Structural color hydrogel microspheres for acute kidney injury multi-element labeling detection and preparation method of structural color hydrogel microspheres

Abstract

The invention discloses a preparation method of structural color hydrogel microspheres for acute kidney injury multi-labeling detection, which comprises the following steps: S1, shearing a silicon dioxide nanoparticle solution into liquid drops, drying the obtained liquid drops, cleaning and calcining to obtain silicon dioxide photonic crystal microspheres; s2, soaking the obtained silicon dioxide photonic crystal microspheres in a hydrogel precursor solution, performing cross-linking curing by ultraviolet irradiation after full incubation, and then stripping and corroding the obtained product to obtain hydrogel structural color microspheres; s3, modifying an acute kidney injury marker antibody to the hydrogel structural color microspheres through chemical coupling, so as to obtain the structural color hydrogel microspheres for acute kidney injury multi-element marker detection. The structural color hydrogel microspheres obtained by the invention can realize simultaneous detection of various acute kidney injury markers, and can enhance fluorescence signals and sensitivity of detection at the same time.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a structural color hydrogel microsphere for multiplex marker detection of acute kidney injury and a preparation method thereof. Background Art

[0002] Acute kidney injury is a common acute disease characterized by a dramatic loss of renal function and is often associated with a higher risk of mortality, prolonged hospitalization, and worsening of chronic kidney disease. Worldwide, the morbidity and mortality of acute kidney injury in hospitalized patients are high, and failure to diagnose acute kidney injury in a timely manner is one of the reasons for the poor prognosis. Although some biomolecules, such as creatinine, urea, cystatin C (CysC), and neutrophil gelatinase-associated lipocalin (NGAL), have been used as biomarkers for the assessment of acute kidney injury, the detection of only one biomarker limits its accuracy, while multiple tests often require more cost and time.

[0003] Therefore, it is necessary to develop a low-cost, time-saving, and highly sensitive method for detecting acute kidney injury. Summary of the invention

[0004] The primary purpose of the present invention is to provide a structural color hydrogel microsphere for multiplex marker detection of acute kidney injury and a preparation method thereof, so as to achieve simultaneous detection of multiple acute kidney injury markers.

[0005] To this end, the present invention provides the following technical solutions.

[0006] One aspect of the present invention provides a method for preparing structural color hydrogel microspheres for multiplex marker detection of acute kidney injury, the method comprising the following steps:

[0007] S1: Shearing the silica nanoparticle solution into droplets, drying the droplets, washing and calcining them to obtain silica photonic crystal microspheres;

[0008] S2: immersing the obtained silica photonic crystal microspheres in a hydrogel precursor solution, incubating sufficiently, and then irradiating with ultraviolet light to cross-link and solidify the photonic crystal / hydrogel composite microspheres, and then peeling and corroding the obtained photonic crystal / hydrogel composite microspheres to obtain hydrogel structural color microspheres;

[0009] S3: The acute kidney injury marker antibody is modified onto the obtained hydrogel structural color microspheres by chemical coupling to obtain structural color hydrogel microspheres for multiplex marker detection of acute kidney injury.

[0010] In a preferred embodiment of the present invention, in step S1, a silica nanoparticle aqueous solution with a mass volume fraction of 5 to 30% is used as the inner phase, and dimethyl silicone oil is used as the outer phase, and the obtained silica nanoparticle solution is sheared into droplets through a microfluidic micro-droplet chip.

[0011] In a preferred embodiment of the present invention, in step S1, the drying process is: first drying at 50-80°C for 6-18 hours, and then drying at 85-95°C for 2-3 hours.

[0012] In a preferred embodiment of the present invention, in step S1, the calcination process is: firstly heating from room temperature to 700-900°C over 3-4 hours and continuously calcining for 4-5 hours, and then cooling to room temperature over 3-4 hours.

[0013] In a preferred embodiment of the present invention, in step S1, the average particle size of the silica photonic crystal microspheres is 200-350 μm.

[0014] In a preferred embodiment of the present invention, in step S2, the hydrogel precursor solution is composed of: polyethylene glycol diacrylate with a mass volume ratio concentration of 5-15%, acrylic acid with a mass volume ratio concentration of 15-30%, photoinitiator with a mass volume ratio concentration of 0.25-2%, and the balance is water.

[0015] In a preferred embodiment of the present invention, the photoinitiator is selected from 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone, or a mixture of the two.

[0016] In a preferred embodiment of the present invention, in step S2, the incubation time is 2 to 8 hours.

[0017] In a preferred embodiment of the present invention, in step S2, the ultraviolet light irradiation conditions are: wavelength 365nm, cross-linking time 10 to 60s.

