CN110240704B

CN110240704B - Preparation method and application of targeted enzyme immobilization carrier based on magnetic molecular imprinting technology

Info

Publication number: CN110240704B
Application number: CN201910452736.5A
Authority: CN
Inventors: 傅强; 陈国宁; 舒花; 王璐; 王燕; 常春
Original assignee: Xian Jiaotong University
Current assignee: Xian Jiaotong University
Priority date: 2019-05-28
Filing date: 2019-05-28
Publication date: 2021-01-19
Anticipated expiration: 2039-05-28
Also published as: CN110240704A

Abstract

The invention discloses a preparation method and application of a targeting enzyme immobilization carrier based on a magnetic molecular imprinting technology, wherein the preparation method comprises the following steps: preparing amino ferroferric oxide; preparing boric acid modified ferroferric oxide; preparing a molecularly imprinted polymer layer by a sol-gel method; removing horseradish peroxidase bound to the molecularly imprinted polymer; and (3) blocking the non-specific adsorption residues on the surface of the polymer to obtain the horseradish peroxidase magnetic molecularly imprinted polymer. The preparation process is simple and low in cost, the obtained magnetic molecularly imprinted polymer not only can specifically identify a target substance horseradish peroxidase, but also can directly fix the horseradish peroxidase in a complex sample through adsorption, and the formed immobilized enzyme can be used for decomposing glucose, sarcosine, uric acid, cholesterol and the like to generate H₂O₂Detection of the substance(s).

Description

Preparation method and application of targeted enzyme immobilization carrier based on magnetic molecular imprinting technology

Technical Field

The invention belongs to the field of analysis and detection, and particularly relates to preparation of a targeted immobilized horseradish peroxidase based on a magnetic molecular imprinting technology and a carrier thereof.

Background

Immobilized enzyme technology is a technology that confines or binds an enzyme within a certain area of a solid support under the condition that the catalytic activity of the enzyme is maintained. Compared with non-immobilized free enzyme, the immobilized enzyme not only maintains the characteristics of high efficiency, specificity and mild enzyme catalytic reaction, but also overcomes the defects of the free enzyme, and has the advantages of high storage stability, easy separation and recovery, repeated use, continuous and controllable operation, simple and convenient process and the like. Immobilized enzymes have been rapidly developed and widely used in the fields of chemistry, biology, medicine, food science, environmental science and the like. The preparation method of the immobilized enzyme comprises two main methods, namely a physical method and a chemical method. The physical methods include physical adsorption, entrapment and the like. The physical method for preparing the immobilized enzyme has the advantages that the enzyme does not participate in chemical reaction, the whole structure is kept unchanged, and the catalytic activity of the enzyme is well reserved. Chemical methods include binding methods, crosslinking methods, and the like. The binding method is a method in which an enzyme is chemically bonded to a natural or synthetic polymer carrier, and the crosslinking method is a method in which the enzyme is crosslinked by a coupling agent via a group on the surface of the enzyme to form an insoluble immobilized enzyme having a relatively large molecular weight. However, the conventional preparation of immobilized enzyme requires the enzyme with higher immobilized purity, the enzyme is protein with poorer stability, the separation and purification process is complex, the separation cost is higher, and the separation process inevitably causes the loss of the enzyme and the reduction of the activity. Therefore, it is of great importance to realize direct immobilization of target enzymes from complex samples.

The molecular imprinting technology is a preparation technology for synthesizing a polymer with specific recognition capability on a specific template molecule. The molecular recognition target molecule has the characteristics of structure pre-determination, recognition specificity, property stability and wide applicability, can recognize target molecules in complex samples, and has been widely applied in the fields of separation analysis, biomimetic catalysis, drug release, biosensors and the like. Therefore, it has the potential to prepare immobilized enzymes directly from complex samples.

At present, the technology of imprinted polymers of small molecules tends to be mature, however, for water-soluble protein molecules (e.g., enzyme molecules), common methods for preparation and identification in organic phases, such as a bulk method, an in-situ method, a precipitation method and the like, are not suitable due to poor tolerance of proteins to organic solvents and acid-base capacity, so that the preparation of the enzyme imprinted polymers has certain difficulty. At present, no report of the application of the molecularly imprinted polymer to immobilized enzyme is found.

