CN114384237A - Double-enzyme co-loaded nanosphere for AFP (alpha-fetoprotein) detection, preparation method thereof and AFP detection method - Google Patents
Double-enzyme co-loaded nanosphere for AFP (alpha-fetoprotein) detection, preparation method thereof and AFP detection method Download PDFInfo
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- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
The invention provides double-enzyme co-loaded nanospheres for Alpha Fetoprotein (AFP) detection, a preparation method, a probe, a kit and an AFP detection method, and belongs to the field of biological detection and enzyme loading materials. The double-enzyme co-loading nanosphere comprises: magnetic mesoporous silica nanospheres with a core-shell structure, and glucose oxidase GoD and horseradish peroxidase HRP distributed in mesoporous channels; wherein the core-shell structure has magnetic core particles and a silica shell structure with mesoporous channels distributed therein; the magnetic core particles are used for enriching AFP in a serum sample through magnetic separation; GoD and HRP were used to catalyze substrate color development. According to the invention, the enzyme loading capacity is improved through the mesoporous nanospheres with the core-shell structure, and meanwhile, the target is enriched through the superparamagnetic property, so that the background interference introduced by a biological sample is reduced, the signal intensity of detection is improved, and the detection sensitivity is improved; meanwhile, the detection stability is ensured, and the detection accuracy and precision are improved.
Description
Technical Field
The invention belongs to the field of biological detection and enzyme loading materials, and particularly relates to a double-enzyme co-loading nanosphere for AFP detection, a preparation method thereof and an AFP detection method.
Background
Alpha-fetoprotein, AFP, consists of 591 amino acids and is a glycoprotein synthesized by the liver and yolk sac during early fetal development. The AFP content in normal human serum is lower and is 10 ng-1-20 ng•mL-1In the meantime. When malignant lesion occurs in liver cells, the concentration of AFP is increased rapidly, the AFP is a common marker for clinically diagnosing primary liver cancer, the diagnosis rate of liver cancer is about 70%, and the AFP is closely related to the occurrence and development of various tumors. When the AFP content in the serum is more than 200 ng/mL within 2 months-1Or more than 400 ng.mL in one month-1When it is determined to be liver cancer, the liver cancer can be determined. Therefore, liver cancer can be diagnosed with the aid of the detection of AFP.
In the prior art, the method for detecting AFP in serum comprises the following steps: Enzyme-Linked Immunosorbent Assay (ELISA) colorimetry, magnetic particle chemiluminescence, cyclic voltammetry, linear sweep voltammetry, and the like. The method comprises the steps of performing chemical luminescence on magnetic particles, performing cyclic voltammetry, performing linear scanning voltammetry and the like, wherein the methods such as the magnetic particle chemiluminescence method, the cyclic voltammetry, the linear scanning voltammetry and the like have the advantages of high detection sensitivity, high automation and the like, but the detection instrument is large in size, the reagent is expensive, and the method is not suitable for single-person or small-batch detection, and especially cannot be popularized and used in large areas in primary hospitals, community clinics, health hospitals and the like with backward economic conditions; in the ELISA method, a 96-well plate is used as a carrier, an antibody is simply fixed as a capture platform, and then the Horseradish Peroxidase (HRP) -labeled antibody is used as a detection probe to catalyze the color development of a substrate, so that the method has the advantages of simplicity in operation, easiness in identification of detection results and the like, but the detection sensitivity is low.
Disclosure of Invention
The embodiment of the invention provides a double-enzyme co-loading nanosphere for AFP detection, a preparation method thereof and an AFP detection method aiming at the problems of low enzyme loading amount, low detection sensitivity and the like in a biological enzyme detection technology.
