Arabinomannan molecularly imprinted electrochemical luminescence sensor as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of electrochemical luminescence biosensing, and particularly relates to a lipid arabinomannan molecularly imprinted electrochemical luminescence sensor as well as a preparation method and application thereof. The preparation method comprises the following steps: firstly, preparing a zirconium-hafnium bimetal organic framework nano luminescent material modified electrode as a nano luminescent substance, then taking lipid arabinomannan as a template molecule, taking acrylic acid and acrylamide as functional monomers, adding a cross-linking agent and a free radical initiator, carrying out thermal polymerization reaction, and then removing the template molecule by adopting an eluent to obtain the zirconium-hafnium bimetal organic framework nano luminescent material modified electrode. The molecularly imprinted electrochemiluminescence sensor capable of specifically recognizing lipid arabinomannan is obtained. The molecularly imprinted electrochemical luminescence sensor provided by the invention has the characteristics of strong affinity, high specificity, high sensitivity and high stability, can realize rapid detection of a target object in an actual clinical sample, proves the practicability of the method, and is suitable for in-vitro detection.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemiluminescent biosensors, and in particular relates to a lipoarabinomannan molecular imprinting electrochemiluminescent sensor and a preparation method and application thereof. Background Art

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis. Rapid and accurate identification of Mycobacterium tuberculosis is the most critical step in the effective prevention and treatment of tuberculosis. However, traditional detection techniques rely on sputum specimens, are invasive, have low sensitivity, are time-consuming, and require professional and complex operations, making them difficult to promote and use. At present, there is still a lack of highly sensitive, rapid, and low-cost detection methods that are generally suitable for the diagnosis and screening of tuberculosis patients. Lipoarabinomannan (LAM) is a lipopolysaccharide on the cell wall of Mycobacterium tuberculosis. It is present in the urine of tuberculosis patients and has an immunomodulatory effect. It plays a key role in the pathogenesis of Mycobacterium tuberculosis infection. Its concentration is related to the load of Mycobacterium tuberculosis and disease progression. Lipoarabinomannan is a diagnostic biomarker recommended by the World Health Organization for tuberculosis. It has a high prospect for clinical application and is of great detection value for tuberculosis patients who cannot obtain sputum.

[0003] Electrochemiluminescence (ECL) is an analytical chemical detection method based on electrochemically triggered light signals. It has the advantages of high sensitivity, strong controllability and low cost. However, most traditional ECL sensing strategies rely on antigen-antibody recognition, which usually has the problems of antigen-antibody inactivation and low antibody specificity, and ECL is easily affected by the environment and sample matrix effects. Molecular imprinting technology, as a polymer that can simulate the interaction between antigens and antibodies, can extract target analytes from various complex biological samples, thereby effectively eliminating the influence of matrix effects. Molecular imprinting polymer (MIP) is a chemically synthesized polymer material developed based on molecular imprinting technology. Compared with biological macromolecules such as antibodies and aptamers, MIP has the advantages of simple preparation, low cost and good stability. It is an effective and efficient method for molecular recognition. Therefore, it is of great practical significance to develop a simple, rapid, low-cost, sensitive and non-invasive electrochemiluminescence detection method for lipoarabinomannan based on MIP.

[0004] Molecular imprinting technology has some shortcomings in detecting lipoarabinomannan, such as limited sensitivity in complex sample detection and time-consuming detection process. The content of LAM in biological samples is usually low, especially in biological samples with a large number of interfering substances, which may be interfered with, making it difficult to achieve high-sensitivity detection of low-concentration LAM, resulting in reduced detection efficiency or inaccurate results. Summary of the invention

[0005] Based on this, in view of the shortcomings of the prior art, the present invention provides a lipoarabinomannan molecular imprinting electrochemiluminescence sensor and a detection method thereof. The sensor uses molecular imprinting polymers instead of antibody recognition, uses electrochemiluminescence signals as readout signals, and utilizes the advantages of electrochemiluminescence, which is simple, low cost, high sensitivity, and fast response speed, to perform rapid, sensitive, and highly selective detection and analysis of lipoarabinomannan in urine samples.

[0006] The objective of the present invention is achieved through the following technical solutions: The first aspect of the present invention provides a molecular imprinting electrochemiluminescence sensor of lipoarabinomannan, using an electrode with a zirconium-hafnium metal organic framework nanoluminescent material and imprinted with lipoarabinomannan as a working electrode, a platinum wire as a counter electrode, and Ag / AgCl as a reference electrode.

