A sensor based on photonic crystal, preparation method and application

Abstract

The present invention belongs to the technical fields of medical materials and biomedical engineering, and particularly relates to a sensor based on photonic crystals, a preparation method and an application thereof. The biosensor of the present invention comprises a recognition element for retinoic acid and a polysaccharide-titanium dioxide hydrogel sensing element, which can specifically recognize and bind to the PML-RARA fusion protein, causing the reflection spectrum to shift, resulting in a change in the color of the photonic crystal, achieving the purpose of visual detection of acute promyelocytic leukemia, enabling real-time detection and real-time diagnosis, and having the advantages of simple operation and fast detection process. The novel detection method provided by the present invention conforms to the concept of green environmental protection advocated by modern times, has low cost and is green and environmentally friendly.

Description

Technical Field

[0001] The present invention belongs to the technical fields of medical materials and biomedical engineering, and particularly relates to a sensor based on photonic crystals, a preparation method and an application thereof. Background Art

[0002] Acute promyelocytic leukemia is a subtype of acute myeloid leukemia and is designated as M3 type of acute myeloid leukemia by the FAB cooperative group. The clinical symptoms of M3 are similar to those of other leukemias, including: anemia, abnormal increase or decrease in the number of white blood cells, lymph node enlargement, bone pain, and bleeding. In addition, M3 also has its special features. Compared with other types of leukemia, M3 has a higher bleeding risk and a greater early death risk. Patients with M3 can have thrombocytopenia at the early stage of onset and are accompanied by abnormal coagulation factors. The fibrinogen in the patient's body is reduced, and the coagulation function is impaired, making it extremely easy to have massive gastrointestinal bleeding or intracranial bleeding. According to statistics, about 20% of M3 patients will die from early bleeding. Secondly, the incidence of disseminated intravascular coagulation (DIC) in acute promyelocytic leukemia is extremely high, and approximately 60% of patients will have DIC in the early stage. Once DIC occurs, the patient has a great chance of death. The early progression of acute promyelocytic leukemia is very rapid. If not detected and treated in time, it will rapidly progress and deteriorate to DIC in a short time, resulting in the death of the patient. Therefore, it is extremely important to make a diagnosis quickly in the early stage.

[0003] The immunophenotype of M3 is the same as that of the rest of myeloid leukemia. Its uniqueness lies in the PML-RARA fusion gene generated by gene mutation. Almost all M3 patients have the expression of this fusion gene. The PML-RARA fusion protein produced by the PML-RARA fusion gene is the cause of the abnormal coagulation function in M3. Due to its specificity, this gene can be used as a marker for the diagnosis of M3. The PML end of this fusion protein can specifically recognize and bind to retinoic acid, which is also the basic principle for retinoic acid to treat M3. Currently, the diagnosis of M3 mainly relies on flow cytometry, FISH, and bone marrow morphology examination. Although flow cytometry and FISH have been widely used in the diagnosis of leukemia and are approaching maturity, they still have certain limitations:

[0004] 1. High requirements for specimen quality. First of all, both flow cytometry and FISH require the best specimen to be bone marrow fluid. Bone marrow puncture is relatively cumbersome and has a relatively high risk compared to venous puncture. Secondly, flow cytometry and FISH must ensure the freshness of the specimen to prevent hemolysis or agglutination of bone marrow fluid. If cell agglutination or massive cell death occurs in the bone marrow fluid specimen, it must be discarded.

[0005] 2. High cost. The addition of antibodies is crucial in flow cytometry, and the quality of the added antibodies can have a significant impact on the final results. High-quality antibodies mean high costs. At the same time, machines such as flow cytometers are expensive, and many hospitals cannot afford them.

[0006] 3. High time cost. The disease progression of M3 is usually relatively rapid. It only takes about ten days from the onset to the advanced stage of the disease. If it cannot be diagnosed and treated in time, the treatment opportunity will be delayed, resulting in immeasurable consequences.

[0007] 4. High requirements for technical personnel. Whether it is flow cytometry or FISH, systematic and professional training is required to master them. It is very difficult to perform relevant operations without systematic training. Due to the influence of the subjective consciousness of the examiner, bone marrow morphology examination can only be used as an auxiliary for the diagnosis of leukemia and cannot be used as a standard for diagnosis.

[0008] Photonic crystals are a new type of dielectric material, which are crystal materials with photonic band gaps formed by the periodic distribution of dielectric media with different dielectric constants in space. It is similar to the semiconductor structure. By assembling two dielectric materials with different dielectric constants in a certain periodic change, due to the periodic arrangement of dielectric materials with different dielectric constants, a certain "potential field" will be generated. This "potential field" will cause Bragg scattering of light waves at the dielectric interface and generate a photonic band gap. The light falling at the band gap cannot be propagated, and this photonic band gap is called the photonic forbidden band. The photonic forbidden band will shift due to changes in volume and internal structure. The reflection spectrum will also change with the movement of the photonic forbidden band. According to the dimension of the photonic forbidden band of the photonic crystal in space, it can be divided into one-dimensional photonic crystals, two-dimensional photonic crystals, and three-dimensional photonic crystals. One-dimensional photonic crystals are the simplest in structure among photonic crystals. One-dimensional photonic crystals have a broad prospect and high research value because of their diverse preparation materials, simple preparation methods, low cost, and excellent optical properties, and they have good sensitivity to temperature, PH, and molecules of different volumes. Summary of the Invention

[0009] Overcoming the deficiencies of the prior art, the present invention provides a biosensor based on photonic crystals and a preparation method, which have certain advantages in detecting acute promyelocytic leukemia.

[0010] Specifically, the technical solution of the present invention is as follows:

[0011] The first object of the present invention is to provide a sensor based on photonic crystals, including:

[0012] (1) A silicon wafer substrate;

[0013] (2) A sensing element: polysaccharide-titanium dioxide hydrogel;

[0014] (3) Recognition element: Specific drug encapsulated in nanocapsules.

[0015] Furthermore, the polysaccharide is chitosan and / or sodium alginate, preferably chitosan.

[0016] Furthermore, the specific drug is one of tretinoin, arsenic trioxide, and arsenous acid, preferably tretinoin.

[0017] Specifically, the photonic crystal-based sensor includes:

[0018] (1) Silicon wafer substrate;

[0019] (2) Sensing element: Chitosan-titanium dioxide hydrogel;

[0020] (3) Recognition element: Tretinoin encapsulated in nanocapsules.

[0021] The second object of the present invention is to provide a method for preparing the above-mentioned photonic crystal-based sensor, including the following steps:

[0022] (1) Preparation of silicon wafer substrate: Cut the silicon wafer into 1 cm 3 square pieces, and soak them in piranha solution for 15 - 24 h, take them out, wash, and dry for later use;

[0023] (2) Preparation of sensing element: Dissolve tetrabutyl titanate and glacial acetic acid in absolute ethanol, seal, and continuously stir at room temperature for 5 h to form titanium dioxide hydrogel. Quickly add an appropriate amount of diluted surfactant, add the polysaccharide aqueous solution, and mix well to obtain polysaccharide-titanium dioxide hydrogel;

[0024] (3) Preparation of recognition element: Dissolve the specific drug in absolute ethanol, and encapsulate the dissolved specific drug with nanocapsules to form a solution of specific drug encapsulated in nanocapsules;

[0025] (4) Assembly of the sensor: First, spin-coat the polysaccharide-titanium dioxide hydrogel evenly on the surface of the silicon wafer in step (1), let it stand and dry, then spin-coat a layer of the solution of specific drug encapsulated in nanocapsules evenly, let it stand and dry in the dark. Repeat the above assembly steps 4 - 7 times to obtain the photonic crystal sensor.

