CN110261604B - Preparation method and application of antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles - Google Patents
Preparation method and application of antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles Download PDFInfo
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Abstract
The preparation method of the antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles comprises the following steps: adding the polyethyleneimine-coated mesoporous silica nanoparticles into a phosphoric acid buffer solution containing pyrroloquinoline quinone, and oscillating for about 12 hours; adding a phosphoric acid buffer solution containing a detection antibody, continuing to shake for reaction for about 1 hour, centrifugally washing, dispersing a lower-layer precipitate in the phosphoric acid buffer solution, and storing at 4 ℃ for later use; before use, the cells were diluted with a phosphate buffer containing bovine serum albumin. The application of the antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles is disclosed, and the antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles are used for antigen detection. The invention has the advantages of simplicity, sensitivity, intuition, high flux, no need of special instruments and the like.
Description
Technical Field
The invention relates to immunoassay detection, in particular to an antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticle for immunoassay detection and a detection method, and belongs to the field of chemistry.
Background
The visual immunoassay method has the characteristics of simple operation, no need of special instruments and the like, and is widely concerned in prevention, diagnosis and treatment of various diseases. At present, standardized visual immunoassay methods usually use enzyme-labeled antibodies to catalyze color reaction, thereby achieving the purpose of quantitative analysis. However, in practical applications, natural enzymes have problems such as poor thermal stability, susceptibility to environmental influences, high preparation and purification costs, low sensitivity, and the like.
With the development of nanotechnology and nanomaterials, the sensitivity and stability of a visualization immunoassay method based on nanomaterials are remarkably improved. Generally, the role of nanomaterials in visual immunoassay experiments can be divided into four types: as a nano-enzyme catalysis color reaction; as a chromogenic substrate, the aggregation of the nanometer material or the change of the shape and the size of the nanometer material is caused by target molecules; as a carrier, a large amount of natural enzyme or color developing agent is loaded; as a precursor, metal ions capable of catalyzing a color development reaction are released. Wherein, the nano enzyme has the advantages of low cost, controllable catalytic activity, high stability and the like. The nano enzyme mainly comprises carbon, metal oxide and other nano materials. In the catalytic reaction of glucose and hydrogen peroxide, they generally function similarly to oxidases, catalases or dismutases. The immunoassay method based on the nano-enzyme is simple and effective, but the nano-enzyme still has some problems in the aspect of the practical application of the visual immunoassay. For example, most nanoenzymes have lower catalytic activity, weak binding capacity and poor substrate specificity compared to native enzymes. In the experimental process, the surface of the nano material needs to be modified so as to perform molecular recognition and reduce nonspecific adsorption. However, these modifying components generally reduce the catalytic activity of nanoenzymes (particularly metal and metal oxide based nanomaterials). Therefore, the construction of a novel nano enzyme or nano catalytic system for visual immunoassay is of great significance.
Disclosure of Invention
The invention aims to overcome the defects in the existing antigen detection method and provides a preparation method and application of an antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticle.
In order to realize the purpose of the invention, the following technical scheme is adopted: the preparation method of the antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles comprises the following steps: adding the polyethyleneimine-coated mesoporous silica nanoparticles into a phosphoric acid buffer solution containing pyrroloquinoline quinone, and oscillating for about 12 hours; adding a phosphoric acid buffer solution containing a detection antibody, continuing to shake for reaction for about 1 hour, centrifugally washing, dispersing a lower-layer precipitate in the phosphoric acid buffer solution, and storing at 4 ℃ for later use; before use, the cells were diluted with a phosphate buffer containing bovine serum albumin.
