CN113138213A - Preparation of signal amplification sensor based on enzyme-like MOF - Google Patents

Abstract

The invention discloses a preparation method of a signal amplification sensor based on similar enzyme MOF, which firstly synthesizes ZnO-ZnIn with good photoelectrochemical signals₂S₄The material is a two-dimensional flower-like structure with large specific surface area, and can effectively increase the area for antibody growth; in addition, the MOF is modified on the secondary antibody to serve as a signal amplification carrier, so that on one hand, the impedance of the sensor is increased, and the photoelectric signal is reduced, on the other hand, the MOF can serve as an electron donor hydrogen peroxide in a catalase-like catalytic sensing system to decompose, so that a photoelectron reagent is reduced, and a photocurrent signal is further reduced, so that the photoelectric chemical sensor for detecting the prostate antigen is constructed through layer-by-layer modification.

Description

Preparation of signal amplification sensor based on enzyme-like MOF

Technical Field

The invention relates to the field of quantitative detection of prostate specific antigen, in particular to preparation of a sensor based on amplification of a similar enzyme MOF signal.

Background

The tumor marker is synthesized and secreted by tumor cells through gene expression, or is a substance abnormally produced by the body due to the reaction to the tumor, and has important significance in early diagnosis of the tumor. Early diagnosis of cancer requires the use of highly selective, highly sensitive, rapid analytical detection methods for its tumor markers. Therefore, a new simple, rapid, sensitive and selective method for detecting the malignant tumor biomarker is established, and the method has very important significance for early discovery and treatment effect evaluation of malignant tumors.

The photoelectrochemistry high sensitivity is combined with the selectivity of a suitable ligand, so that the photoelectrochemistry aptamer sensor becomes a new research hotspot in the field of analytical chemistry, a generated sensing signal is converted into an outputtable photoelectrochemical signal through a signal converter, and the signal and the concentration of a target analyte present a certain proportional relation, so that the quantitative analysis of the target substance can be realized. The photoelectrochemical immunosensor has the advantages of high specificity, simple operation, high sensitivity, high selectivity, high analysis speed and the like of immunoreaction, and is widely applied to the fields of clinical medicine, environmental pollutant detection, food analysis, biotechnology and the like.

Disclosure of Invention

The invention aims to construct a preparation of a MOF-based released lactic acid signal amplification sensor.

In order to solve the technical problem, the invention is realized by the following measures: preparation of a sensor based on amplification of a MOF-like signal, which is characterized by comprising the following steps:

(1) and (3) synthesizing ZnO: cutting conductive glass ITO into strips of 4.0 multiplied by 0.5 cm, ultrasonically cleaning the strips for 5 min by using an acetone solution, secondary distilled water and absolute ethyl alcohol in sequence, naturally drying the strips under nitrogen, spin-coating a layer of 0.25 mol/mL zinc acetate solution on the surface of the ITO by using a spin-coating method, annealing the surfaces of the ITO at 350 ℃ for 30 min, immersing the annealed ITO into a mixed solution of 0.025 mol/mL hexamethylenetetramine solution and 0.025 mol/mL zinc nitrate, placing the mixed solution at 95 ℃ for 6 h, naturally cooling the mixed solution to room temperature, washing the mixed solution three times by using absolute ethyl alcohol and ultrapure water respectively, and drying the washed solution at 60 ℃ for 12 h to obtain ZnO;

(2) synthesis of ZnO-ZnIn₂S₄: adding 0.4 mmol of ZnSO₄·7H₂O、0.8 mmol InCl₃·4H₂Dissolving O and 1.6 mmol of thioacetamide in 15 mL of water, stirring for 15 min at normal temperature, placing the mixture in a reaction kettle, placing a ZnO base growing on the ITO in the step (1) in the reaction kettle, heating for 12 h at 160 ℃, then washing with absolute ethyl alcohol and ultrapure water for three times respectively, and drying at 60 DEG CDrying for 12 h to obtain ZnO-ZnIn₂S₄；