[0026] Furthermore, step (3) specifically includes the following steps:

[0027] a. Wash the SiO2 particles of 300 - 500 nm with pure water, centrifuge, soak in PAA solution for 5 - 10 min, centrifuge at 500 - 1500 rpm, soak in bPEI solution for 5 - 10 min, centrifuge at 500 - 1500 rpm, and repeat the above operations 2 times. Obtain SiO2-(PAA / bPEI);

[0028] b. Immerse SiO2-(PAA / bPEI) in an HPF6 solution for 20 - 30 min, centrifuge at 2000 - 5000 rmp, wash and centrifuge 2 - 3 times to obtain hollow nanocapsules;

[0029] c. Dissolve the specific drug in absolute ethanol to make a specific drug - ethanol solution with a concentration of 0.5 - 2 mg / ml. Freeze the aqueous solution of nanocapsules in a refrigerator for 1 - 2 h to make it freeze, quickly place the frozen nanocapsules in a vacuum freeze dryer and freeze - dry for 15 - 24 h. Dissolve the completely dried nanocapsules in the specific drug - ethanol solution and store them in a refrigerator in the dark for 15 - 24 h to make specific drugs encapsulated by nanocapsules.

[0030] Furthermore, the surfactant is 8% 2 - undecyl - N - carboxymethyl - N - hydroxyethyl imidazoline (w / v) and 5% poloxamer (w / v).

[0031] Specifically, the volume ratio of 8% 2 - undecyl - N - carboxymethyl - N - hydroxyethyl imidazoline (w / v) to 5% poloxamer (w / v) is 1 - 5:1, preferably 3:1.

[0032] Furthermore, the HPF6 solution is 2% - 10% (w / v), preferably 5% (w / v). A large number of experimental studies have shown that hexafluorophosphoric acid has a strong ability to remove SiO2 particles, and the effect is better than that of hydrofluoric acid. However, due to the strong corrosiveness of hexafluorophosphoric acid, if a high - concentration hexafluorophosphoric acid is used when preparing the recognition element coating, it will cause certain corrosion to the silicon wafer substrate, affecting the sensitivity of the sensing response. If the concentration of hexafluorophosphoric acid is too low, the SiO2 particles cannot be completely removed, and the SiO2 particles invade the inside of the nanocapsules, hindering the loading of specific drugs and reducing the specificity. In addition, the concentration of HPF6 also has a great impact on the stability of retinoic acid nanocapsules. Therefore, the selection of the concentration of hexafluorophosphoric acid is crucial.

[0033] Preferably, the method for preparing the above - mentioned photonic - crystal - based sensor is as follows:

[0034] (1) Preparation of the silicon wafer substrate: Cut the silicon wafer into square pieces about 1 cm 3 in size, and immerse them in a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:7 for 15 - 24 h, rinse the surface of the concentrated sulfuric acid with pure water, wash repeatedly with absolute ethanol 2 - 3 times, and dry with nitrogen;

[0035] (2) Preparation of sensing element: Take 4 ml of tetrabutyl titanate and dissolve it in 16 ml of absolute ethanol, and slowly add 4 ml of glacial acetic acid dropwise thereto. Seal the bottle mouth and stir magnetically at room temperature for 5 h to obtain a TiO2 colloidal solution. Add 9 ml of 8% 2-undecyl-N-carboxymethyl-N-hydroxyethyl imidazoline (w / v) and 3 ml of 5% poloxamer (w / v) to the TiO2 colloidal solution, and add 40 - 48 ml of chitosan solution (CS) to prepare chitosan-titanium dioxide hydrogel (CS-TiO2);

[0036] (3) Preparation of recognition element:

[0037] a. Using pure water as a solvent, prepare 8% polyacrylic acid (PAA) solution and 15% branched polyethylenimine (bPEI) solution. Wash SiO2 particles with a size of 300 - 500 nm with pure water, centrifuge to obtain clean SiO2 cores. Immerse the SiO2 cores in an aqueous polyacrylic acid PAA solution for 5 - 10 min, centrifuge at 500 - 1500 rpm, then immerse in an aqueous branched polyethylenimine bPEI solution for 5 - 10 min, centrifuge at 500 - 1500 rpm, and repeat the above operations 2 times to obtain SiO2-(PAA / bPEI);

[0038] b. Immerse the obtained SiO2-(PAA / bPEI) in a 5% HPF6 solution for 20 - 30 min, centrifuge at 2000 - 5000 rmp, then wash and centrifuge 2 - 3 times to obtain hollow nanocapsules;

[0039] c. Dissolve tretinoin in absolute ethanol to prepare a 0.5 - 2 mg / ml tretinoin ethanol solution. Place the nanocapsule aqueous solution in the refrigerator and freeze it for 1 - 2 h to make it freeze, and quickly place the frozen nanocapsules in a vacuum freeze dryer and freeze-dry for 15 - 24 h. Then dissolve the completely dried nanocapsules in the above tretinoin ethanol solution, and store them in the refrigerator in the dark for 15 - 24 h to prepare Nanocapsules-Va;

[0040] (4) Assembly of the sensor: Using the spin coating method, first spin coat the CS-TiO2 gel evenly on the surface of the silicon wafer in step (1), let it stand and dry, then spin coat a layer of Nanocapsules-Va evenly, let it stand and dry in the dark. The spin coating speed for each layer is 2000 - 4000 rmp, and the spin coating time is 15 - 30 s. Take one layer of CS-TiO2 gel and one layer of Nanocapsules-Va as a spin coating cycle, and spin coat 4 - 7 cycles to prepare a tretinoin-one-dimensional photonic crystal film encapsulated by nanocapsules.

[0041] The third object of the present invention is to provide the application of the sensor of the photonic crystal in the detection of acute promyelocytic leukemia.

[0042] The fourth object of the present invention is to provide an application of the sensor of the photonic crystal in detecting PML-RARA fusion protein.

[0043] Based on the fact that one-dimensional photonic crystals have very good sensitivity to molecular volume, the present invention utilizes this characteristic of one-dimensional photonic crystals to prepare a one-dimensional photonic crystal thin film that can sensitively sense and bind to a specific substance, resulting in a change in its own volume, for detecting the PML-RARA fusion protein, which is a specific diagnostic marker in acute promyelocytic leukemia.

[0044] The detection mechanism is specifically as follows: The PML-RARA fusion protein can specifically recognize and bind to retinoic acid. The photonic crystal sensor provided by the present invention includes a recognition element containing retinoic acid and a polysaccharide-titanium dioxide hydrogel sensing element. This sensor can specifically recognize and bind to the PML-RARA fusion protein, thereby detecting whether the specific diagnostic marker of promyelocytic leukemia - the PML-RARA fusion protein exists, and determining whether a person has promyelocytic leukemia.

[0045] Since retinoic acid is extremely unstable under light, high temperature, and acidic conditions and is prone to decomposition and loss of its original structure, it is encapsulated with nanocapsule retinoic acid to avoid contact with light, high temperature, acid, etc. Then, the nanocapsule-encapsulated retinoic acid (Nanocapsules-Va) is assembled with CS-TiO2 to form an integral whole, which we call the "fusion protein sensor". CS-TiO2 serves as the conduction element of the "sensor", while Nanocapsules-Va serves as the recognition element. The recognition element can accurately recognize the target protein and bind tightly to it. The binding of the protein to the recognition element directly causes changes in the internal structure and volume of the photonic crystal. The conduction element can sensitively sense the change in the internal structure, ultimately resulting in a shift in the photonic band gap inside the photonic crystal and a change in the reflection spectrum, achieving accurate detection with high sensitivity and good specificity.

[0046] The fifth object of the present invention is to provide a kit for detecting acute promyelocytic leukemia, which comprises the above-mentioned photonic crystal sensor. When in use, a blood sample is dropped onto the biosensing membrane in the kit. If the sensing membrane changes color, it proves that there is a specific diagnostic marker for promyelocytic leukemia in the blood sample, namely the PML-RARA fusion protein, and it is determined that the patient has promyelocytic leukemia; if it does not change color, it means that there is no specific diagnostic marker for promyelocytic leukemia, namely the PML-RARA fusion protein, in the blood sample, and it is determined that the patient does not have promyelocytic leukemia. This kit has the advantages of simple operation and fast detection process. Compared with the complex flow cytometry and cell gene immunofluorescence hybridization techniques, the preparation and operation of the present invention are very simple, do not require professional personnel, and do not require systematic learning. The configuration of the test laboratories in general hospitals can meet the standards for preparation and detection. The present invention can also achieve real-time detection and real-time diagnosis without long waiting. For patients, the diagnosis can be clearly made in time and treatment can be carried out in time, effectively avoiding the regret of delaying the disease due to untimely diagnosis and missing the best treatment opportunity.

