Detection kit for 4-hydroxybenzoic acid

Abstract

The invention relates to the technical field of biological detection, in particular to a detection kit for 4-hydroxybenzoic acid. The detection kit for 4-hydroxybenzoic acid comprises an Au-Pt / Fe3O4 / POPs capture probe for detecting a target sample, and a preparation method of the Au-Pt / Fe3O4 / POPs capture probe comprises the following steps: S1, preparation and pretreatment of POPs: S101, preparation of POPs; s102, carrying out pretreatment on the POPs; s2, preparation of a Fe3O4 / POPs compound; s3, preparing a precursor solution; and (S4) Au-Pt / Fe3O4 / POPs preparation is carried out. The invention provides the detection kit aiming at the 4-hydroxybenzoic acid, the detection kit can realize rapid and sensitive detection of the gastric cancer marker 4-hydroxybenzoic acid, the detection method is simple and high in accuracy, and the detection kit has a relatively high application value.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a detection kit for 4-hydroxybenzoic acid. Background Art

[0002] Gastric cancer is a common malignant tumor caused by lesions in the gastric mucosal epithelial cells. In the early stages of gastric cancer, there are usually no clinical symptoms or only unclear symptoms. About two-thirds of gastric cancer patients are diagnosed with gastric cancer in the late stage, and surgical treatment is too late.

[0003] At present, there are various methods for early diagnosis of gastric cancer, but there are many shortcomings, which cannot be applied to the convenient and fast detection of the general public. For example, gastroscopy and pathological biopsy methods both cause certain trauma to patients, and require professional equipment and technicians. The examination costs are relatively high and are not suitable for large-scale screening; CT examination and X-ray barium meal examination have limited qualitative diagnosis capabilities for lesions, and the diagnostic accuracy is relatively low.

[0004] At present, a variety of quantitative detection methods for gastric cancer markers have been proposed clinically, but the detection steps of these markers are complicated, low in sensitivity, and often accompanied by high costs. In related technologies, studies have found that there are a series of differential metabolites in the serum of gastric cancer patients compared with healthy controls, among which the small molecule 4-hydroxybenzoic acid (4-Hydroxybenzoic acid) is an ideal marker for early diagnosis of gastric cancer. However, the content of 4-hydroxybenzoic acid in blood samples is usually very low, and common detection techniques such as electrochemical methods, high-performance liquid chromatography, fluorescence, and mass spectrometry have low sensitivity and high detection limits, which cannot meet the detection requirements for disease diagnosis. Summary of the invention

[0005] The present application provides a detection kit for 4-hydroxybenzoic acid, which can realize rapid and sensitive detection of 4-hydroxybenzoic acid, a gastric cancer marker. The detection method is simple and highly accurate, and has high application value.

[0006] To achieve the above object, the present application provides a detection kit for 4-hydroxybenzoic acid, which comprises:

[0007] Au-Pt / Fe for detecting target samples 3 O 4 / POPs capture probe;

[0008] The Au-Pt / Fe 3 O 4 The preparation method of the POPs capture probe comprises the following steps:

[0009] S1. POPs preparation and pretreatment:

[0010] S101, POPs preparation: TCT, anhydrous piperazine and terephthalaldehyde were mixed in a molar mass ratio of 2:3:1, dissolved in 1,4-dioxane, and K 2 CO 3 Ultrasonic dispersion for 10 to 20 minutes, heating at 130°C to reflux to obtain a dispersed solution, wherein the K 2 CO 3 The molar mass of 1,4-dioxane is three times that of TCT, and the molar mass ratio of 1,4-dioxane to TCT is 200:1;

[0011] After the dispersed solution is cooled to room temperature, it is distilled under reduced pressure for 20 to 40 minutes, and under continuous stirring, the remaining product is poured into excess methanol and stirred for 10 minutes, and centrifuged to obtain a precipitate, and the precipitate is dried to obtain POPs;

[0012] S102, POPs pretreatment: The POPs obtained in S101 are immersed in a 0.5 mol / L sodium hydroxide solution at a mass volume ratio of 1:(40-50) at 60°C for 3-4 hours, and then rinsed with deionized water until neutral to obtain treated POPs;

[0013] S2、Fe 3 O 4 / POPs complex preparation;

[0014] S3. Preparation of precursor solution:

[0015] S301, 0.01 mol / L HAuCl 4 solution and 0.005 mol / L H 2 PtCl 6 The solutions are mixed in a volume ratio of 2:(0.5-1) to obtain a mixed solution;

[0016] S302, slowly add 0.1 mol / L hexadecyltrimethylammonium bromide solution to the mixed solution under stirring conditions, and stir to react for 10 minutes. The hexadecyltrimethylammonium bromide solution and HAuCl 4 The ratio of the solution is 1:1;