[0018] In a preferred embodiment of the present invention, in step S2, the etching method is: etching with hydrofluoric acid having a mass volume ratio concentration of 1 to 8% for 0.5 to 3 hours.

[0019] In a preferred embodiment of the present invention, in step S3, the chemical coupling method is: using 40-300mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 40-750mM N-hydroxysuccinimide to activate the obtained hydrogel structural color microspheres for 10-40min, wherein the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to N-hydroxysuccinimide is 1:1 to 1:2.5.

[0020] Another aspect of the present invention also provides a use of structural color hydrogel microspheres prepared according to the preparation method as described above in the preparation of a multi-marker detection product for acute kidney injury.

[0021] By means of the above technical solution, the present invention has at least the following advantages:

[0022] The present invention firstly emulsifies silica nanoparticles into droplets, then dries and calcines to obtain structural color microspheres-silica photonic crystal microspheres; then immerses the obtained silica photonic crystal microspheres in a hydrogel precursor solution, fully incubates, and then irradiates with ultraviolet light to induce chemical crosslinking and curing to obtain photonic crystal / hydrogel composite microspheres, and removes the silica template by stripping and corroding the obtained photonic crystal / hydrogel composite microspheres to obtain hydrogel structural color microspheres; finally, the acute kidney injury marker antibody is modified to the hydrogel microsphere by chemical coupling. The present invention utilizes the mutually penetrating nanopores and huge specific surface area provided by the structural color hydrogel microspheres to increase the modification density of the antibody, thereby enhancing the fluorescent signal and sensitivity of the detection. At the same time, the encoding function of the structural color is utilized to realize the simultaneous detection of multiple acute kidney injury markers. The structural color hydrogel microspheres coupled with the acute kidney injury marker antibody obtained by the present invention have high sensitivity and specificity in detecting acute kidney injury markers.

[0023] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the detection principle of the structural color hydrogel microspheres for multivariate detection of acute kidney injury of the present invention;

[0025] Figure 2 is a scanning electron microscope image of the silicon dioxide photonic crystal microsphere of Example 1;

[0026] Figure 3 is a scanning electron microscope image of the inverse opal structure hydrogel microspheres of Example 1;

[0027] Figure 4 The wavelength-reflectivity curves of the photonic crystal microspheres with different structural colors in Example 1;

[0028] Figure 5 It is a fluorescence intensity diagram of the antibody-coupled inverse opal structure hydrogel microspheres of Example 1 in different concentration solutions of the corresponding substrate;

[0029] Figure 6 This is a fluorescence intensity diagram of the antibody-coupled inverse opal structure hydrogel microspheres of Example 1 in different substrate solutions;

[0030] Figure 7 The figure shows the correlation comparison between the CysC and NGAL bioassay method of the inverse opal structure hydrogel microspheres of Example 1 and the traditional ELISA method in clinical samples. DETAILED DESCRIPTION

[0031] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the percentage content involved in the present invention refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0033] Unless otherwise specified, the percentage concentrations referred to in the present invention all refer to final concentrations, which refer to the percentage of the added component in the system after the addition of the component.

[0034] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0035] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] Example 1: Preparation of structural color hydrogel microspheres for multivariate marker detection of acute kidney injury

[0038] (1) Synthesis of silica photonic crystal microspheres:

[0039] A 20% mass-to-volume concentration of silica nanoparticles in water was loaded into a syringe as the inner phase solution, and a syringe filled with dimethyl silicone oil (500 cSt) was used as the outer phase solution. The two syringes were then fixed on a microfluidic pump (LSP01-1A, Baoding Lange Constant Flow Pump Co., Ltd.). The inner phase solution and the outer phase solution were respectively introduced into the inner phase port and the outer phase port of a PE capillary microfluidic device through the microfluidic pump. The flow rate was controlled by the microfluidic device (1 mL / h for the inner phase solution and 6 mL / h for the outer phase solution), and microdroplets of uniform size were obtained at the confluence of the two phase outlets, namely, silica nanoparticle emulsion droplets.

[0040] The collected silica nanoparticle droplets were placed in a 65°C oven overnight (12h), and the temperature was adjusted to 85°C and maintained for 2h the next day to ensure complete evaporation of water. Then, the excess dimethyl silicone oil on the droplets was removed by repeated washing with n-hexane, and the droplets were collected in a crucible and washed five times to ensure that there was no residual silicone oil. Finally, the washed droplets were transferred to a muffle furnace for calcination: first, the temperature was raised from 30°C to 800°C after 3h and calcined for 4h, and then the temperature was lowered from 800°C to 30°C after 3h. After calcination, solid silica photonic crystal microspheres were obtained.