Further, the method comprises the following steps of; the polyethyleneimine-coated mesoporous silica nano particle is prepared by the following method:
a: preparation of mesoporous silica nanoparticles: dissolving cetyl trimethyl ammonium bromide in ultrapure water, adding a sodium hydroxide solution, stirring and reacting at 80 ℃ for 15 min, slowly adding a tetraethoxysilane solution, stirring vigorously for 20 min, carrying out suction filtration on the obtained white precipitate, washing with water and methanol in sequence, and drying at 80 ℃; dispersing the dried solid in a mixed solution of hydrochloric acid and methanol, carrying out reflux reaction for about 12 hours, washing with water and methanol again, and carrying out vacuum drying at 60 ℃ to obtain the silicon dioxide nano-particles with the mesoporous structure;
b: preparation of polyethyleneimine-coated mesoporous silica nanoparticles: adding the mesoporous silica nano particles prepared in the step (1) into a polyethyleneimine water solution, stirring and reacting for about 12 hours, centrifugally washing, and carrying out vacuum drying at 60 ℃.
The application of the antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles is disclosed, and the antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles are used for antigen detection.
Further, the method comprises the following steps of; the specific method for detecting the antigen comprises the following steps: adding a phosphate buffer solution containing a biotin-functionalized capture antibody into a streptavidin-functionalized magnetic ball, reacting for 30-60 minutes, separating by using a magnet, washing by using the phosphate buffer solution, discarding the supernatant, and dispersing the lower precipitate in the phosphate buffer solution; then adding phosphate buffer solution containing antigen, mixing, shaking and incubating; magnetic separation and washing, dispersing the lower layer precipitate in a phosphoric acid buffer solution, adding a mixed solution of the detection antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles and bovine serum albumin, and carrying out oscillation reaction; and (3) performing magnetic separation and washing, dispersing the lower precipitate in a phosphoric acid buffer solution containing tris (2-carboxyethyl) phosphine and ferric ion-phenazine complex, and observing the color change of the solution by naked eyes or measuring the absorption value of the solution at 562 nm by an ultraviolet-visible spectrophotometer.
Further, the method comprises the following steps of; the antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticle is used for detecting prostate specific antigen, and the specific detection method comprises the following steps: adding a phosphate buffer solution containing a capture antibody of a biotin-functionalized prostate specific antigen into a streptavidin-functionalized magnetic ball, reacting for 30-60 minutes, separating by using a magnet, washing by using the phosphate buffer solution, discarding the supernatant, and dispersing the lower precipitate in the phosphate buffer solution; adding the prepared magnetic ball modified by the capture antibody into a phosphate buffer solution containing the prostate specific antigen, mixing and oscillating, and incubating for about 30 minutes; magnetic separation and washing, adding a mixed solution of pyrroloquinoline quinone-loaded mesoporous silica nanoparticles and bovine serum albumin for detecting antibody functionalization, wherein an antibody in the pyrroloquinoline quinone-loaded mesoporous silica nanoparticles for detecting antibody functionalization is an antibody of prostate specific antigen, and carrying out a concussion reaction for about 30 minutes; the precipitate was dispersed in a phosphoric acid buffer solution containing tris (2-carboxyethyl) phosphine and a ferric ion-phenazine complex, and the color change of the solution was observed visually or the absorbance of the solution at 562 nm was measured with a spectrophotometer.
The technical invention has the positive and beneficial technical effects that: (1) the mesoporous silica nanoparticles can support a large amount of supported pyrroloquinoline quinone; (2) through the redox cycling reaction between the pyrroloquinoline quinone catalyzed tris (2-carboxyethyl) phosphine and the ferric ion-phenanthroline complex, the colorimetric analysis method for antigen detection is established, and the colorimetric analysis method has the advantages of simplicity, sensitivity, intuition, high flux, no need of special instruments and the like, and the successful development of the technology can realize the detection of different types of antigens.
Drawings
Fig. 1 is a transmission electron microscope image of antibody functionalized pyrroloquinoline quinone supported mesoporous silica nanoparticles.
Fig. 2 is a transmission electron microscope image of streptavidin-functionalized magnetic spheres after the processing steps of biotin-functionalized capture antibody, prostate specific antigen, antibody-functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles.