[0007] The second aspect of the present invention provides a method for preparing a working electrode of a molecularly imprinted electrochemiluminescent sensor of lipoarabinomannan, comprising the following steps: A. Preparation of zirconium-hafnium bimetallic organic framework nanoluminescent material modified electrode: A1. Disperse 9,10-anthracene acyl bis(benzoic acid), zirconium tetrachloride, hafnium tetrachloride and benzoic acid in N,N-dimethylformamide solvent, stir at 100 °C for 20 minutes to obtain a solid-liquid mixture, collect by centrifugation, wash and vacuum dry to obtain a zirconium-hafnium bimetallic organic framework material; A2. Weigh the zirconium-hafnium bimetallic organic framework nanomaterial, perform ultrasound in ionized water to form a 1 mg / mL dispersion, take 3-5 μL of it and drop it on the surface of the glassy carbon electrode, and after drying, obtain the zirconium-hafnium bimetallic organic framework nanoluminescent material modified electrode; B. Preparation of lipoarabinomannan molecular imprinting membrane on modified electrode: B1, dissolving the template molecule lipoarabinomannan and the functional monomer in acetonitrile solvent to obtain a prepolymerization solution a; B2, adding a crosslinking agent and a free initiator to the prepolymer solution a to obtain a mixed solution b; B3. Take 10-15 μl of the mixed solution b and drop it on the surface of the modified electrode obtained in step A. Place the electrode in an oven for thermal polymerization reaction. After the reaction, use an eluent to elute to remove the template molecules and obtain a lipoarabinomannan molecular imprinting membrane.

[0008] Furthermore, in step A1, the molar ratio of zirconium tetrachloride to hafnium tetrachloride is 3:(1-9), preferably 3:1.

[0009] Furthermore, in step B1, the functional monomers are acrylic acid and acrylamide in a molar ratio of 1:1, and the concentration of the functional monomers is 10-150 mM, preferably 60 mM; in step B2, the crosslinking agent and free radical initiator are ethylenediaminetetraacetic acid and ammonium persulfate, respectively.

[0010] Furthermore, in step B3, the temperature of the thermal polymerization reaction is 30-80°C, preferably 70°C.

[0011] Furthermore, in step B3, the eluent is a mixed solution of methanol and acetic acid, and the volume ratio of methanol to acetic acid is 9:(1~81); the specific steps of elution are: immersing the electrode in the eluent for 20~30 minutes, and then immersing it in deionized water for another 15~20 minutes.

[0012] The third aspect of the present invention provides a method for detecting lipoarabinomannan using a molecular imprinting electrochemiluminescence sensor, comprising the following steps: (1) sequentially immersing the prepared molecular imprinting electrochemiluminescence sensor in a series of concentrations of lipoarabinomannan standard solutions for incubation, then using a PBS solution containing 30 mM hydrogen peroxide as a detection base solution, and measuring the intensity of the electrochemiluminescence signal under the voltage condition of 0~+1.8V with a photomultiplier tube voltage of 800~900 V, and drawing a standard working curve; (2) selecting glucose, urea, bovine serum albumin, and maltose as interferents of lipoarabinomannan to evaluate the selectivity of the molecular imprinting electrochemiluminescence sensor; and (3) using the drawn standard working curve to detect the lipoarabinomannan content in the pre-treated analyte.

[0013] Furthermore, the incubation time in step (1) is 10 to 40 minutes, preferably 30 minutes.

