB and also had peroxidase activity, which could catalyze H 2 O 2 Oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) generates blue oxTMB. Finally, the color change intensity is analyzed by smartphone software to determine the content of ALB in urine. The method established in this study does not require antibodies and biological enzymes, but is based on the specificity of epitope-imprinted microplates and Fe 3 O 4 The dual recognition of boron affinity and selectivity of MNPs@BA ensures high specificity of detection. 3 O 4 MNPs@BA has catalytic activity. Due to the epitope imprinting strategy, ALB retains more binding sites and can label more nanozymes, thereby improving the sensitivity of detection. Combined with a smartphone, it can achieve instant detection, which has great application prospects in the early diagnosis of CKD.

[0049] Specifically, the method for detecting urinary microalbumin based on molecular imprinting polymer and nanozyme proposed in the present invention is operated by the following experimental steps:

[0050] 1. Experimental operation

[0051] 1.1 Preparation of amino-modified microplates

[0052] Concentrated nitric acid and concentrated sulfuric acid (v / v, 1:3) were fully mixed and added to the microplate, placed at room temperature for 30 minutes, then washed with distilled water until neutral, and then placed in a 50°C oven to dry to obtain an activated microplate. Then, aired to room temperature, 10% APTES aqueous solution was added to the microplate, shaken at room temperature for 2 hours, placed in a 62°C oven, and heated for 2 hours to solidify the amino group, and finally washed three times with distilled water and anhydrous ethanol, and then dried to obtain an amino-modified microplate.

[0053] 1.2 Preparation of Boronic Acid Modified Microplate

[0054] Add 150 μL of 10 mg / mL FPBA and 150 μL of 10 mg / mL NaBH to the amino-modified microplate. 3 The methanol solution of CN was sealed and shaken at room temperature for 24 h, and washed with water and anhydrous ethanol three times each to obtain a boric acid-modified microplate.

[0055] 1.3 Preparation of glutaraldehyde-modified microplates

[0056] 250 μL of 5% glutaraldehyde aqueous solution was added to the amino-modified microplate, and the microplate was washed 6 times with distilled water after being shaken at room temperature for 2 h, and then dried in an oven to obtain a glutaraldehyde-modified microplate.

[0057] 1.4 Preparation of microplates for epitope imprinting

[0058] (1) Immobilization of glycosylated epitopes: 200 μL of phosphate buffer (10 mM, pH 7.4) containing 2 μg / mL of the C-terminal glycosylated epitope of ALB was added to a boric acid-modified microplate. After incubation at room temperature for 2 h, the microplate was washed three times with phosphate buffer (10 mM, pH 7.4) to prepare a microplate bound with glycosylated epitopes.

[0059] (2) Directed blotting: 160 μL of an ethanol solution containing 4.5 μL of 28% ammonia and 10 μL of distilled water was added to the microplate to which the glycosylated epitope was bound, and then 40 μL of an ethanol solution of 0.5% APTES:UPTES:IBTES:TEOS with a molar ratio of 10:10:30:50 was added. After reacting for 50 min, the plate was washed three times with anhydrous ethanol.

[0060] (3) Elimination of glycosylated epitopes: Add 200 μL of acetonitrile: water: glacial acetic acid (v / v / v, 50:49:1) to the above microplate, shake at room temperature for 20 min, repeat the above experimental steps three times, then wash with water and anhydrous ethanol three times each, and finally dry at room temperature to obtain an epitope-imprinted microplate. The preparation process of the corresponding non-imprinted microplate is the same except that the C-terminal glycosylated epitope template of ALB is not added.

[0061] 1.5Fe 3 O 4 MNPs@NH 2 Preparation

[0062] 4.0g anhydrous sodium acetate, 2.0g FeCl 3 6H 2 O, 13.0 g 1,6-hexanediamine and 60 ml ethylene glycol were added to a 250 ml three-necked flask, and mechanically stirred at 300 rpm until the solid was completely dissolved. The solution was then transferred to a high-temperature and high-pressure reactor and reacted at 198 ° C for 6 h. After cooling to room temperature, the product was transferred to a beaker to obtain amino-modified Fe 3 O 4 Magnetic nanoparticles (Fe 3 O 4 MNPs@NH 2 ) After magnetic separation, the samples were washed three times with distilled water and anhydrous ethanol respectively, and finally dried in a 50°C oven overnight.