Figure 3 is a graph of the uv-vis absorption spectra of solutions in the presence of different concentrations of prostate specific antigen.
FIG. 4 is a graph of absorbance versus prostate specific antigen concentration.
FIG. 5 is a selective illustration of the assay.
Detailed Description
In order to more fully explain the implementation of the present invention, examples of the implementation of the present invention are provided. These examples are merely illustrative of the process and do not limit the scope of the present invention, and the present invention is described by the following examples, but not limited to the following examples, and any modified embodiments are included in the technical scope of the present invention.
The antibodies of the invention are antibodies to prostate specific antigens. The following examples are specifically illustrated by way of example of prostate specific antigen. To further explain the attached drawings, fig. 1 is a transmission electron micrograph of antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles. As can be seen from FIG. 1, the synthesized nanoparticles have good dispersibility, and the size is about 80 nm.
Fig. 2 is a projection electron microscope image of streptavidin-functionalized magnetic spheres after the processing step of biotin-functionalized capture antibody, prostate specific antigen, antibody-functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles. As can be seen from the figure, the antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles are captured by magnetic beads.
FIG. 3 is a graph of the UV-VIS absorption spectra of solutions in the presence of varying concentrations of prostate specific antigen, which are 0, 0.005,0.05, 0.125, 0.25, 0.5, 1, 2 ng/mL in that order. FIG. 4 is a graph of absorbance versus concentration of prostate specific antigen, which is 0.005,0.05, 0.125, 0.25, 0.5, 1, 2 ng/mL in order, with a linear range of 0.005-0.5 ng/mL (see inset in FIG. 4). FIG. 5 shows the effect of different proteins on the UV absorption values, which correspond in sequence from 1 to 6 to: immunoglobulin G at 50 ng/mL, alpha-fetoprotein at 50 ng/mL, carcinoembryonic antigen at 50 ng/mL, thrombin at 50 ng/mL, blank sample, prostate specific antigen at 2.5 ng/mL. The polyethyleneimine-coated mesoporous silica nanoparticles have a mature technical scheme in the prior literature technology.
Firstly, preparing a polypeptide functionalized magnetic ball:
a: preparation of mesoporous silica nanoparticles: dissolving 50 mg of hexadecyl trimethyl ammonium bromide in 200 mL of ultrapure water, then adding 1.75 mL of 2M sodium hydroxide solution, stirring and reacting for 15 min at 80 ℃, then slowly adding 2.5 mL of tetraethoxysilane solution, stirring vigorously for 20 min, carrying out suction filtration on the obtained white precipitate, washing with water and methanol in sequence, and drying at 80 ℃; dispersing the dried solid in a mixed solution of hydrochloric acid (0.5 mL) and methanol (50 mL), carrying out reflux reaction for about 12 hours, washing with water and methanol again, and carrying out vacuum drying at 60 ℃ to obtain the silicon dioxide nano-particles with the mesoporous structure;
b: preparation of polyethyleneimine-coated mesoporous silica nanoparticles: adding 500 mg of the mesoporous silica nanoparticles prepared in the step (1) into 300 mg of polyethyleneimine water solution, stirring and reacting for about 12 hours, centrifuging and washing, and performing vacuum drying at 60 ℃.
Secondly, preparing the antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles:
adding 1 mg of polyethyleneimine-coated mesoporous silica nanoparticles into 1 mL of phosphoric acid buffer solution containing pyrroloquinoline quinone (12 mu M), and oscillating for about 12 hours; then adding 1 mL of phosphoric acid buffer solution containing detection antibody (50 mug) of prostate specific antigen, continuing to shake for reaction for about 1 hour, centrifuging for 15 min under the condition of 5000 rpm, washing with ultrapure water, dispersing the lower-layer precipitate in 1 mL of phosphoric acid buffer solution (50 mM, pH 7.4), and storing at 4 ℃ for later use; before use, the cells were diluted with a phosphate buffer containing bovine serum albumin (0.1% w/v).