(3) Synthesis of MOF: dispersing 40 mg of 2, 2-bipyridine-5, 5-dicarboxylic acid in 18 mL of N-N dimethylformamide, adding 3.06 mg of zirconium chloride into the solution, carrying out ultrasound treatment for 10 min, heating the solution at 90 ℃ for 18 h, cooling to room temperature, centrifuging at 8000 rmp for 10 min, dispersing in 50 mL of tetrahydrofuran, and then dispersing 106 mg of CuCl₂·2H₂Adding O into the solution, performing ultrasonic treatment for 10 min, and stirring for 2 h to obtain a required MOF material;

(4) synthesis of SA-MOF: dissolving 4 mg of MOF synthesized in the step (3) in 4 mL of ethanol solution, adding 200 μ L of 3-aminopropyltriethoxysilane, performing ultrasound treatment for 1 h to obtain an aminated MOF material, centrifuging the obtained material, dispersing the centrifuged material in 2 mL of PBS, adding 0.2 mL of glutaraldehyde solution, incubating at room temperature for 1 h, adding 0.2 mL of 2 mg/mL of streptavidin, incubating at 4 ℃ for 1 h, washing, centrifuging, and dissolving the obtained precipitate in 2 mL of PBS to obtain SA-MOF;

(5) synthesis of SA-Ab₂-a MOF: 1 mL of a secondary antibody, Ab, at a concentration of 10. mu.g/mL₂Adding into the synthesized product of step (4), incubating at 4 deg.C for 2 h, washing 3 times with pH 7.4 phosphate buffer solution to remove non-complexed Ab₂Obtaining the SA-Ab₂-MOF；

(6) Construction of the photoelectrochemical sensor: 6 mu L of primary antibody Ab1 with the concentration of 10 mu g/mL is dripped in the synthetic ZnO-ZnIn₂S₄Incubating for 16 h at 4 ℃, and thoroughly washing for 3 times by using a phosphate buffer solution with pH of 7.4; continuously dripping 20 mu L of 3% bovine serum albumin to block the non-specific binding sites, thoroughly washing the non-specific binding sites for 3 times by using a phosphate buffer solution with pH 7.4, dripping 20 mu L of prostate antigens with different concentrations onto the surface of the electrode, incubating for 30 min at room temperature, and washing for 3 times by using a phosphate buffer solution with pH 7.4; continuously dropwise adding 20 mu L of SA-Ab synthesized in the step (5)₂-MOF, incubation for 2 h at room temperature;

(7) photoelectrochemical detection of sandwich-type signal amplification biosensors: and (3) taking the modified electrode processed in the step (6) as a working electrode, taking the counter electrode as a platinum wire electrode, taking the reference electrode as an Ag/AgCl electrode, taking the bias voltage value as 0V, taking a xenon lamp as a light source for stimulation, taking a phosphate buffer solution system (1 mol/L hydrogen peroxide) with the pH value of 7.4 as an electrolytic cell, and measuring a current I-T curve to detect the photoelectric property.

The invention has the beneficial effects that:

(1) the method has the advantages of low cost, simple experimental operation and easy control of reaction conditions.

(2) Synthetic ZnO-ZnIn₂S₄The antibody is in a two-dimensional flower-shaped structure, has a large specific surface area, and can effectively increase the area for antibody growth.

(3) The MOF is used as a signal amplification carrier and has large impedance, so that the signal can be reduced, and meanwhile, the synthesized catalase-like enzyme can catalyze the decomposition of the electron donor hydrogen peroxide in the solution, so that the electron donor is reduced, and the signal is further reduced.

Detailed Description

In order to further understand the invention, the technical scheme is implemented by combining the embodiment, and the specific implementation mode is given:

(1) and (3) synthesizing ZnO: cutting conductive glass ITO into strips of 4.0 multiplied by 0.5 cm, ultrasonically cleaning the strips for 5 min by using an acetone solution, secondary distilled water and absolute ethyl alcohol in sequence, naturally drying the strips under nitrogen, spin-coating a layer of 0.25 mol/mL zinc acetate solution on the surface of the ITO by using a spin-coating method, annealing the surfaces of the ITO at 350 ℃ for 30 min, immersing the annealed ITO into a mixed solution of 0.025 mol/mL hexamethylenetetramine solution and 0.025 mol/mL zinc nitrate, placing the mixed solution at 95 ℃ for 6 h, naturally cooling the mixed solution to room temperature, washing the mixed solution three times by using absolute ethyl alcohol and ultrapure water respectively, and drying the washed solution at 60 ℃ for 12 h to obtain ZnO;