[0014] Furthermore, in step (3), the analyte is a urine sample, and the pretreatment method thereof comprises the following steps: centrifuging the collected urine sample from the tuberculosis patient at 12000 r / min for 5 minutes, and collecting the supernatant for testing.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. Acrylic acid and acrylamide are used as bifunctional monomers for molecular imprinting to synthesize molecular imprinting polymers, which realize the rapid separation of target substances in complex matrices by molecular imprinting polymers. The immobilized template and bifunctional monomer molecular imprinting system realizes precise and controllable surface molecular imprinting with high specificity and high imprinting efficiency; 2. As the electrochemiluminescence substrate, the bimetallic organic framework significantly enhances the electrochemiluminescence signal compared with the single metal organic framework, achieving highly sensitive detection of the target; 3. The detection method provided by the present invention uses molecular imprinting polymers instead of expensive and difficult-to-preserve antibodies, and has the characteristics of low cost, high stability and high specificity. At the same time, the sensor has a wide linear detection range and high sensitivity. Analysis of urine samples shows that this detection method has potential application prospects in the field of tuberculosis diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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 embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0017] Figure 1 is a schematic diagram of molecular imprinting electrochemiluminescence sensor for detecting lipoarabinomannan; Figure 2 is a scanning electron microscope image of the zirconium-hafnium bimetallic organic framework material with a molar ratio of 3:1 in Example 1; Figure 3 This is a graph showing the intensity changes of electrochemiluminescence signals of the zirconium-hafnium bimetallic organic framework under different molar ratios of metal ions; Figure 4 is the difference in electrochemiluminescence signal intensity before and after elution of MIP and NIP formed by different functional monomer concentrations in Example 3; Figure 5 is the difference in electrochemiluminescence signal intensity of lipoarabinomannan before and after elution at different polymerization temperatures in Example 4; Figure 6 is the difference in electrochemiluminescence signal intensity before and after elution in PBS detection base solution of different pH in Example 5; Figure 7 is the electrochemical impedance diagram of different modified electrodes in Example 6; Figure 8 is the difference in electrochemiluminescent signal intensity after elution and rebinding of lipoarabinomannan at different incubation times in Example 7; Fig. 9 is an electrochemiluminescence intensity graph corresponding to the concentration range of 10 fg / mL-200 pg / mL of the target lipoarabinomannan detected in Example 8; Fig.10 is the quantitative analysis working curve corresponding to the concentration range of 10fg / mL-200pg / mL of the target lipoarabinomannan detected in Example 8; Fig.11 It is a selectivity test of the method for detecting lipoarabinomannan based on molecular imprinting electrochemiluminescence in Example 9; Fig.12 This is the actual urine sample test result in Example 10. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The specific examples described herein are only used to explain the present invention, rather than to limit the present invention. In the absence of conflict, the embodiments in this application and the features in the embodiments can be combined with each other. It should also be noted that, for ease of description, only the parts related to the present invention rather than all structures are shown in the accompanying drawings. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several modifications and improvements can also be made, which should also be regarded as belonging to the protection scope of the present invention.

[0019] Example

[0020] Example 1 This example provides the preparation of zirconium-hafnium bimetallic organic framework materials with different molar ratios: (1) Synthesis of zirconium-hafnium bimetallic organic framework materials with a molar ratio of 3:1: 20.9 mg of 9,10-anthracene acyl bis(benzoic acid), 5.24 mg of zirconium tetrachloride, 2.4 mg of hafnium tetrachloride, and 100 mg of benzoic acid were dispersed in 5 mL of N,N-dimethylformamide. The mixture was stirred at 100 °C for 20 minutes to obtain a solid-liquid mixture, which was collected by centrifugation, washed with N,N-dimethylformamide, and vacuum dried to obtain a zirconium-hafnium bimetallic organic framework material.

[0021] (2) Synthesis of zirconium-hafnium bimetallic organic framework materials with a molar ratio of 1:3: 20.9 mg of 9,10-anthracene acyl bis(benzoic acid), 1.75 mg of zirconium tetrachloride, 7.2 mg of hafnium tetrachloride, and 100 mg of benzoic acid were dispersed in 5 mL of N,N-dimethylformamide. The mixture was stirred at 100 °C for 20 minutes to obtain a solid-liquid mixture, which was collected by centrifugation, washed with N,N-dimethylformamide, and vacuum dried to obtain a zirconium-hafnium bimetallic organic framework material.

[0022] (3) Synthesis of zirconium-hafnium bimetallic organic framework materials with a molar ratio of 1:1: 20.9 mg of 9,10-anthracene acyl bis(benzoic acid), 3.5 mg of zirconium tetrachloride, 4.8 mg of hafnium tetrachloride, and 100 mg of benzoic acid were dispersed in 5 mL of N,N-dimethylformamide. The mixture was stirred at 100 °C for 20 minutes to obtain a solid-liquid mixture, which was collected by centrifugation, washed with N,N-dimethylformamide, and vacuum dried to obtain a zirconium-hafnium bimetallic organic framework material.