Detection of prostate specific antigen
Adding a phosphate buffer solution containing l mL of 20 mug/mL biotin-functionalized prostate specific antigen capture antibody into l mL of 200 mug/mL streptavidin-functionalized magnetic spheres, reacting for about 30-60 minutes, separating by using a magnet, washing by using the phosphate buffer solution, discarding the supernatant, and dispersing the lower precipitate in 4 mL of phosphate buffer solution; taking 90 mu L of prepared magnetic balls modified by capture antibodies, adding 10 mu L of phosphoric acid buffer solution containing prostate specific antigens, mixing and oscillating, and incubating for about 30 minutes; magnetic separation and washing, adding 50 mu L of detection antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nano-particles and bovine serum albumin mixed solution, and carrying out shake reaction for about 30 minutes; magnetic separation and washing were performed twice, and the lower precipitate was dispersed in 100. mu.L of a phosphate buffer solution (pH 8.6) containing tris (2-carboxyethyl) phosphine (0.5 mM) and a ferric ion-phenazine complex (0.2 mM), and finally the color change of the solution was observed with the naked eye or the absorbance of the solution at 562 nm was measured with a spectrophotometer. FIG. 3 is a graph showing the UV-VIS absorption spectrum of a solution in the presence of prostate specific antigens of different concentrations, showing that the greater the concentration of prostate specific antigens, the more red the solution color and the stronger the intensity of the absorption spectrum of the solution, the absorption being due to the ferrous ion-phenazine complex formed in the solution, and the change in the solution color being observable with the naked eye when the concentration of prostate specific antigens is higher than 0.05 ng/mL; FIG. 4 is a graph showing the relationship between the absorption value of the solution at 562 nm and the detection of prostate specific antigen with different concentrations, and it can be seen that the larger the absorption value of the solution with the increase of the concentration, the greater the absorption value of the solution, the concentration of prostate specific antigen can be determined according to the change of the absorption intensity of the generated ferrous ion-phenazine complex, and 0.005 ng/mL prostate specific antigen can be detected by using a spectrophotometer, which indicates that the method can be used for colorimetric analysis and detection of prostate specific antigen, and the linear range is 0.005-0.5 ng/mL.
Example 3: selectivity is
The procedure is as in example 2, the prostate specific antigen is replaced with the substance to be tested, the conditions are not changed in the other procedures, and the experimental results are shown in FIG. 5. The result shows that only prostate specific antigen can generate stronger absorption value, which causes the solution to turn into purple red, other substances do not cause the color change of the solution, and the change of ultraviolet absorption value caused by the substances can be ignored, which shows that the method has better selectivity for detecting the prostate specific antigen.
The mechanism discussion and analysis of the invention are as follows: pyrroloquinoline quinone can be reduced by tris (2-carboxyethyl) phosphine, and the reduction product further reduces the colorless ferric iron ion-phenanthroline complex into a purple-red ferrous iron ion-phenanthroline complex; and the generated pyrroloquinoline quinone is reduced by the tri (2-carboxyethyl) phosphine again, and then begins to react with the ferric ion-phenanthroline complex again, and after multiple cycles, a large amount of ferrous ion-phenanthroline complex is generated, so that a purple red solution is formed. Based on this principle, when prostate specific antigen is present in the sample, the pyrroloquinoline quinone-loaded mesoporous silica nanoparticles will be captured by the magnetic beads, and the loaded pyrroloquinoline quinone can reduce the colorless ferric iron ion-phenazine complex to form a purple ferrous iron ion-phenazine complex. This change can be monitored visually or by a spectrophotometer. The specific technical scheme is as follows: adding a phosphate buffer solution containing a biotin-functionalized capture antibody into a streptavidin-functionalized magnetic ball, reacting for 30-60 minutes, separating by using a magnet, washing by using the phosphate buffer solution, discarding the supernatant, and dispersing the lower precipitate in the phosphate buffer solution; then adding phosphate buffer solution containing antigen, mixing, shaking and incubating; magnetic separation and washing, dispersing the lower layer precipitate in a phosphoric acid buffer solution, adding a mixed solution of the detection antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles and bovine serum albumin, and carrying out oscillation reaction; and (3) performing magnetic separation and washing, dispersing the lower precipitate in a phosphoric acid buffer solution containing tris (2-carboxyethyl) phosphine and ferric ion-phenazine complex, and observing the color change of the solution by naked eyes or measuring the absorption value of the solution at 562 nm by an ultraviolet-visible spectrophotometer.