Disclosure of Invention

The invention aims to provide a preparation method and application of a targeting enzyme immobilization carrier based on a magnetic molecular imprinting technology.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of a horseradish peroxidase immobilized carrier comprises the following steps:

1) preparation of amino Fe₃O₄Magnetic nanoparticles;

2) using amino Fe₃O₄Preparation of boric acid modified Fe by magnetic nanoparticles₃O₄Magnetic nanoparticles;

3) horse radish peroxidase is taken as a template molecule, and the template molecule is fixed in boric acid modified Fe₃O₄On the magnetic nanoparticles, 3-aminopropyltriethoxysilane is used as a functional monomer and tetraethoxysilane is used as a crosslinking monomer in a water phase system, and the boric acid-modified Fe coated by the coating is prepared by a sol-gel method₃O₄A molecularly imprinted polymer layer on the magnetic nanoparticles to which the template molecules are bound;

4) removing the template molecules (horse radish peroxidase) combined in the molecularly imprinted polymer layer to obtain a horse radish peroxidase magnetic molecularly imprinted polymer;

5) and (3) sealing non-specific adsorption residues on the surface of the horseradish peroxidase magnetic molecularly imprinted polymer to obtain the horseradish peroxidase immobilized carrier.

Preferably, the amino group Fe₃O₄The magnetic nanoparticles are prepared by a hot solvent method, and the method specifically comprises the following steps: 0.5-2.0g FeCl₃·6H₂O, 1.0-4.0g of anhydrous sodium acetate and 3-15g of 1, 6-hexamethylene diamine are dispersed in 20-60mL of ethylene glycol to obtain a mixture, the mixture is stirred at the temperature of 20-40 ℃ until the mixture becomes a transparent solution, then the transparent solution is transferred to a high-pressure reaction kettle with a polytetrafluoroethylene lining, then the reaction is carried out for 6-12h at the temperature of 160-200 ℃, a reaction product is collected by a magnet, and the obtained product is washed and dried.

Preferably, the boronic acid modifies Fe₃O₄The magnetic nano-particle takes 4-formylphenylboronic acid as a boric acid reagent, and uses formyl and amino Fe thereof₃O₄The amino group on the surface of the magnetic nanoparticle reacts to generate imine, and the imine is reduced to amine by using cyano sodium borohydride as a reducing agent, so that the method specifically comprises the following steps: 200-1000mg of amino Fe₃O₄Adding magnetic nanoparticles and 10-50mg of 4-formylphenylboronic acid into 10-50mL of anhydrous methanol, reacting for 16-24h at 20-40 ℃, collecting a reaction product by using a magnet, washing the obtained product to obtain an unstable nanoparticle modified product, adding the obtained nanoparticle modified product and 10-50mg of sodium cyanoborohydride into 10-50mL of anhydrous methanol, continuing reacting for 18-24h at 20-40 ℃, collecting the reaction product by using the magnet, and washing and drying the obtained product (the nanoparticle modified product with a stable structure).

Preferably, the immobilization of the template molecule specifically comprises the following steps: modification of Fe with boric acid₃O₄The magnetic nanoparticles adsorb horseradish peroxidase dispersed in a solvent, wherein the dispersion concentration of the horseradish peroxidase is 125-1000 mu g-mL^-1The solvent is phosphate buffer solution with pH of 8.0-9.5, the adsorption temperature is 20-40 ℃, and the adsorption time is 0.5-4 h.

Preferably, the water phase system is composed of ethyl orthosilicate, 3-aminopropyltriethoxysilane and boric acid modified Fe immobilized with template molecules₃O₄The magnetic nanoparticle comprises magnetic nanoparticles and 0.005-0.1% of Tween aqueous solution in volume fraction, wherein the volume ratio of ethyl orthosilicate to 3-aminopropyltriethoxysilane is 1:2-1:16, the total volume of the ethyl orthosilicate and the 3-aminopropyltriethoxysilane is 5-20 mu L, the volume of the Tween aqueous solution is 5-20mL, and the Tween aqueous solution is used for fixing boric acid modified Fe of template molecules₃O₄The mass of the magnetic nanoparticles is 5-50 mg.

Preferably, the reaction conditions of the sol-gel method are as follows: reacting for 12-24h at 20-40 ℃.