[0023] The three zirconium-hafnium bimetallic organic framework materials with different molar ratios prepared above were dispersed in deionized water to form a 1 mg / mL solution. 4 μL of the solution was dropped on the surface of a clean glassy carbon electrode and dried. A PBS buffer solution containing 30 mM hydrogen peroxide (pH = 7.4) was used as the detection base solution. The scanning voltage was 0~+1.8V, and the photomultiplier tube voltage was 900 V. The electrochemiluminescence intensity of the zirconium-hafnium bimetallic organic framework nanoluminescent materials was measured.

[0024] Figure 2 This is a scanning electron microscope image of the zirconium-hafnium bimetallic organic framework nanomaterial with a molar ratio of 3:1 prepared in Example 1. It can be seen from the figure that the zirconium-hafnium bimetallic organic framework nanomaterial with a molar ratio of 3:1 is a uniformly dispersed spherical particle with a particle size of about 200 nanometers.

[0025] Comparative Example 1 This comparative example provides the preparation of a single metal organic framework material: (1) Synthesis of zirconium metal-organic framework materials: 20.9 mg of 9,10-anthracene acyl bis(benzoic acid), 6.99 mg of zirconium tetrachloride, and 100 mg of benzoic acid were dispersed in 5 mL of N,N-dimethylformamide. The mixture was stirred at 100 °C for 20 minutes to obtain a solid-liquid mixture, which was collected by centrifugation, washed with N,N-dimethylformamide, and vacuum dried to obtain a zirconium metal organic framework material.

[0026] (2) Synthesis of Hafnium Metal-Organic Framework Materials: 20.9 mg of 9,10-anthracene acyl bis(benzoic acid), 9.61 mg of hafnium tetrachloride, and 100 mg of benzoic acid were dispersed in 5 mL of N,N-dimethylformamide. The mixture was stirred at 100 °C for 20 minutes to obtain a solid-liquid mixture, which was collected by centrifugation, washed with N,N-dimethylformamide, and vacuum dried to obtain a hafnium metal organic framework material.

[0027] The two metal organic framework materials prepared above were dispersed in deionized water to form a 1 mg / mL solution. 4 μL of the solution was dropped on the surface of a clean glassy carbon electrode and dried. PBS buffer solution containing 30 mM hydrogen peroxide (pH = 7.4) was used as the detection base solution. The scanning voltage was 0~+1.8V, and the photomultiplier tube voltage was 900 V. The electrochemiluminescence intensity of the metal organic framework nanoluminescent materials was measured respectively.

[0028] Figure 3The graphs show the changes in electrochemical luminescence signal intensity of the zirconium-hafnium bimetallic organic framework with different molar ratios (1:0, 3:1, 1:1, 1:3, 0:1) in Example 1 and Comparative Example 1. Figure 3 It can be seen that the electrochemical signal intensity first increases and then decreases with the addition of hafnium metal. When the molar ratio is 3:1, the electrochemical luminescence signal intensity is the highest, which is the optimal molar ratio.

[0029] Example 2 This embodiment provides a method for preparing a lipoarabinomannan molecular imprinted electrochemiluminescence sensor, comprising the following steps: A. Preparation of zirconium-hafnium bimetallic organic framework nanoluminescent material modified electrode: The zirconium-hafnium bimetallic organic framework material prepared in Example 1 with a molar ratio of 3:1 was dispersed in deionized water to form a 1 mg / mL solution, and 4 μL of the solution was drop-coated on the surface of the glassy carbon electrode. After drying, a zirconium-hafnium bimetallic organic framework nanoluminescent material modified electrode was obtained; B. Preparation of lipoarabinomannan molecular imprinting membrane on modified electrode: B1, take 50 μL of 200 pg / mL template molecule lipoarabinomannan, 0.013 g acrylic acid and 0.0126 g acrylamide and dissolve them in 3 mL acetonitrile to obtain prepolymerization solution a; B2, adding 0.003 g of ethylenediaminetetraacetic acid and 0.0056 g of ammonium persulfate to the prepolymer solution a to obtain a mixed solution b; B3. Take 15 μL of the mixed solution b and drop it on the surface of the modified electrode obtained in step A. Place the electrode in an oven at 70°C for thermal polymerization. After the reaction, immerse the electrode in 10 mL of a mixed eluent of methanol and acetic acid (volume ratio of 7:3) for 20 minutes. Then immerse the electrode in deionized water for another 15 minutes. After drying, obtain the lipoarabinomannan molecular imprinting membrane.