[0063] 1.6Fe 3 O 4 Preparation of MNPs@BA

[0064] In a 250 ml three-necked flask, add 0.2 g Fe 3 O 4 MNPs@NH 2 After ultrasonic dispersion with 80 ml of methanol, 0.4 g of NaBH 3 CN and 0.4 g FPBA were added to the above solution, and then mechanically stirred at 300 rpm for 24 h at room temperature. 3 O 4 After magnetic separation, MNPs@BA was washed three times with distilled water and anhydrous ethanol respectively, and finally dispersed in 20 ml phosphate buffer solution (10 mM, pH 7.4) for use.

[0065] 1.7 Optimization of monomer ratio

[0066] 200 μL of 0.1 mg / mL ALB in phosphate buffer (10 mM, pH 7.4) was added to each of the epitope-imprinted microplates prepared at different molar ratios of APTES / UPTES / IBTES / TEOS (5:5:30:60, 5:5:40:50, 10:10:30:50, 10:10:40:40, 15:15:30:40, 15:15:40:30), and after shaking at room temperature for 40 min, the microplates were washed three times with phosphate buffer (10 mM, pH 7.4). Then, 200 μL of 2 mg / mL Fe 3 O 4 MNPs@BA were shaken at room temperature for 40 min and then washed three times with phosphate buffer solution (10 mM, pH 7.4). Finally, TMB colorimetric solution (0.6 MH 2 O 2 , 0.4 mM TMB, pH 4), shaken at room temperature for 20 min, and the absorbance was read at 650 nm. The experimental procedure for non-blotted microplates was the same as above. The control experiment was the same as above except that ALB was not added to the extraction solution. The experiments were repeated three times.

[0067] 1.8 Optimization of blotting time

[0068] 200 μL of 0.1 mg / mL ALB in phosphate buffer (10 mM, pH 7.4) was added to each of the microplates prepared for epitope imprinting at different imprinting times (30, 40, 50, 60, 70, 80 min), shaken at room temperature for 40 min, and then washed three times with phosphate buffer (10 mM, pH 7.4). Then, 200 μL of 2 mg / mL Fe 3 O 4 MNPs@BA were shaken at room temperature for 40 min and then washed three times with phosphate buffer solution (10 mM, pH 7.4). Finally, TMB colorimetric solution (0.6 MH 2 O 2 , 0.4 mM TMB, pH 4), shaken at room temperature for 20 min, and the absorbance was read at 650 nm. The experimental procedure for non-blotted microplates was the same as above. The control experiment was the same as above except that ALB was not added to the extraction solution. The experiments were repeated three times.

[0069] 1.9Fe 3 O 4 Study on the oxidase-like activity and steady-state kinetics of MNPs@BA

[0070] 200 μL of 0.1 mg / mL ALB in phosphate buffer solution (10 mM, pH 7.4) was added to each of the glutaraldehyde-modified microplates, shaken at room temperature for 2 h, and then washed three times with phosphate buffer solution (10 mM, pH 7.4). 3 O 4 MNPs@BA were shaken at room temperature for 40 min and then washed three times with phosphate buffer solution (10 mM, pH 7.4). 2 O 2 , (0.1, 0.2, 0.4, 0.6, 0.8, 1 mM), TMB colorimetric solution with a pH of 4 was shaken at room temperature for 20 min, and the absorbance was read at a wavelength of 650 nm to investigate the Fe 3 O 4 Steady-state kinetic constants of MNPs@BA. All the steps of the control experiment were the same as above except that ALB was not added to the extraction solution. The experiments were repeated three times. 2 O 2 When Fe 3 O 4 The steady-state kinetic constants of MNPs@BA were investigated using (0.1, 0.2, 0.4, 0.6, 0.8, 1 M) H 2 O 2 The steps were the same except for the color developing solution of 0.4 mM TMB and pH 4.