[0017] S303, adding 0.1 mol / L ascorbic acid solution to the solution obtained in S302, and continuing stirring for 20 to 30 minutes to obtain a precursor solution, wherein the volume ratio of the ascorbic acid solution to the hexadecyltrimethylammonium bromide solution is 1:1;

[0018] S4, Au-Pt / Fe 3 O 4 / POPs Preparation:

[0019] The Fe 3 O 4 The Au-Pt / Fe / POPs composite was mixed with the precursor solution in a mass-volume ratio of 1:(10-20), and after ultrasonic dispersion for 30 minutes, stirring was continued for 1 hour, and the product was collected by magnetic separation. The product was washed alternately with ultrapure water and ethanol for no less than three times, and the washed product was dispersed in water to obtain Au-Pt / Fe / POPs composite. 3 O 4 / POPs capture probe.

[0020] Preferably, after collecting the product by magnetic separation, the method further comprises:

[0021] The product was stirred and reacted with 2 mol / L polydopamine solution at a mass volume ratio of 1:(5-6) for 2-3 hours.

[0022] Preferably, the S2 includes:

[0023] S201, preparation of Fe 3 O 4 Nanoparticle dispersion: Fe 3 O 4 Nanoparticles were dispersed in 1,4-dioxane to obtain 0.1 g / mL Fe 3 O 4 Nanoparticle dispersion;

[0024] S202, the treated POPs and Fe 3 O 4 The nanoparticles were mixed in a mass ratio of 1:(1.5-2.5), anhydrous piperazine was added, stirred for 15-30 min, refluxed with nitrogen at 120 °C for 36 h, and Fe was obtained after magnetic separation. 3 O 4 / POPs complex, the anhydrous piperazine is combined with POPs and Fe 3 O 4 The total mass ratio of the nanoparticles is 1:10.

[0025] Preferably, after S202, the following steps are further included:

[0026] Dichloromethane, anhydrous ethanol and ultrapure water were used to 3 O 4 / POPs complexes were washed by centrifugation several times;

[0027] The washed Fe 3 O 4 / POPs composites were vacuum freeze-dried, and the freeze-dried Fe 3 O4 / POPs complex crushing treatment.

[0028] Preferably, the Fe is prepared in S2 3 O 4 / POPs complex, the Fe 3 O 4 Fe 3 O 4 Nanoparticles, the Fe 3 O 4 The method for preparing nanoparticles includes:

[0029] FeCl 2 ·4H 2 O and FeCl 3 6H 2 O is stirred and mixed in a ratio of 1:(1-2) of iron ion molar amount, and heated from room temperature to 50-65°C in a water bath, and then the heating is stopped;

[0030] The heated mixture was stirred, and ammonia water was added dropwise to the mixture at a volume ratio of one tenth of the volume of the mixture to collect Fe 3 O 4 The particles were precipitated, washed three times with anhydrous ethanol and deionized water until neutral, and dried to obtain Fe 3 O 4 Nanoparticles.

[0031] Preferably, the FeCl 2 ·4H 2 O and FeCl 3 6H 2 O and stirred in a 1:1 ratio of molar amount of iron ions.

[0032] Preferably, after S2 and before S3, the following steps are further included:

[0033] Fe 3 O 4 / POPs complex modification:

[0034] The Fe 3 O 4 The / POPs complex was mixed with 0.5 mol / L maleic anhydride solution in a mass volume ratio of 1:(3-6), heated to 60°C, stirred for 0.5-1 h under nitrogen protection, and then centrifuged to obtain the modified Fe 3 O 4 / POPs complex.

[0035] The beneficial effects of the technical solution provided by this application include:

[0036] The present application provides a detection kit for 4-hydroxybenzoic acid, which utilizes the good framework structure and specific selective adsorption of POPs, and combines with magnetic Fe 3 O 4 Compound, the obtained Fe 3 O 4 / POPs has a strong adsorption capacity for 4-hydroxybenzoic acid (4-HBA), and Au and Pt provide more active sites, enhancing the interaction with 4-HBA molecules, thereby improving the adsorption enrichment capacity and detection sensitivity; POPs are deprotonated by sodium hydroxide pretreatment to introduce more negative charges, and the negative charges produce electrostatic repulsion with the carboxyl groups in the 4-HBA molecules, making it easier for 4-HBA molecules to enter the pore structure of POPs, increasing their contact opportunities with the active sites inside POPs and improving the adsorption effect, thereby achieving rapid and sensitive detection of 4-hydroxybenzoic acid, a gastric cancer marker. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0038] Figure 1 Au-Pt / Fe in the detection kit provided in this application 3 O 4 / Schematic diagram of the preparation method of POPs capture probe;