Preferably, in the step 4), the template molecules are removed by using an elution solvent (elution time is 16-32h), the elution solvent is a mixed solution of acetonitrile and an acetic acid aqueous solution, wherein the volume ratio of the acetonitrile to the acetic acid aqueous solution is 2:8-4:6, and the volume fraction of the acetic acid aqueous solution is 2% -10%.

Preferably, in the step 5), a solution of gelatin, bovine serum albumin or casein (e.g., 1-5 mg. multidot.mL) is used^-1BSA solution) blocking (15-60 min at 20-40 ℃) the non-specific adsorption residues (e.g., horseradish peroxidase non-specific adsorption residues, etc.).

A preparation method of immobilized horseradish peroxidase comprises the following steps:

1) preparing the horseradish peroxidase immobilized carrier;

2) mixing the above horseradish peroxidase-immobilized carrier with a complex sample (e.g., horseradish extract) containing horseradish peroxidase, reacting at 20-40 deg.C for 20-120min, and magnetically separating to obtain immobilized horseradish peroxidase.

The horseradish peroxidase immobilized carrier prepared by the method is applied to preparation of immobilized horseradish peroxidase.

The immobilized horseradish peroxidase can be decomposed in glucose, sarcosine, uric acid, cholesterol, etc. to generate H₂O₂And the like.

The invention has the beneficial effects that:

the invention respectively takes 3-aminopropyl triethoxysilane (APTES) and Tetraethoxysilane (TEOS) as a functional monomer and a crosslinking monomer, adopts a non-catalytic sol-gel technology to prepare a horse radish peroxidase molecularly imprinted polymer with high selectivity and low cost in a water phase, and takes the molecularly imprinted polymer as an enzyme immobilization carrier to realize the direct immobilization of horse radish peroxidase in a complex sample.

The preparation process is simple and low in cost, the obtained magnetic molecularly imprinted polymer not only can specifically identify a target substance horseradish peroxidase, but also can directly fix the horseradish peroxidase in a complex sample through adsorption, and the formed immobilized enzyme can be used for decomposing glucose, sarcosine, uric acid, cholesterol and the like to generate H₂O₂Detection of the substance(s).

Furthermore, in the non-catalytic sol-gel method provided by the invention, in a reaction system containing tween, a silane reagent can be emulsified into a uniform reaction system to accelerate hydrolytic polymerization reaction to obtain a uniform polymer, so that the problem that the traditional sol-gel method needs hydrolytic polymerization in an organic phase under the catalysis of acid or alkali is overcome, and the preparation of the water-soluble protein (such as enzyme molecules such as horseradish peroxidase) molecularly imprinted polymer can be realized under mild reaction conditions.

Drawings

FIG. 1 shows the characterization results of horseradish peroxidase molecularly imprinted polymer: a is X-ray powder diffraction pattern (a is Fe)₃O₄-NH₂B is MIPs); b is magnetic hysteresis (a is MIPs, B is Fe)₃O₄-NH₂) (ii) a C is boric acid modified Fe₃O₄XPS characterization diagrams of magnetic nanoparticles and MIPs; d is boric acid modified Fe₃O₄Characteristic peak B of XPS characterization chart of magnetic nanoparticle; e is boric acid modified Fe₃O₄XPS characterization of magnetic nanoparticles N characteristic peak.

FIG. 2 is a diagram of the results of selective investigation of horseradish peroxidase molecularly imprinted polymer.

FIG. 3 is a graph showing the result of horseradish peroxidase immobilization in horseradish.

FIG. 4 is a Linerweaver-Burk reciprocal double plot showing the catalytic kinetics of immobilized and free enzymes: a is an immobilized enzyme enzymatic power curve; b is a Linerweaver-Burk double reciprocal curve of A; c is a non-immobilized enzyme (free enzyme) enzymatic kinetic curve; d is a Linerweaver-Burk double reciprocal curve of C.

FIG. 5 shows standard curves for glucose and sarcosine determination and results of interference experiments: a is a glucose determination standard curve; b is the comparison of the interfering substance with the glucose measurement; c is a sarcosine determination standard curve; d is the comparison of the interfering substance with the sarcosine measurement.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples.