After the embodiments of the present invention have been described in detail, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention, and it is intended that all simple modifications, equivalent changes and modifications made to the above embodiments based on the technical spirit of the present invention shall fall within the technical scope of the present invention, and the present invention shall not be limited to the embodiments illustrated in the description.
Claims (2)
1. The application of the antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticle is characterized in that the antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticle is prepared by the following method: adding the polyethyleneimine-coated mesoporous silica nanoparticles into a phosphoric acid buffer solution containing pyrroloquinoline quinone, and oscillating for about 12 hours; adding a phosphoric acid buffer solution containing a detection antibody, continuing to shake for reaction for about 1 hour, centrifugally washing, dispersing a lower-layer precipitate in the phosphoric acid buffer solution, and storing at 4 ℃ for later use; before use, the solution is diluted by phosphate buffer solution containing bovine serum albumin, and is characterized in that: the application of the antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles in antigen detection is used for the purpose of non-disease diagnosis, and the specific method for the antigen detection comprises the following steps: adding a phosphate buffer solution containing a capture antibody of a biotin-functionalized antigen into a streptavidin-functionalized magnetic ball, reacting for 30-60 minutes, separating by using a magnet, washing by using the phosphate buffer solution, discarding the supernatant, and dispersing the lower precipitate in the phosphate buffer solution; then adding phosphate buffer solution containing antigen, mixing, shaking and incubating; magnetic separation and washing, dispersing the lower layer precipitate in a phosphoric acid buffer solution, adding a mixed solution of the detection antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles and bovine serum albumin, and carrying out oscillation reaction; and (3) performing magnetic separation and washing, dispersing the lower precipitate in a phosphoric acid buffer solution containing tris (2-carboxyethyl) phosphine and ferric ion-phenazine complex, and observing the color change of the solution by naked eyes or measuring the absorption value of the solution at 562 nm by an ultraviolet-visible spectrophotometer.
2. Use of antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles according to claim 1, characterized in that: the application of the antibody functionalized pyrroloquinoline quinone-loaded mesoporous silica nanoparticles in the detection of prostate specific antigens comprises the following specific detection steps: adding a phosphate buffer solution containing a capture antibody of a biotin-functionalized prostate specific antigen into a streptavidin-functionalized magnetic ball, reacting for 30-60 minutes, separating by using a magnet, washing by using the phosphate buffer solution, discarding the supernatant, and dispersing the lower precipitate in the phosphate buffer solution; adding the prepared magnetic ball modified by the capture antibody into a phosphate buffer solution containing the prostate specific antigen, mixing and oscillating, and incubating for 30 minutes; magnetic separation and washing, adding a mixed solution of pyrroloquinoline quinone-loaded mesoporous silica nanoparticles and bovine serum albumin for detecting antibody functionalization, wherein an antibody in the pyrroloquinoline quinone-loaded mesoporous silica nanoparticles for detecting antibody functionalization is an antibody of prostate specific antigen, and performing concussion reaction for 30 minutes; the precipitate was dispersed in a phosphoric acid buffer solution containing tris (2-carboxyethyl) phosphine and a ferric ion-phenazine complex, and the color change of the solution was observed visually or the absorbance of the solution at 562 nm was measured with a spectrophotometer.
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