In order to achieve the above purpose, the embodiment of the invention adopts the following technical scheme:
in a first aspect, an embodiment of the present invention provides a double-enzyme co-loaded nanosphere for AFP detection, comprising: magnetic mesoporous silica nanospheres with a core-shell structure, and glucose oxidase GoD and horseradish peroxidase HRP distributed in mesoporous channels;
the core-shell structure is provided with magnetic core particles and a silicon dioxide spherical shell structure distributed with mesoporous pore canals; the magnetic core particles are used for enriching AFP in a serum sample through magnetic separation; the glucose oxidase GoD and the horseradish peroxidase HRP are used for catalyzing substrate color development.
When the carrier loaded with the enzyme is used for biological detection, the loading capacity of the enzyme can directly influence the detection sensitivity. In the carrier used in the prior art, enzyme is generally loaded on the surface of the nanoparticle, and the amount of the loaded enzyme is limited by the specific surface area of the nanoparticle, so that the detection sensitivity is not high, and the accuracy and precision of a detection result are influenced.
In this embodiment, the double enzymes for AFP detection are loaded in the mesoporous channels of the magnetic mesoporous silica nanospheres having the core-shell structure, so that more enzymes are loaded through the ultra-large specific surface area of the mesopores, and meanwhile, since the core of the core-shell structure has the magnetic property, the AFP can be separated and enriched for the target object, and the detection sensitivity for the target object is greatly improved. Therefore, the magnetic mesoporous silica nanosphere with the core-shell structure magnetically separates and enriches a target substance AFP in serum, the loaded double-enzyme catalysis substrate develops color, the AFP content is detected by biologically coupling an AFP capture antibody, the biological labeling process is simple, and the sensitivity is high. The magnetic core particles adopt Fe3O4Magnetic nanoparticles.
Further, the particle size of the magnetic core particle is 230-250nm, and the thickness of the silica shell structure layer distributed with the mesoporous channels is 50-200 nm.
Further, the feed ratio of the GoD to the HRP is 1: 1.
The embodiment of the invention also provides a preparation method of the double-enzyme co-loaded nanosphere for AFP detection, which comprises the following steps:
step S101, dissolving ferric trichloride, trisodium citrate and sodium acetate in ethylene glycol, and synthesizing 230-250nm Fe through a solvothermal method3O4Magnetic nanoparticles;
step S102, preparing a mesoporous silica shell structure layer outside the magnetic nanoparticles by adopting an interface co-assembly method; specifically, the Fe is added3O4Dispersing magnetic nano particles in an ethanol-water mixed solution, taking tetraethyl orthosilicate (TEOS) as a silicon source, Cetyl Trimethyl Ammonium Bromide (CTAB) as a template agent, an organic reagent as a pore-expanding agent and an alkaline reagent as a catalyst, and performing a water-bath reaction under the condition of mechanical stirring to obtain magnetic mesoporous silica nanospheres with a core-shell structure; in the step, the nanospheres can be modified by adopting epoxy groups, and the specific process is as follows: modifying the core-shell structure magnetic mesoporous silica nanospheres with 3- (2, 3-epoxypropane) propyl trimethoxy silane (GLYMO) to obtain epoxy group functionalized core-shell structure magnetic macroporous mesoporous silica nanospheres;
step S103, coupling glucose oxidase GoD and horseradish peroxidase HRP into the mesoporous pore canal of the shell structure layer by adopting a covalent coupling method. The specific coupling parameters are: the reaction was carried out at 37 ℃ for 2 h.
In a second aspect, the embodiments of the present invention further provide a probe for AFP detection, where the probe includes the nanosphere and an AFP capture antibody coupled to the nanosphere.
Further, the content of the AFP capture antibody in the probe is 1% by mass.
In a third aspect, an embodiment of the present invention further provides an elisa plate for AFP detection, which is obtained by:
with 0.05 mol.L-1phosphate buffer pH 8.6 as coating solution, AFP detecting antibody was immobilized on an ELISA plate, and then washing solution (pH 7.4 PBS added with 0.5% Tween 20, PBST)Washing the plate, and blocking with Bovine Serum Albumin (BSA); the dried sample is placed in a constant temperature room (25 ℃) and dried to obtain an ELISA plate for alpha fetoprotein AFP detection; the ELISA plate was sealed in vacuum and stored at 4 ℃.
