Alkaline phosphatase-responsive hydrogel photonic crystal microspheres and their preparation method and application

Abstract

The present invention discloses an alkaline phosphatase-responsive hydrogel photonic crystal microsphere and its preparation method and application. The preparation method comprises the following steps: step 1, preparing positive structure photonic crystal microspheres assembled with silica nanoparticles, immersing them in a photocrosslinkable hard hydrogel precursor solution, and irradiating and curing them to obtain a single-shot glue-cast photonic crystal microsphere; step 2, corroding the silica nanoparticles in the single-shot glue-cast photonic crystal microsphere to obtain an inverse opal photonic crystal microparticle scaffold; step 3, mixing a fluorescent probe and a sodium alginate solution, placing the inverse opal photonic crystal microparticle scaffold in the mixed solution, pouring a salt solution containing copper ions, and ion crosslinking and curing to obtain the alkaline phosphatase-responsive hydrogel photonic crystal microsphere. The present invention has the advantages of convenient detection, wide detection range, sensitivity, and accuracy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials and relates to a hydrogel photonic crystal microsphere, in particular to an alkaline phosphatase-responsive hydrogel photonic crystal microsphere and a preparation method and application thereof. Background Art

[0002] Alkaline phosphatase (ALP) is one of the most widely distributed enzymes in biological species. It plays a key role in the dephosphorylation of nucleic acids, proteins, and other biomolecules, and therefore plays an important role in regulating cellular function and signal transduction. Furthermore, a wealth of evidence indicates that abnormal ALP levels in the human body are a sign and diagnostic indicator of a variety of diseases, such as those of the liver, endocrine system, and bone tissue. Therefore, highly sensitive and specific ALP detection methods are needed to assist in the assessment of clinical physiological and pathological conditions. To date, researchers have established a variety of ALP detection strategies, such as chemiluminescence, electrochemistry, Raman spectroscopy, liquid chromatography, and fluorescent probes. Among them, fluorescence-based assays, which rely on fluorescent dyes, offer high sensitivity and specificity and are a preferred approach. While these fluorescence-based assays have achieved multi-molecule coverage and promising results, they still require specialized instrumentation and complex procedures for signal readout. To address this limitation, researchers have constructed molecular sensors using hydrogel materials as carriers for fluorescent dyes. These hydrogel sensors can undergo phase transitions upon external stimuli, converting the target molecule signal into an optical readout, significantly simplifying the detection process. However, conventional sensors made solely from hydrogels still face the challenge of long reaction times, and the accuracy and reliability of detection are limited by a single signal. Therefore, it is necessary to develop a new ALP detection platform that is fast, efficient, and accurate.

[0003] Inverse opal photonic crystal microparticles refer to a type of 3D material with microscopic ordered voids. They are covered with highly interconnected pores on the surface and inside, providing a huge specific surface area and abundant molecular binding sites. In addition, the periodic porous structure of the inverse opal particles produces a photonic bandgap effect, thereby exhibiting rich structural colors visible to the naked eye. Hydrogel photonic crystal microspheres constructed by combining the above-mentioned phase-change responsive hydrogel with inverse opal photonic crystal microparticles can effectively sense specific molecular stimuli through phase change. Therefore, hydrogel photonic crystal microspheres have been widely used to detect various targets, including ions, small molecules and biomacromolecules. However, most hydrogels are not responsive to ALP, making them difficult to use directly for ALP detection. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the deficiencies of the above-mentioned prior art and provide an alkaline phosphatase-responsive hydrogel photonic crystal microsphere, a preparation method thereof, and an application in alkaline phosphatase detection. The method uses a hard inverse opal photonic crystal as a scaffold, on which is covered a hydrogel that can produce a specific phase change response, to prepare microspheres that can simultaneously exhibit fluorescence and structural color, thereby solving the problems of long reaction time and low sensitivity and accuracy existing in traditional detection relying on the hydrogel body.

