CN110702670B - Sarcosine detection method based on metal organic framework material - Google Patents
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
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Abstract
The invention relates to a sarcosine detection method based on a metal organic framework material, which is characterized in that sarcosine oxidase and catalase are wrapped on the basis of the metal organic framework material, so that cascade enzymes are limited in a small space, and the content of sarcosine is determined by adopting a chemiluminescence method after cascade reaction. The invention adopts ZIF-8 to wrap SOX and HRP. ZIF-8 has larger specific surface area and porous pore channels, is beneficial to reaction substrates in solution to pass through the pore channels to react with SOX and HRP, is beneficial to the enrichment of the substrates around the enzyme, and increases the local substrate concentration. Meanwhile, the package of the ZIF-8 limits the SOX and the HRP within the range of hundreds of nanometers, the distance between the SOX and the HRP is regulated, hydrogen peroxide generated by catalyzing sarcosine by the SOX can be effectively transmitted to the HRP for color reaction, and the reaction rate of the reaction cascade enzyme is improved.
Description
Technical Field
The invention relates to a detection method for determining specific target sarcosine of prostate cancer, in particular to a sarcosine detection method based on a metal organic framework material. The invention belongs to the field of chemical detection.
Background
The prostate cancer is one of common male malignant tumors, and particularly, with the increasing aging of the Chinese society, the diagnosis rate and the mortality rate of the prostate cancer are increased, so that the survival rate of a patient can be remarkably improved and improved by early screening of the prostate cancer patient. The existing prostate cancer diagnosis method is mainly based on Prostate Specific Antigen (PSA), the detection sensitivity and specificity of the method have great problems, the false positive rate and the false negative rate are both high, and therefore, many patients carry out unnecessary biopsy, and great pain is caused. Therefore, a new marker detection method and an effective screening means are urgently needed.
In 2009, a pilot study created a non-invasive method to identify the presence and progression of prostate cancer and showed that the glycine metabolite, sarcosine, can be detected in the urine and blood of patients and can be used as a predictive marker for prostate cancer. The traditional sarcosine determination methods are: an electrochemical method (Chinese patent: an electrochemical detection method for sarcosine, publication No. CN 106596693A.); electrophoresis method: (Chinese patent: a method for detecting sarcosine, publication No. CN 101718746A.); fluorescence spectroscopy: (Chinese patent: method for quantitative determination of sarcosine content and reaction kit, publication No. CN 101587076A.); chromatographic mass spectrometry (A reproducible and high-throughput HPLC/MS method to separate and purify both from alpha-and beta-alkane and to quantify the carbohydrate in human serum and urine, anal. chem. 83(2011): 5735 and 5740). The main defects of the technologies are high instrument cost, complicated sample preparation, requirements of skilled operators and unsuitability for daily operation and analysis, so that the development of a sensitive and stable sarcosine detection method is urgently needed.
Direct measurement of Sarcosine is difficult, and Sarcosine content can be measured by using a cascade enzyme of Sarcosine Oxidase (SOX) and Horseradish Peroxidase (HRP) to catalyze the production of glycine, formaldehyde and hydrogen peroxide from Sarcosine by SOX and the production of chromogenic reaction from hydrogen peroxide by HRP. However, SOX and HRP in a disordered state in the solution, random substrate collisions significantly reduce the catalytic efficiency of the cascade reaction. Metal Organic Frameworks (MOFs) are a class of crystalline porous materials with periodic network structures formed by the interconnection of inorganic metal centers (metal ions or metal clusters) and organic ligands through self-assembly. The special ZIF-8 material has the advantages of large specific surface area, good biocompatibility, stable chemical and physical properties, and has great potential in the fields of drug loading, catalysis, biosensing, substance enrichment, separation and determination and the like. Therefore, the ZIF-8 material is used for wrapping SOX and HRP, a structure of a domain-restricted cascade enzyme is designed, and the detection of sarcosine has higher research significance and application value.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a sarcosine detection method based on a metal-organic framework material.