[0047] The present invention provides a new detection method, which is very in line with the concept of green environmental protection advocated by modern society. Its raw materials are simple and the cost is low. No toxic substances will be produced during the preparation process, which is green and environmentally friendly. Compared with traditional detection methods such as flow cytometry and FISH, photonic crystals do not require expensive detection machines or expensive kits. Its basic raw materials, tetrabutyl titanate, ethanol, acetic acid, and chitosan, are all low-cost raw materials, greatly reducing the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Photonic crystals of different colors prepared (from left to right are the photonic crystal thin film sensors prepared in Example 4, Example 1, Example 5, and Example 6)

[0049] Figure 2 Spectrum change and color change diagram of visual detection of acute promyelocytic leukemia by the sensor based on one-dimensional photonic crystal prepared in Example 1

[0050] Figure 3 Comprehensive performance evaluation of the photonic crystal sensor DETAILED DESCRIPTION OF THE INVENTION

[0051] In order to make the objects and technical solutions of the present invention clearer, the following will further explain the present invention with reference to the embodiments. However, the protection scope of the present invention is not limited to these embodiments, and the embodiments are only used to explain the present invention. Those skilled in the art should understand that any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0052] Preparation Method of CS-TiO2 / Nanocapsules-Va One-Dimensional Photonic Crystal Thin Film Sensor

[0053] (1) Preparation of silicon wafer substrate: Cut the silicon wafer into square pieces about 1 cm, soak them in a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:7 for 18 h, rinse the surface of concentrated sulfuric acid with pure water, wash repeatedly with absolute ethanol 3 times, and dry with nitrogen; 3 Soak them in a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:7 for 18 h, rinse the surface of concentrated sulfuric acid with pure water, wash repeatedly with absolute ethanol 3 times, and dry with nitrogen;

[0054] (2) Preparation of sensing element: Take 4 ml of tetrabutyl titanate and dissolve it in 16 ml of absolute ethanol, and slowly drop 4 ml of glacial acetic acid into it. Seal the bottle mouth and stir magnetically at room temperature for 5 h to obtain TiO2 colloidal solution. Add 9 ml of 8% 2-undecyl-N-carboxymethyl-N-hydroxyethyl imidazoline (w / v) and 3 ml of 5% poloxamer (w / v) to the TiO2 colloidal solution, and add 45 ml of chitosan solution (CS) to make chitosan-titanium dioxide hydrogel (CS-TiO2);

[0055] (3) Preparation of recognition element:

[0056] a. Using pure water as the solvent, prepare 8% polyacrylic acid (PAA) solution and 15% branched polyethyleneimine (bPEI) solution. Wash SiO2 particles with a size of 300 - 500 nm with pure water and centrifuge to obtain clean SiO2 cores. Soak the SiO2 cores in an aqueous solution of polyacrylic acid PAA for 8 min, centrifuge at 1000 rpm, then soak in an aqueous solution of branched polyethyleneimine bPEI for 8 min, centrifuge at 1000 rpm, and repeat the above operations 2 times to obtain SiO2-(PAA / bPEI);

[0057] b. Soak the obtained SiO2-(PAA / bPEI) in 5% HPF6 solution for 25 min, centrifuge at 3500 rmp, then wash and centrifuge 3 times to obtain hollow nanocapsules;

[0058] c. Dissolve tretinoin in absolute ethanol to make a 1.2 mg / ml tretinoin ethanol solution. Put the aqueous solution of nanocapsules into the refrigerator and freeze it for 1.5 h to make it freeze, quickly place the frozen nanocapsules in a vacuum freeze dryer and freeze-dry for 18 h. Then dissolve the completely dried nanocapsules in the above tretinoin ethanol solution, and store them in the refrigerator in the dark for 18 h to make Nanocapsules-Va;

[0059] (4) Assembly of the sensor: Using the spin-coating method, first spin-coat the CS-TiO2 gel evenly on the surface of the silicon wafer in step (1), let it stand for drying, then spin-coat a layer of Nanocapsules-Va evenly, and let it stand for drying in the dark. The spin-coating speed for each layer is 3000 rmp, and the spin-coating time is 20 s. Taking one layer of CS-TiO2 gel and one layer of Nanocapsules-Va as a spin-coating cycle, spin-coat 6 cycles to prepare a retinoic acid one-dimensional photonic crystal thin film sensor encapsulated by nanocapsules.

[0060] Example 2 Preparation method of CS-TiO2 / Nanocapsules-Va one-dimensional photonic crystal thin film sensor

[0061] (1) Preparation of the silicon wafer substrate: Cut the silicon wafer into square pieces about 1 cm 3 in size, and immerse them in a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:7 for 15 h, rinse the surface of the concentrated sulfuric acid with pure water, wash repeatedly with absolute ethanol 2 times, and dry with nitrogen;

[0062] (2) Preparation of the sensing element: Take 4 ml of tetrabutyl titanate and dissolve it in 16 ml of absolute ethanol, and slowly drop 4 ml of glacial acetic acid into it. Seal the bottle mouth and stir magnetically at room temperature for 5 h to obtain a TiO2 colloidal solution. Add 5 ml of 8% 2-undecyl-N-carboxymethyl-N-hydroxyethyl imidazoline (w / v) and 5 ml of 5% poloxamer (w / v) to the TiO2 colloidal solution, and add 40 ml of chitosan solution (CS) to make chitosan-titanium dioxide hydrogel (CS-TiO2);

[0063] (3) Preparation of the recognition element:

[0064] a. Using pure water as the solvent, prepare 8% polyacrylic acid (PAA) solution and 15% polyethylenimine (bPEI) solution. Wash the SiO2 particles with a size of 300 - 500 nm with pure water and centrifuge to obtain clean SiO2 cores. Immerse the SiO2 cores in the polyacrylic acid PAA aqueous solution for 5 min, centrifuge at 500 rpm, then immerse them in the polyethylenimine bPEI aqueous solution for 5 min, and centrifuge at 500 rpm. Repeat the above operations 2 times to obtain SiO2-(PAA / bPEI);

[0065] b. Immerse the obtained SiO2-(PAA / bPEI) in 2% HPF6 solution for 20 min, centrifuge at 2000 rmp, then wash and centrifuge 2 times to obtain hollow nanocapsules;

[0066] c. Dissolve tretinoin in absolute ethanol to prepare a 0.5 mg / ml tretinoin ethanol solution. Place the aqueous solution of nanocapsules in the refrigerator and freeze it for 1 h to make it freeze. Quickly place the frozen nanocapsules in a vacuum freeze dryer and freeze-dry them for 15 h. Then dissolve the completely dried nanocapsules in the above-mentioned tretinoin ethanol solution and store them in the refrigerator in the dark for 15 h to prepare Nanocapsules-Va;

[0067] (4) Assemble the sensor: Using the spin-coating method, first spin-coat the CS-TiO2 gel evenly on the surface of the silicon wafer in step (1), let it stand and dry, then spin-coat a layer of Nanocapsules-Va evenly, let it stand and dry in the dark. The spin-coating speed for each layer is 2000 - 4000 rmp, and the spin-coating time is 15 s. Take one layer of CS-TiO2 gel and one layer of Nanocapsules-Va as a spin-coating cycle, and spin-coat 4 cycles to prepare a tretinoin one-dimensional photonic crystal thin film sensor encapsulated by nanocapsules.