In a fourth aspect, the embodiment of the present invention further provides an enzyme-linked immunosorbent assay ELISA kit for AFP detection, which comprises the probe, an ELISA plate coated with an AFP detection antibody, a working solution (phosphate buffered saline, PBS at pH 7.4), a glucose solution, a chromogenic substrate (3, 3',5,5' -tetramethylbenzidine, TMB), a stop solution and a washing solution (PBS with 0.5% tween 20 added, PBST).
The ELISA kit provided in this example is different from the HRP-labeled ELISA kit. HRP-labeled ELISA kit in the presence of H2O2In the ELISA kit provided by the embodiment, the double-enzyme co-supported nanosphere is simultaneously loaded with GoD and HRP, wherein the GoD firstly catalyzes the substrate glucose to be oxidized to generate H2O2Then HRP re-catalyzes H2O2The color change is generated by oxidizing the chromogenic substrate, and the kit has better stability and small environmental interference compared with an ELISA kit marked by HRP. Meanwhile, the double-enzyme co-loading nanosphere has a superparamagnetic characteristic, and can enrich target objects in a sample, so that background interference introduced by a biological sample is reduced, and the detection sensitivity is remarkably improved.
When the ELISA kit is used for detecting alpha-fetoprotein AFP, the method comprises the following steps:
step S201, carrying out oxidative color development on a color substrate in an ELISA kit catalyzed by glucose oxidase GoD and horse radish peroxidase HRP on the double-enzyme co-carried nanospheres;
step S202, terminating the color development by using the stop solution, and reading a color development signal.
Further, the step S201 of the oxidation color development process specifically includes: diluting the probe with a working solution, mixing the diluted probe with a serum sample to be detected, adding the mixture into an AFP detection antibody coated ELISA plate, incubating for 1-3 h, washing for 3 times with a washing solution, and then adding a glucose solution; hydrogen peroxide generation by oxidation of glucose with GoD catalysis（H2O2) HRP catalyzed H2O2The oxidation color development substrate such as TMB generates color signals, and the signals can be interpreted by naked eyes or an enzyme-labeling instrument can be used for quantitatively reading the absorbance value.
The invention has the following beneficial effects:
according to the double-enzyme co-loading nanosphere for AFP detection, the probe, the ELISA kit for enzyme-linked immunosorbent assay and the AFP detection method, double enzymes are loaded in the magnetic macroporous mesoporous silica nanospheres with the core-shell structure, so that more enzymes are loaded through the oversized specific surface area of the macroporous mesoporous silica, and meanwhile, a target object is enriched through the superparamagnetic property, so that background interference introduced by a biological sample is reduced, the signal intensity of biological enzyme detection is improved, and the detection sensitivity is improved; and the method has better stability for detecting alpha fetoprotein AFP, is less interfered by environment, and improves the detection accuracy and precision.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 shows Fe synthesized in example 1 of the present invention3O4Scanning electron microscopy of magnetic cores.
FIG. 2 shows Fe synthesized in example 1 of the present invention3O4Transmission electron microscopy of magnetic cores.
Fig. 3 is a scanning electron microscope image of the magnetic mesoporous silica nanosphere with the core-shell structure synthesized in example 1 of the present invention.
Fig. 4 is a transmission electron microscope image of the magnetic mesoporous silica nanosphere with the core-shell structure synthesized in example 1 of the present invention.
FIG. 5 is a standard curve of AFP detection by a probe with a silica shell thickness of 60nm in example 1 of the present invention.
Fig. 6 is a transmission electron microscope image of the magnetic mesoporous silica nanosphere with the core-shell structure synthesized in example 2 of the present invention.
Detailed Description
The technical problems, aspects and advantages of the invention will be explained in detail below with reference to exemplary embodiments. The following exemplary embodiments are merely illustrative of the present invention and are not to be construed as limiting the invention.
