CN111440355B - Preparation method and application of magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis - Google Patents
Preparation method and application of magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis, wherein a silicon dioxide photonic crystal microstructure is taken as a template, hydrogel with good mechanical property is filled in pores of nanoparticles, and the template is removed after solidification to obtain the microcarrier with an inverse opal structure; and preparing functional pre-gel mixed with magnetic nano particles, filling the functional pre-gel into gaps of the inverse opal structure, and polymerizing by ultraviolet light to obtain the periodically ordered magnetic structure color hydrogel microcarrier similar to the template. The invention has the advantages of low preparation cost, good repeatability, simple and convenient operation and the like, the prepared magnetic structure color hydrogel microcarrier has the characteristics of resisting nonspecific protein combination, supplying probe coupling active groups, directional magnetic control effect and the like, and has the performance advantages of high sensitivity and specificity when being used for the multivariate analysis of the bladder cancer protein besides the performances of good coding stability, high flexibility and the like of a liquid phase chip.
Description
Technical Field
The invention relates to the field of biomedical materials, in particular to a preparation method and application of a magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis.
Background
Bladder cancer is the most common malignant tumor of the urinary system, and the incidence and the fatality rate of the bladder cancer are at the front of the common tumors in the world at present, thus seriously threatening the life and health. Bladder cancer has obvious multicentric occurrence characteristics and has common relapse. Therefore, effective early diagnosis and prognostic monitoring methods are of great significance for the diagnosis and treatment of bladder cancer. The method for detecting the tumor protein marker has the potential of predicting tumor progression and evaluating the malignancy degree of tumors.
At present, the tumor protein marker quantitative determination methods commonly used in clinical laboratories include enzyme-linked immunoassay, electrochemiluminescence, and the like. However, the common disadvantages of these diagnostic techniques are that a single test is only for a single biomarker and the cost of the test is high, the throughput is low, and the clinical diagnosis and treatment requirements are difficult to meet. The existing multi-path analysis technology, such as the traditional solid phase chip and microarray, has the defects of large steric hindrance, easy cross contamination, poor repeatability and the like by fixing probe molecules on a planar substrate and coding according to the arrangement positions of the probe molecules.
To solve the above problems, photonic crystal microcarriers used as microcoded carriers have been developed. The periodic ordered nano structure of the photonic crystal can generate a photonic band gap effect, so that a bright structural color is presented, and a characteristic reflection peak of the photonic crystal is used for a coding element of multivariate analysis. The liquid phase code has excellent spectroscopic characteristics, has good stability in biological multivariate coding, and has the advantage of high flexibility of liquid phase coding.
Disclosure of Invention
The invention aims to solve the defects of large steric hindrance, easy cross contamination and poor repeatability of the traditional solid phase chip and provides a preparation method and application of a magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a preparation method of a magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis is characterized by comprising the following steps:
s1: taking the monodisperse silicon dioxide nanoparticle suspension as an internal phase and the dimethyl fluorinated silicone oil as an external phase, injecting the external phase and the internal phase into corresponding channels of a microfluidic device, collecting the external phase and the internal phase in a container through an output channel of the microfluidic device, and forming silicon dioxide photonic crystal microspheres through solidification and crystallization; then collecting the silicon dioxide photonic crystal microspheres in a crucible, washing the microspheres for multiple times by using normal hexane, completely absorbing the normal hexane after the washing is finished, and placing the crucible in a muffle furnace for high-temperature treatment to obtain a coding microcarrier template;
s2: soaking the encoded microcarrier template prepared in the step S1 in a solution of ethylene glycol dimethacrylate, and performing photopolymerization curing, stripping and corrosion to obtain microspheres with inverse opal structures;
s3: and (2) preparing functional pre-gel mixed with magnetic nanoparticles, pouring the functional pre-gel into the microspheres with inverse opal structures prepared in the step S2, and performing photopolymerization to obtain the magnetic structure color hydrogel microcarrier, wherein the magnetic structure color hydrogel microcarrier integrates photonic crystal coding and directional magnetic control effects into a whole, so that biomolecule coding and multivariate analysis are realized.
In order to optimize the technical scheme, the specific measures adopted further comprise:
in step S1, the flow rates of the external phase and the internal phase in the corresponding channels of the microfluidic device were 3.0 mL/h and 1.5 mL/h, respectively.
In the step S1, the concentration of the silica nanoparticles in the monodisperse silica nanoparticle suspension is 20 m/v%; wherein the particle size of the silica nanoparticles includes any one of 215nm, 237nm and 247nm, and the silica nanoparticles are dispersed in ethanol. According to the particle size of the monodisperse silicon dioxide nano particles, the wavelength of the reflection peak of the prepared photonic crystal microsphere can be calculated through a Bragg diffraction equation.