[0071] 1.10Fe 3 O 4 Optimization of MNPs@BA catalytic conditions

[0072] Investigation of the pH value of the reaction system: 200 μL of 0.1 mg / mL ALB in phosphate buffer solution (10 mM, pH 7.4) was added to each microplate modified with glutaraldehyde, shaken at room temperature for 2 h, and then washed three times with phosphate buffer solution (10 mM, pH 7.4). Then 200 μL of 2 mg / mL Fe 3 O 4 MNPs@BA were shaken at room temperature for 40 min and then washed three times with phosphate buffer solution (10 mM, pH 7.4). 2 O 2, 0.4 mM TMB, pH 2, 3, 4, 5, 6, 7, 8, 9 color solution, shake at room temperature for 20 min, and read the absorbance value at 650 nm. The control experiment was the same as above except that ALB was not added to the extraction solution. The experiments were repeated three times.

[0073] Investigation of catalytic time: 200 μL of 0.1 mg / mL ALB in phosphate buffer solution (10 mM, pH 7.4) was added to each microplate modified with glutaraldehyde, shaken at room temperature for 2 h, and then washed three times with phosphate buffer solution (10 mM, pH 7.4). Then 200 μL of 2 mg / mL Fe 3 O 4 MNPs@BA were shaken at room temperature for 40 min and then washed three times with phosphate buffer solution (10 mM, pH 7.4). 200 μL of TMB colorimetric solution (0.6 MH 2 O 2 , 0.4 mM TMB, pH 4), shake at room temperature, and read the absorbance at 650 nm every 5 min. The control experiment was the same as above except that ALB was not added to the extraction solution. The experiment was repeated three times. 3 O 4 Stability investigation of MNPs@BA

[0074] For the investigation of pH stability: 200 μL of 0.1 mg / mL ALB in phosphate buffer solution (10 mM, pH 7.4) was added to each microplate modified with glutaraldehyde, shaken at room temperature for 2 h, and then washed three times with phosphate buffer solution (10 mM, pH 7.4). Then 200 μL of 2 mg / mL Fe 3 O 4 MNPs@BA (placed in an environment with pH values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 for 2 h) were shaken at room temperature for 40 min and then washed three times with phosphate buffer solution (10 mM, pH 7.4). Finally, TMB colorimetric solution (0.6 MH 2 O 2 , 0.4 mM TMB, pH 4), shaken at room temperature for 20 min, and the absorbance was read at 650 nm. The control experiment was the same as above except that ALB was not added to the extraction solution. The experiment was repeated three times.

[0075] Temperature stability test: 200 μL of 0.1 mg / mL ALB in phosphate buffer solution (10 mM, pH 7.4) was added to each microplate modified with glutaraldehyde, shaken at room temperature for 2 h, and then washed three times with phosphate buffer solution (10 mM, pH 7.4). Then 200 μL of 2 mg / mL Fe 3 O 4 MNPs@BA (placed at 4, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75°C for 2 h) were shaken at room temperature for 40 min and then washed three times with phosphate buffer solution (10 mM, pH 7.4). Finally, TMB colorimetric solution (0.6 MH 2 O 2 , 0.4 mM TMB, pH 4), shaken at room temperature for 20 min, and read the absorbance at 650 nm. The control experiment was the same as above except that ALB was not added to the extraction solution. The experiments were repeated three times.

[0076] 1.12 Investigation of boron affinity selectivity

[0077] Boron affinity selectivity of microplates: 200 μL of HRP phosphate buffer solution (pH 4.7 and 8.5) was added to each of the untreated microplates, amino-modified microplates, and boric acid-modified microplates. After shaking at room temperature for 30 minutes, the plates were washed three times with phosphate buffer solutions of the corresponding pH. Finally, 200 μL of TMB colorimetric solution was added to each of the above microplates. After shaking at room temperature for 15 minutes, the absorbance was read at a wavelength of 450 nm. All steps of the control experiment were the same as above except that HRP was not added to the extraction solution. The experiments were repeated three times.