[0005] To achieve the above object, the present invention provides a method for preparing alkaline phosphatase-responsive hydrogel photonic crystal microspheres, which has the following characteristics:

[0006] Step 1: preparing positive-structure photonic crystal microspheres assembled with silica nanoparticles through a microfluidic chip, immersing the positive-structure photonic crystal microspheres in a photocrosslinkable hard hydrogel precursor solution, then irradiating and curing, and peeling off to obtain a single-gel-filled photonic crystal microsphere;

[0007] Step 2: etching away the silica nanoparticles in the primary-gel-cast photonic crystal microspheres to obtain a porous inverse opal photonic crystal microparticle scaffold consisting only of hard hydrogel;

[0008] Step 3: Mix the fluorescent probe and sodium alginate solution, place the inverse opal photonic crystal microparticle scaffold in the mixed solution, pour in the salt solution containing copper ions, and keep shaking until it is completely gelled (i.e., after ionic crosslinking and curing). The microparticles are peeled off to form alkaline phosphatase-responsive hydrogel photonic crystal microspheres. The outer layer of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres is a cured copper alginate hydrogel containing the fluorescent probe.

[0009] Furthermore, the present invention provides a method for preparing alkaline phosphatase-responsive hydrogel photonic crystal microspheres, which may also have the following characteristics: wherein, in step 1, the microfluidic chip is selected from one of a capillary assembly chip, a glass / silicon material chip, an organic rubber and a plastic material chip.

[0010] Furthermore, the present invention provides a method for preparing alkaline phosphatase-responsive hydrogel photonic crystal microspheres, which may also have the following characteristics: in step 1, the preparation method of the hard hydrogel precursor solution is: adding a photoinitiator to the hard hydrogel precursor, the volume ratio of the hard hydrogel precursor to the photoinitiator is 100:1, and then stirring and mixing thoroughly.

[0011] Furthermore, the present invention provides a method for preparing alkaline phosphatase-responsive hydrogel photonic crystal microspheres, which may also have the following characteristics: wherein, in step 1, the hard hydrogel precursor is selected from one of ethoxylated trimethylolpropane triacrylate (ETPTA), polyethylene glycol (diol) diacrylate (PEGDA), and ethylene glycol dimethacrylate (EGDMA).

[0012] Furthermore, the present invention provides a method for preparing alkaline phosphatase-responsive hydrogel photonic crystal microspheres, which may also have the following characteristics: wherein, in step 2, hydrofluoric acid is used to etch away the silica particles.

[0013] Furthermore, the present invention provides a method for preparing alkaline phosphatase-responsive hydrogel photonic crystal microspheres, which may also have the following characteristics: wherein, in step three, the fluorescent probe is selected from one of fluorescein probes, inorganic ion fluorescent probes, fluorescent quantum dots, and molecular beacons.

[0014] Furthermore, the present invention provides a method for preparing alkaline phosphatase-responsive hydrogel photonic crystal microspheres, which may also have the following characteristics: wherein, in step three, the salt solution containing copper ions is selected from one of aqueous solutions of copper chloride, copper sulfate, copper nitrate, copper carbonate, and basic copper carbonate.

[0015] Furthermore, the present invention provides a method for preparing alkaline phosphatase-responsive hydrogel photonic crystal microspheres, which may also have the following characteristics: wherein, in step three, the concentration of the sodium alginate solution is 1wt%; the volume ratio of the fluorescent probe to the sodium alginate solution is 1:1; the concentration of the salt solution with copper ions is 2mM; and the volume ratio of the sodium alginate solution to the salt solution with copper ions is 1:2.

[0016] The present invention also provides alkaline phosphatase-responsive hydrogel photonic crystal microspheres prepared by the preparation method.

[0017] The present invention also provides the use of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres in alkaline phosphatase detection, which has the following characteristics: pyrophosphate ions are introduced as an intermediate medium for quantitative detection of alkaline phosphatase.