[0068] Example 3 Preparation method of CS-TiO2 / Nanocapsules-Va one-dimensional photonic crystal thin film sensor

[0069] (1) Preparation of silicon wafer substrate: Cut the silicon wafer into square pieces about 1 cm 3 in size, and immerse them in a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:7 for 24 h. Rinse the surface of the concentrated sulfuric acid with pure water, wash it repeatedly with absolute ethanol 2 - 3 times, and dry it with nitrogen;

[0070] (2) Preparation of sensing element: Take 4 ml of tetrabutyl titanate and dissolve it in 16 ml of absolute ethanol, and slowly drop 4 ml of glacial acetic acid into it. Seal the bottle mouth and stir magnetically at room temperature for 5 h to obtain a TiO2 colloidal solution. Add 10 ml of 8% 2-undecyl-N-carboxymethyl-N-hydroxyethyl imidazoline (w / v) and 1 ml of 5% poloxamer (w / v) to the TiO2 colloidal solution, and add 48 ml of chitosan solution (CS) to make chitosan-titanium dioxide hydrogel (CS-TiO2);

[0071] (3) Preparation of recognition element:

[0072] a. Using pure water as a solvent, prepare an 8% polyacrylic acid (PAA) solution and a 15% branched polyethyleneimine (bPEI) solution. Wash the SiO2 particles with a size of 300 - 500 nm with pure water and centrifuge them to obtain clean SiO2 cores. Immerse the SiO2 cores in an aqueous solution of polyacrylic acid PAA for 10 min, centrifuge at 1500 rpm, then immerse them in an aqueous solution of branched polyethyleneimine bPEI for 10 min, centrifuge at 1500 rpm, and repeat the above operations 2 times to obtain SiO2-(PAA / bPEI);

[0073] b. Immerse the obtained SiO2-(PAA / bPEI) in a 10% HPF6 solution for 30 min, centrifuge at 5000 rmp, then wash and centrifuge 3 times to obtain hollow nanocapsules;

[0074] c. Dissolve tretinoin in absolute ethanol to prepare a 2 mg / ml tretinoin ethanol solution. Place the nanocapsule aqueous solution in the refrigerator and freeze it for 2 h to make it freeze. Quickly place the frozen nanocapsules in a vacuum freeze dryer and freeze-dry for 24 h. Then dissolve the completely dried nanocapsules in the above tretinoin ethanol solution and store them in the refrigerator in the dark for 24 h to prepare Nanocapsules-Va;

[0075] (4) Assemble the sensor: Using the spin-coating method, first spin-coat the CS-TiO2 gel evenly on the surface of the silicon wafer in step (1), let it stand and dry, then spin-coat a layer of Nanocapsules-Va evenly, let it stand and dry in the dark. The spin-coating speed for each layer is 4000 rmp, and the spin-coating time is 30 s. Taking one layer of CS-TiO2 gel and one layer of Nanocapsules-Va as a spin-coating cycle, spin-coat 7 cycles to prepare a tretinoin-one-dimensional photonic crystal thin film sensor encapsulated by nanocapsules.

[0076] Example 4 Preparation method of sodium alginate-TiO2 / Nanocapsules-Va one-dimensional photonic crystal thin film sensor

[0077] (1) Preparation of silicon wafer substrate: Cut the silicon wafer into square pieces about 1 cm 3 in size, and immerse them in a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:7 for 18 h. Rinse the surface of the concentrated sulfuric acid with pure water, wash it repeatedly with absolute ethanol 3 times, and dry it with nitrogen;

[0078] (2) Preparation of sensing element: Take 4 ml of tetrabutyl titanate and dissolve it in 16 ml of absolute ethanol, and slowly drop 4 ml of glacial acetic acid into it. Seal the bottle mouth and stir magnetically at room temperature for 5 h to obtain a TiO2 colloidal solution. Add 9 ml of 8% 2-undecyl-N-carboxymethyl-N-hydroxyethyl imidazoline (w / v) and 3 ml of 5% poloxamer (w / v) to the TiO2 colloidal solution, and add 45 ml of sodium alginate solution to make a sodium alginate-titanium dioxide hydrogel;

[0079] (3) Preparation of recognition element:

[0080] a. Using pure water as the solvent, prepare an 8% polyacrylic acid (PAA) solution and a 15% branched polyethyleneimine (bPEI) solution. Wash SiO2 particles with a size of 300 - 500 nm with pure water and centrifuge to obtain clean SiO2 cores. Immerse the SiO2 cores in the aqueous PAA solution for 8 min, centrifuge at 1000 rpm, then immerse in the aqueous bPEI solution for 8 min and centrifuge at 1000 rpm. Repeat the above operations twice to obtain SiO2-(PAA / bPEI);

[0081] b. Immerse the obtained SiO2-(PAA / bPEI) in a 5% HPF6 solution for 25 min, centrifuge at 3500 rmp, then wash and centrifuge 3 times to obtain hollow nanocapsules;

[0082] c. Dissolve tretinoin in absolute ethanol to make a 1.2 mg / ml tretinoin ethanol solution. Place the aqueous nanocapsule solution in the refrigerator and freeze it for 1.5 h to make it freeze. Quickly place the frozen nanocapsules in a vacuum freeze dryer and freeze-dry for 18 h. Then dissolve the completely dried nanocapsules in the above tretinoin ethanol solution and store them in the refrigerator in the dark for 18 h to make Nanocapsules-Va;

[0083] (4) Assemble the sensor: Using the spin-coating method, first spin-coat the sodium alginate-TiO2 gel evenly on the surface of the silicon wafer in step (1), let it stand and dry, then spin-coat a layer of Nanocapsules-Va evenly, let it stand and dry in the dark. The spin-coating speed for each layer is 3000 rmp and the spin-coating time is 20 s. Take a layer of sodium alginate-TiO2 gel and a layer of Nanocapsules-Va as a spin-coating cycle, and spin-coat 6 cycles to prepare a tretinoin one-dimensional photonic crystal thin film sensor encapsulated by nanocapsules.

[0084] Example 5 Preparation method of CS-TiO2 / Nanocapsules-As2O3 one-dimensional photonic crystal thin film sensor

[0085] (1) Preparation of silicon wafer substrate: Cut the silicon wafer into square pieces about 1 cm 3 in size, and immerse them in a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:7 for 18 h. Rinse the surface of the concentrated sulfuric acid with pure water, wash repeatedly with absolute ethanol 3 times, and dry with nitrogen;

[0086] (2) Preparation of sensing element: Take 4 ml of tetrabutyl titanate and dissolve it in 16 ml of absolute ethanol, and slowly add 4 ml of glacial acetic acid dropwise thereto. Seal the bottle mouth and stir magnetically at room temperature for 5 h to obtain TiO2 colloidal solution. Add 9 ml of 8% 2-undecyl-N-carboxymethyl-N-hydroxyethyl imidazoline (w / v) and 3 ml of 5% poloxamer (w / v) to the TiO2 colloidal solution, and add 45 ml of chitosan solution (CS) to prepare chitosan-titanium dioxide hydrogel (CS-TiO2);

[0087] (3) Preparation of recognition element:

[0088] a. Using pure water as a solvent, prepare 8% polyacrylic acid (PAA) solution and 15% branched polyethyleneimine (bPEI) solution. Wash SiO2 particles with a size of 300 - 500 nm with pure water and centrifuge to obtain clean SiO2 cores. Immerse the SiO2 cores in an aqueous solution of polyacrylic acid PAA for 8 min, centrifuge at 1000 rpm, then immerse in an aqueous solution of branched polyethyleneimine bPEI for 8 min, centrifuge at 1000 rpm, and repeat the above operations 2 times to obtain SiO2-(PAA / bPEI);

[0089] b. Immerse the obtained SiO2-(PAA / bPEI) in 5% HPF6 solution for 25 min, centrifuge at 3500 rmp, then wash and centrifuge 3 times to obtain hollow nanocapsules;

[0090] c. Dissolve As2O3 in absolute ethanol to prepare a 1.2 mg / ml As2O3 ethanol solution. Place the aqueous solution of nanocapsules in the refrigerator and freeze it for 1.5 h to make it freeze, and quickly place the frozen nanocapsules in a vacuum freeze dryer and freeze-dry for 18 h. Then dissolve the completely dried nanocapsules in the above As2O3 ethanol solution and store them in the refrigerator in the dark for 18 h to prepare Nanocapsules-As2O3;

[0091] (4) Assembly of sensor: Using the spin coating method, first spin coat the CS-TiO2 gel evenly on the surface of the silicon wafer in step (1), let it stand and dry, then spin coat a layer of Nanocapsules-As2O3 evenly, let it stand and dry in the dark. The spin coating speed for each layer is 3000 rmp, and the spin coating time is 20 s. Taking one layer of CS-TiO2 gel and one layer of Nanocapsules-As2O3 as a spin coating cycle, spin coat 6 cycles to prepare a one-dimensional photonic crystal film sensor of As2O3 encapsulated by nanocapsules.