[0078] Fe 3 O 4 Boron affinity selectivity of MNPs@BA: 200 μL of 0.1 mg / mL ALB and BSA in phosphate buffer solution (10 mM, pH 7.4) were added to the glutaraldehyde-modified microplate, respectively, and after shaking at room temperature for 2 h, the microplate was washed three times with phosphate buffer solution (10 mM, pH 7.4). Then, 200 μL of 2 mg / mL Fe 3 O 4 MNPs@BA were shaken at room temperature for 40 min and then washed three times with phosphate buffer solution (10 mM, pH 7.4). Finally, TMB colorimetric solution (0.6 MH 2 O 2, 0.4 mM TMB, pH 4), shaken at room temperature for 20 min, and the absorbance was read at 650 nm. The control experiment was the same as above except that no protein was added to the extraction solution. The experiment was repeated three times.

[0079] 1.13 Optimization of extraction time

[0080] 200 μL of 0.1 mg / mL ALB in phosphate buffer (10 mM, pH 7.4) was added to each well of the epitope-imprinted microplate, and the wells were shaken at room temperature for 10, 20, 30, 40, 50, and 60 min, respectively, and then washed three times with phosphate buffer (10 mM, pH 7.4). 3 O 4 MNPs@BA were shaken at room temperature for 40 min and then washed three times with phosphate buffer solution (10 mM, pH 7.4). Finally, TMB colorimetric solution (0.6 MH 2 O 2 , 0.4 mM TMB, pH 4), shaken at room temperature for 20 min, and the absorbance was read at 650 nm. The control experiment was the same as above except that ALB was not added to the extraction solution. The experiment was repeated three times.

[0081] 1.14 Optimization of marking time

[0082] 200 μL of 0.1 mg / mL ALB in phosphate buffer (10 mM, pH 7.4) was added to each well of the epitope-imprinted microplate, shaken at room temperature for 40 min, and then washed three times with phosphate buffer (10 mM, pH 7.4). 3 O 4 MNPs@BA were shaken at room temperature for 20, 30, 40, 50, 60, and 70 min, respectively, and then washed three times with phosphate buffer solution (10 mM, pH 7.4). Finally, TMB colorimetric solution (0.6 MH 2 O 2 , 0.4 mM TMB, pH 4), shaken at room temperature for 20 min, and the absorbance was read at 650 nm. The control experiment was the same as above except that ALB was not added to the extraction solution. The experiment was repeated three times.

[0083] 1.15 Specificity Study

[0084] 200 μL of 0.1 mg / mL phosphate buffer solution (10 mM, pH 7.4) of ALB, BSA, TRF, OVA, and LYZ were added to the microplate for epitope imprinting, respectively, and after shaking at room temperature for 40 min, the microplate was washed three times with phosphate buffer solution (10 mM, pH 7.4). 3 O 4 MNPs@BA were shaken at room temperature for 40 min and then washed three times with phosphate buffer solution (10 mM, pH 7.4). Finally, TMB colorimetric solution (0.6 MH 2 O 2 , 0.4 mM TMB, pH 4), shaken at room temperature for 20 min, and the absorbance was read at 650 nm. The control experiment was the same as above except that no protein was added to the extraction solution. The experiment was repeated three times.

[0085] 1.16 Stability Study

[0086] 200 μL of 0.1 mg / mL ALB in phosphate buffer (10 mM, pH 7.4) was added to each well of the epitope-imprinted microplate, shaken at room temperature for 40 min, and then washed three times with phosphate buffer (10 mM, pH 7.4). 3 O 4 MNPs@BA were shaken at room temperature for 40 min and then washed three times with phosphate buffer solution (10 mM, pH 7.4). Finally, TMB colorimetric solution (0.6 MH 2 O 2 , 0.4 mM TMB, pH 4), and after shaking at room temperature for 20 min, the absorbance was read at a wavelength of 650 nm. All steps of the control experiment were the same as above except that ALB was not added to the extraction solution. The experiments were repeated three times. 3 O 4 The above experiments were carried out on MNPs@BA every other month for six consecutive months.