[0030] The electrode with zirconium-hafnium metal organic framework nanoluminescent material and imprinted with lipoarabinomannan was used as the working electrode, platinum wire as the counter electrode, Ag / AgCl as the reference electrode, pH=7.4 PBS buffer solution (containing 30 mM H2O2 as a co-reactant) as the detection base solution, the scanning voltage was 0~+1.8V, and the photomultiplier tube voltage was 900 V. The electrochemiluminescence signal before and after elution of the zirconium-hafnium bimetallic organic framework nanoluminescent material and lipoarabinomannan molecular imprinting polymer system was measured.

[0031] Example 3 In this example, referring to the method of Example 2, different molar ratios of functional monomers acrylic acid and acrylamide were selected to prepare lipoarabinomannan molecular imprinting polymers MIPs, and correspondingly the same method was used to prepare non-molecular imprinting polymers NIPs without adding template molecules. The other steps were the same as in Example 2.

[0032] Specifically, in step B1, acrylic acid and acrylamide with concentrations of 15, 30, 60, 90, and 120 mM, respectively, were selected as functional monomers and dissolved in 3 mL of acetonitrile together with 50 μL of 200 pg / mL template molecule lipoarabinomannan. Before and after the elution of the MIPs prepared with different molar ratios of bifunctional monomers, the electrochemiluminescence signals of the system based on the zirconium-hafnium bimetallic organic framework nanoluminescent material and lipoarabinomannan molecular imprinting polymer were measured. Table 1 records the electrochemiluminescence signal intensity values ​​before and after the elution of the system. Figure 4 is the difference in electrochemiluminescence signals before and after elution of the system. It can be concluded that the optimal ratio of acrylic acid to acrylamide is 60 mM.

[0033] Table 1 Electrochemical luminescence intensity values ​​of the system before and after elution at different concentrations

[0034] Example 4 In this embodiment, the method of Example 2 is referred to, but the molecular imprinting membrane is formed by polymerization reaction at different temperatures, and the other steps are the same as Example 2. Specifically, in step B3, the modification layer 2 is formed by polymerization reaction at 30°C, 40°C, 50°C, 60°C, 70°C and 80°C respectively. Before and after elution of the MIPs prepared by polymerization at different temperatures, the electrochemiluminescence signals of the zirconium-hafnium bimetallic organic framework nanoluminescent material and lipoarabinomannan molecular imprinting polymer system are measured before and after elution, and the difference is calculated. Table 2 records the electrochemiluminescence signal intensity values ​​before and after elution of the system at different temperatures. Figure 5 As shown, it can be seen that the difference in electrochemiluminescence signals before and after elution is the largest at 70°C, so 70°C is selected as the optimal polymerization temperature.

[0035] Table 2 Electrochemical luminescence intensity values ​​of the system before and after elution at different temperatures

[0036] Example 5 In this example, referring to the method of Example 2, PBS buffer solutions of different pH values ​​(containing 30 mM H2O2 as a co-reactant) were used as the detection base solution, and the other steps were the same as those of Example 2. Specifically, the electrode obtained in step B was placed in PBS solutions with pH values ​​of 5.0, 6.0, 7.4, 8.0, and 8.5, respectively, to measure the electrochemiluminescence signal before and after elution of the zirconium-hafnium bimetallic organic framework nanoluminescent material and lipoarabinomannan molecular imprinting polymer system. Table 3 records the electrochemiluminescence signal intensity values ​​before and after elution of the system at different pH values. The optimal pH value was determined by comparing the difference in electrochemiluminescence signals obtained at different pH values, such as Figure 6 As shown, for the electrode of this example, the optimal pH value is 7.4.

[0037] Table 3 Electrochemical luminescence intensity values ​​of the system before and after elution at different pH values

[0038] Example 6 In this example, the electrochemical impedance spectra of the bare electrode (GCE), the electrode modified with the zirconium-hafnium metal organic framework (ZrHf MOF) prepared in step A of Example 2, the electrode before and after MIPs elution prepared in step B of Example 2, and the electrode recombined with MIPs and lipoarabinomannan were measured. Figure 7 As shown in the figure, compared with before MIPs elution, after lipoarabinomannan was eluted, the decrease in the semicircle diameter indicated that the charge transfer impedance on the electrode surface decreased. This is because the imprinted cavity after elution can promote electron transfer, confirming the formation of the lipoarabinomannan cavity. Table 4 records the electrochemical impedance spectroscopy data of different modified electrodes. After lipoarabinomannan was recognized and captured, the transfer of electrons was restricted, resulting in an increase in resistance and an increase in the semicircle diameter, confirming the feasibility of the method for detecting lipoarabinomannan based on molecular imprinting electrochemiluminescence detection method.