(2) synthesis of ZnO-ZnIn₂S₄: adding 0.4 mmol of ZnSO₄·7H₂O、0.8 mmol InCl₃·4H₂Dissolving O and 1.6 mmol thioacetamide in 15 mL water, stirring at normal temperature for 15 min, placing in a reaction kettle, placing a ZnO base growing on the ITO in the step (1) in the reaction kettle, heating at 160 ℃ for 12 h, washing with absolute ethyl alcohol and ultrapure water for three times respectively, and drying at 60 ℃ for 12 h to obtain ZnO-ZnIn₂S₄；

(3) Synthesis of MOF: dispersing 40 mg of 2, 2-bipyridine-5, 5-dicarboxylic acid in 18 mL of N-N dimethylformamide, adding 3.06 mg of zirconium chloride into the solution, carrying out ultrasound treatment for 10 min, heating the solution at 90 ℃ for 18 h, cooling to room temperature, centrifuging at 8000 rmp for 10 min, dispersing in 50 mL of tetrahydrofuran, and then dispersing 106 mg of CuCl₂·2H₂Adding O into the solution, performing ultrasonic treatment for 10 min, and stirring for 2 h to obtain a required MOF material;

(4) synthesis of SA-MOF: dissolving 4 mg of MOF synthesized in the step (3) in 4 mL of ethanol solution, adding 200 μ L of 3-aminopropyltriethoxysilane, performing ultrasound treatment for 1 h to obtain an aminated MOF material, centrifuging the obtained material, dispersing the centrifuged material in 2 mL of PBS, adding 0.2 mL of glutaraldehyde solution, incubating at room temperature for 1 h, adding 0.2 mL of 2 mg/mL of streptavidin, incubating at 4 ℃ for 1 h, washing, centrifuging, and dissolving the obtained precipitate in 2 mL of PBS to obtain SA-MOF;

(5) synthesis of SA-Ab₂-a MOF: 1 mL of a secondary antibody, Ab, at a concentration of 10. mu.g/mL₂Adding into the synthesized product of step (4), incubating at 4 deg.C for 2 h, washing 3 times with pH 7.4 phosphate buffer solution to remove non-complexed Ab₂Obtaining the SA-Ab₂-MOF；

(6) Construction of the photoelectrochemical sensor: 6 mu L of primary antibody Ab1 with the concentration of 10 mu g/mL is dripped in the synthetic ZnO-ZnIn₂S₄Incubating for 16 h at 4 ℃, and thoroughly washing for 3 times by using a phosphate buffer solution with pH of 7.4; continuously dripping 20 mu L of 3% bovine serum albumin to block the non-specific binding sites, thoroughly washing the non-specific binding sites for 3 times by using a phosphate buffer solution with pH 7.4, dripping 20 mu L of prostate antigens with different concentrations onto the surface of the electrode, incubating for 30 min at room temperature, and washing for 3 times by using a phosphate buffer solution with pH 7.4; continuously dropwise adding 20 mu L of SA-Ab synthesized in the step (5)₂-MOF, incubation for 2 h at room temperature;

(7) photoelectrochemical detection of sandwich-type signal amplification biosensors: and (3) taking the modified electrode treated in the step (6) as a working electrode, taking the counter electrode as a platinum wire electrode, taking the reference electrode as an Ag/AgCl electrode, taking the bias voltage value as 0V, taking a xenon lamp as a light source for stimulation, taking an electrolytic cell as a phosphate buffer solution system (1 mol/L of hydrogen peroxide) with the pH of 7.4, measuring a current I-T curve to detect the photoelectric property, obtaining a linear equation of I = -4.21 log (c) -64.24, a correlation coefficient of 0.996 and a detection limit of 0.05 pg/mL, and realizing the high-sensitivity detection of the prostate specific antigen.