[0039] Figure 2 The Au-Pt / Fe provided in Example 3 of the present application 3 O 4 / POPs substrate detection of different concentrations of 4-HBA at 1072 cm -1 SERS spectrum at ;

[0040] Figure 3 The Au-Pt / Fe provided in Example 3 of the present application 3 O 4 / POPs base detection of different concentrations of 4-HBA in serum at 1072cm -1 SERS spectrum at ;

[0041] Figure 4 Au-Pt / Fe 3 O 4 Schematic diagram of Zeta potential during the synthesis of POPs. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0043] See also Figure 1 to Figure 4 As shown, the present application provides a detection kit for 4-hydroxybenzoic acid, which can realize rapid and sensitive detection of 4-hydroxybenzoic acid, a gastric cancer marker, and the detection method is simple and highly accurate, and has high application value.

[0044] Au-Pt / Fe 3 O 4 The method for preparing a POPs capture probe comprises the following steps:

[0045] S1. POPs preparation and pretreatment:

[0046] S101, POPs preparation: TCT, anhydrous piperazine and terephthalaldehyde were mixed in a molar mass ratio of 2:3:1, dissolved in 1,4-dioxane, and K 2 CO 3 Ultrasonic dispersion was performed for 10 to 20 minutes, and then heated to reflux at 130°C to obtain a dispersed solution. 2 CO 3 The molar mass of 1,4-dioxane is three times that of TCT, and the molar mass ratio of 1,4-dioxane to TCT is 200:1;

[0047] After the dispersed solution is cooled to room temperature, it is distilled under reduced pressure for 20 to 40 minutes. Under continuous stirring, the remaining product is poured into excess methanol and stirred for 10 minutes. The precipitate is obtained by centrifugation, and the precipitate is dried to obtain POPs.

[0048] S102, POPs pretreatment: The POPs obtained in S101 are immersed in a 0.5 mol / L sodium hydroxide solution at a mass volume ratio of 1:(40-50) at 60°C for 3-4 hours, and then rinsed with deionized water until neutral to obtain treated POPs;

[0049] S2、Fe 3 O 4 / POPs complex preparation;

[0050] Optionally, S2 includes:

[0051] S201, preparation of Fe 3 O 4Nanoparticle dispersion: Fe 3 O 4 Nanoparticles were dispersed in 1,4-dioxane to obtain 0.1 g / mL Fe 3 O 4 Nanoparticle dispersion;

[0052] S202, the treated POPs and Fe 3 O 4 The nanoparticles were mixed in a mass ratio of 1:(1.5-2.5), anhydrous piperazine was added, stirred for 15-30 min, refluxed with nitrogen at 120 °C for 36 h, and Fe was obtained after magnetic separation. 3 O 4 / POPs complex, anhydrous piperazine with POPs and Fe 3 O 4 The total mass ratio of the nanoparticles is 1:10.

[0053] In some embodiments, after S202, the process further includes:

[0054] Dichloromethane, anhydrous ethanol and ultrapure water were used to 3 O 4 / POPs complexes were washed by centrifugation several times;

[0055] The washed Fe 3 O 4 / POPs composites were vacuum freeze-dried, and the freeze-dried Fe 3 O 4 / POPs complex crushing treatment.

[0056] In some embodiments, Fe 3 O 4 / POPs complex, the Fe 3 O 4 Fe 3 O 4 Nanoparticles, Fe 3 O 4 The method for preparing nanoparticles includes:

[0057] FeCl 2 ·4H 2 O and FeCl 3 6H 2 O is stirred and mixed in a ratio of 1:(1-2) of iron ion molar amount, and heated from room temperature to 50-65°C in a water bath, and then the heating is stopped;

[0058] Stir the heated mixture and add one tenth of the volume of ammonia water to the mixture according to the volume ratio to collect Fe 3 O 4The particles were precipitated, washed three times with anhydrous ethanol and deionized water until neutral, and dried to obtain Fe 3 O 4 Nanoparticles.

[0059] In some embodiments, FeCl 2 ·4H 2 O and FeCl 3 6H 2 O and stirred in a 1:1 ratio of molar amount of iron ions.