The purpose of the invention is realized by the following scheme: a sarcosine detection method based on a metal organic framework material is characterized in that sarcosine oxidase and catalase are wrapped on the basis of the metal organic framework material, so that a pair of cascade enzymes are limited in a small space, and the content of sarcosine is measured by adopting a chemiluminescence method after cascade reaction, and the method specifically comprises the following steps:
(1) ZIF-8 encapsulation of SOX and HRP:
weighing 0.1-0.5 mg of SOX and 0.2-0.5 mg of HRP, dissolving in 1 mL of 2-methylimidazole (2-MIM, 1M) aqueous solution to make the mass ratio of SOX to HRP 1: 1-1: 5, mixing uniformly at room temperature, incubating for 10 min, then, adding 1 mL zinc acetate or zinc nitrate (20 mM) solution into the mixed solution rapidly, mixing uniformly, mechanically stirring at room temperature overnight, centrifuging the mixed solution at 5000 rpm/min for 15 min, washing with ethanol for 2 times, and centrifuging the precipitate by using solution containing 2% PVP (w/w), mechanically stirring for 1 hr, then, centrifugation was carried out at 5000 rpm/min for 20 min, PVP-stabilized SOX-HRP @ ZIF-8 was collected, the precipitate was dispersed in 2 mL of phosphate buffer solution (PBS, pH 7.4), obtaining a PVP-SOX-HRP @ ZIF-8 solution;
(2) determination of sarcosine content:
preparing a chromogenic reaction substrate 2, 2-dinitrogen-di (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS, 10 mM) by using PBS, preparing a sarcosine (5-100 mu M) solution, storing in an ice bath, opening an ultraviolet spectrophotometer, setting parameters, correcting a base line, sequentially taking 10 mu L sarcosine solution, 10 mu L ABTS chromogenic solution and 70 mu L PBS solution (pH 7.4), uniformly mixing, transferring to a cuvette, then adding 10 mu L PVP-SOX-HRP @ ZIF-8 solution, reacting for 10 min after uniformly mixing, detecting the change of absorbance at 414 nm, and drawing a standard curve of the sarcosine concentration.
Based on the scheme, the better mass ratio of SOX to HRP is 1: 3.
In the technical scheme of the invention, the ZIF-8 material is designed to wrap sarcosine oxidase and horseradish peroxidase, and the sarcosine oxidase can catalyze sarcosine to generate glycine, formaldehyde and hydrogen peroxide. Further, catalase may catalyze hydrogen peroxide to oxidize 2, 2-diaza-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) to generate blue-green free radical cations (ABTS).+) Thereby realizing the output of the detection signal.
The invention relates to a method for determining sarcosine by a domain-limited cascade enzyme structure, wherein sarcosine oxidase and horseradish peroxidase are wrapped by MOFs materials to enable SOX and HRP to be limited in a space of hundreds of nanometers, and a chemiluminescence signal generated by enzymatic cascade reaction is used for quantifying the concentration of the sarcosine. The method can regulate and control the distance of the cascade enzyme, improve the reaction rate of the cascade enzyme, has higher sensitivity, and can quickly carry out quantitative detection on the content of the sarcosine in the blood and the urine.
The invention has the advantages that:
(1) the invention adopts ZIF-8 to wrap SOX and HRP. ZIF-8 has larger specific surface area and porous pore channels, is beneficial to reaction substrates in the solution to pass through the pore channels to react with SOX and HRP, is beneficial to the enrichment of the substrates around the enzyme, and increases the local substrate concentration;
(2) meanwhile, the package of the ZIF-8 limits the SOX and the HRP within the range of hundreds of nanometers, the distance between the SOX and the HRP is regulated, hydrogen peroxide generated by catalyzing sarcosine by the SOX can be effectively transmitted to the HRP for color reaction, and the reaction rate of the reaction cascade enzyme is improved.