Preparation of horseradish peroxidase magnetic molecularly imprinted polymer

1) Amino Fe₃O₄Nano particle (Fe)₃O₄-NH₂) Preparation of

Weighing FeCl₃·6H₂1.0g of O, 2.0g of anhydrous sodium acetate and 6.5g of 1, 6-hexanediamine were dispersed in 30mL of ethylene glycol in this order. Stir vigorously at room temperature until the mixture becomes a clear solution. The transparent solution is transferred into a high-pressure reaction kettle with a polytetrafluoroethylene lining and reacts for 6 hours at the temperature of 200 ℃. Collecting the product with magnet, washing the obtained product with ethanol and water, and drying at 50 deg.C to obtain amino Fe with average particle diameter of about 20nm₃O₄Nanoparticles for use.

2) Boric acid modified Fe₃O₄Preparation of

Taking the amino Fe₃O₄Dispersing 500mg of nanoparticles in 20mL of anhydrous methanol, adding 20mg of 4-formylphenylboronic acid into the obtained dispersion system, shaking for reaction at room temperature for 18h, collecting the nanoparticles by using a magnet, washing the nanoparticles three times by using the anhydrous methanol (washing away unreacted substances), re-dispersing the obtained nanoparticles in 20mL of anhydrous methanol, adding 20mg of sodium cyanoborohydride into the obtained dispersion system, continuing shaking for reaction at room temperature for 22h, collecting the nanoparticles by using the magnet, washing away the unreacted substances by using methanol and water, and drying the nanoparticles at 50 ℃ to obtain the boric acid modified Fe₃O₄And then standby.

3) Preparation of horse radish peroxidase magnetic molecularly imprinted polymer

Taking the boric acid modified Fe₃O₄10mg, added to 1mL of a dispersion having a concentration of 500. mu.g.mL^-1Adsorbing with phosphate buffer solution (pH 8.5) of horseradish peroxidase (HRP) under shaking at room temperature for 1h, collecting with magnet, washing off unbound HRP molecules with phosphate buffer solution (pH 8.5), re-dispersing with 5mL of 0.05% volume fraction Tween (Tween-20) aqueous solution, adding TEOS (tetraethylorthosilicate) 10 μ L and APTES (3-aminopropyltriethoxysilane) 2.5 μ L into the obtained dispersion, reacting under shaking at room temperature for 16h, collecting the product with magnet, eluting template molecule HRP with acetonitrile: 5% acetic acid aqueous solution (30:70, v/v) for 24h to obtain horseradish peroxidase magnetic solutionAnd (4) dispersing the molecular imprinted polymer (marked as MIPs) by using ethanol solution with the volume fraction of 30%, and storing (room temperature) for later use.

Non-molecularly imprinted polymers (designated NIPs) were prepared in the same manner as the MIPs described above, except that no template molecule, horseradish peroxidase, was added.

Referring to FIG. 1A, prepared amino Fe₃O₄The nanoparticles have characteristic diffraction peaks at 2 theta of 30.20 degrees, 35.40 degrees, 43.14 degrees, 53.56 degrees, 57.02 degrees and 62.70 degrees, and the characteristic peaks can be matched with Fe in powder diffraction data set₃O₄The characteristic diffraction peaks (220, 311, 400, 422, 511, and 440) coincided, indicating that the magnetic carrier (Fe) was prepared₃O₄-NH₂) Containing Fe₃O₄. With amino groups Fe₃O₄MIPs prepared by using nanoparticles as carriers and magnetic carriers (Fe)₃O₄-NH₂) Have similar diffraction peaks, which indicates that the MIPs polymerization process does not affect Fe₃O₄The crystal structure of (1).

See FIG. 1B, amino Fe₃O₄The saturation magnetic strengths of the nanoparticles and the magnetic Molecularly Imprinted Polymers (MIPs) are 65.6emu g respectively^-1And 62.3emu g^-1Amino group Fe₃O₄The imprinted polymer coating on the surface of the nanoparticle does not cause the saturation magnetic strength of the MIPs to be greatly reduced, and the prepared MIPs still have high saturation magnetic strength, so that the rapid separation in subsequent experiments can be ensured. In addition, it can be seen that the amino group Fe₃O₄The magnetization of the nanoparticles and the MIPs has obvious reversibility, namely amino Fe₃O₄The nanoparticles and MIPs showed no significant hysteresis, indicating that both show superparamagnetism.