[0018] Furthermore, the present invention provides the use of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres in alkaline phosphatase detection, which may also have the following characteristics: wherein the quantitative detection method of alkaline phosphatase comprises the following steps:

[0019] Step 1: Thoroughly mix the pyrophosphate solution with alkaline phosphatase solutions of different concentration gradients, where the concentration of the alkaline phosphatase solution is 0 to 400 mU / mL; incubate at 37°C for 30 minutes; then add the alkaline phosphatase-responsive hydrogel photonic crystal microspheres to the mixed solution, shake at 150 rpm at room temperature, and react for 2 hours; remove the alkaline phosphatase-responsive hydrogel photonic crystal microspheres, and use a fiber optic spectrometer to measure the fluorescence and reflectance spectra of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres; plot the relationship between the detected fluorescence intensity and reflection peak shift value and the concentration of the alkaline phosphatase solution into a standard curve;

[0020] Step 2: Fully mix the pyrophosphate solution with the test solution containing alkaline phosphatase and incubate at 37°C for 30 minutes; then add the alkaline phosphatase-responsive hydrogel photonic crystal microspheres for detection to the mixed solution, shake at 150 rpm at room temperature, and react for 2 hours; remove the alkaline phosphatase-responsive hydrogel photonic crystal microspheres for detection, detect the fluorescence intensity and reflection peak shift value of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres for detection, and then substitute them into the standard curve to finally calculate the alkaline phosphatase content in the test solution to achieve dual optical signal conversion;

[0021] The concentration of the pyrophosphate solution in step 2 is equal to that of the pyrophosphate solution in step 1 and is less than 10 mM.

[0022] The beneficial effects of the present invention lie in the following: By constructing a rigid inverse opal photonic crystal, using its porous structure as a scaffold, and embedding a hydrogel capable of producing a specific phase transition response, the invention produces microspheres that exhibit both fluorescence and structural color. This invention not only utilizes the fluorescence intensity changes within the microsphere's outer hydrogel layer, but also indicates the target alkaline phosphatase content through the shift in the inverse opal structural color and characteristic reflection peak. This dual-signal detection method overcomes the long reaction time and low sensitivity and accuracy associated with traditional detection methods that rely on the hydrogel itself. Specifically:

[0023] 1. The present invention uses a specifically responsive copper alginate hydrogel as a molecular sensor to convert target molecular signals into dual optical signals triggered by phase change, without the need for complex reading equipment and operating procedures.

[0024] 2. The photonic crystal microspheres prepared by the present invention are used as detection carriers, which greatly shortens the reaction time of bulk hydrogels. At the same time, due to the large number of through holes inherent in the inverse opal structure, the specific surface area of ​​the spheres can be increased, providing sufficient molecular binding sites, thereby effectively reducing the detection limit and broadening the target detection range.

[0025] Third, this invention not only utilizes changes in fluorescence intensity within the microsphere's outer hydrogel layer but also indicates the target alkaline phosphatase content through shifts in the inverse opal structural color and characteristic reflectance peaks. This dual-signal detection method innovatively combines the specificity of fluorescence detection with the stability of structural color, further enhancing its sensitivity and accuracy. The photonic crystal microspheres prepared in this invention are expected to become ideal carriers for clinical alkaline phosphatase testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the preparation method of alkaline phosphatase-responsive hydrogel photonic crystal microspheres of the present invention and their use in alkaline phosphatase detection, wherein a is the process of obtaining alkaline phosphatase-responsive hydrogel photonic crystal microspheres by the method of glue filling-corrosion-refilling, and b is the mechanism of introducing pyrophosphate ions to detect alkaline phosphatase;

[0027] Figure 2 The electron microscopy characterizations of the surfaces and interiors of different microspheres are shown in Figure 1. a and d are the surface and interior images of positive structure photonic crystal microspheres, b and e are the surface and interior images of inverse opal photonic crystal microparticle scaffolds, and c and f are the surface and interior images of alkaline phosphatase-responsive hydrogel photonic crystal microspheres.