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FIG. 1 is a transmission electron microscopy characterization of PVP-SOX-HRP @ ZIF-8 prepared in example 1.
Detailed Description
The technical solution of the present invention is further described below by specific examples. The following examples are further illustrative of the present invention and do not limit the scope of the present invention.
Example 1
A sarcosine detection method based on a metal organic framework material is characterized in that sarcosine oxidase and catalase are wrapped on the basis of the metal organic framework material, so that a pair of cascade enzymes are limited in a small space, and the content of sarcosine is measured by adopting a chemiluminescence method after cascade reaction, and the method comprises the following steps:
(1) ZIF-8 encapsulation of SOX and HRP:
weighing 0.1 mg of SOX and 0.2 mg of HRP, dissolving in 1 mL of 1M 2-methylimidazole (2-MIM) water solution, uniformly mixing at room temperature, and incubating for 10 minutes to obtain a mixed solution; subsequently, 1 mL of 20 mM zinc acetate solution was added to the mixture quickly, and after mixing well, the mixture was mechanically stirred at room temperature overnight. Centrifuging the mixed solution at 5000 rpm/min for 15 min, washing with ethanol for 2 times, centrifuging the precipitate with solution containing 2% PVP (w/w), and mechanically stirring for 1 hr; and then, centrifuging at the rotating speed of 5000 rpm/min for 20 min, collecting PVP stable SOX-HRP @ ZIF-8, and dispersing the precipitate in 2 mL phosphate buffer solution (PBS, pH 7.4) to obtain a PVP-SOX-HRP @ ZIF-8 solution. The resulting PVP-SOX-HRP @ ZIF-8 nanostructure was characterized by TEM with a size in the range of about 130 nm. FIG. 1 shows a transmission electron microscopy characterization of PVP-SOX-HRP @ ZIF-8 prepared in this example.
(2) Determination of sarcosine content:
preparing a 10 mM ABTS solution by using PBS, preparing a sarcosine solution of 5-100 mu M, and storing in an ice bath; opening an ultraviolet spectrophotometer, setting parameters and correcting a base line; sequentially taking 10 muL sarcosine solutions with various concentrations, respectively and uniformly mixing 10 muL ABTS chromogenic solutions and 70 muL PBS solutions (pH 7.4), and transferring the mixture to a cuvette; then, 10 muL of PVP-SOX-HRP @ ZIF-8 solution is added, after uniform mixing, reaction is carried out for 10 min, absorbance change at 414 nm is detected, and a standard curve of sarcosine concentration is drawn.
Example 2
A sarcosine detection method based on a metal organic framework material is similar to that of the embodiment 1, and comprises the following steps:
(1) ZIF-8 encapsulation of SOX and HRP:
weighing 0.1 mg of SOX and 0.3 mg of HRP, dissolving in 1 mL of 2-methylimidazole (2-MIM) aqueous solution with the concentration of 1M, uniformly mixing at room temperature, and incubating for 10 minutes to obtain a mixed solution; then, rapidly adding 1 mL of 20 mM zinc acetate solution into the mixed solution, uniformly mixing, and mechanically stirring at room temperature overnight; centrifuging the mixed solution at 5000 rpm/min for 15 min to obtain precipitate, washing with ethanol for 2 times, re-centrifuging the precipitate with solution containing 2% PVP (w/w), and mechanically stirring for 1 hr; and then, centrifuging at the rotating speed of 5000 rpm/min for 20 min, collecting PVP stable SOX-HRP @ ZIF-8, and dispersing the precipitate in 2 mL phosphate buffer solution (PBS, pH 7.4) to obtain a PVP-SOX-HRP @ ZIF-8 solution.