It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
Example 1
This example provides a double-enzyme co-loaded nanosphere for alpha-fetoprotein (AFP) detection, comprising: magnetic mesoporous silica nanospheres with a core-shell structure, and glucose oxidase GoD and horseradish peroxidase HRP distributed in mesoporous channels; the core-shell structure has magnetic core particles and a silica shell structure with mesoporous channels distributed therein; the magnetic core particles are used for enriching AFP in a serum sample, and the AFP is enriched in a shell structure and a mesoporous pore channel; the glucose oxidase GoD and the horseradish peroxidase HRP are used for catalyzing chromogenic substrates.
The magnetic core particles are Fe3O4The magnetic nano-particles are synthesized by the following steps: FeCl is added3(0.65 g, 4.0 mmol) and trisodium citrate (0.20 g, 0.68 mmol) were dissolved in ethylene glycol (20 mL) and then 1.20 g of sodium acetate was added by stirring; the mixture is stirred vigorously for 30min and then sealed in a stainless steel autoclave with polytetrafluoroethylene lining and 50 mL capacity; the autoclave was heated and held at 200 ℃ for 10h and then cooled to room temperature to give a black product. Washing the black product with ethanol and deionized water for several times to obtain Fe3O4Magnetic nanoparticlesAnd (4) adding the active ingredients. As shown in fig. 1 and 2.
The thickness of the silicon dioxide shell structure layer is 60nm, and the preparation process is as follows: the core-shell magnetic macroporous mesoporous silica nanospheres with proper magnetism and shell thickness are synthesized by adopting an interface co-assembly method. First, 100 mg of Fe was ultrasonically treated3O4The nanoparticles were dispersed in 50 mL CTAB solution (10% aqueous, w/v). Then, 1mL of concentrated aqueous ammonia (28 wt%), 30 mL of cyclohexane, and 1mL of ethyl orthosilicate were added to the dispersion solution overnight under continuous magnetic stirring at 45 ℃. Finally, the synthesized nanospheres were collected after washing three times with 50% ethanol. As shown in fig. 3 and 4, the magnetic mesoporous silica nanosphere with the core-shell structure having the magnetic core particles and the silica spherical shell structure with the mesoporous channels distributed therein is obtained through the above process.
The glucose oxidase GoD and the horseradish peroxidase HRP distributed in the mesoporous pore canal are prepared by the following steps: dispersing 15 mg of magnetic macroporous mesoporous silica nanospheres with core-shell structures in 1mL of NH4HCO3Solution (25 mmol.L)-1pH =8, containing 1.5mgGoD and 1.5 mgHRP).
Based on the AFP detection double-enzyme co-loaded nanosphere, the embodiment also provides a probe for AFP detection. The preparation process of the probe is based on the NH dispersed with the double-enzyme co-loaded nanospheres4HCO3Solution mixture to which 0.5 mg and 1mg of afp capture antibody were added, respectively, and gently shaken at 37 ℃ for 2h in the dark. After removal of excess GoD, HRP, and AFP capture antibodies, nanospheres loaded with GoD, HRP, and AFP capture antibodies were collected by magnetic separation and dispersed in 3 mL PBS (10 mmol-1pH 7.4; 5% bovine serum albumin (w/v%)) for further use.
Based on the probe, the embodiment also provides an ELISA plate for AFP detection, and the preparation process of the ELISA plate is as follows: with 0.05 mol.L-1The AFP detection antibody (a commercially available finished product) is diluted to 15 mug.mL by taking phosphate buffer solution with pH of 8.6 as coating solution-1Coating overnight at 4 ℃ in 100 muL/hole; the plate was washed 3 times with PBST and then PBS (10 mmol. L.)-1pH 7.4) plates were washed once with 3% BSA (dissolved in 10 mmol. L.) at 37 deg.C-1PBS, pH 7.4, 300. mu.L/well) and the microplate was blocked for 1 hour, followed by three PBST washes with PBS (10 mmol. L.)-1pH 7.4) was washed once. Then placing the enzyme-labeled plate in a constant temperature room (25 ℃) for airing to obtain an enzyme-labeled plate; after the sample is qualified, the ELISA plate is sealed in vacuum and then stored at 4 ℃.