[0039] Table 4 Electrochemical impedance spectroscopy data of different modified electrodes

[0040] Example 7 In this example, the electrode obtained in Example 2 was incubated with a 200 pg / mL lipoarabinomannan standard solution at room temperature for 0, 10, 20, 30, and 40 minutes, respectively. A PBS buffer solution with a pH of 7.4 (containing 30 mM H2O2 as a co-reactant) was used as the detection base solution. The photomultiplier tube voltage was 900 V. Under the voltage condition of 0~+1.8V, the electrochemiluminescence signal before and after incubation was measured, and the difference was calculated.

[0041] like Figure 8 It can be seen that in the present invention, under the same concentration conditions of target lipoarabinomannan, as the incubation time changes, the difference in electrochemiluminescence signals before and after incubation (after elution and rebinding) is the largest when the incubation time is 30 minutes. Therefore, in this embodiment, 30 minutes is used as the optimal incubation time.

[0042] Example 8 In this example, the electrode obtained in Example 2 was incubated with target lipoarabinomannan standard samples of different concentrations (10 fg / mL, 50 fg / mL, 100 fg / mL, 200 fg / mL, 2 pg / mL, 20 pg / mL, 200 pg / mL) at room temperature for 30 minutes. A PBS buffer solution with pH = 7.4 (containing 30 mM H2O2 as a co-reactant) was used as the detection base solution, the photomultiplier tube voltage was 900 V, and under the voltage condition of 0~+1.8V, the electrochemiluminescence intensity before and after incubation was measured, and the difference was calculated. The concentration of lipoarabinomannan was used as the horizontal axis, and the difference in electrochemiluminescence signal before and after incubation was used as the vertical axis to construct an analytical working curve.

[0043] like Fig. 9 and Fig.10 As shown, the molecular imprinting electrochemiluminescence detection method of the present invention has a good linear response to lipoarabinomannan in the range of 10fg / mL to 200pg / mL: Y=1753x+25463, R 2 =0.9871. Where x is the concentration of lipoarabinomannan (g / mL), and Y is the electrochemiluminescence signal intensity (au). After calculation, the detection limit of the sensor is 3.3 fg / mL.

[0044] Example 9 This embodiment provides a selective test for detecting lipoarabinomannan based on a molecular imprinting electrochemiluminescence detection method, which specifically includes the following steps: Glucose (Glu), urea (Urea), bovine serum albumin (BSA), and maltose (Maltose) were selected as interfering substances for the selectivity test. The electrode obtained in Example 2 was incubated with the same concentration of interfering substances and lipoarabinomannan at room temperature for 30 minutes, respectively. A PBS buffer solution with a pH of 7.4 (containing 30 mM H2O2 as a co-reactant) was used as the detection base solution. The photomultiplier tube voltage was 900 volts. Under the voltage condition of 0~+1.8V, the total electrochemiluminescence intensity was measured.

[0045] like Fig.11As shown, glucose (Glu), urea (Urea), bovine serum albumin (BSA), and maltose (Maltose) did not cause substantial changes, confirming the high selectivity of the molecular imprinting electrochemiluminescence method.

[0046] Example 10 In this example, the target lipoarabinomannan standard samples of different concentrations in Example 8 were replaced with pre-treated urine samples of tuberculosis patients (the test samples were centrifuged at 12000 r / min for 5 minutes, and the supernatant was collected for testing). The electrochemiluminescence intensity before and after incubation was measured under the same conditions, and the difference was calculated. Finally, the lipoarabinomannan concentration in the urine sample was calculated according to the standard working curve drawn in Example 8.

[0047] like Fig.12 As shown, urine samples of tuberculosis patients clinically confirmed as positive by the gold standard had obvious electrochemiluminescence signals, indicating that the preparation method of lipoarabinomannan molecular imprinting electrochemiluminescence sensor has certain clinical detection feasibility and clinical application potential.

[0048] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A molecular imprinting electrochemiluminescence sensor of lipoarabinomannan, characterized in that: The molecular imprinting electrochemiluminescence sensor uses an electrode with a zirconium-hafnium metal organic framework nano-luminescent material and imprinted lipoarabinomannan as a working electrode, a platinum wire as a counter electrode, and Ag / AgCl as a reference electrode.