[0092] Example 6 Preparation method of CS-TiO2 / Nanocapsules-H3AsO3 one-dimensional photonic crystal film sensor

[0093] (1) Preparation of silicon wafer substrate: Cut the silicon wafer into 1 cm3 About square pieces, soaked in a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:7 for 18 h, rinsed with pure water to remove the concentrated sulfuric acid on the surface, washed repeatedly with absolute ethanol 3 times, and dried with nitrogen;

[0094] (2) Preparation of sensing element: Dissolve 4 ml of tetrabutyl titanate in 16 ml of absolute ethanol, and slowly add 4 ml of glacial acetic acid dropwise thereto. Seal the bottle mouth and stir magnetically at room temperature for 5 h to obtain a TiO2 colloidal solution. Add 9 ml of 8% 2-undecyl-N-carboxymethyl-N-hydroxyethyl imidazoline (w / v) and 3 ml of 5% poloxamer (w / v) to the TiO2 colloidal solution, and add 45 ml of chitosan solution (CS) to prepare chitosan-titanium dioxide hydrogel (CS-TiO2);

[0095] (3) Preparation of recognition element:

[0096] a. Using pure water as a solvent, prepare 8% polyacrylic acid (PAA) solution and 15% polyethylenimine (bPEI) solution. Wash SiO2 particles with a size of 300 - 500 nm with pure water, centrifuge to obtain clean SiO2 cores, soak the SiO2 cores in an aqueous solution of polyacrylic acid PAA for 8 min, centrifuge at 1000 rpm, then soak in an aqueous solution of polyethylenimine bPEI for 8 min, centrifuge at 1000 rpm, and repeat the above operations 2 times to obtain SiO2-(PAA / bPEI);

[0097] b. Soak the obtained SiO2-(PAA / bPEI) in 5% HPF6 solution for 25 min, centrifuge at 3500 rmp, then wash and centrifuge 3 times to obtain hollow nanocapsules;

[0098] c. Dissolve H3AsO3 in absolute ethanol to prepare a 1.2 mg / ml H3AsO3 ethanol solution. Place the aqueous solution of nanocapsules in the refrigerator and freeze it for 1.5 h to make it freeze, quickly place the frozen nanocapsules in a vacuum freeze dryer and freeze-dry for 18 h. Then dissolve the completely dried nanocapsules in the above H3AsO3 ethanol solution, store it in the refrigerator in the dark for 18 h to prepare Nanocapsules-H3AsO3;

[0099] (4) Assembly of the sensor: Using the spin coating method, first spin coat the CS-TiO2 gel evenly on the surface of the silicon wafer in step (1), let it stand and dry, then spin coat a layer of Nanocapsules-H3AsO3 evenly, let it stand and dry in the dark. The spin coating speed for each layer is 3000 rmp, and the spin coating time is 20 s. Taking one layer of CS-TiO2 gel and one layer of Nanocapsules-H3AsO3 as a spin coating cycle, spin coat 6 cycles to prepare a nanocapsule-encapsulated tretinoin one-dimensional photonic crystal film sensor.

[0100] Preparation Method of CS-TiO2 / Nanocapsules-Va One-Dimensional Photonic Crystal Film Sensor in Comparative Example 1

[0101] (1) Preparation of silicon wafer substrate: Cut the silicon wafer into square pieces about 1 cm 3 in size, and immerse them in a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:7 for 18 h. Rinse the surface of the concentrated sulfuric acid with pure water, wash it repeatedly with absolute ethanol 3 times, and dry it with nitrogen;

[0102] (2) Preparation of sensing element: Dissolve 4 ml of tetrabutyl titanate in 16 ml of absolute ethanol, and slowly add 4 ml of glacial acetic acid dropwise. Seal the bottle mouth and stir magnetically at room temperature for 5 h to obtain a TiO2 colloidal solution. Add 9 ml of 8% 2-undecyl-N-carboxymethyl-N-hydroxyethyl imidazoline (w / v) and 3 ml of 5% poloxamer (w / v) to the TiO2 colloidal solution, and add 45 ml of chitosan solution (CS) to prepare chitosan-titanium dioxide hydrogel (CS-TiO2);

[0103] (3) Preparation of recognition element:

[0104] a. Using pure water as a solvent, prepare 8% polyacrylic acid (PAA) solution and 15% branched polyethyleneimine (bPEI) solution. Wash SiO2 particles with a size of 300 - 500 nm with pure water and centrifuge to obtain clean SiO2 cores. Immerse the SiO2 cores in an aqueous polyacrylic acid PAA solution for 8 min, centrifuge at 1000 rpm, then immerse them in an aqueous branched polyethyleneimine bPEI solution for 8 min, and centrifuge at 1000 rpm. Repeat the above operations 2 times to obtain SiO2-(PAA / bPEI);

[0105] b. Immerse the obtained SiO2-(PAA / bPEI) in 5% HF solution for 25 min, centrifuge at 3500 rmp, then wash and centrifuge 3 times to obtain hollow nanocapsules;

[0106] c. Dissolve tretinoin in absolute ethanol to prepare a 1.2 mg / ml tretinoin ethanol solution. Freeze the nanocapsule aqueous solution in the refrigerator for 1.5 h to make it freeze, and quickly place the frozen nanocapsules in a vacuum freeze dryer and freeze-dry for 18 h. Then dissolve the completely dried nanocapsules in the above tretinoin ethanol solution, and store them in the refrigerator in the dark for 18 h to prepare Nanocapsules-Va;

[0107] (4) Assemble the sensor: Using the spin coating method, first spin coat the CS-TiO2 gel evenly on the surface of the silicon wafer in step (1), let it stand for drying, then spin coat a layer of Nanocapsules-Va evenly, let it stand in the dark for drying. The spin coating speed for each layer is 3000 rmp, and the spin coating time is 20 s. Taking one layer of CS-TiO2 gel and one layer of Nanocapsules-Va as a spin coating cycle, spin coat for 6 cycles to prepare a retinoic acid one-dimensional photonic crystal thin film sensor encapsulated with nanocapsules.