[0041] The obtained silica photonic crystal microspheres were analyzed by scanning electron microscope. Figure 2 .like Figure 2 As shown, the silica photonic crystal microspheres are self-assembled from multiple nanoparticles of uniform size, and the average particle size of the microspheres is between 200-350 μm. The surface and interior of the obtained silica photonic crystal microspheres have three-dimensional interconnected nano-network gaps, and the porous structure can be used to fill the hydrogel precursor solution.

[0042] (2) Preparation of inverse opal structured hydrogel microspheres:

[0043] Polyethylene glycol diacrylate (hereinafter referred to as PEGDA), acrylic acid and 2-hydroxy-2methyl-1-phenyl-1-propanone (hereinafter referred to as HMPP) are added to water and fully dissolved to prepare a hydrogel precursor solution, wherein the mass volume ratio concentrations of PEGDA, acrylic acid and HMPP in the hydrogel precursor solution are 10%, 15% and 1%, respectively. Subsequently, the 20 silica photonic crystal microspheres obtained in step (1) are added to a 200 μL centrifuge tube containing the hydrogel precursor solution and incubated for 4 hours. The incubated microspheres are irradiated with a 365nm ultraviolet light source for 20 seconds to obtain photonic crystal / hydrogel composite microspheres. The obtained photonic crystal / hydrogel composite microspheres are peeled off and corroded with 4% (w / v) HF for 2 hours to obtain inverse opal structure hydrogel microspheres, i.e., hydrogel structural color microspheres, whose scanning electron microscope image is shown in FIG. Figure 3 .

[0044] By selecting silica nanoparticles of different sizes for assembly, photonic crystal microsphere templates with different structural colors can be obtained. The optical photos and corresponding spectra are shown in Figure 4 .

[0045] (3) Implementation of multivariate detection:

[0046] by Figure 4The different structural color photonic crystal microspheres shown in the figure are used as templates, and the inverse opal structure hydrogel microspheres with different structural colors are prepared according to the methods described in (1) and (2) above. Then, the obtained inverse opal structure hydrogel microspheres are activated, that is, 35 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (hereinafter referred to as EDC) and 55 mg / mL N-hydroxysuccinimide (hereinafter referred to as NHS) are used to activate the obtained inverse opal structure hydrogel microspheres for 30 minutes to obtain activated inverse opal structure hydrogel microspheres. The activated inverse opal structure hydrogel microspheres are added with CysC and NGAL primary antibodies and incubated for 24 hours. After the incubation, they are blocked with BSA for 6 hours to obtain structural color microspheres labeled with two antibodies (CysC and NGAL primary antibodies). The obtained structural color microspheres labeled with the two antibodies are added to the sample to be tested and incubated for 2 hours. After the incubation, they are incubated again with fluorescently labeled CysC and NGAL secondary antibodies for 2 hours. After the incubation, the microspheres were washed and the fluorescence intensity was recorded and analyzed under a fluorescence microscope. Figure 5 and 6 .like Figure 5 As shown in the figure, with the increase of the concentration of each substrate (CysC and NGAL), the detection fluorescence intensity of each structural color hydrogel microsphere showed a significant enhancement trend, and the detection limit of the structural color hydrogel microspheres labeled with two antibodies can reach 10ng / mL. Figure 6 As shown, the structural color hydrogel microspheres labeled with antibodies can only undergo fluorescence reaction in the corresponding substrate solution, while the structural color hydrogel microspheres labeled with two antibodies can undergo fluorescence reaction in both substrate mixed solutions. This result shows that the antibody-labeled structural color hydrogel microspheres of the present invention have good specificity.

[0047] Embodiment 2:

[0048] The preparation method is the same as that of Example 1, except that in step (2), the mass volume ratios of PEGDA, acrylic acid and HMPP in the hydrogel precursor solution are 5%, 20% and 1%, respectively.

[0049] Embodiment 3:

[0050] The preparation method is the same as that of Example 1, except that in step (2), the mass volume ratios of PEGDA, acrylic acid and HMPP in the hydrogel precursor solution are 5%, 25% and 1.5%, respectively.

[0051] According to the method described in Example 1, the sensitivity and specificity of the antibody-labeled structural color gel microspheres obtained in Examples 2 and 3 were tested respectively, and the results obtained were consistent with the detection results of the antibody-labeled structural color gel microspheres obtained in Example 1. This result shows that the antibody-labeled structural color hydrogel microspheres obtained by the preparation method of the present invention have high sensitivity and good specificity for the detection of acute kidney injury markers.