[0060] Specifically, the present application adopts a coprecipitation method to prepare Fe3O4 nanoparticles, and the reaction formula is:

[0061] Fe 2+ +Fe 3+ +8NH 3 ·H 2 O=Fe 3 O 4 +8HN 4 + +4H 2 O

[0062] S3. Preparation of precursor solution:

[0063] S301, 0.01 mol / L HAuCl 4 solution and 0.005 mol / L H 2 PtCl 6 The solutions are mixed in a mass ratio of 2:(0.5-1) to obtain a mixed solution;

[0064] S302, slowly add 0.1 mol / L hexadecyl trimethyl ammonium bromide solution to the mixed solution under stirring conditions, and stir to react for 10 minutes. The hexadecyl trimethyl ammonium bromide solution and HAuCl 4 The ratio of the solution is 1:1;

[0065] S303. Add 0.1 mol / L ascorbic acid solution to the solution obtained in S302, and continue stirring for 20 to 30 minutes to obtain a precursor solution, wherein the volume ratio of the ascorbic acid solution to the hexadecyltrimethylammonium bromide solution is 1:1.

[0066] In some embodiments, after S2 and before S3, the following steps are further included:

[0067] Fe 3 O 4 / POPs complex modification:

[0068] Fe 3 O 4The / POPs complex was mixed with 0.5 mol / L maleic anhydride solution in a mass volume ratio of 1:(3-6), heated to 60°C, stirred for 0.5-1 h under nitrogen protection, and then centrifuged to obtain the modified Fe 3 O 4 / POPs complex.

[0069] S4, Au-Pt / Fe 3 O 4 / POPs Preparation:

[0070] Fe 3 O 4 The Au-Pt / Fe / POPs complex was mixed with the precursor solution in a mass-volume ratio of 1:(10-20), and after ultrasonic dispersion for 30 min, stirring was continued for 1 h. The product was collected by magnetic separation, and the product was washed alternately with ultrapure water and ethanol for no less than three times. The washed product was dispersed in water to obtain Au-Pt / Fe 3 O 4 / POPs capture probe.

[0071] In some embodiments, after collecting the product by magnetic separation, the method further comprises:

[0072] The product was stirred and reacted with 2 mol / L polydopamine solution at a mass volume ratio of 1:(5-6) for 2-3 hours.

[0073] Polydopamine is wrapped on the probe surface, which can increase the adsorption sites and adjust the hydrophilicity and hydrophobicity of the capture probe, making it more conducive to the adsorption and enrichment of 4-HBA.

[0074] The target sample was mixed with Au-Pt / Fe 3 O 4 The analyte solution was obtained by mixing the POPs / POPs capture probes in a volume ratio of 1:1 and incubating with ultrasound for 0.5 to 2 hours. The analyte solution was measured to collect the Raman signal of the gastric cancer marker 4-hydroxybenzoic acid.

[0075] Example 1

[0076] In this embodiment, Au-Pt / Fe 3 O 4 The method for preparing a POPs capture probe comprises the following steps:

[0077] S1. POP preparation and pretreatment:

[0078] S101, POPs preparation: 1.1064 g (6 mmol) TCT, 9 mmol (0.7752 g) anhydrous piperazine and 0.432 g (3 mmol) terephthalaldehyde were mixed and dissolved in 100 mL 1,4-dioxane, and 2.4879 g (18 mmol) K 2 CO 3 Ultrasonic dispersion was performed for 10 min, and a dispersion solution was obtained after heating under reflux at 130°C;

[0079] After the dispersed solution was cooled to room temperature, it was distilled under reduced pressure for 20 minutes. Under continuous stirring, the remaining product was poured into excess methanol and stirred for 10 minutes. The precipitate was obtained by centrifugation, and the precipitate was dried to obtain POPs.

[0080] S102, POPs pretreatment: 2 g of POPs were immersed in 80 mL of 0.5 mol / L sodium hydroxide solution at 60° C. for 3 h, and then rinsed with deionized water until neutral to obtain treated POPs.

[0081] S2、Fe 3 O 4 Preparation of / POPs complex:

[0082] S201, preparation of Fe 3 O 4 Nanoparticle dispersion: Fe 3 O 4 Nanoparticles were dispersed in 1,4-dioxane to obtain 0.1 g / mL Fe 3 O 4 Nanoparticle dispersion;

[0083] S202, 1g of the treated POPs and 20mL of the above Fe 3 O 4 After the nanoparticle dispersion was mixed, 30 g of anhydrous piperazine was added, stirred for 20 min, and refluxed with nitrogen at 120 °C for 36 h. Fe 3 O 4 / POPs complex.

[0084] Among them, Fe 3 O 4 Fe 3 O 4 Nanoparticles, Fe 3 O 4 The method for preparing nanoparticles includes:

[0085] 1.057 g of FeCl 2 ·4H 2 O and 1.148 g of FeCl 3 6H2 O was stirred and mixed in 30 mL of deionized water, and heated from room temperature to 60 °C in a water bath, then the heating was stopped;

[0086] Stir the heated mixture and add 3 mL of ammonia water to the mixture according to the volume ratio. Use a permanent magnet to collect Fe 3 O 4 The pellet was precipitated and washed three times with anhydrous ethanol and deionized water until neutral. The resulting precipitate was dried in a vacuum freeze-drying oven to obtain Fe 3 O 4 Nanoparticles.