[0108] Comparative Example 2 Preparation method of CS-TiO2 / Nanocapsules-Va one-dimensional photonic crystal thin film sensor

[0109] (1) Preparation of silicon wafer substrate: Cut the silicon wafer into square pieces about 1 cm 3 in size, and immerse them in a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:7 for 18 h, rinse the surface of the concentrated sulfuric acid with pure water, wash repeatedly with absolute ethanol 3 times, and dry with nitrogen;

[0110] (2) Preparation of sensing element: Take 4 ml of tetrabutyl titanate and dissolve it in 16 ml of absolute ethanol, and slowly drop 4 ml of glacial acetic acid into it. Seal the bottle mouth and stir magnetically at room temperature for 5 h to obtain a TiO2 colloidal solution. Add 9 ml of 8% 2-undecyl-N-carboxymethyl-N-hydroxyethyl imidazoline (w / v) and 3 ml of 5% poloxamer (w / v) to the TiO2 colloidal solution, and add 45 ml of chitosan solution (CS) to make chitosan-titanium dioxide hydrogel (CS-TiO2);

[0111] (3) Preparation of recognition element:

[0112] a. Using pure water as the solvent, prepare 8% polyacrylic acid (PAA) solution and 15% branched polyethyleneimine (bPEI) solution. Wash the SiO2 particles with a size of 300 - 500 nm with pure water and centrifuge to obtain clean SiO2 cores. Immerse the SiO2 cores in the polyacrylic acid PAA aqueous solution for 8 min, centrifuge at 1000 rpm, then immerse in the branched polyethyleneimine bPEI aqueous solution for 8 min, centrifuge at 1000 rpm, and repeat the above operations 2 times to obtain SiO2-(PAA / bPEI);

[0113] b. Immerse the obtained SiO2-(PAA / bPEI) in 1% HPF6 solution for 25 min, centrifuge at 3500 rmp, then wash and centrifuge 3 times to obtain hollow nanocapsules;

[0114] c. Dissolve tretinoin in absolute ethanol to prepare a tretinoin ethanol solution with a concentration of 1.2 mg / ml. Place the aqueous solution of nanocapsules in a refrigerator and freeze it for 1.5 h until it freezes. Quickly place the frozen nanocapsules in a vacuum freeze dryer and freeze-dry them for 18 h. Then dissolve the completely dried nanocapsules in the above-mentioned tretinoin ethanol solution and store them in a refrigerator protected from light for 18 h to prepare Nanocapsules-Va;

[0115] (4) Assemble the sensor: Using the spin-coating method, first spin-coat the CS-TiO2 gel evenly on the surface of the silicon wafer in step (1), let it stand and dry, then spin-coat a layer of Nanocapsules-Va evenly, let it stand and dry in the dark. The spin-coating speed for each layer is 3000 rmp, and the spin-coating time is 20 s. Take one layer of CS-TiO2 gel and one layer of Nanocapsules-Va as a spin-coating cycle, and spin-coat 6 cycles to prepare a tretinoin one-dimensional photonic crystal thin film sensor encapsulated by nanocapsules.

[0116] Preparation method of CS-TiO2 / Nanocapsules-Va one-dimensional photonic crystal thin film sensor in Comparative Example 3

[0117] (1) Preparation of silicon wafer substrate: Cut the silicon wafer into square pieces about 1 cm 3 in size, and immerse them in a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:7 for 18 h. Rinse the surface of the concentrated sulfuric acid with pure water, wash it repeatedly with absolute ethanol 3 times, and dry it with nitrogen;

[0118] (2) Preparation of sensing element: Take 4 ml of tetrabutyl titanate and dissolve it in 16 ml of absolute ethanol, and slowly drop 4 ml of glacial acetic acid into it. Seal the bottle mouth and stir magnetically at room temperature for 5 h to obtain a TiO2 colloidal solution. Add 9 ml of 8% 2-undecyl-N-carboxymethyl-N-hydroxyethyl imidazoline (w / v) and 3 ml of 5% poloxamer (w / v) to the TiO2 colloidal solution, and add 45 ml of chitosan solution (CS) to prepare chitosan-titanium dioxide hydrogel (CS-TiO2);

[0119] (3) Preparation of recognition element:

[0120] a. Using pure water as a solvent, prepare an 8% polyacrylic acid (PAA) solution and a 15% branched polyethyleneimine (bPEI) solution. Wash the SiO2 particles with a size of 300 - 500 nm with pure water and centrifuge them to obtain clean SiO2 cores. Immerse the SiO2 cores in an aqueous solution of polyacrylic acid PAA for 8 min, centrifuge at 1000 rpm, then immerse them in an aqueous solution of branched polyethyleneimine bPEI for 8 min, and centrifuge at 1000 rpm. Repeat the above operations 2 times to obtain SiO2-(PAA / bPEI);

[0121] b. Immerse the obtained SiO2-(PAA / bPEI) in a 15% HPF6 solution for 25 min, centrifuge at 3500 rmp, then wash and centrifuge 3 times to obtain hollow nanocapsules;

[0122] c. Dissolve tretinoin in absolute ethanol to prepare a 1.2 mg / ml tretinoin ethanol solution. Place the aqueous solution of nanocapsules in the refrigerator and freeze it for 1.5 h to make it freeze, and quickly place the frozen nanocapsules in a vacuum freeze dryer and freeze-dry for 18 h. Then dissolve the completely dried nanocapsules in the above tretinoin ethanol solution and store them in the refrigerator in the dark for 18 h to prepare Nanocapsules-Va;

[0123] (4) Assemble the sensor: Using the spin-coating method, first spin-coat the CS-TiO2 gel evenly on the surface of the silicon wafer in step (1), let it stand and dry, then spin-coat a layer of Nanocapsules-Va evenly, let it stand and dry in the dark. The spin-coating speed for each layer is 3000 rmp, and the spin-coating time is 20 s. Take a layer of CS-TiO2 gel and a layer of Nanocapsules-Va as a spin-coating cycle, and spin-coat 6 cycles to prepare a tretinoin-one-dimensional photonic crystal film sensor encapsulated by nanocapsules.

[0124] Comparative Example 4 Preparation method of CS-TiO2 / Nanocapsules-Va one-dimensional photonic crystal film sensor

[0125] (1) Preparation of silicon wafer substrate: Cut the silicon wafer into square pieces about 1 cm 3 in size, and immerse them in a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:7 for 18 h, rinse the surface concentrated sulfuric acid with pure water, wash repeatedly with absolute ethanol 3 times, and dry with nitrogen;

[0126] (2) Preparation of sensing element: Take 4 ml of tetrabutyl titanate and dissolve it in 16 ml of absolute ethanol, and slowly drop 4 ml of glacial acetic acid into it. Seal the bottle mouth and stir magnetically at room temperature for 5 h to obtain a TiO2 colloidal solution. Add 12 ml of 5% poloxamer (w / v) to the TiO2 colloidal solution, and add 45 ml of chitosan solution (CS) to make chitosan-titanium dioxide hydrogel (CS-TiO2);

[0127] (3) Preparation of recognition element:

[0128] a. Using pure water as the solvent, prepare an 8% polyacrylic acid (PAA) solution and a 15% branched polyethylenimine (bPEI) solution. Wash SiO2 particles with a size of 300 - 500 nm with pure water and centrifuge to obtain clean SiO2 cores. Immerse the SiO2 cores in the aqueous PAA solution for 8 min, centrifuge at 1000 rpm, then immerse in the aqueous bPEI solution for 8 min and centrifuge at 1000 rpm. Repeat the above operations twice to obtain SiO2-(PAA / bPEI);

[0129] b. Immerse the obtained SiO2-(PAA / bPEI) in a 5% HPF6 solution for 25 min, centrifuge at 3500 rmp, then wash and centrifuge 3 times to obtain hollow nanocapsules;

[0130] c. Dissolve tretinoin in absolute ethanol to prepare a 1.2 mg / ml tretinoin ethanol solution. Place the aqueous nanocapsule solution in the refrigerator and freeze it for 1.5 h to make it freeze. Quickly place the frozen nanocapsules in a vacuum freeze dryer and freeze-dry for 18 h. Then dissolve the completely dried nanocapsules in the above tretinoin ethanol solution and store them in the refrigerator in the dark for 18 h to prepare Nanocapsules-Va;

[0131] (4) Assemble the sensor: Using the spin-coating method, first spin-coat the CS-TiO2 gel evenly on the surface of the silicon wafer in step (1), let it stand and dry, then spin-coat a layer of Nanocapsules-Va evenly, let it stand and dry in the dark. The spin-coating speed for each layer is 3000 rmp and the spin-coating time is 20 s. Take one layer of CS-TiO2 gel and one layer of Nanocapsules-Va as a spin-coating cycle, and spin-coat 6 cycles to prepare a tretinoin one-dimensional photonic crystal thin film sensor encapsulated by nanocapsules.