[0028] Figure 3 Schematic diagrams and light microscopy images of different microspheres, among which column a is positive structure photonic crystal microspheres, column b is one-time glue-casting photonic crystal microspheres, column c is inverse opal photonic crystal microparticle scaffolds, and column d is alkaline phosphatase-responsive hydrogel photonic crystal microspheres; the second row of light microscopy images are positive structure photonic crystal microspheres and their derivative microspheres assembled based on 215nm silica nanoparticles, the third row of light microscopy images are positive structure photonic crystal microspheres and their derivative microspheres assembled based on 260nm silica nanoparticles, and the fourth row of light microscopy images are positive structure photonic crystal microspheres and their derivative microspheres assembled based on 305nm silica nanoparticles;

[0029] Figure 4 The fluorescence intensity and reflection peak of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres change with the concentration of added pyrophosphate ions, and the fluorescence spectrum and reflection spectrum are presented, where a is the fluorescence spectrum and b is the reflection spectrum;

[0030] Figure 5Figure 2 shows the optical microscopy and fluorescence images of alkaline phosphatase-responsive hydrogel photonic crystal microspheres before and after the addition of pyrophosphate ions, wherein ai and aii are the optical microscopy and fluorescence images of alkaline phosphatase-responsive hydrogel photonic crystal microspheres with a reflection peak of 570 nm, bi and bii are the optical microscopy and fluorescence images of alkaline phosphatase-responsive hydrogel photonic crystal microspheres with a reflection peak of 600 nm, and ci and cii are the optical microscopy and fluorescence images of alkaline phosphatase-responsive hydrogel photonic crystal microspheres with a reflection peak of 650 nm; aiii and aiv are the optical microscopy and fluorescence images of alkaline phosphatase-responsive hydrogel photonic crystal microspheres with a reflection peak shifted to 540 nm after the addition of pyrophosphate ions, bii and biv are the optical microscopy and fluorescence images of alkaline phosphatase-responsive hydrogel photonic crystal microspheres with a reflection peak shifted to 576 nm after the addition of pyrophosphate ions, and ciii and civ are the optical microscopy and fluorescence images of alkaline phosphatase-responsive hydrogel photonic crystal microspheres with a reflection peak shifted to 622 nm after the addition of pyrophosphate ions;

[0031] Figure 6 Figure 3 shows the spectral changes of alkaline phosphatase-responsive hydrogel photonic crystal microspheres after adding quantitative pyrophosphate ions and different concentrations of alkaline phosphatase. a is the fluorescence spectrum after adding different concentrations of alkaline phosphatase, b is the corresponding fluorescence intensity curve, c is the reflectance spectrum after adding different concentrations of alkaline phosphatase, and d is the corresponding reflection peak shift value curve. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to specific embodiments.

[0033] Example 1

[0034] This embodiment provides an alkaline phosphatase-responsive hydrogel photonic crystal microsphere based on a PEGDA scaffold, and the preparation method thereof is as follows: Figure 1 As shown in a, the following steps are specifically included:

[0035] Step 1: Prepare positive structure photonic crystal microspheres assembled from silica nanoparticles using a microfluidic chip, such as Figure 2 As shown in a, d. The positive structure photonic crystal microspheres are then immersed in a hard hydrogel precursor solution, which is a polyethylene glycol (diol) diacrylate (PEGDA) hard hydrogel precursor with a photoinitiator added. The volume ratio of the hard hydrogel precursor to the photoinitiator is 100:1. Let it stand for 4 hours to ensure that the hard hydrogel precursor solution fully penetrates into the nanopores of the positive structure photonic crystal microspheres. Next, the mixture of the positive structure photonic crystal microspheres and the hard hydrogel precursor solution is exposed to ultraviolet light to cure the mixed system. After curing, the microspheres are peeled off and the excess cured hard hydrogel is removed to obtain a single-shot glue-filled photonic crystal microsphere.

[0036] Step 2: Use 4wt% hydrofluoric acid solution to corrode the silica particles in the primary glue-filled photonic crystal microspheres to obtain a porous inverse opal photonic crystal microparticle scaffold composed only of PEGDA hydrogel, such as Figure 2 As shown in b and e.

[0037] Step 3: Mix the fluorescent quantum dots with an equal volume of 1 wt% sodium alginate solution, place the inverse opal photonic crystal microparticle scaffold in the mixed solution, shake for 30 minutes, then pour in 2 times the volume of 2 mM copper chloride solution, keep shaking until all gelled, and peel off the copper alginate composite microspheres that have been cast twice from the surrounding hydrogel to obtain alkaline phosphatase-responsive hydrogel photonic crystal microspheres based on PEGDA scaffold, as shown in FIG. Figure 2 As shown in c,f.