With reference to FIG. 1C and FIGS. 1D and 1E, modification of boric acid with XPS for Fe₃O₄And MIPs, fig. 1C, fig. 1D and fig. 1E are boric acid modified Fe₃O₄The result shows that the surface of the material has absorption peaks of C, O, Fe, N and B elements, B in figure 1C is MIPs, the surface of the material has absorption peaks of C, O, Si and N elements, and boric acid modified Fe₃O₄Surface element composition phaseIn comparison, the MIPs surface has no Fe element absorption peak, and a stronger Si element absorption peak appears, which proves that the boric acid modifies Fe₃O₄Has silica polymerization, and at the same time, the absorption peak of the N element in the MIPs is obviously enhanced due to the enhancement of the absorption peak of the N element caused by the amino group in the APTES. XPS results show that polymer layers in MIPs are successfully coated on Fe₃O₄A surface.

(II) evaluation of Selectivity of horse radish peroxidase magnetic molecularly imprinted polymers (experiments after MIPs blocking)

Human Serum Albumin (HSA), Ovalbumin (OVA), cytochrome C (Cyt), lysozyme (Lyz) and bromelain (Bro) are selected as control substances of a selectivity experiment (Blank is a Blank experiment group, no competitive control substance is added), and compete with horseradish peroxidase to bind to a magnetic molecularly imprinted polymer at the same concentration, TMB developing solution is added for developing color, an absorbance value is read, and the selectivity of the prepared magnetic molecularly imprinted polymer on horseradish peroxidase is evaluated by comparing the interference degree of the added interference substances (control substances) on the binding of the magnetic molecularly imprinted polymer on the horseradish peroxidase.

Referring to fig. 2, human serum albumin, ovalbumin, cytochrome C, lysozyme and bromelain with the same concentration almost do not interfere with the adsorption of horseradish peroxidase, indicating that the prepared magnetic molecularly imprinted polymer has good specific adsorption capacity for horseradish peroxidase. The method also shows that in the preparation process of the magnetic molecularly imprinted polymer, the addition of the template molecule horseradish peroxidase can form a three-dimensional structure matched with horseradish peroxidase in a spatial structure and a binding site, and the adsorption performance of the magnetic molecularly imprinted polymer to other proteins with similar structures is poor due to the difference of the spatial structure and the action site.

(III) immobilization of peroxidase in Horseradish

Taking 1g of fresh horseradish, adding 10mL of PBST solution (PBS solution containing 0.05% Tween-20), homogenizing, extracting at 4 deg.C for 4h, filtering, and storing the filtrate, i.e. horseradish extract, at-20 deg.C for use, and diluting by 50 times before use; taking molecularly imprinted polymers (i.e. MIPs)2Sealing with 2mg/mL Bovine Serum Albumin (BSA) at 200 μ L for 30min at room temperature, adding the diluted horse radish extract 150 μ L, standing at room temperature for 30min (MIPs selectively adsorb horse radish peroxidase molecules in the extract), collecting the product with a magnet, and washing with PBST solution to obtain immobilized horse radish peroxidase (immobilized enzyme for short) with the sealed MIPs as carrier; adding TMB developing solution 150 μ L into the immobilized enzyme, developing at room temperature for 30min, adding 2M H₂SO₄The reaction was stopped with 50. mu.L of the solution, the immobilized enzyme was separated by a magnet, and then 160. mu.L of the reaction solution was read for absorbance under a microplate reader. The same immobilization and color development treatments were used for non-molecularly imprinted polymers (i.e., NIPs).

Referring to FIG. 3, since there is no specific recognition site in the NIPs, the immobilization amount of the NIPs to horseradish peroxidase is much less than that of MIPs, and after chromogenic substrate is added, the absorbance intensity corresponding to MIPs is much higher than that corresponding to the NIPs. The result shows that the prepared MIPs can identify, adsorb and fix the horseradish peroxidase from the complex horseradish extract after being sealed, and other substances in the horseradish have small interference on the immobilization process.