[0087] S3. Preparation of precursor solution:

[0088] S301, 20mL, 0.01mol / L HAuCl 4 solution and 10mL of 0.005mol / L H 2 PtCl 6 The solutions are mixed to obtain a mixed solution;

[0089] S302, slowly adding 20 mL of 0.1 mol / L hexadecyltrimethylammonium bromide solution to the mixed solution under stirring, and stirring for 10 min;

[0090] S303. Add 20 mL of 0.1 mol / L ascorbic acid solution to the solution obtained in S302, and continue stirring for 20 min to obtain a precursor solution.

[0091] S4, Au-Pt / Fe 3 O 4 / POPs Preparation:

[0092] 1.5 g of Fe 3 O 4 The Au-Pt / Fe / POPs complex was mixed with 15 mL of the precursor solution and ultrasonically dispersed for 30 min. The mixture was stirred for 1 h and the product was collected by magnetic separation. The product was washed alternately with ultrapure water and ethanol for no less than three times. The washed product was dispersed in water to obtain Au-Pt / Fe / POPs. 3 O 4 / POPs capture probe.

[0093] Example 2

[0094] In this embodiment, Au-Pt / Fe 3 O 4 The preparation method of the POPs capture probe comprises the following steps:

[0095] S1. POP preparation and pretreatment:

[0096] S101, POPs preparation: 1.1064 g (6 mmol) TCT, 9 mmol (0.7752 g) anhydrous piperazine and 0.432 g (3 mmol) terephthalaldehyde were mixed and dissolved in 100 mL 1,4-dioxane, and 2.4879 g (18 mmol) K 2 CO 3 Ultrasonic dispersion was performed for 20 min, and a dispersion solution was obtained after heating under reflux at 130°C;

[0097] After the dispersed solution was cooled to room temperature, it was distilled under reduced pressure for 20 minutes. Under continuous stirring, the remaining product was poured into excess methanol and stirred for 10 minutes. The precipitate was obtained by centrifugation, and the precipitate was dried to obtain POPs.

[0098] S102, POPs pretreatment: 2 g of POPs were immersed in 100 mL of 0.5 mol / L sodium hydroxide solution at 60° C. for 3.5 h, and then rinsed with deionized water until neutral to obtain treated POPs.

[0099] S2、Fe 3 O 4 Preparation of POPs / POPs complex:

[0100] S201, preparation of Fe 3 O 4 Nanoparticle dispersion: Fe 3 O 4 Nanoparticles were dispersed in 1,4-dioxane to obtain 0.1 g / mL Fe 3 O 4 Nanoparticle dispersion;

[0101] S202, 1g of the treated POPs and 15mL of the above Fe 3 O 4 After the nanoparticle dispersion was mixed, 25 g of anhydrous piperazine was added, stirred for 15 min, and refluxed with nitrogen at 120 °C for 36 h. Fe 3 O 4 / POPs complex.

[0102] Dichloromethane, anhydrous ethanol and ultrapure water were used to 3 O 4 / POPs complexes were washed by centrifugation three times;

[0103] The washed Fe 3 O 4 / POPs composites were vacuum freeze-dried, and the freeze-dried Fe 3 O 4 / POPs complex crushing treatment.

[0104] Among them, Fe 3 O 4 Fe 3 O 4 Nanoparticles, Fe 3 O 4 The method for preparing nanoparticles includes:

[0105] 1g of FeCl 2 ·4H 2 O and 1.36 g of FeCl 3 6H 2 O was stirred and mixed in 30 mL of deionized water, and heated from room temperature to 50 °C in a water bath, then the heating was stopped;

[0106] Stir the heated mixture and add 3 mL of ammonia water to the mixture according to the volume ratio. Use a permanent magnet to collect Fe 3 O 4 The pellet was precipitated and washed three times with anhydrous ethanol and deionized water until neutral. The resulting precipitate was dried in a vacuum freeze-drying oven to obtain Fe 3 O 4 Nanoparticles.

[0107] S3. Preparation of precursor solution:

[0108] S301, 40mL, 0.01mol / L HAuCl 4 solution and 10mL of 0.005mol / L H 2 PtCl 6 The solutions are mixed to obtain a mixed solution;

[0109] S302, slowly adding 40 mL of 0.1 mol / L hexadecyltrimethylammonium bromide solution to the mixed solution under stirring, and stirring for 10 min;

[0110] S303. Add 40 mL of 0.1 mol / L ascorbic acid solution to the solution obtained in S302, and continue stirring for 30 min to obtain a precursor solution.