(2) Determination of sarcosine content:
preparing 10 mM ABTS solution by PBS, preparing 5-100 mu M sarcosine solution, and storing in ice bath; opening an ultraviolet spectrophotometer, setting parameters and correcting a base line; sequentially taking 10 muL sarcosine solution, 10 muL ABTS chromogenic solution and 70 muL PBS solution (pH 7.4), uniformly mixing, and transferring to a cuvette; then, 10 muL of PVP-SOX-HRP @ ZIF-8 solution is added, after uniform mixing, reaction is carried out for 10 min, absorbance change at 414 nm is detected, and a standard curve of sarcosine concentration is drawn.
Example 3
A sarcosine detection method based on a metal organic framework material is similar to that of the embodiment 1, and comprises the following steps:
(1) ZIF-8 encapsulation of SOX and HRP:
weighing 0.2 mg of SOX and 0.3 mg of HRP, dissolving in 1 mL of 1M 2-methylimidazole (2-MIM) water solution, uniformly mixing at room temperature, and incubating for 10 minutes to obtain a mixed solution; then, rapidly adding 1 mL of 20 mM zinc acetate solution into the mixed solution, uniformly mixing, and mechanically stirring at room temperature overnight; centrifuging the mixed solution at 5000 rpm/min for 15 min to obtain precipitate, washing with ethanol for 2 times, re-centrifuging the precipitate with solution containing 2% PVP (w/w), and mechanically stirring for 1 hr; then, centrifuging at the rotating speed of 5000 rpm/min for 20 min, collecting PVP stable SOX-HRP @ ZIF-8, and dispersing the precipitate in 2 mL phosphate buffer (PBS, pH 7.4) to obtain a PVP-SOX-HRP @ ZIF-8 solution;
(2) determination of sarcosine content:
preparing 10 mM ABTS solution by PBS, preparing 5-100 mu M sarcosine solution, and storing in ice bath; opening an ultraviolet spectrophotometer, setting parameters and correcting a base line; sequentially taking 10 muL sarcosine solution with each concentration, respectively and uniformly mixing with 10 muL ABTS chromogenic solution and 70 muL PBS solution (pH 7.4), and transferring to a cuvette; then, 10 muL of PVP-SOX-HRP @ ZIF-8 solution is added, after uniform mixing, reaction is carried out for 10 min, absorbance change at 414 nm is detected, and a standard curve of sarcosine concentration is drawn.
Example 4
A sarcosine detection method based on a metal organic framework material is similar to that in example 1, and comprises the following steps:
(1) ZIF-8 encapsulation of SOX and HRP:
0.2 mg of SOX and 0.5 mg of HRP are weighed and dissolved in 1 mL of 2-methylimidazole (2-MIM, 1M) water solution, mixed uniformly at room temperature and incubated for 10 minutes; then, 1 mL of zinc acetate (20 mM) solution is rapidly added into the mixed solution, and after uniform mixing, mechanical stirring is carried out at room temperature overnight; centrifuging the mixed solution at 5000 rpm/min for 15 min, washing with ethanol for 2 times, re-spinning the precipitate with solution containing 2% PVP (w/w), and mechanically stirring for 1 hr; then, centrifuging at the rotating speed of 5000 rpm/min for 20 min, collecting PVP stable SOX-HRP @ ZIF-8, and dispersing the precipitate in 2 mL phosphate buffer (PBS, pH 7.4) to obtain a PVP-SOX-HRP @ ZIF-8 solution;
(2) determination of sarcosine content:
preparing 10 mM ABTS solution by PBS, preparing 5-100 mu M sarcosine solution, and storing in ice bath; and opening the ultraviolet spectrophotometer, setting parameters and correcting the base line. Sequentially taking 10 muL sarcosine solution, 10 muL ABTS chromogenic solution and 70 muL PBS solution (pH 7.4), uniformly mixing, and transferring to a cuvette; then, 10 muL of PVP-SOX-HRP @ ZIF-8 solution is added, after uniform mixing, reaction is carried out for 10 min, absorbance change at 414 nm is detected, and a standard curve of sarcosine concentration is drawn.