[0038] The positive structure photonic crystal microspheres, one-time glue-cast photonic crystal microspheres, inverse opal photonic crystal microparticle scaffolds and alkaline phosphatase-responsive hydrogel photonic crystal microspheres were characterized by optical microscopy, showing different structural colors, such as Figure 3 shown.

[0039] Example 2

[0040] This embodiment provides an alkaline phosphatase-responsive hydrogel photonic crystal microsphere based on an ETPTA scaffold, and the preparation method thereof specifically comprises the following steps:

[0041] Step 1: Prepare positive-structure photonic crystal microspheres assembled with silica nanoparticles through a microfluidic chip. The positive-structure photonic crystal microspheres are then immersed in a hard hydrogel precursor solution, which is an ethoxylated trimethylolpropane triacrylate (ETPTA) hard hydrogel precursor with a photoinitiator added thereto. The volume ratio of the hard hydrogel precursor to the photoinitiator is 100:1. Let it stand for 4 hours to ensure that the hard hydrogel precursor solution fully penetrates into the nanopores of the positive-structure photonic crystal microspheres. Next, the mixture of the positive-structure photonic crystal microspheres and the hard hydrogel precursor solution is exposed to ultraviolet light to cure the mixed system. After curing, the microspheres are peeled off and the excess cured hard hydrogel is removed to obtain one-time glue-filled photonic crystal microspheres.

[0042] Step 2: Use 4 wt % hydrofluoric acid solution to corrode the silica particles in the primary-cast photonic crystal microspheres to obtain a porous inverse opal photonic crystal microparticle scaffold consisting only of ETPTA hydrogel.

[0043] Step 3: Mix the fluorescent quantum dots with an equal volume of 1wt% sodium alginate solution, place the inverse opal photonic crystal microparticle scaffold in the mixed solution, shake for 30 minutes, then pour in 2 times the volume of 2mM copper chloride solution, keep shaking until all gelled, and peel off the copper alginate composite microspheres that have been cast twice from the surrounding hydrogel to obtain alkaline phosphatase-responsive hydrogel photonic crystal microspheres based on the ETPTA scaffold.

[0044] Example 3

[0045] This embodiment provides an alkaline phosphatase-responsive hydrogel photonic crystal microsphere based on an EGDMA scaffold, and the preparation method thereof specifically comprises the following steps:

[0046] Step 1: Prepare positive-structure photonic crystal microspheres assembled with silica nanoparticles through a microfluidic chip. The positive-structure photonic crystal microspheres are then immersed in a hard hydrogel precursor solution, which is a hard hydrogel precursor of ethylene glycol dimethacrylate (EGDMA) with the addition of a photoinitiator. The volume ratio of the hard hydrogel precursor to the photoinitiator is 100:1. Let it stand for 4 hours to ensure that the hard hydrogel precursor solution fully penetrates into the nanopores of the positive-structure photonic crystal microspheres. Next, the mixture of the positive-structure photonic crystal microspheres and the hard hydrogel precursor solution is exposed to ultraviolet light to solidify the mixed system. After solidification, the microspheres are peeled off and the excess solidified hard hydrogel is removed to obtain one-time glue-filled photonic crystal microspheres.

[0047] Step 2: Use 4 wt % hydrofluoric acid solution to corrode the silica particles in the primary-cast photonic crystal microspheres to obtain a porous inverse opal photonic crystal microparticle scaffold consisting only of EGDMA hydrogel.

[0048] Step 3: Mix the fluorescent quantum dots with an equal volume of 1wt% sodium alginate solution, place the inverse opal photonic crystal microparticle scaffold in the mixed solution, shake for 30 minutes, then pour in 2 times the volume of 2mM copper chloride solution, keep shaking until all gelled, and peel off the copper alginate composite microspheres that have been cast twice from the surrounding hydrogel to obtain alkaline phosphatase-responsive hydrogel photonic crystal microspheres based on the EGDMA scaffold.