Based on the probe and the ELISA plate, the embodiment also provides an ELISA kit for AFP detection, which comprises the probe, the ELISA plate coated with the AFP detection antibody, a phosphate buffer solution with the pH value of 7.4, a glucose solution, 3',5,5' -tetramethylbenzidine TMB, a stop solution and a washing solution PBST formed by PBS added with 0.5% Tween 20.
When the ELISA kit is used for detecting alpha-fetoprotein AFP, the specific process is as follows: 50 mu L of double-enzyme co-loading core-shell structure magnetic macroporous mesoporous silica nanosphere (5 mu g.mL) modified by AFP capture antibody-1) And 50. mu.L of serially diluted AFP standard solution or serum (PBS, 10 mmol.L.)-1pH 7.4, 50 μ L/well) was added to the wells and left at 37 ℃ for 2 h. After washing three times again with PBST and once with ultrapure water, a glucose solution (50 mmol. L.) was added-1) 100 μ L/well for 45 min. Finally, ethanol diluted TMB substrate (10 mmol. l.) was added-150. mu.L/well) for 20 min, followed by addition of 650 nm TMB chromogenic stop solution (50. mu.L/well) to the wells. Subsequently, the absorbance at 650 nm was either read by visual interpretation or recorded on a Cytation 3 microplate reader.
As shown in FIG. 5, the detection sensitivity of the prepared ELISA kit was calculated by substituting the absorbance value into the established standard curve, and the detection limit of AFP detection using the core-shell structure magnetic macroporous mesoporous silica nanosphere probe with a shell thickness of 60nm was 0.08 ng.mL-1。
Example 2
This example is essentially the same as example 1, except that the silica shell structure layer has a thickness of 200 nm and is preparedThe process is as follows: the core-shell magnetic macroporous mesoporous silica nanospheres with proper magnetism and shell thickness are synthesized by adopting an interface co-assembly method. First, 100 mg of Fe was ultrasonically treated3O4The nanoparticles were dispersed in 50 mL CTAB solution (10% aqueous, w/v). Then, 1mL of concentrated aqueous ammonia (28 wt%), 30 mL of cyclohexane, and 4mL of ethyl orthosilicate were added to the dispersion solution overnight under continuous magnetic stirring at 45 ℃. Finally, the synthesized nanospheres were collected after three washes with 50% ethanol, as shown in fig. 6.
Example 3
This example is substantially the same as example 1, except that the prepared silica shell structure layer with a thickness of 60nm is functionalized by epoxy group GLYMO, and the specific process is as follows: 0.2g of magnetic macroporous/mesoporous silica nanospheres with a core-shell structure, wherein the thicknesses of the silica shells of the magnetic macroporous/mesoporous silica nanospheres are 60nm and 200 nm respectively, are dispersed in a mixture of 100mL of toluene and 0.2g/0.8 g of GLYMO respectively. The mixture was stirred at room temperature for 1 h, then heated to 65 ℃ and stirring continued for 8 h. Washing the obtained product with absolute ethyl alcohol for several times, drying the product in vacuum at 50 ℃ overnight, and collecting the epoxy group functionalized magnetic macroporous mesoporous silica nanospheres with the core-shell structure for further use.
While the foregoing is directed to the preferred embodiment of the present invention, it is understood that the invention is not limited to the exemplary embodiments disclosed, but is made merely for the purpose of providing those skilled in the relevant art with a comprehensive understanding of the specific details of the invention. It will be apparent to those skilled in the art that various modifications and adaptations of the present invention can be made without departing from the principles of the invention and the scope of the invention is to be determined by the claims.