[0087] 1.17 Practical Application

[0088] 200 μL of 24-hour urine sample was added to each well of the epitope-imprinted microplate, shaken at room temperature for 40 min, and then washed three times with phosphate buffer solution (10 mM, pH 7.4). Then, 200 μL of 2 mg / mL Fe 3 O 4MNPs@BA were shaken at room temperature for 40 min and then washed three times with phosphate buffer solution (10 mM, pH 7.4). Finally, TMB colorimetric solution (0.6 MH 2 O 2 , 0.4 mM TMB, pH 4), and after shaking at room temperature for 20 min, the color intensity was read by a smartphone. The control experiment was the same as above except that phosphate buffer (10 mM, pH 7.4) was used instead of urine samples. The experiments were repeated three times.

[0089] 2. Results Analysis

[0090] 2.1 Material characterization

[0091] To specifically identify ALB, we prepared an epitope-imprinted microplate, and to label ALB, we synthesized Fe 3 O 4 MNPs@BA. The morphology of the epitope-imprinted microplate was characterized by SEM. Figure 2 As shown in Figures ac, the surface of the untreated microplate is very flat, while the surface of the epitope-imprinted microplate is rough and uneven, and the non-imprinted microplate is relatively flat. This shows that an imprinting layer has been successfully formed on the surface of the epitope-imprinted microplate. 3 O 4 The morphology of MNPs@BA was characterized. Figure 2 de shown, Fe 3 O 4 MNPs@NH 2 , Fe 3 O 4 MNPs@BA are in the shape of flower clusters, with uniform size and good dispersibility, and the particle size is 150nm.

[0092] FTIR analysis results are as follows Figure 2 f, 570cm -1 A strong single peak appears at 1078cm, which is caused by the stretching vibration of the Fe-O bond. -1 The absorption peak at 1538cm is attributed to the stretching vibration of the CN bond. -1 3353cm -1 The absorption peaks at 2 These absorption peaks prove that the amino group is successfully modified on Fe 3 O 4 MNPs. 3 O 4 MNPs@NH 2 In comparison, Fe 3 O 4The infrared spectrum of MNPs@BA is at 1362 cm -1 The stretching vibration absorption peak of BO appears at , indicating that the boric acid group is successfully modified on the nanozyme.

[0093] In order to prove that Fe 3 O 4 MNPs@BA has peroxidase activity and selects TMB as the oxidation substrate. 2 O 2 The absorbance at wavelengths of 500 to 800 nm was measured in the presence and absence of Figure 2 g shows that the four reaction systems include TMB+H 2 O 2 +Fe 3 O 4 MNPs@BA,TMB+Fe 3 O 4 MNPs@BA, TMB+H 2 O 2 and TMB alone, the latter three systems all showed weak absorbance, while H 2 O 2 +TMB+Fe 3 O 4 The MNPs@BA system showed a strong absorbance at 650 nm, indicating that only Fe 3 O 4 In the presence of MNPs@BA, TMB can be absorbed by H 2 O 2 Oxidized to blue product oxTMB.

[0094] 2.2Fe 3 O 4 Steady-state kinetics of MNPs@BA

[0095] To further investigate Fe 3 O 4 The catalytic activity of MNPs@BA was investigated by using TMB and H 2 O 2 The kinetic parameters were investigated as reaction substrates. Figure 3 The Michaelis-Menten curve and the corresponding Lineweaver-Burk double reciprocal curve are shown. 3 O 4 MNPs@BA follows the Michaelis-Menten equation. Using the Lineweaver-Burk double reciprocal curve, TMB and H 2 O 2The relevant kinetic parameters Km were 0.27mM, 336.30mM, and Vmax were 4.33×10 -8 M.s -1 , 5.39×10 -8 M.s -1 These results are comparable to the catalytic performance of bioenzymes and nanozymes described in the literature (Table 1), indicating that Fe 3 O 4 MNPs@BA has high peroxidase activity.

[0096] Table 1. Fe 3 O 4 Comparison of Km and Vmax values ​​of MNPs@BA with biological enzymes and other nanozymes.

[0097]

[0098]

[0099] 2.3Fe 3 O 4 Optimization of MNPs@BA catalytic conditions

[0100] The pH value of the system will significantly affect the catalytic activity of the nanozyme, so the pH value of the system was investigated. Figure 4 As shown in a, when pH is 4, Fe 3 O 4 MNPs@BA obtained the maximum catalytic activity, and higher or lower pH affected the Fe 3 O 4 The catalytic effect of MNPs@BA. The catalytic time will also affect the catalytic efficiency, so the catalytic time was investigated. The results are as follows Figure 4 As shown in b, with the increase of catalytic time, the concentration of catalytic products continued to increase, but in order to save time and ensure that the absorbance intensity meets the detection requirements, 20 min was selected as the catalytic reaction time.