In step S3, the functional pre-gel is prepared from polyethylene glycol diacrylate, acrylic acid, and Fe3O4Nano particles and a photoinitiator; wherein the concentration of the polyethylene glycol diacrylate is 10-30 v/v%, the concentration of the acrylic acid is 0-20 v/v%, and the Fe content is3O4The concentration of the nano particles is Fe3O41-8 m/v%, and the concentration of the photoinitiator is 0.1 m/v%.
And after the step S3, performing probe molecule modification on the surface of the prepared magnetic structure color hydrogel microcarrier, capturing and marking the bladder cancer protein by using a double-antibody sandwich principle, and performing multivariate analysis on the bladder cancer protein by using a fluorescence microscope. The coded signal is used for marker identification, and the fluorescence intensity is used for marker quantification, so that the multivariate analysis of the magnetic structure color hydrogel microcarrier on the bladder cancer protein is realized.
The bladder cancer protein includes any one of BTA protein molecule, NMP22 protein molecule or FDP protein molecule.
The above molecules include any one of an anti-BTA antibody, an anti-NMP22 antibody, or an anti-FDP antibody; wherein the concentration of the probe molecules is 0.001-0.5 mg/mL, and the reaction time for capturing the bladder cancer protein by the probe molecules is 20-120 min.
The invention also protects the application of the magnetic structure color hydrogel microcarrier prepared by the method in preparing a bladder cancer protein marker material.
The invention has the beneficial effects that:
1) the invention is based on the microfluidic technology, takes the silicon dioxide photonic crystal microspheres as the microcarrier template, has simple and convenient preparation method, is quick and efficient, and can regulate and control the microcarrier preparation method according to the application and functional requirements.
2) The invention applies liquid phase coding, has the advantages of good stability and high flexibility, and provides a high-efficiency method for biological multivariate analysis.
3) The magnetic structure color hydrogel microcarrier prepared by the invention has the characteristics of nonspecific protein combination resistance, probe coupling active group supply, directional magnetic control effect and the like, has the performances of good coding stability, high flexibility and the like of a liquid phase chip, and also has the performance advantages of high sensitivity and specificity when being used for the multivariate analysis of the bladder cancer protein.
Drawings
FIG. 1 is a schematic diagram of the preparation process of the magnetic structure color hydrogel microcarrier of the invention.
FIG. 2 is a representation of the nano-microstructure of the magnetic structure color hydrogel microcarrier of the invention.
FIG. 3 is a schematic diagram showing the change of concentration of polyethylene glycol diacrylate with the intensity of the encoding signal.
FIG. 4 is a graph showing the change in acrylic acid concentration with the intensity of the coding signal.
FIG. 5 is a graph showing the dilution gradient of the antibody probe as a function of fluorescence intensity.
FIG. 6 is a graph showing the reaction time of the probe molecules to capture bladder cancer protein as a function of fluorescence intensity.
FIG. 7 is a performance characterization diagram of magnetic structure color hydrogel microcarrier for multiplex analysis of bladder cancer protein markers.
Detailed Description
The invention is further illustrated by the following figures and examples. The examples, in which specific conditions are not specified, were conducted according to conventional conditions well known in the art or conditions recommended by the manufacturer, and the apparatus or reagents used are not specified by the manufacturer, and are all conventional products commercially available.
Example 1
Preparation of magnetic structure color hydrogel microcarrier
(1) Referring to the preparation process of fig. 1, silica nanoparticles with a particle size of 215nm are respectively selected, monodisperse silica nanoparticle suspensions with a concentration of 20m/v% are prepared as an internal phase, simethicone (50 cSt) is used as an external phase, the external phase and the internal phase are injected into corresponding channels of a microfluidic device, the external phase and the internal phase pass through an output channel of the microfluidic device and are collected in a container, droplets of the silica nanoparticles are self-assembled along with water evaporation at 65 ℃, and the silica photonic crystal microspheres are formed through 12h solidification and crystallization; heating to 95 ℃ to solidify the silicon dioxide photonic crystal microspheres for 2 hours; then collecting the silicon dioxide photonic crystal microspheres in a crucible, washing the microspheres for multiple times by using normal hexane, completely absorbing the normal hexane after the washing is finished, and slowly placing the crucible in a muffle furnace for high-temperature treatment to improve the mechanical stability of the crucible to obtain a coding microcarrier template;
(2) dehydrating and drying the prepared coding microcarrier template into milk white by alcohol, soaking the template into 100v/v% ethylene glycol dimethacrylate solution, adding 0.1m/v% photoinitiator, and curing the hydrogel by ultraviolet irradiation for 30 s; transferring to a clean culture dish, and placing under a microscope to strip the microspheres from the cured hydrogel; collecting the microspheres in an EP tube, and corroding the template by using a low-concentration hydrofluoric acid solution to obtain microspheres with an inverse opal structure;
(3) 20v/v% of polyethylene glycol diacrylate, 15 v/v% of acrylic acid and 4 m/v% of Fe3O4Adding nano particles and 0.1m/v% of photoinitiator into deionized water to prepare a functional pre-gel mixed with magnetic nano particles, soaking microspheres with an inverse opal structure into the functional pre-gel until the communicated pores of the inverse opal structure are completely filled, and curing the hydrogel by ultraviolet irradiation for 30 seconds to obtain the magnetic structure colored hydrogel microcarrier.