[0111] S4, Au-Pt / Fe 3 O 4 / POPs Preparation:

[0112] 1.5 g of Fe 3 O 4 The / POPs complex was mixed with 30 mL of the precursor solution, and after ultrasonic dispersion for 30 min, stirring was continued for 1 h, and the product was collected by magnetic separation;

[0113] The product was mixed with 7.5 mL of 2 mol / L polydopamine solution and stirred for 3 h, and then the product was separated by magnetic separation. Ultrapure water and ethanol were used to wash the product three times each, and the washed product was dispersed in water to obtain Au-Pt / Fe 3 O 4 / POPs capture probe.

[0114] Example 3

[0115] In this embodiment, Au-Pt / Fe 3 O 4 The method for preparing a POPs capture probe comprises the following steps:

[0116] S1. POP preparation and pretreatment:

[0117] S101, POPs preparation: 1.1064 g (6 mmol) TCT, 9 mmol (0.7752 g) anhydrous piperazine and 0.432 g (3 mmol) terephthalaldehyde were mixed and dissolved in 100 mL 1,4-dioxane, and 2.4879 g (18 mmol) K 2 CO 3 Ultrasonic dispersion was performed for 15 min, and a dispersion solution was obtained after heating under reflux at 130°C;

[0118] After the dispersed solution was cooled to room temperature, it was distilled under reduced pressure for 20 minutes. Under continuous stirring, the remaining product was poured into excess methanol and stirred for 10 minutes. The precipitate was obtained by centrifugation, and the precipitate was dried to obtain POPs.

[0119] S102, POPs pretreatment: 2 g of POPs were immersed in 90 mL of 0.5 mol / L sodium hydroxide solution at 60° C. for 4 h, and then rinsed with deionized water until neutral to obtain treated POPs.

[0120] S2、Fe 3 O 4 Preparation of / POPs complex:

[0121] S201, preparation of Fe 3 O 4 Nanoparticle dispersion: Fe 3 O 4 Nanoparticles were dispersed in 1,4-dioxane to obtain 0.1 g / mL Fe 3 O 4 Nanoparticle dispersion;

[0122] S202, 1g of the treated POPs and 25mL of the above Fe 3 O 4After the nanoparticle dispersion was mixed, 35 g of anhydrous piperazine was added, stirred for 30 min, and refluxed with nitrogen at 120 °C for 36 h. Fe 3 O 4 / POPs complex.

[0123] Dichloromethane, anhydrous ethanol and ultrapure water were used to 3 O 4 / POPs complexes were washed by centrifugation three times;

[0124] The washed Fe 3 O 4 / POPs composites were vacuum freeze-dried, and the freeze-dried Fe 3 O 4 / POPs complex crushing treatment.

[0125] Among them, Fe 3 O 4 Fe 3 O 4 Nanoparticles, Fe 3 O 4 The method for preparing nanoparticles includes:

[0126] 1.057 g of FeCl 2 ·4H 2 O and 1.148 g of FeCl 3 6H 2 O was stirred and mixed in 30 mL of deionized water, and heated from room temperature to 65 °C in a water bath, then the heating was stopped;

[0127] Stir the heated mixture and add 3 mL of ammonia water to the mixture according to the volume ratio. Use a permanent magnet to collect Fe 3 O 4 The pellet was precipitated and washed three times with anhydrous ethanol and deionized water until neutral. The resulting precipitate was dried in a vacuum freeze-drying oven to obtain Fe 3 O 4 Nanoparticles.

[0128] Fe 3 O 4 / POPs complex modification:

[0129] Fe 3 O 4 The / POPs complex was mixed with 6 mL of 0.5 mol / L maleic anhydride solution, heated to 60 °C, stirred for 0.5 h under nitrogen protection, and then centrifuged to obtain the modified Fe 3 O 4 / POPs complex.

[0130] S3. Preparation of precursor solution:

[0131] S301, 20mL, 0.01mol / L HAuCl 4 solution and 10mL of 0.005mol / L H 2 PtCl 6 The solutions are mixed to obtain a mixed solution;

[0132] S302, slowly adding 20 mL of 0.1 mol / L hexadecyltrimethylammonium bromide solution to the mixed solution under stirring, and stirring for 10 min;

[0133] S303. Add 20 mL of 0.1 mol / L ascorbic acid solution to the solution obtained in S302, and continue stirring for 25 min to obtain a precursor solution.