[0101] 2.4Fe 3 O 4 Stability of MNPs@BA

[0102] Good stability is one of the most important advantages of nanozymes compared to natural enzymes. 3 O 4 The stability of MNPs@BA was investigated, and the results were as follows Figure 5 As shown, Fe 3 O 4The relative activity of MNPs@BA varies little in the range of pH 2-12 and temperature 4-75°C, and is above 80%. 3 O 4 MNPs@BA exhibits good stability under acid, alkali and high temperature conditions, making it easy to store and use in various environments.

[0103] 2.5 Boron Affinity Selectivity

[0104] Directed blotting of immobilized glycosylated epitope templates on boronate-modified microplates, and Fe 3 O 4 The boric acid group in MNPs@BA plays a crucial role in the labeling of ALB, so it is necessary to investigate the boron affinity selectivity of the two materials. Glycoprotein HRP (pH values ​​of 4.7 and 8.5, respectively) was captured using an untreated microplate, an amino-modified microplate, and a boric acid-modified microplate. The results are shown in Figure 2. Figure 6 As shown in a, only the microplate modified with boric acid under alkaline conditions has good selectivity for HRP. This is because the boric acid group can bind to cis-dihydroxy compounds under alkaline conditions and dissociate under acidic conditions. Therefore, the microplate modified with boric acid under alkaline conditions can bind to HRP through boron affinity, thereby catalyzing the TMB color development reaction. The untreated and amino-modified microplates cannot capture HRP due to the lack of boric acid groups and can only bind a small amount of HRP through nonspecific adsorption. 3 O 4 MNPs@BA labeled glycoprotein ALB and non-glycoprotein BSA respectively. The results are shown in Figure 6 As shown in b, Fe 3 O 4 MNPs@BA has better selectivity for ALB due to the Fe 3 O 4 MNPs@BA can bind to the sugar chains on ALB through boron affinity, but cannot bind to BSA which lacks glycosylation sites. 3 O 4 MNPs have been successfully modified with boronic acid groups.

[0105] 2.6 Optimization of blotting conditions

[0106] In order to enhance the affinity of the epitope-imprinted microplate, APTES, UPTES, and IBTES were selected as functional monomers, which can generate electrostatic, hydrogen bonding, hydrophobic and other interactions with the ALB C-terminal epitope sequence, respectively. Although a larger functional monomer ratio can increase specific recognition sites, it will also increase nonspecific adsorption, greatly reducing the reliability of detection. Therefore, it is necessary to investigate the monomer ratio and optimize it according to the imprinting factor (IF), where IF is the ratio of the capture amount of the target analyte by the epitope-imprinted microplate and the non-imprinted microplate. IF is one of the most important parameters for evaluating imprinting performance. The larger the value, the better the imprinting effect. The results are shown in Figure 7 As shown in a, when the molar ratio of APTES / UPTES / IBTES / TEOS is 10:10:30:50, the IF value reaches the maximum.

[0107] The imprinting time determines the thickness of the imprinting layer. A too shallow imprinting layer will result in a lack of sufficient binding sites and complementary three-dimensional structures in the imprinting cavity, thus affecting the specificity of the microplate for epitope imprinting. A too deep imprinting layer will result in the template being embedded and unable to form an effective imprinting cavity. Therefore, it is necessary to examine the imprinting time. The results are as follows Figure 7 As shown in (b), with the increase of imprinting time, the adsorption amount of ALB by the epitope-imprinted microplate first increased and then decreased, while the adsorption amount of ALB by the non-imprinted microplate remained almost unchanged. When the imprinting time was 50 min, the IF value reached the maximum (8.0).