(IV) evaluation of immobilized Horseradish peroxidase

The enzymatic kinetic behavior of immobilized enzyme and non-immobilized enzyme on a substrate under the same condition is researched, the Km value of the Michaelis constant of the immobilized enzyme and the non-immobilized enzyme is calculated by a Linerweaver-Burk method, and the affinity of the immobilized enzyme and the non-immobilized enzyme on the substrate is evaluated. Referring to fig. 4A and 4C, the enzymatic kinetic results of the immobilized enzyme and the non-immobilized enzyme on the substrate Tetramethylbenzidine (TMB) indicate that the immobilized enzyme and the non-immobilized enzyme have similar enzymatic kinetic behavior; further, the Km values of the Michaelis constants of the immobilized enzyme and the non-immobilized enzyme were 0.36mM and 0.29mM respectively (FIGS. 4B and 4D) as determined by the Linerweaver-Burk method, and it can be seen that the Michaelis constants of the immobilized enzyme and the non-immobilized enzyme are not much different, indicating that the affinity of the immobilized horseradish peroxidase and the non-immobilized horseradish peroxidase with the substrate is equivalent.

(V) determination of Glucose (Glucose) content in blood

The standard curve for glucose determination is established as follows: in 100. mu.L of a composition containingSame concentration (1. mu.g. mL)^-1、5μg·mL^-1、10μg·mL^-1、50μg·mL^-1、100μg·mL^-1) Citric acid-sodium acetate buffer for glucose and 100. mu.L of TMB (400. mu.g.mL)^-1) To the mixed solution of (1 mg. about.mL), a glucose oxidase solution was added^-1) Reacting 10 μ L with immobilized enzyme 20 μ g at room temperature for 1h, adding 2M H₂SO₄Stopping reaction with 50 μ L of solution, separating immobilized enzyme with magnet, reading absorbance value of 160 μ L of reaction solution with enzyme labeling instrument, and establishing standard curve of response relationship between glucose concentration and absorbance value with absorbance value as ordinate and standard substance concentration as abscissa, as shown in FIG. 5A.

The method for detecting the glucose content in the blood plasma comprises the following steps: the plasma sample was diluted 50-fold with a citric acid-sodium acetate buffer solution before use, and 100. mu.L of the diluted plasma sample solution and a TMB solution (400. mu.g.mL) were taken^-1) Mixing 100 μ L, adding glucose oxidase solution (1 mg. mL)^-1) Reacting 10 μ L with immobilized enzyme 20 μ g at room temperature for 1h, adding 2M H₂SO₄Stopping reaction with 50 μ L of solution, separating immobilized enzyme with magnet, reading absorbance value with 160 μ L of reaction solution under enzyme labeling instrument, and calculating glucose concentration in blood plasma according to the standard curve.

In the experiment, the accuracy and precision of glucose are inspected and determined by using the standard recovery rate and the relative standard deviation, and the method comprises the following specific steps: respectively taking the high, medium and low (50 mug. mL)^-1、25μg·mL^-1、5μg·mL^-1) And 3 parts of glucose standard-added plasma solution with three concentrations are prepared, the absorbance values of different standard-added samples are obtained according to the glucose content determination operation steps, and the recovery rate and the relative standard deviation are calculated by combining the drawn standard curve, so that the accuracy and precision of the method are inspected. The results (see Table 1) show that the recovery rate is between 114.5% and 119.7% and the precision RSD value is less than 4.2% under the standard concentration. The standard recovery rate and precision experiment shows that the method for detecting the glucose content in the blood plasma has good accuracy and precision and can be used for detecting the glucose in an actual sample.

Table 1 accuracy and precision of glucose assay (n ═ 3)

In order to examine the selectivity of the method for measuring glucose content in plasma, vitamin c (vitamin c), Sucrose (Sucrose), Glycine (Glycine), and Mannitol (Mannitol) were selected as control substances in the experiment, and the selectivity of the method was investigated. The specific operation is as follows: and respectively preparing solutions with the concentration of 10 times of the reference glucose concentration for interference experiments, respectively determining according to the glucose content detection method, and judging the interference condition of the solutions on glucose determination according to the obtained absorbance values. The results (see FIG. 5B) show that even when the concentration of the control substance was 10 times higher than the glucose concentration, the color produced by the detection thereof did not interfere with the glucose measurement, indicating that the glucose content detection method was constructed with good selectivity.