[0134] S4, Au-Pt / Fe 3 O 4 / POPs Preparation:

[0135] 1.5 g of the modified Fe 3 O 4 The / POPs complex was mixed with 18 mL of the precursor solution, and after ultrasonic dispersion for 30 min, stirring was continued for 1 h, and the product was collected by magnetic separation;

[0136] The product was mixed with 9 mL of 2 mol / L polydopamine solution and stirred for 2 h, and then the product was separated by magnetic separation. Ultrapure water and ethanol were used to wash the product three times each, and the washed product was dispersed in water to obtain Au-Pt / Fe 3 O 4 / POPs capture probe.

[0137] See also Figure 4 As shown in the figure, the Fe 3 O 4 / POPs has a potential of 35.7 mV, Au-Pt / Fe 3 O 4 The Zeta potential of / POPs is 12.7 mV, and the molecule to be tested 4-HBA has a negative potential, indicating that it can be adsorbed on Au-Pt / Fe by electrostatic interaction. 3 O 4 / POPs for easy detection.

[0138] Example 4

[0139] The difference from Example 3 is that Fe 3 O 4 When the / POPs complex is modified, Fe3 O 4 / POPs complex is 2 g, and the volume of maleic anhydride solution is 12 mL.

[0140] The kit includes 100 μL ultrapure water and 100 μL Au-Pt / Fe 3 O 4 / POPs capture probe, Au-Pt / Fe 3 O 4 / POPs capture probe concentration was 5 mg / mL.

[0141] Detection of 4-hydroxybenzoic acid using the above kit comprises the following steps:

[0142] The target sample was mixed with Au-Pt / Fe 3 O 4 / POPs capture probes were mixed in a volume ratio of 1:1 and incubated with ultrasound for 30 min to obtain a mixed sample;

[0143] The mixed sample was magnetically separated and rinsed with ultrapure water, and dispersed in 100 μL of ultrapure water to obtain an analyte solution, which was then aspirated onto the surface of a glass slide for measurement, and the Raman signal was collected.

[0144] Example 5

[0145] The detection was performed using the detection kit for 4-hydroxybenzoic acid in Example 3.

[0146] 100 μL of target sample (4-HBA standard solution, concentration of 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 and 10 -12 mol / L) and Au-Pt / Fe 3 O 4 / POPs capture probe (5 mg / mL, 100 μL) detection kit was ultrasonically incubated for 30 min;

[0147] After magnetic separation and washing with ultrapure water, the analyte was evenly dispersed in 100 μL of ultrapure water in the detection kit; finally, the analyte solution was drawn up with a capillary and placed on the surface of a glass slide for three parallel measurements to collect Raman signals.

[0148] Figure 2 Figure 2 shows the SERS spectra of 4-HBA at different concentrations. The Raman intensity of 4-HBA decreases as the concentration decreases. When the concentration of 4-HBA decreases to 10-10 mol / L, 1072cm -1 The peak at shows the higher sensitivity of the base.

[0149] Example 6

[0150] The detection was performed using the detection kit for 4-hydroxybenzoic acid in Example 3.

[0151] Different concentrations of 4-HBA were added to the serum collected from healthy people to obtain 100 μL target samples. The concentrations of 4-HBA in these target samples were 10 -8 , 10 -9 , 10 -10 , 10 -11 mol / L.

[0152] The above target samples containing 100 μL of different concentrations were mixed with Au-Pt / Fe 3 O 4 / POPs capture probe (5mg·mL -1 , 100 μL) of the detection kit and incubate with ultrasound for 30 min;

[0153] After magnetic separation and washing with ultrapure water, the analyte was evenly dispersed in 100 μL ultrapure water; finally, the analyte solution was drawn up with a capillary and placed on the surface of a glass slide for three parallel measurements to collect Raman signals.

[0154] See also Figure 3 As shown in the figure, the SERS spectra of serum samples with different concentrations of 4-HBA added show that the Au-Pt / Fe 3 O 4 / POPs substrates can be detected at concentrations as low as 10 -10 mol / L of 4-HBA, indicating that this method can be used for the detection of 4-HBA in actual serum.