Claims (1)

1. The preparation of the sensor based on the amplification of the MOF signal of the enzyme is characterized by comprising the following steps:

(1) and (3) synthesizing ZnO: cutting conductive glass ITO into strips of 4.0 multiplied by 0.5 cm, ultrasonically cleaning the strips for 5 min by using an acetone solution, secondary distilled water and absolute ethyl alcohol in sequence, naturally drying the strips under nitrogen, spin-coating a layer of 0.25 mol/mL zinc acetate solution on the surface of the ITO by using a spin-coating method, annealing the surfaces of the ITO at 350 ℃ for 30 min, immersing the annealed ITO into a mixed solution of 0.025 mol/mL hexamethylenetetramine solution and 0.025 mol/mL zinc nitrate, placing the mixed solution at 95 ℃ for 6 h, naturally cooling the mixed solution to room temperature, washing the mixed solution three times by using absolute ethyl alcohol and ultrapure water respectively, and drying the washed solution at 60 ℃ for 12 h to obtain ZnO;

(2) synthesis of ZnO-ZnIn₂S₄: adding 0.4 mmol of ZnSO₄·7H₂O、0.8 mmol InCl₃·4H₂Dissolving O and 1.6 mmol thioacetamide in 15 mL water, stirring at normal temperature for 15 min, placing in a reaction kettle, placing a ZnO base growing on the ITO in the step (1) in the reaction kettle, heating at 160 ℃ for 12 h, washing with absolute ethyl alcohol and ultrapure water for three times respectively, and drying at 60 ℃ for 12 h to obtain ZnO-ZnIn₂S₄；

(3) Synthesis of MOF: dispersing 40 mg of 2, 2-bipyridine-5, 5-dicarboxylic acid in 18 mL of N-N dimethylformamide, adding 3.06 mg of zirconium chloride into the solution, carrying out ultrasound treatment for 10 min, heating the solution at 90 ℃ for 18 h, cooling to room temperature, centrifuging at 8000 rmp for 10 min, dispersing in 50 mL of tetrahydrofuran, and then dispersing 106 mg of CuCl₂·2H₂Adding O into the solution, performing ultrasonic treatment for 10 min, and stirring for 2 h to obtain a required MOF material;

(4) synthesis of SA-MOF: dissolving 4 mg of MOF synthesized in the step (3) in 4 mL of ethanol solution, adding 200 μ L of 3-aminopropyltriethoxysilane, performing ultrasound treatment for 1 h to obtain an aminated MOF material, centrifuging the obtained material, dispersing the centrifuged material in 2 mL of PBS, adding 0.2 mL of glutaraldehyde solution, incubating at room temperature for 1 h, adding 0.2 mL of 2 mg/mL of streptavidin, incubating at 4 ℃ for 1 h, washing, centrifuging, and dissolving the obtained precipitate in 2 mL of PBS to obtain SA-MOF;

(5) synthesis of SA-Ab₂-a MOF: 1 mL of a secondary antibody, Ab, at a concentration of 10. mu.g/mL₂Adding into the synthesized product of step (4), incubating at 4 deg.C for 2 h, washing 3 times with pH 7.4 phosphate buffer solution to remove non-complexed Ab₂Obtaining the SA-Ab₂-MOF；

(6) Construction of the photoelectrochemical sensor: 6 mu L of primary antibody Ab1 with the concentration of 10 mu g/mL is dripped in the synthetic ZnO-ZnIn₂S₄Incubating for 16 h at 4 ℃, and thoroughly washing for 3 times by using a phosphate buffer solution with pH of 7.4; continuously dripping 20 mu L of 3% bovine serum albumin to block the non-specific binding sites, thoroughly washing the non-specific binding sites for 3 times by using a phosphate buffer solution with pH 7.4, dripping 20 mu L of prostate antigens with different concentrations onto the surface of the electrode, incubating for 30 min at room temperature, and washing for 3 times by using a phosphate buffer solution with pH 7.4; continuously dropwise adding 20 mu L of SA-Ab synthesized in the step (5)₂-MOF, incubation for 2 h at room temperature;

(7) photoelectrochemical detection of sandwich-type signal amplification biosensors: and (3) taking the modified electrode processed in the step (6) as a working electrode, taking the counter electrode as a platinum wire electrode, taking the reference electrode as an Ag/AgCl electrode, taking the bias voltage value as 0V, taking a xenon lamp as a light source for stimulation, taking a phosphate buffer solution system (1 mol/L hydrogen peroxide) with the pH value of 7.4 as an electrolytic cell, and measuring a current I-T curve to detect the photoelectric property.