(VI) determination of sarcosine (Creatine) content in urine

The standard curve for sarcosine determination is established as follows: at 100. mu.L, different concentrations (0.1. mu.g.mL)^-1、0.5μg·mL^-1、1μg·mL^-1、5μg·mL^-1、10μg·mL^-1) Sarcosine oxidase solution (1 mg. multidot.mL) was added to sarcosine PBS buffer^-1)10 μ L, reacted at room temperature for 30min, and added with TMB solution (400 μ g. mL)^-1) Reacting 100 μ L with immobilized enzyme 20 μ g at room temperature for 30min, adding 2M H₂SO₄Stopping reaction with 50 μ L of solution, separating immobilized enzyme with magnet, reading absorbance value with 160 μ L of reaction solution under microplate reader, and establishing standard curve of response relationship between sarcosine concentration and absorbance value, as shown in FIG. 5C.

The method for detecting the content of sarcosine in urine comprises the following steps: the urine sample was diluted 20-fold with PBS buffer before use, and 100. mu.L of the diluted urine sample solution was added with sarcosine oxidase solution (1 mg. multidot.mL)^-1)10 μ L, reacted at room temperature for 30min, and added with TMB solution (400 μ g. mL)^-1) Reacting 100 μ L with immobilized enzyme 20 μ g at room temperature for 30min, adding 2M H₂SO₄Stop 50. mu.L of solutionAnd (3) reacting, separating the immobilized enzyme by using a magnet, reading an absorbance value of 160 mu L of reaction liquid under an enzyme-labeling instrument, and calculating the sarcosine content in the urine according to the established standard curve.

In the experiment, the accuracy and precision of the method for measuring the sarcosine are inspected by using the standard recovery rate and the relative standard deviation, and the method comprises the following specific steps: respectively taking the high, middle and low (5 mug. mL)^-1、1μg·mL^-1、0.5μg·mL^-1) And (3) preparing 3 parts of sarcosine standard-added urine with three concentrations, obtaining the absorbance values of different standard-added samples according to the sarcosine content measurement operation steps, and calculating the recovery rate and the relative standard deviation by combining the drawn standard curve so as to investigate the accuracy and precision of the method for detecting the sarcosine content in the urine. The results (see Table 2) show that the recovery rate is between 87.4% and 93.8% and the precision RSD value is less than 6.7% under the standard concentration. The standard recovery rate and precision experiment shows that the method for detecting the content of the sarcosine in the urine has good accuracy and precision, and can be used for detecting the sarcosine in an actual sample.

TABLE 2 accuracy and precision of sarcosine test (n ═ 3)

In order to examine the selectivity of the method for detecting the content of sarcosine in urine, Glucose (Glucose), vitamin c (vitamin c), Glycine (Glycine), Phenylalanine (Phenylalanine), and Arginine (Arginine) were selected as control substances in the experiment, and the selectivity of the method was examined. The specific operation is as follows: solutions with ten times of reference sarcosine concentration are respectively prepared for interference experiments, the detection is respectively carried out according to the detection method of the sarcosine content in urine, and the interference condition of the detection on the sarcosine is judged according to the obtained absorbance value. The results (see FIG. 5D) show that even when the concentration of the control substance is 10 times higher than the concentration of sarcosine, the color generated by the detection does not interfere with the measurement of sarcosine, indicating that the constructed method for detecting the content of sarcosine has good selectivity.

Claims

1. A preparation method of a horseradish peroxidase immobilized carrier is characterized by comprising the following steps: the method comprises the following steps:

1) preparation of amino Fe₃O₄Magnetic nanoparticles;

3) horse radish peroxidase is taken as a template molecule, and the template molecule is fixed in boric acid modified Fe₃O₄On the magnetic nanoparticles, 3-aminopropyltriethoxysilane is used as a functional monomer and tetraethoxysilane is used as a crosslinking monomer in a water phase system, and the Fe coated with boric acid modified Fe is prepared by adopting a non-catalytic sol-gel method₃O₄A molecularly imprinted polymer layer on the magnetic nanoparticles to which the template molecules are bound;

4) removing the template molecules combined in the molecularly imprinted polymer layer to obtain a horseradish peroxidase magnetic molecularly imprinted polymer;