Claims (10)
1. A double-enzyme co-loaded nanosphere for Alpha Fetoprotein (AFP) detection, which is characterized by comprising: magnetic mesoporous silica nanospheres with a core-shell structure, and glucose oxidase GoD and horseradish peroxidase HRP distributed in mesoporous channels; wherein,
the core-shell structure has magnetic core particles and a silica shell structure with mesoporous channels distributed therein; the magnetic core particles are used for enriching AFP in a serum sample through magnetic separation; the glucose oxidase GoD and the horseradish peroxidase HRP are used for catalyzing substrate color development.
2. The double-enzyme co-loaded nanosphere for Alpha Fetoprotein (AFP) detection as claimed in claim 1, wherein the particle size of the magnetic core particle is 230-250nm, and the thickness of the silica spherical shell structure layer distributed with mesoporous channels is 50-200 nm.
3. The double-enzyme co-loaded nanosphere for Alpha Fetoprotein (AFP) detection according to claim 1, wherein the GoD and HRP are added at a ratio of 1: 1.
4. A preparation method of a double-enzyme co-loaded nanosphere for Alpha Fetoprotein (AFP) detection is characterized by comprising the following steps:
step S101, dissolving ferric trichloride, trisodium citrate and sodium acetate in ethylene glycol, and synthesizing 230-250nm Fe through a solvothermal method3O4Magnetic nanoparticles;
step S102, preparing a mesoporous silica shell structure layer outside the magnetic nanoparticles by adopting an interface co-assembly method; specifically, the Fe is added3O4Dispersing magnetic nanoparticles in an ethanol-water mixed solution, and performing a water-bath reaction under the condition of mechanical stirring by using tetraethyl orthosilicate TEOS as a silicon source, cetyl trimethyl ammonium bromide CTAB as a template agent, an organic reagent as a pore-expanding agent and an alkaline reagent as a catalyst to obtain magnetic mesoporous silica nanospheres with a core-shell structure;
step S103, coupling glucose oxidase GoD and horseradish peroxidase HRP into the mesoporous pore canal of the shell structure layer by adopting a covalent coupling method.
5. The preparation method according to claim 4, wherein in the step S102, the magnetic mesoporous silica nanospheres are modified by epoxy groups, and the specific process is as follows: and modifying the magnetic mesoporous silica nanospheres with the core-shell structure by using 3- (2, 3-epoxypropane) propyl trimethoxy silane to obtain the magnetic macroporous mesoporous silica nanospheres with the core-shell structure and functionalized by epoxy groups.
6. A probe for the detection of alpha-fetoprotein, AFP, characterized in that it comprises a nanosphere according to any of claims 1-3, and an AFP capture antibody coupled to the nanosphere.
7. The probe for Alpha Fetoprotein (AFP) detection according to claim 6, wherein the content of said AFP capture antibody in the probe is 1% o by mass.
8. An ELISA kit for enzyme-linked immunosorbent assay for AFP detection, which is characterized by comprising the probe as described in claim 6 or 7, an ELISA plate coated with an AFP detection antibody, a working solution, a glucose solution, a chromogenic substrate, a stop solution and a washing solution.
9. A method of AFP detection using an ELISA kit of claim 8 comprising the steps of:
step S201, carrying out oxidative color development on a chromogenic substrate in an ELISA kit through glucose oxidase GoD and Horse Radish Peroxidase (HRP) which are carried by a probe and are on a double-enzyme co-carrier nanosphere;
step S202, terminating the color development by using the stop solution, and reading a color development signal.
10. An AFP detection method according to claim 9 wherein said step S201 of oxidative coloration specifically comprises: diluting the probe with a working solution, mixing the diluted probe with a serum sample to be detected, adding the mixture into an AFP detection antibody coated ELISA plate, incubating for 1-3 h, washing with a washing solution, and adding a glucose solution; hydrogen peroxide H produced by oxidation of glucose under catalysis of GoD2O2And then H is catalyzed by HRP2O2Oxidation colour-developing substrate productGenerating a signal, adding the stop solution, and judging the AFP content through a color signal.
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