[0052] Application Example 1: Comparison with Standard ELISA Test Kit

[0053] In order to further verify the detection accuracy of the structural color hydrogel microspheres for multi-label detection of the present invention, this application example compares the antibody-labeled structural color hydrogel microspheres obtained in Example 1 with the ELISA kit of Beijing Solebow Technology Co., Ltd. to verify the reliability of the antibody-coupled anti-opal structural hydrogel microspheres of the present invention in detecting clinical serum samples. The specific operation is as follows: 5 μL of the serum to be tested is taken with an EP tube, and pure water is used as a control. The antibody-coupled anti-opal structural hydrogel microspheres obtained in Example 1 are placed in the EP tube and incubated at 37°C for 40 minutes. Then, after washing the microspheres 3 times, the corresponding fluorescein isothiocyanate-labeled antibodies are added to the EP tube, and the reaction is continued for 30 minutes under light-proof conditions. After the reaction is completed, the antibody-coupled anti-opal structural hydrogel microspheres are thoroughly washed more than 3 times to remove unreacted substances. Finally, the fluorescence intensity is observed and recorded using a fluorescence microscope. The test results are compared with the results of the ELISA kit. The results are shown in the figure. Figure 7 .like Figure 7 As shown, the detection effect of the structural color hydrogel microspheres labeled with two antibodies on the target substrate is significantly consistent with that of the ELISA method. This result shows that the structural color hydrogel microspheres labeled with antibodies of the present invention have good reliability and application potential in detecting the target substrate.

[0054] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing structural color hydrogel microspheres for multiplex marker detection of acute kidney injury, characterized in that: The method comprises the following steps: S1: Shearing the silica nanoparticle solution into droplets, drying the droplets, washing and calcining them to obtain silica photonic crystal microspheres; S2: immersing the obtained silica photonic crystal microspheres in a hydrogel precursor solution, incubating them sufficiently, and then irradiating them with ultraviolet light for cross-linking and curing to obtain photonic crystal / hydrogel composite microspheres, and then peeling and corroding the obtained photonic crystal / hydrogel composite microspheres to obtain hydrogel structural color microspheres; S3: The acute kidney injury marker antibody is modified onto the obtained hydrogel structural color microspheres by chemical coupling to obtain structural color hydrogel microspheres for multiplex marker detection of acute kidney injury.

2. The preparation method according to claim 1, characterized in that: In step S1, a silica nanoparticle aqueous solution with a mass volume fraction of 5 to 30% is used as an inner phase, dimethyl silicone oil is used as an outer phase, and the obtained silica nanoparticle solution is sheared into droplets through a microfluidic micro-droplet chip.

3. The preparation method according to claim 1, characterized in that: In step S1, the drying process is: first drying at 50-80°C for 6-18 hours, and then drying at 85-95°C for 2-3 hours.

4. The preparation method according to claim 1, characterized in that: In step S1, the calcination process is: firstly, the temperature is raised from room temperature to 700-900°C over 3-4 hours and the calcination is continued for 4-5 hours, and then the temperature is lowered to room temperature over 3-4 hours.

5. The preparation method according to claim 1, characterized in that: In step S1, the average particle size of the silicon dioxide photonic crystal microspheres is 200-350 μm.

6. The preparation method according to claim 1, characterized in that: In step S2, the hydrogel precursor solution is composed of: polyethylene glycol diacrylate with a mass volume ratio of 5-15%, acrylic acid with a mass volume ratio of 15-30%, photoinitiator with a mass volume ratio of 0.25-2%, and the balance is water.

7. The preparation method according to claim 6, characterized in that: The photoinitiator is selected from one of 2-hydroxy-2-methyl-1-phenyl-1-propanone and 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone or a mixture of the two.

8. The preparation method according to claim 1, characterized in that: In step S2, the incubation time is 2 to 8 hours; The ultraviolet irradiation conditions are: wavelength 365nm, cross-linking time 10 to 60s; The etching method comprises: etching with hydrofluoric acid having a mass volume ratio concentration of 1 to 8% for 0.5 to 3 hours.

9. The preparation method according to claim 1, characterized in that: In step S3, the chemical coupling method is: using 40-300mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 40-750mM N-hydroxysuccinimide to activate the obtained hydrogel structural color microspheres for 10-40min, wherein the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to N-hydroxysuccinimide is 1:1-1:2.

5.

10. Use of the structural color hydrogel microspheres prepared by the preparation method according to any one of claims 1 to 9 in preparing a multi-marker detection product for acute kidney injury.

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