[0132] Preparation method of hyaluronic acid-TiO2 / Nanocapsules-Va one-dimensional photonic crystal thin film sensor in Comparative Example 5

[0133] (1) Preparation of silicon wafer substrate: Cut the silicon wafer into square pieces about 1 cm 3 in size, and immerse them in a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:7 for 18 h. Rinse the surface of the concentrated sulfuric acid with pure water, wash repeatedly with absolute ethanol 3 times, and dry with nitrogen;

[0134] (2) Preparation of sensing element: Take 4 ml of tetrabutyl titanate and dissolve it in 16 ml of absolute ethanol. Slowly add 4 ml of glacial acetic acid dropwise thereto. Seal the bottle mouth and stir magnetically at room temperature for 5 h to obtain a TiO2 colloidal solution. Add 9 ml of 8% 2-undecyl-N-carboxymethyl-N-hydroxyethyl imidazoline (w / v) and 3 ml of 5% poloxamer (w / v) to the TiO2 colloidal solution, and add 45 ml of hyaluronic acid solution to prepare a hyaluronic acid-titanium dioxide hydrogel;

[0135] (3) Preparation of recognition element:

[0136] a. Using pure water as a solvent, prepare an 8% polyacrylic acid (PAA) solution and a 15% branched polyethylenimine (bPEI) solution. Wash SiO2 particles with a size of 300 - 500 nm with pure water and centrifuge to obtain clean SiO2 cores. Immerse the SiO2 cores in an aqueous polyacrylic acid PAA solution for 8 min, centrifuge at 1000 rpm, then immerse in an aqueous branched polyethylenimine bPEI solution for 8 min, and centrifuge at 1000 rpm. Repeat the above operations twice to obtain SiO2-(PAA / bPEI);

[0137] b. Immerse the obtained SiO2-(PAA / bPEI) in a 5% HPF6 solution for 25 min, centrifuge at 3500 rmp, then wash and centrifuge 3 times to obtain hollow nanocapsules;

[0138] c. Dissolve tretinoin in absolute ethanol to prepare a 1.2 mg / ml tretinoin ethanol solution. Place the aqueous nanocapsule solution in the refrigerator and freeze it for 1.5 h to make it freeze. Quickly place the frozen nanocapsules in a vacuum freeze dryer and freeze-dry for 18 h. Then dissolve the completely dried nanocapsules in the above tretinoin ethanol solution and store them in the refrigerator in the dark for 18 h to prepare Nanocapsules-Va;

[0139] (4) Assembly of sensor: Using the spin coating method, first spin coat the hyaluronic acid-TiO2 gel evenly on the surface of the silicon wafer in step (1), let it stand and dry, then spin coat a layer of Nanocapsules-Va evenly, let it stand and dry in the dark. The spin coating speed for each layer is 3000 rmp, and the spin coating time is 20 s. Using one layer of hyaluronic acid-TiO2 gel and one layer of Nanocapsules-Va as a spin coating cycle, spin coat 6 cycles to prepare a tretinoin one-dimensional photonic crystal film sensor encapsulated with nanocapsules.

[0140] Preparation method of the comparative example 6 CS-TiO2 / Nanocapsules-vesnarinone one-dimensional photonic crystal film sensor

[0141] (1) Preparation of silicon wafer substrate: Cut the silicon wafer into 1 cm 3Squares on the left and right were placed in a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 3:7 and soaked for 18 h. The surface concentrated sulfuric acid was rinsed off with pure water, and then washed repeatedly with absolute ethanol three times and dried with nitrogen;

[0142] (2) Preparation of the sensing element: 4 ml of tetrabutyl titanate was dissolved in 16 ml of absolute ethanol, and 4 ml of glacial acetic acid was slowly added dropwise thereto. The bottle mouth was sealed and magnetically stirred at room temperature for 5 h to obtain a TiO2 colloidal solution. 9 ml of 8% 2-undecyl-N-carboxymethyl-N-hydroxyethyl imidazoline (w / v) and 3 ml of 5% poloxamer (w / v) were added to the TiO2 colloidal solution, and 45 ml of chitosan solution (CS) was added to prepare chitosan-titanium dioxide hydrogel (CS-TiO2);

[0143] (3) Preparation of the recognition element:

[0144] a. Using pure water as the solvent, 8% polyacrylic acid (PAA) solution and 15% branched polyethyleneimine (bPEI) solution were prepared. SiO2 particles with a size of 300 - 500 nm were washed with pure water and centrifuged to obtain clean SiO2 cores. The SiO2 cores were soaked in an aqueous solution of polyacrylic acid PAA for 8 min, centrifuged at 1000 rpm, then soaked in an aqueous solution of branched polyethyleneimine bPEI for 8 min, and centrifuged at 1000 rpm. The above operations were repeated twice to obtain SiO2-(PAA / bPEI);

[0145] b. The obtained SiO2-(PAA / bPEI) was soaked in a 5% HPF6 solution for 25 min and centrifuged at 3500 rmp, then washed and centrifuged three times to obtain hollow nanocapsules;

[0146] c. Vesnarinone was dissolved in absolute ethanol to prepare a 1.2 mg / ml vesnarinone ethanol solution. The aqueous solution of nanocapsules was placed in a refrigerator and frozen for 1.5 h to freeze it, and the frozen nanocapsules were quickly placed in a vacuum freeze dryer and freeze-dried for 18 h. Then the completely dried nanocapsules were dissolved in the above vesnarinone ethanol solution and stored in a refrigerator in the dark for 18 h to prepare Nanocapsules-vesnarinone;

[0147] (4) Assembly of the sensor: Using the spin coating method, first, the CS-TiO2 gel was evenly spin-coated on the surface of the silicon wafer in step (1), allowed to stand and dry, and then a layer of Nanocapsules-vesnarinone was evenly spin-coated, allowed to stand and dry in the dark. The spin coating speed for each layer was 3000 rmp, and the spin coating time was 20 s. Taking one layer of CS-TiO2 gel and one layer of Nanocapsules-vesnarinone as a spin coating cycle, spin coating was performed for 6 cycles to prepare a vesnarinone one-dimensional photonic crystal thin film sensor encapsulated with nanocapsules.

[0148] Verification example

[0149] I. Comprehensive Performance Evaluation

[0150] 1. Scratch Resistance

[0151] Insert the photonic crystal thin film sensors prepared in Examples 1 - 6 and Comparative Examples 1 - 6 into fine sand grains 30 times respectively, observe the number of scratches under a microscope, and evaluate their scratch resistance according to the scoring criteria in Table 1.

[0152] Table 1 Scratch Resistance Scoring Criteria

[0153] Number of scratches Score 0-4 5-6 5-9 4-5 11-14 3-4 15-19 2-3 ≥20 1-2

[0154] 2. Antibacterial Ability

[0155] Place the photonic crystal thin film sensors prepared in Examples 1 - 6 and Comparative Examples 1 - 6 in an environment with a temperature of 40°C and a relative humidity of 75% ± 5% at the 0th month and the 6th month, detect Staphylococcus aureus on the surface of the thin film sensors of each example, and score according to the criteria in Table 2.

[0156] Table 2 Antibacterial Performance Scoring Criteria

[0157] Antibacterial ability Score ≥99.9％ 5-6 98.0％-99.9％ 4-5 96.0％-97.9％ 3-4 94.0％-95.9％ 2-3 ＜94.0％ 1-2

[0158] 3. Impact Resistance

[0159] Conduct a notched impact test on the photonic crystal thin film sensors prepared in Examples 1 - 6 and Comparative Examples 1 - 6. Accurately place the sample on the loading platform of the testing machine, ensure that the axial direction of the pendulum is consistent with the center line of the notch, start the testing machine to make the pendulum fall, impact the specimen, and record the impact absorption work A kv . Score according to the criteria in Table 3.