[0049] Example 4

[0050] This embodiment provides an application of alkaline phosphatase-responsive hydrogel photonic crystal microspheres in alkaline phosphatase detection.

[0051] The application method is to quantitatively detect alkaline phosphatase by introducing pyrophosphate ions as an intermediate medium. The detection principle is that pyrophosphate solutions with different concentration gradients are added to hydrogel photonic crystal microspheres respectively. Due to the strong competitive coordination effect between pyrophosphate ions and copper ions, the alginate copper hydrogel on the outer layer of the microspheres undergoes different degrees of phase change and disintegration, resulting in a certain amount of fluorescent probe being released into the external solution; at the same time, due to the change in the refractive index of the microspheres, their structural color and characteristic reflection peak change accordingly. Then, by dual measurement of the fluorescence intensity and characteristic reflection peak of the microspheres, the initial concentration of pyrophosphate ions can be quantified. The addition of alkaline phosphatase can catalyze the hydrolysis of pyrophosphate ions and thus hinder the above-mentioned competitive coordination effect. Therefore, the addition of pyrophosphate solution and alkaline phosphatase can achieve quantitative detection of the latter.

[0052] The above mechanism was verified by introducing sodium pyrophosphate disintegration hydrogel experiments and the threshold concentration of pyrophosphate solution was obtained. The specific experiments are as follows:

[0053] At room temperature, the alkaline phosphatase-responsive hydrogel photonic crystal microspheres with reflection peaks of 570 nm, 600 nm, and 650 nm were added to 500 μL of sodium pyrophosphate aqueous solution with different concentration gradients (0-3 mM) and kept shaking at 150 rpm for 2 hours. Subsequently, the alkaline phosphatase-responsive hydrogel photonic crystal microspheres were taken out, and the fluorescence and reflection spectra of each alkaline phosphatase-responsive hydrogel photonic crystal microsphere were measured using a fiber optic spectrometer. The relationship between the fluorescence intensity and reflection peak offset value obtained by the detection and the concentration of the pyrophosphate solution was plotted into a standard curve, as shown in FIG. Figure 4 As shown in FIG. 1 , the threshold value of the pyrophosphate solution concentration is obtained to be 10 mM.

[0054] After the addition of pyrophosphate ions, the hydrogel disintegrated, causing the reflection peak of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres to blue shift and the fluorescence intensity to decrease. Figure 5 As shown, the reflection peaks of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres with three structural colors shifted to 540 nm, 576 nm, and 622 nm, respectively, and the structural colors changed visibly to the naked eye.

[0055] The quantitative detection method of alkaline phosphatase specifically comprises the following steps:

[0056] Step 1: Thoroughly mix 10 mM pyrophosphate solution with alkaline phosphatase solutions of different concentration gradients, where the concentration of the alkaline phosphatase solution is 0 to 400 mU / mL; incubate at 37°C for 30 minutes; then add the alkaline phosphatase-responsive hydrogel photonic crystal microspheres to the mixed solution, shake at 150 rpm at room temperature, and react for 2 hours; remove the alkaline phosphatase-responsive hydrogel photonic crystal microspheres, and use a fiber optic spectrometer to measure the fluorescence and reflection spectra of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres; plot the relationship between the detected fluorescence intensity and reflection peak shift value and the concentration of the alkaline phosphatase solution into a standard curve, as shown in FIG. Figure 6 As shown;

[0057] Step 2: Fully mix the 10 mM pyrophosphate solution with the test solution containing alkaline phosphatase, and incubate at 37°C for 30 minutes; then add the alkaline phosphatase-responsive hydrogel photonic crystal microspheres for detection to the mixed solution, shake at 150 rpm at room temperature, and react for 2 hours; remove the alkaline phosphatase-responsive hydrogel photonic crystal microspheres for detection, detect the fluorescence intensity and reflection peak shift value of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres for detection, and then substitute them into the standard curve to finally calculate the alkaline phosphatase content in the test solution to achieve dual optical signal conversion;