Referring to fig. 2, panel a is an SEM characterization of a photonic crystal microcarrier; b, image is SEM representation of inverse opal structure; the figure c is the SEM representation of the magnetic structure color hydrogel microcarrier, and the figure shows that the interior of the silicon dioxide photonic crystal microsphere presents a periodic ordered nano structure, and the hydrogel prepared by taking the periodic ordered nano structure as a template through copying has a similar highly ordered inverse opal structure; with the re-pouring of the functional pre-gel, the average refractive index of the microcarrier is changed, but the microcarrier still has periodically ordered nano-arrangement and is used as the structural basis for the photonic crystal to exert the coding effect through the photon forbidden band principle.
Example 2
Preparation of magnetic structure color hydrogel microcarrier
(1) Referring to the preparation process of fig. 1, silica nanoparticles with a particle size of 237nm are respectively selected, monodisperse silica nanoparticle suspensions with a concentration of 20m/v% are prepared as an internal phase, simethicone (50 cSt) is used as an external phase, the external phase and the internal phase are injected into corresponding channels of a microfluidic device, the external phase and the internal phase pass through an output channel of the microfluidic device and are collected in a container, droplets of the silica nanoparticles are self-assembled along with water evaporation at 65 ℃, and the silica photonic crystal microspheres are formed through 12h solidification and crystallization; heating to 95 ℃ to solidify the silicon dioxide photonic crystal microspheres for 2 hours; then collecting the silicon dioxide photonic crystal microspheres in a crucible, washing the microspheres for multiple times by using normal hexane, completely absorbing the normal hexane after the washing is finished, and slowly placing the crucible in a muffle furnace for high-temperature treatment to improve the mechanical stability of the crucible to obtain a coding microcarrier template;
(2) dehydrating and drying the prepared coding microcarrier template into milk white by alcohol, soaking the template into 100v/v% ethylene glycol dimethacrylate solution, adding 0.1m/v% photoinitiator, and curing the hydrogel by ultraviolet irradiation for 30 s; transferring to a clean culture dish, and placing under a microscope to strip the microspheres from the cured hydrogel; collecting the microspheres in an EP tube, and corroding the template by using a low-concentration hydrofluoric acid solution to obtain microspheres with an inverse opal structure;
(3) 10v/v% of polyethylene glycol diacrylate, 20v/v% of acrylic acid and 1m/v% of Fe3O4Adding nano particles and 0.1m/v% of photoinitiator into deionized water to prepare a functional pre-gel mixed with magnetic nano particles, soaking microspheres with an inverse opal structure into the functional pre-gel until the communicated pores of the inverse opal structure are completely filled, and curing the hydrogel by ultraviolet irradiation for 30 seconds to obtain the magnetic structure colored hydrogel microcarrier.