2. The method for preparing a molecularly imprinted electrochemiluminescent sensor of lipoarabinomannan according to claim 1, characterized in that: The method for preparing the working electrode of the molecular imprinting electrochemiluminescence sensor comprises the following steps: A. Preparation of zirconium-hafnium bimetallic organic framework nanoluminescent material modified electrode: A1. Disperse 9,10-anthracene acyl bis(benzoic acid), zirconium tetrachloride, hafnium tetrachloride and benzoic acid in N,N-dimethylformamide solvent, stir at 100 °C for 20 minutes to obtain a solid-liquid mixture, collect by centrifugation, wash and vacuum dry to obtain a zirconium-hafnium bimetallic organic framework material; A2. Weigh the zirconium-hafnium bimetallic organic framework nanomaterial, perform ultrasound in ionized water to form a 1 mg / mL dispersion, take 3-5 μL of it and drop it on the surface of the glassy carbon electrode, and after drying, obtain the zirconium-hafnium bimetallic organic framework nanoluminescent material modified electrode; B. Preparation of lipoarabinomannan molecular imprinting membrane on modified electrode: B1, dissolving the template molecule lipoarabinomannan and the functional monomer in acetonitrile solvent to obtain a prepolymerization solution a; B2, adding a crosslinking agent and a free radical initiator to the prepolymer solution a to obtain a mixed solution b; B3. Take 10-15 μl of the mixed solution b and drop it on the surface of the modified electrode obtained in step A. Place the electrode in an oven for thermal polymerization reaction. After the reaction, use an eluent to elute to remove the template molecules and obtain a lipoarabinomannan molecular imprinting membrane.

3. The method for preparing a molecularly imprinted electrochemiluminescent sensor of lipoarabinomannan according to claim 2, characterized in that: In step A1, the molar ratio of zirconium tetrachloride to hafnium tetrachloride is 3:(1-9).

4. The method for preparing a molecularly imprinted electrochemiluminescent sensor of lipoarabinomannan according to claim 2, characterized in that: In step B1, the functional monomers are acrylic acid and acrylamide in a molar ratio of 1:1, and the concentration of the functional monomers is 10-150 mM, preferably 60 mM; In step B2, the crosslinking agent and the free radical initiator are ethylenediaminetetraacetic acid and ammonium persulfate respectively.

5. The method for preparing a molecularly imprinted electrochemiluminescent sensor of lipoarabinomannan according to claim 2, characterized in that: In step B3, the temperature of the thermal polymerization reaction is 30-80°C.

6. The method for preparing a molecularly imprinted electrochemiluminescent sensor of lipoarabinomannan according to claim 2, characterized in that: In step B3, the eluent is a mixed solution of methanol and acetic acid, and the volume ratio of methanol to acetic acid is 9:(1-81); The specific steps of elution are as follows: immersing the electrode in the eluent for 20 to 30 minutes, and then immersing it in deionized water for another 15 to 20 minutes.

7. An application of the molecular imprinting electrochemiluminescence sensor according to claim 1 in the detection of lipoarabinomannan, characterized in that: The method comprises the following steps: (1) sequentially immersing the prepared molecular imprinting electrochemiluminescence sensor in a series of concentrations of lipoarabinomannan standard solutions for incubation, and then using a PBS solution containing 30 mM hydrogen peroxide as the detection base solution, the photomultiplier tube voltage is 800-900 V, and under the voltage condition of 0-+1.8 V, the intensity of the electrochemiluminescence signal is measured to draw a standard working curve; (2) glucose, urea, bovine serum albumin, and maltose are selected as interferents of lipoarabinomannan to evaluate the selectivity of the molecular imprinting electrochemiluminescence sensor; (3) the lipoarabinomannan content in the pre-treated analyte is detected using the drawn standard working curve.

8. Use of a molecular imprinting electrochemiluminescence sensor in the detection of lipoarabinomannan according to claim 7, characterized in that: The incubation time in step (1) is 10 to 40 minutes.

9. Use of a molecular imprinting electrochemiluminescence sensor in the detection of lipoarabinomannan according to claim 7, characterized in that: In step (3), the test object is a urine sample, and the pretreatment method thereof comprises the following steps: centrifuging the collected urine sample from a tuberculosis patient at 12000 r / min for 5 minutes, and collecting the supernatant for testing.

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