[0155] The above is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A detection kit for 4-hydroxybenzoic acid, characterized in that: It includes: Au-Pt / Fe3O4 / POPs capture probe for detecting target samples; The preparation method of the Au-Pt / Fe3O4 / POPs capture probe comprises the following steps: S1. POPs preparation and pretreatment: S101, preparation of POPs: TCT, anhydrous piperazine and terephthalaldehyde are mixed in a molar mass ratio of 2:3:1, and dissolved in 1,4-dioxane, and K2CO3 is added for ultrasonic dispersion for 10 to 20 minutes, and heated under reflux at 130° C. to obtain a dispersed solution, wherein the molar mass of the K2CO3 is three times that of TCT, and the molar mass ratio of the 1,4-dioxane to TCT is 200:1; After the dispersed solution is cooled to room temperature, it is distilled under reduced pressure for 20 to 40 minutes, and under continuous stirring, the remaining product is poured into excess methanol and stirred for 10 minutes, and centrifuged to obtain a precipitate, and the precipitate is dried to obtain POPs; S102, POPs pretreatment: The POPs obtained in S101 are immersed in a 0.5 mol / L sodium hydroxide solution at a mass volume ratio of 1:(40-50) at 60°C for 3-4 hours, and then rinsed with deionized water until neutral to obtain treated POPs; Preparation of S2 and Fe3O4 / POPs composites; S3. Preparation of precursor solution: S301, mixing 0.01 mol / L HAuCl4 solution and 0.005 mol / L H2PtCl6 solution in a volume ratio of 2:(0.5-1) to obtain a mixed solution; S302, slowly adding 0.1 mol / L hexadecyltrimethylammonium bromide solution to the mixed solution under stirring conditions, stirring and reacting for 10 minutes, wherein the ratio of the hexadecyltrimethylammonium bromide solution to the HAuCl4 solution is 1:1; S303, adding 0.1 mol / L ascorbic acid solution to the solution obtained in S302, and continuing stirring for 20 to 30 minutes to obtain a precursor solution, wherein the volume ratio of the ascorbic acid solution to the hexadecyltrimethylammonium bromide solution is 1:1; Preparation of S4, Au-Pt / Fe3O4 / POPs: The Fe3O4 / POPs complex is mixed with the precursor solution in a mass-to-volume ratio of 1:(10-20), and after ultrasonic dispersion for 30 minutes, stirring is continued for 1 hour, and the product is collected by magnetic separation. The product is washed alternately with ultrapure water and ethanol, and the washing times are not less than three times. The washed product is dispersed in water to obtain an Au-Pt / Fe3O4 / POPs capture probe.

2. The detection kit for 4-hydroxybenzoic acid according to claim 1, characterized in that: After collecting the product by magnetic separation, it also includes: The product was stirred and reacted with 2 mol / L polydopamine solution at a mass volume ratio of 1:(5-6) for 2-3 hours.

3. The detection kit for 4-hydroxybenzoic acid according to claim 1, characterized in that: The S2 includes: S201, preparing a Fe3O4 nanoparticle dispersion: dispersing Fe3O4 nanoparticles in 1,4-dioxane to obtain a 0.1 g / mL Fe3O4 nanoparticle dispersion; S202. The treated POPs and Fe3O4 nanoparticles are mixed in a mass ratio of 1:(1.5-2.5), anhydrous piperazine is added, stirred for 15-30 minutes, refluxed with nitrogen at 120°C for 36 hours, and a Fe3O4 / POPs complex is obtained after magnetic separation, wherein the total mass ratio of anhydrous piperazine to POPs and Fe3O4 nanoparticles is 1:

10.

4. The detection kit for 4-hydroxybenzoic acid according to claim 3, characterized in that: After S202, the following steps are also included: The Fe3O4 / POPs composite was washed by centrifugation several times using dichloromethane, anhydrous ethanol, and ultrapure water; The washed Fe3O4 / POPs composite is freeze-dried in vacuum, and the freeze-dried Fe3O4 / POPs composite is pulverized.

5. The detection kit for 4-hydroxybenzoic acid according to claim 1, characterized in that: When preparing the Fe3O4 / POPs composite in S2, the Fe3O4 used is Fe3O4 nanoparticles, and the preparation method of the Fe3O4 nanoparticles includes: FeCl2·4H2O and FeCl3·6H2O are stirred and mixed in a ratio of 1:(1-2) of iron ion molar amount, and heated in a water bath from room temperature to 50-65°C, and then the heating is stopped; The heated mixture was stirred, and ammonia water in an amount of one tenth of the volume of the mixture was added dropwise to the mixture according to a volume ratio. The Fe3O4 particle precipitate was collected, washed three times with anhydrous ethanol and deionized water until neutral, and dried to obtain Fe3O4 nanoparticles.

6. The detection kit for 4-hydroxybenzoic acid as claimed in claim 5, characterized in that: The FeCl2·4H2O and FeCl3·6H2O are stirred and mixed in a ratio of 1:1 in terms of molar amount of iron ions.

7. The detection kit for 4-hydroxybenzoic acid according to claim 1, characterized in that: After S2 and before S3, the following further includes: Modification of Fe3O4 / POPs composites: The Fe3O4 / POPs complex is mixed with 0.5 mol / L maleic anhydride solution in a mass volume ratio of 1:(3-6), the temperature is raised to 60°C, and the mixture is stirred for reaction for 0.5-1h under nitrogen protection and then centrifuged to obtain a modified Fe3O4 / POPs complex.

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