Example 3
Preparation of magnetic structure color hydrogel microcarrier
(1) Referring to the preparation process of fig. 1, silica nanoparticles with a particle size of 247nm are respectively selected, monodisperse silica nanoparticle suspensions with a concentration of 20m/v% are prepared as an internal phase, simethicone (50 cSt) is used as an external phase, the external phase and the internal phase are injected into corresponding channels of a microfluidic device, the external phase and the internal phase pass through an output channel of the microfluidic device and are collected in a container, droplets of the silica nanoparticles are self-assembled along with water evaporation at 65 ℃, and the silica photonic crystal microspheres are formed through 12h solidification and crystallization; heating to 95 ℃ to solidify the silicon dioxide photonic crystal microspheres for 2 hours; then collecting the silicon dioxide photonic crystal microspheres in a crucible, washing the microspheres for multiple times by using normal hexane, completely absorbing the normal hexane after the washing is finished, and slowly placing the crucible in a muffle furnace for high-temperature treatment to improve the mechanical stability of the crucible to obtain a coding microcarrier template;
(2) dehydrating and drying the prepared coding microcarrier template into milk white by alcohol, soaking the template into 100v/v% ethylene glycol dimethacrylate solution, adding 0.1m/v% photoinitiator, and curing the hydrogel by ultraviolet irradiation for 30 s; transferring to a clean culture dish, and placing under a microscope to strip the microspheres from the cured hydrogel; collecting the microspheres in an EP tube, and corroding the template by using a low-concentration hydrofluoric acid solution to obtain microspheres with an inverse opal structure;
(3) 30v/v% of polyethylene glycol diacrylate and 8 m/v% of Fe3O4Adding nano particles and 0.1m/v% of photoinitiator into deionized water to prepare a functional pre-gel mixed with magnetic nano particles, soaking microspheres with an inverse opal structure into the functional pre-gel until the communicated pores of the inverse opal structure are completely filled, and curing the hydrogel by ultraviolet irradiation for 30 seconds to obtain the magnetic structure colored hydrogel microcarrier.
Examples of the experiments
The preparation of the magnetic structure color hydrogel microcarrier influences the biocompatibility and the magnetic control effect of the magnetic structure color hydrogel microcarrier, and the bioanalysis performance of the magnetic structure color hydrogel microcarrier is optimized by comparing the working conditions in the hydrogel construction process. Referring to fig. 3, as the concentration of the polyethylene glycol diacrylate increases, the intensity of the encoded signal of the magnetic structure color hydrogel microcarrier decreases accordingly; referring to fig. 4, as the concentration of acrylic acid increases, the intensity of the encoded signal of the magnetic structure color hydrogel microcarrier increases accordingly; said Fe3O4The concentration of the nano particles can directly change the magnetic effect of the magnetic structure color hydrogel microcarrier, and the frequency of vibration of the magnetic structure color hydrogel microcarrier can be judged macroscopically by naked eyes, so that the Fe is increased within a certain range3O4The concentration ratio of the nano particles and the directional magnetic control effect of the magnetic structure color hydrogel microcarrier are correspondingly enhanced; by setting polyethylene glycol diacrylate, acrylic acid and Fe3O4Ratio of nanoparticlesFor example, the encoded signal intensity is measured as a preferred standard, combined with its diffusion efficiency and mechanical properties in perfusing an inverse opal structure, with 20v/v% PEGDA, 15 v/v% AA, and 4 m/v% Fe3O4The nano-particle is used as the preferable condition for constructing the detection platform.
Application example
And modifying the surface of the prepared magnetic structure color hydrogel microcarrier with probe molecules, capturing and marking the bladder cancer protein by a double-antibody sandwich principle, and performing multiplex analysis on the bladder cancer protein by a fluorescence microscope. Selecting BTA, NMP22 and FDP three protein molecules approved by FDA to be applied to clinical tumor markers from bladder cancer protein; the probe molecules comprise anti-BTA antibodies, anti-NMP22 antibodies and anti-FDP antibodies, and are used for capturing the bladder cancer proteins BTA, NMP22 and FDP.
After APTES amination and succinic anhydride carboxylation are carried out on the magnetic structural color hydrogel microcarrier, carboxyl is activated by NHS and EDC in MES (pH 6.0) buffer solution, and covalent reaction is carried out on the carboxyl and amino of Anti-BTA, Anti-NMP22 and Anti-FDP of target protein antibodies, so that the antibodies are modified on the microcarrier. Referring to fig. 5 and 6, setting an antibody probe dilution gradient of 0.001-0.5 mg/mL and a reaction time of probe molecules for capturing bladder cancer protein of 20-120 min, and measuring signal intensity under different experimental conditions to serve as an optimization standard. Selecting the probe antibody concentration of 0.01 mg/mL and the protein marker reaction time of 60min as the optimal condition for biological analysis of the magnetic structure color hydrogel microcarrier.
Referring to FIG. 7, under optimized conditions, the bladder cancer protein markers BTA, NMP22 and FDP were quantitatively added, the concentration gradient thereof was set to be 0.001 ng/mL-0.01 mg/mL, and the detection curve of the platform was fitted by concentration-signal intensity correlation analysis. The detection signal intensity is correspondingly increased along with the increase of the concentration of the protein markers BTA, NMP22 and FDP, and when the concentration of the protein markers BTA, NMP22 and FDP is increased from 1.0 ng/mL to 1000 ng/mL, the concentration-signal intensity correlation coefficient R is20.9970, 0.9925 and 0.9912, respectively. Meanwhile, in the multiplex analysis of the protein markers, the cross-reactivity is evaluated by increasing the concentration (0 ng/mL-100 ng/mL) of other protein markers in the detection solution,the signal intensity change amplitude of the specific marker (20 ng/mL) is analyzed to be less than or equal to 5 percent, which indicates that the cross reaction of the multivariate analysis of the system does not influence the detection specificity of the system.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.