Example 5
A sarcosine detection method based on a metal organic framework material is similar to that of the embodiment 1, and comprises the following steps:
(1) ZIF-8 encapsulation of SOX and HRP:
0.1 mg of SOX and 0.4mg of HRP were dissolved in 1 mL of an aqueous solution of 2-methylimidazole (2-MIM, 1M), mixed well at room temperature, and incubated for 10 minutes. Subsequently, 1 mL of zinc acetate (20 mM) solution was added to the mixture quickly, mixed well, and then mechanically stirred at room temperature overnight. The mixture was centrifuged at 5000 rpm/min for 15 min, washed with ethanol 2 times, and the precipitate was re-spun with a solution containing 2% PVP (w/w) and mechanically stirred for 1 hr. And then, centrifuging at the rotating speed of 5000 rpm/min for 20 min, collecting PVP stable SOX-HRP @ ZIF-8, and dispersing the precipitate in 2 mL phosphate buffer solution (PBS, pH 7.4) to obtain a PVP-SOX-HRP @ ZIF-8 solution.
(2) Determination of sarcosine content:
preparing 10 mM ABTS solution by PBS, preparing 5-100 mu M sarcosine solution, and storing in ice bath. And opening the ultraviolet spectrophotometer, setting parameters and correcting the base line. Sequentially taking 10 muL sarcosine solution, 10 muL ABTS chromogenic solution and 70 muL PBS solution (pH 7.4), uniformly mixing, and transferring to a cuvette. Then, 10 muL of PVP-SOX-HRP @ ZIF-8 solution is added, after uniform mixing, reaction is carried out for 10 min, absorbance change at 414 nm is detected, and a standard curve of sarcosine concentration is drawn.
Claims (2)
1. A sarcosine detection method based on a metal organic framework material is characterized in that sarcosine oxidase and catalase are wrapped on the basis of the metal organic framework material, so that a pair of cascade enzymes are limited in a small space, and the content of sarcosine is measured by adopting a chemiluminescence method after cascade reaction, and the method comprises the following steps:
(1) the zinc-based metal organic framework structure ZIF-8 wraps sarcosine oxidase SOX and horseradish peroxidase HRP:
weighing 0.1-0.5 mg SOX and 0.2-0.5 mg HRP, dissolving in 1 mL 2-methylimidazole (2-MIM) water solution with concentration of 1M, the mass ratio of SOX to HRP is 1: (1-5), uniformly mixing at room temperature, incubating for 10 minutes to obtain a mixed solution, and then, adding 1 mL of 20 mM zinc acetate or zinc nitrate solution into the mixed solution rapidly, mixing uniformly, mechanically stirring at room temperature overnight, centrifuging at 5000 rpm/min for 15 min to obtain precipitate, washing with ethanol for 2 times, and centrifuging the precipitate by using solution containing 2% PVP (w/w), mechanically stirring for 1 hr, then, the solution was centrifuged at 5000 rpm/min for 20 min, and SOX-HRP @ ZIF-8 stabilized with PVP was collected, and the precipitate was dispersed in 2 mL of phosphate buffered saline PBS, pH 7.4, obtaining a PVP-SOX-HRP @ ZIF-8 solution;
(2) determination of sarcosine content:
preparing 10 mM chromogenic reaction substrate 2, 2-linked nitrogen-di (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt ABTS by using PBS, preparing 5-100 mu M sarcosine solution, storing in ice bath, opening an ultraviolet spectrophotometer, setting parameters, correcting a base line, sequentially taking 10 mu L sarcosine solution, 10 mu L ABTS chromogenic solution and 70 mu L PBS solution, pH 7.4, uniformly mixing, transferring to a cuvette, then adding 10 mu L PVP-SOX-HRP @ ZIF-8 solution, reacting for 10 min after uniformly mixing, detecting the absorbance change at 414 nm, and drawing a standard curve of the sarcosine concentration.
2. The method for detecting sarcosine based on a metal-organic framework material as claimed in claim 1, wherein the method comprises the steps of: the mass ratio of SOX to HRP is 1: 3.
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