5) sealing non-specific adsorption residues on the surface of the horseradish peroxidase magnetic molecularly imprinted polymer to obtain a horseradish peroxidase immobilized carrier;

the water phase system is composed of ethyl orthosilicate, 3-aminopropyl triethoxysilane and boric acid modified Fe fixed with template molecules₃O₄The magnetic nanoparticle comprises magnetic nanoparticles and 0.005-0.1% of Tween aqueous solution in volume fraction, wherein the volume ratio of ethyl orthosilicate to 3-aminopropyltriethoxysilane is 1:2-1:16, the total volume of the ethyl orthosilicate and the 3-aminopropyltriethoxysilane is 5-20 mu L, the volume of the Tween aqueous solution is 5-20mL, and the Tween aqueous solution is used for fixing boric acid modified Fe of template molecules₃O₄The mass of the magnetic nanoparticles is 5-50 mg;

the reaction conditions of the sol-gel method are as follows: reacting for 12-24h at 20-40 ℃;

horse radish peroxidase in horse radish is immobilized by using a horse radish peroxidase immobilized carrier, and the affinity of the immobilized horse radish peroxidase and the immobilized horse radish peroxidase are equivalent to the substrate.

2. The method for preparing a horseradish peroxidase immobilized carrier according to claim 1, which is characterized in that: said amino group Fe₃O₄The magnetic nanoparticles are prepared by a hot solvent method.

3. The method for preparing a horseradish peroxidase immobilized carrier according to claim 1, which is characterized in that: the boric acid modified Fe₃O₄The magnetic nano-particle is prepared by taking 4-formylphenylboronic acid as a boric acid reagent.

4. The method for preparing a horseradish peroxidase immobilized carrier according to claim 1, which is characterized in that: the immobilization of the template molecule specifically comprises the following steps: modification of Fe with boric acid₃O₄The magnetic nanoparticles adsorb horseradish peroxidase dispersed in a solvent, wherein the dispersion concentration of the horseradish peroxidase is 125-1000 mu g-mL^-1The solvent is phosphate buffer solution with pH of 8.0-9.5, the adsorption temperature is 20-40 ℃, and the adsorption time is 0.5-4 h.

5. The method for preparing a horseradish peroxidase immobilized carrier according to claim 1, which is characterized in that: in the step 4), the template molecules are removed by using an elution solvent, wherein the elution solvent is a mixed solution of acetonitrile and an acetic acid aqueous solution, the volume ratio of the acetonitrile to the acetic acid aqueous solution is 2:8-4:6, and the volume fraction of the acetic acid aqueous solution is 2% -10%.

6. The method for preparing a horseradish peroxidase immobilized carrier according to claim 1, which is characterized in that: in the step 5), the non-specific adsorption residue is blocked by using a solution of gelatin, bovine serum albumin or casein.

7. A preparation method of immobilized horseradish peroxidase is characterized by comprising the following steps: the method comprises the following steps:

1) preparation of Horseradish peroxidase immobilized Carrier

Preparation of amino Fe₃O₄Magnetic nanoparticles; using amino Fe₃O₄Preparation of boric acid modified Fe by magnetic nanoparticles₃O₄Magnetic nanoparticles; horse radish peroxidase is taken as a template molecule, and the template molecule is fixed in boric acid modified Fe₃O₄On the magnetic nanoparticles, 3-aminopropyltriethoxysilane is used as a functional monomer and tetraethoxysilane is used as a crosslinking monomer in a water phase system, and the Fe coated with boric acid modified Fe is prepared by adopting a non-catalytic sol-gel method₃O₄A molecularly imprinted polymer layer on the magnetic nanoparticles to which the template molecules are bound; removing the template molecules combined in the molecularly imprinted polymer layer to obtain a horseradish peroxidase magnetic molecularly imprinted polymer; sealing non-specific adsorption residues on the surface of the horseradish peroxidase magnetic molecularly imprinted polymer to obtain a horseradish peroxidase immobilized carrier;

2) mixing the horseradish peroxidase immobilized carrier with a complex sample containing horseradish peroxidase, reacting at 20-40 ℃ for 20-120min, and then obtaining the immobilized horseradish peroxidase by magnetic separation, wherein the affinity of the immobilized horseradish peroxidase and the non-immobilized horseradish peroxidase with a substrate is equivalent.