[0058] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions based on the principles of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Application of alkaline phosphatase-responsive hydrogel photonic crystal microspheres in alkaline phosphatase detection, characterized in that: Pyrophosphate ions were introduced as an intermediate medium for the quantitative detection of alkaline phosphatase; The preparation method of alkaline phosphatase-responsive hydrogel photonic crystal microspheres comprises the following steps: Step 1: preparing positive-structure photonic crystal microspheres assembled with silica nanoparticles through a microfluidic chip, immersing the positive-structure photonic crystal microspheres in a photocrosslinkable hard hydrogel precursor solution, then irradiating and curing, and peeling off to obtain a single-gel-filled photonic crystal microsphere; The hard hydrogel precursor solution is prepared by adding a photoinitiator to the hard hydrogel precursor at a volume ratio of 100:1, and then stirring and mixing thoroughly; the hard hydrogel precursor is selected from one of ethoxylated trimethylolpropane triacrylate, polyethylene glycol (glycol) diacrylate, and ethylene glycol dimethacrylate; Step 2: etching away the silica nanoparticles in the primary-gel-cast photonic crystal microspheres to obtain a porous inverse opal photonic crystal microparticle scaffold consisting only of hard hydrogel; Step 3: Mix the fluorescent probe and sodium alginate solution, place the inverse opal photonic crystal microparticle scaffold in the mixed solution, pour in the salt solution containing copper ions, keep shaking until all gelled, and peel off the microparticles to obtain alkaline phosphatase-responsive hydrogel photonic crystal microspheres.

2. The use of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres according to claim 1 in alkaline phosphatase detection, characterized in that: in, In step 2, hydrofluoric acid is used to etch away the silicon dioxide particles.

3. The use of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres according to claim 1 in alkaline phosphatase detection, characterized in that: in, In step three, the fluorescent probe is selected from one of fluorescein probes, inorganic ion fluorescent probes, fluorescent quantum dots, and molecular beacons.

4. The use of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres according to claim 1 in alkaline phosphatase detection, characterized in that: in, In step 3, the salt solution containing copper ions is selected from one of aqueous solutions of copper chloride, copper sulfate, copper nitrate, copper carbonate, and basic copper carbonate.

5. The use of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres according to claim 1 in alkaline phosphatase detection, characterized in that: in, In step 3, the concentration of the sodium alginate solution is 1wt%; The volume ratio of fluorescent probe to sodium alginate solution was 1:1; The concentration of the salt solution with copper ions is 2 mM; The volume ratio of the sodium alginate solution to the salt solution containing copper ions is 1:

2.

6. Use of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres according to claim 1 in alkaline phosphatase detection, characterized in that: in, The quantitative detection method of alkaline phosphatase comprises the following steps: Step 1: Thoroughly mix a pyrophosphate solution with an alkaline phosphatase solution of different concentration gradients, where the concentration of the alkaline phosphatase solution is 0 to 400 mU / mL; incubate at 37°C for 30 minutes; then add the alkaline phosphatase-responsive hydrogel photonic crystal microspheres to the mixed solution, shake at 150 rpm at room temperature, and react for 2 hours; remove the alkaline phosphatase-responsive hydrogel photonic crystal microspheres, and measure the fluorescence and reflectance spectra of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres; plot the relationship between the detected fluorescence intensity and reflectance peak shift value and the concentration of the alkaline phosphatase solution into a standard curve; Step 2: Thoroughly mix the pyrophosphate solution with the test solution containing alkaline phosphatase and incubate at 37°C for 30 minutes; then add the alkaline phosphatase-responsive hydrogel photonic crystal microspheres for detection to the mixed solution, shake at 150 rpm at room temperature, and react for 2 hours; remove the alkaline phosphatase-responsive hydrogel photonic crystal microspheres for detection, detect the fluorescence intensity and reflection peak shift value of the alkaline phosphatase-responsive hydrogel photonic crystal microspheres for detection, and then substitute them into the standard curve to finally calculate the content of alkaline phosphatase in the test solution; The concentration of the pyrophosphate solution in step 2 is equal to that of the pyrophosphate solution in step 1 and is less than 10 mM.

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