[0108] 2.7 Optimization of extraction time and labeling time

[0109] In order to obtain the strongest detection signal in the shortest operating time, the extraction time and Fe 3 O 4 The labeling time of MNPs@BA was investigated respectively. Figure 8 As shown in Figure 2, the binding capacity of epitope-imprinted microplate to ALB gradually increased with the increase of extraction time and reached equilibrium at 40 min. 3 O 4 The binding amount of MNPs@BA to ALB gradually increased with the increase of labeling time and reached equilibrium at 40 min. Therefore, 40 min was the optimal extraction time and labeling time.

[0110] 2.8 Specificity

[0111] The specificity of the method established in this study was investigated by selecting ALB as the target protein and BSA, OVA, LYZ, and TRF as the competing proteins, and the cross-reactivity was evaluated, where the cross-reactivity was the ratio of the adsorption amount of the competing protein to the target protein on the epitope-imprinted microplate. Fig. 9As shown in Figure 2, the method showed excellent specificity for ALB, but almost no recognition for competing proteins, with the maximum cross-reactivity of only 4.4%, which was mainly due to the epitope-imprinted microplate and Fe 3 O 4 Dual recognition effect of MNPs@BA.

[0112] 2.9 Stability

[0113] In this study, epitope-imprinted microplates were used instead of antibodies to detect Fe 3 O 4 MNPs@BA replaces biological enzymes, so the stability of the synthetic material is of great significance in the detection process, which can not only affect the recognition performance of the epitope-imprinted microplate and Fe 3 O 4 The catalytic activity of MNPs@BA can also affect the accuracy of the detection method. Therefore, the stability of the method established in this study was investigated by testing ALB every month for six months. Fig.10 As shown, this method has good stability, and its relative absorbance can still be maintained above 89.4% after 6 months, which is convenient for transportation and storage, providing a guarantee for subsequent home testing.

[0114] 2.10 Performance analysis of smartphone-assisted immunosandwich assay based on MIPs and nanozymes

[0115] With the continuous advancement of technology, smartphone cameras are becoming more and more accurate in color recognition and have been widely used in portable detection. The analysis was performed using the Microplate Reader application, which reads the color signal intensity. A standard curve was obtained by plotting the color intensity against the concentration of the target analyte. The concentration of ALB corresponding to a signal-to-noise ratio of 3:1 was the LOD, and the concentration of ALB corresponding to a signal-to-noise ratio of 10:1 was the LOQ. The results are shown in Fig.11 As shown in the figure, the linear equation for smartphone detection of ALB is y=0.0195x+9.2275, r=0.9977, and the linearity is good in the range of 2.5-1000μg / mL. The LOD is 0.3μg / ml and the LOQ is 1μg / ml. The results show that the detection method has high sensitivity.

[0116] 2.11 Actual Sample Application

[0117] The method established in this study was used to determine the ALB content in the 24-hour urine of CKD patients and compared with the test results of the ELISA kit. The results are shown in Table 2. The ALB content in the 24-hour urine of three CKD patients determined by the immunosandwich method based on MIPs and nanozymes was 19.6μg / mL, 84.2μg / mL, and 499.6μg / mL, respectively. The relative error with the ALB content determined by the ELISA kit was less than 5%, the sample recovery rate was 95.7% to 105.1%, and RSD ≤ 1.32% (n = 3). The above results show that this method is accurate and reliable and can be used for early diagnosis of CKD.

[0118] Table 2. Determination of ALB content in 24-h urine of CKD patients using the immunosandwich method based on MIPs and nanozymes (n=3).

[0119]

[0120] 2.12 Comparison with commercial ELISA kits

[0121] At present, ELISA kit is a classic method for detecting urine microalbumin in clinical practice. Therefore, the method established in this study was compared with ELISA. The results are shown in Table 3. This method has the advantages of fast detection speed, wide linear range, high sensitivity, etc., and does not require antibodies and biological enzymes, which reduces the cost of detection. In combination with smartphones, instant detection can be achieved. These results show that the method established in this study is an efficient, convenient and accurate method for detecting urine microalbumin, which has significant advantages in the early diagnosis of CKD.

[0122] Table 3. Performance comparison of the immunosandwich assay based on MIPs and nanozymes and commercial ELISA kits.