Claims (7)
1. A preparation method of a magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis is characterized by comprising the following steps:
s1: taking the monodisperse silicon dioxide nanoparticle suspension as an internal phase and the dimethyl fluorinated silicone oil as an external phase, injecting the external phase and the internal phase into corresponding channels of a microfluidic device, collecting the external phase and the internal phase in a container through an output channel of the microfluidic device, and forming silicon dioxide photonic crystal microspheres through solidification and crystallization; then collecting the silicon dioxide photonic crystal microspheres in a crucible, washing the microspheres for multiple times by using normal hexane, completely absorbing the normal hexane after the washing is finished, and placing the crucible in a muffle furnace for high-temperature treatment to obtain a coding microcarrier template;
s2: soaking the encoded microcarrier template prepared in the step S1 in a solution of ethylene glycol dimethacrylate, and performing photopolymerization curing, stripping and corrosion to obtain microspheres with inverse opal structures;
s3: preparing functional pre-gel mixed with magnetic nano particles, pouring the functional pre-gel into the microspheres with the inverse opal structure prepared in the step S2, and performing photopolymerization to obtain the magnetic structure color hydrogel microcarrier, wherein the functional pre-gel is prepared from polyethylene glycol diacrylate, acrylic acid and Fe3O4Nano particles and a photoinitiator; wherein the concentration of the polyethylene glycol diacrylate is 10-30 v/v%, the concentration of the acrylic acid is 0-20 v/v%, and the Fe content is3O4The concentration of the nano particles is Fe3O41-8 m/v%, and the concentration of the photoinitiator is 0.1 m/v%.
2. The method for preparing the magnetic structural color hydrogel microcarrier for the multiplex analysis of bladder cancer protein according to claim 1, wherein the method comprises the following steps: in step S1, the flow rates of the external phase and the internal phase in the corresponding channels of the microfluidic device are 3.0 mL/h and 0.5 mL/h, respectively.
3. The method for preparing the magnetic structural color hydrogel microcarrier for the multiplex analysis of bladder cancer protein according to claim 1, wherein the method comprises the following steps: in the step S1, the concentration of the silica nanoparticles in the monodisperse silica nanoparticle suspension is 20 m/v%; wherein the particle size of the silica nanoparticles includes any one of 215nm, 237nm and 247nm, and the silica nanoparticles are dispersed in ethanol.
4. The method for preparing the magnetic structural color hydrogel microcarrier for the multiplex analysis of bladder cancer protein according to claim 1, wherein the method comprises the following steps: and step S3, performing probe molecule modification on the surface of the prepared magnetic structure color hydrogel microcarrier, capturing and marking the bladder cancer protein by using a double-antibody sandwich principle, and performing multivariate analysis on the bladder cancer protein by using a fluorescence microscope.
5. The method for preparing the magnetic structural color hydrogel microcarrier for the multiplex analysis of bladder cancer protein according to claim 4, wherein the method comprises the following steps: the bladder cancer protein comprises any one of BTA protein molecules, NMP22 protein molecules or FDP protein molecules.
6. The method for preparing the magnetic structural color hydrogel microcarrier for the multiplex analysis of bladder cancer protein according to claim 5, wherein the method comprises the following steps: the probe molecule comprises any one of an anti-BTA antibody, an anti-NMP22 antibody or an anti-FDP antibody; wherein the concentration of the probe molecules is 0.001-0.5 mg/mL, and the reaction time for capturing the bladder cancer protein by the probe molecules is 20-120 min.
7. Use of the magnetic structure color hydrogel microcarrier prepared by the method of claim 1 in preparation of a material for bladder cancer protein labeling, wherein the surface of the magnetic structure color hydrogel microcarrier is modified with probe molecules, the bladder cancer protein is captured and labeled by a double antibody sandwich principle, and the bladder cancer protein is subjected to multiplex analysis by a fluorescence microscope, wherein the probe molecules comprise any one of anti-BTA antibody, anti-NMP22 antibody or anti-FDP antibody; the bladder cancer protein comprises any one of BTA protein molecules, NMP22 protein molecules or FDP protein molecules, wherein the concentration of the probe molecules is 0.001-0.5 mg/mL, and the reaction time for capturing the bladder cancer protein by the probe molecules is 20-120 min.
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