[0123]

[0124] Compared with the related art, the method for detecting urine microalbumin based on molecular imprinting polymer and nanozyme provided by the present invention has the following beneficial effects:

[0125] The present invention provides a method for detecting urinary microalbumin based on molecular imprinting polymer and nanozyme, by studying the use of epitope imprinted microplate to replace antibodies to specifically capture ALB, and using Fe 3 O 4 MNPs@BA replaced biological enzymes to label captured ALB and catalyzed TMB color development. Combined with smartphones, an immunosandwich method based on MIPs and nanozymes was constructed for instant detection of urinary microalbumin.

[0126] Compared with commercial ELISA kits, the method established in this study has the advantages of simple preparation, low price, good stability, high sensitivity, and wide linear range. It does not require professional personnel and equipment and can be easily extended to the detection of other glycoprotein biomarkers.

[0127] Therefore, this method provides a powerful detection tool for the early diagnosis of CKD and has important clinical application value.

[0128] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for detecting urinary microalbumin based on molecular imprinting polymer and nanozyme, characterized in that: The steps include: S1. Preparation of molecular imprinted polymer: amino modification, boric acid modification, and epitope imprinting are performed on the microplate in sequence; S2, preparing boronic acid modified nanozymes; S3. Detection operation: S3.1, mixing the pretreated urine sample with the prepared molecularly imprinted polymer to allow albumin to specifically bind to the molecularly imprinted polymer; S3.2, adding boric acid-modified nanozymes to label the captured albumin; S3.3, triggering the catalytic activity of the nanozyme by adding a substrate, so that the nanozyme catalyzes the substrate to produce a detectable signal (such as a color change), and the signal intensity is proportional to the albumin content in the sample; S3.

4. Use smartphone software to analyze the intensity of the color change and calculate the concentration of urine microalbumin using a preset algorithm; S3.

5. Based on the test results and combined with clinical guidelines, provide a basis for early diagnosis of chronic kidney disease; S4. Verify specificity and stability.

2. The method for detecting urinary microalbumin based on molecular imprinting polymer and nanozyme according to claim 1, characterized in that: In S1, the specific steps of preparing the molecularly imprinted polymer include: Preparation of amino-modified microplate: Concentrated nitric acid and concentrated sulfuric acid were mixed in a volume fraction ratio of 1:3 and added to the microplate, left at room temperature and then washed with water until neutral, then dried, and then aired to room temperature, followed by adding 10% APTES aqueous solution, shaking at room temperature and then heating to solidify the amino group, and finally washed with distilled water and anhydrous ethanol three times each, and dried to obtain the amino-modified microplate; Preparation of boronic acid-modified microplate: 150 μL of 10 mg / mL FPBA and 150 μL of 10 mg / mL NaBH3CN methanol solution were added to the amino-modified microplate, respectively, and the plate was sealed and shaken at room temperature, and then washed with water and anhydrous ethanol for 3 times each to obtain a boronic acid-modified microplate; Preparation of epitope-imprinted microplate: The above-mentioned boronic acid-modified microplate is subjected to operations of fixing glycosylated epitopes, directional imprinting, and clearing glycosylated epitopes to obtain an epitope-imprinted microplate.

3. The method for detecting urinary microalbumin based on molecular imprinting polymer and nanozyme according to claim 1, characterized in that: The synthesis steps of the nanozyme include preparing amino-modified ferroferric oxide nanoparticles by a solvothermal method and then modifying the boric acid group.

4. The method for detecting urinary microalbumin based on molecularly imprinted polymer and nanozyme according to claim 1, characterized in that: In S4, the specificity test method is: using albumin as the target protein and other proteins as competitive proteins, each protein is tested separately to verify the specific recognition ability of the detection method for urinary microalbumin.

5. The method for detecting urinary microalbumin based on molecular imprinting polymer and nanozyme according to claim 1, characterized in that: In S4, the method for stability testing is: conducting a long-term stability test on the prepared imprinted polymer and nanozyme at room temperature to ensure long-term maintenance of the detection performance.

6. The method for detecting urinary microalbumin based on molecular imprinting polymer and nanozyme according to claim 1, characterized in that: The detection method is suitable for early diagnosis of chronic kidney disease.

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