[0160] Table 3 Impact Resistance Scoring Criteria

[0161] <![CDATA[A kv > Score ≥70 5-6 60-69 4-5 50-59 3-4 40-49 2-3 ＜40 1-2

[0162] 4. Uniformity Evaluation

[0163] Divide the 1 - cm square pieces of the photonic crystal thin film sensors prepared in Examples 1 - 6 and Comparative Examples 1 - 6 into 9 columns on average, measure the thickness of the thin film sensors in each column respectively, calculate the average value and the standard deviation, calculate the RSD, and score according to the criteria in Table 4. 3

[0164] Table 4 Uniformity Scoring Criteria

[0165]

[0166]

[0167] ​5. Evaluation of membrane / substrate bonding strength

[0168] Using the acoustic emission method, a hard needle with a known tip radius (usually a diamond cone indenter) is placed vertically on the film surface, and the vertical load is gradually increased and moved along the film surface until the film is scratched. The load that can just scratch the film is called the critical load and is used as a measure of the film / substrate bonding strength, and is scored according to the standards in Table 5.

[0169] Table 5 Membrane / substrate bonding strength scoring criteria

[0170] <![CDATA[Critical detachment load L C Normal Force (N) at Score ≥4.0 5-6 3.0-4.0 4-5 2.0-3.0 3-4 1.0-2.0 2-3 ＜1.0 1-2

[0171] The above five performance tests were performed on the photonic crystal thin film sensors prepared in Examples 1-6 and Comparative Examples 1-6 to characterize their comprehensive performance. Figure 3 It is shown that the film / substrate bonding strength, uniformity, impact resistance, antibacterial ability and scratch resistance of the photonic crystal thin film sensor prepared in the embodiment of the present invention are better than those of the photonic crystal thin film sensor prepared in the comparative embodiment.

[0172] 2. Test of stability of retinoic acid

[0173] Since retinoic acid is extremely unstable under light, high temperature, and acidic conditions and is easily decomposed and loses its original structure, the retinoic acid is wrapped in nanocapsules to prevent it from being exposed to light, high temperature, acid, etc. Therefore, in this experiment, the retinoic acid nanocapsules prepared in step (3) were placed at a temperature of 40±2°C and a relative humidity of 75%±5% for 6 months, and samples were taken at the end of the first, second, third, and sixth months of the test period to determine the retinoic acid content.

[0174] The determination was carried out by high performance liquid chromatography in a light-proof manner.

[0175] The specific method is:

[0176] Take about 10 mg of this product, accurately weigh it, place it in a 100 ml brown volumetric flask, add 10 ml of isopropanol to dissolve it, dilute it to the scale with methanol, shake it well, accurately measure 5 ml, place it in a 50 ml brown volumetric flask, dilute it to the scale with methanol, shake it well, and use it as the test solution.

[0177] Take about 10 mg of retinoic acid reference substance, weigh it accurately, put it in a 100 ml brown volumetric flask, add 10 ml of isopropanol to dissolve it, dilute it to the scale with methanol, shake it well, accurately measure 5 ml, put it in a 50 ml brown volumetric flask, dilute it to the scale with methanol, shake it well, and use it as the reference substance solution.

[0178] Weigh appropriate amounts of tretinoin reference substance and isotretinoin reference substance respectively, dissolve them with a small amount of isopropanol, and dilute with methanol to prepare a mixed solution containing about 10 μg of each per 1 ml as the system suitability solution.

[0179] Chromatographic conditions: Octadecylsilane chemically bonded silica gel is used as the filler; methanol - 2% glacial acetic acid solution (81:19) is used as the mobile phase; the detection wavelength is 350 nm; the injection volume is 20 μl.

[0180] Assay method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate by the external standard method based on the peak area.

[0181] Table 6 Content of tretinoin nanocapsules in the examples and comparative examples (%)

[0182]

[0183] Table 6 shows that in the preparation scheme of the examples of the present invention, the tretinoin nanocapsules have high stability, are not easily decomposed, and do not lose their original structure.

[0184] III. Application of the photonic crystal sensor of the present invention in the detection of acute promyelocytic leukemia

[0185] Taking the sensor prepared in Example 1 as an example, Figure 2 It shows that the CS-TiO2 / Nanocapsules-Va one-dimensional photonic crystal thin film sensor of Example 1 can specifically recognize and bind to the PML-RARA fusion protein. The recognition element Nanocapsules-Va can accurately recognize the target PML-RARA fusion protein and tightly bind to it, directly resulting in changes in the internal structure and volume of the photonic crystal. The conduction element CS-TiO2 can sensitively sense the change in the internal structure, ultimately leading to a shift in the photonic band gap inside the photonic crystal and a change in the reflection spectrum, achieving accurate detection with high sensitivity and good specificity. Based on this principle, it is used to detect whether the specific diagnostic marker of acute promyelocytic leukemia - the PML-RARA fusion protein exists, and to determine whether a patient has acute promyelocytic leukemia.

[0186] The CS-TiO2 / Nanocapsules-vesnarinone one-dimensional photonic crystal thin film sensors prepared in Comparative Examples 5 and 6 have no response to the PML-RARA fusion protein.

[0187] IV. Kit for detecting acute promyelocytic leukemia

[0188] The kit for detecting acute promyelocytic leukemia contains the one-dimensional photonic crystal sensor prepared in Example 1, Example 2, Example 3 or Example 4. Taking the one-dimensional photonic crystal sensor prepared in Example 1 as an example, when in use, a blood sample is dropped onto the biosensing membrane in the kit, and the sensing membrane changes color (such as the display of the color change in Figure 2 ), which proves the existence of the specific diagnostic marker of promyelocytic leukemia, PML-RARA fusion protein, in the blood sample, and it is determined that the patient has promyelocytic leukemia. This kit has the advantages of simple operation, fast detection process and high visualization degree.

Claims

1. A method for preparing a photonic crystal-based sensor, characterized in that, It includes the following steps: (1)Silicon wafer substrate preparation: Cut the silicon wafer into square pieces about 1 cm 3 in size, and immerse them in piranha solution for 15 - 24 h, then take them out, wash and dry for standby; (2) Preparation of the sensing element: Dissolve tetrabutyl titanate and glacial acetic acid in absolute ethanol, seal it, and continuously stir for 4 - 6 h at room temperature to prepare titanium dioxide hydrogel. Quickly add an appropriate amount of diluted surfactant, add an aqueous polysaccharide solution, and mix well to obtain a polysaccharide - titanium dioxide hydrogel; (3) Preparation of the recognition element: Dissolve the specific drug in absolute ethanol, and wrap the dissolved specific drug with nanocapsules to prepare a solution of specific drug encapsulated by nanocapsules; (4) Assembly of the sensor: First, spin - coat the polysaccharide - titanium dioxide hydrogel evenly on the surface of the silicon wafer in step (1), let it stand and dry. Then, spin - coat a layer of the solution of specific drug encapsulated by nanocapsules evenly, let it stand and dry in the dark. Take one cycle of spin - coating a layer of polysaccharide - titanium dioxide hydrogel and a layer of the solution of specific drug encapsulated by nanocapsules, and repeat the spin - coating 4 - 7 cycles to obtain the photonic crystal sensor; The said step (3) includes: a. Wash SiO2 particles with a size of 300 - 500 nm with pure water, centrifuge them, soak them in PAA solution for 5 - 10 min, centrifuge at 500 - 1500 rpm, soak them in bPEI solution for 5 - 10 min, centrifuge at 500 - 1500 rpm, and repeat the above operations 2 times to obtain SiO2-(PAA / bPEI); b. Soak SiO2-(PAA / bPEI) in an HPF6 solution with a concentration of 2% - 10% (w / v) for 20 - 30 min, centrifuge at 2000 - 5000 rmp, wash and centrifuge 2 - 3 times to obtain hollow nanocapsules; c. Dissolve the specific drug in absolute ethanol to prepare a specific drug - ethanol solution with a concentration of 0.5 - 2 mg / ml. Freeze the aqueous nanocapsule solution in the refrigerator for 1 - 2 h to make it freeze, quickly place the frozen nanocapsules in a vacuum freeze - dryer and freeze - dry for 15 - 24 h. Dissolve the completely dried nanocapsules in the specific drug - ethanol solution, and store it in the refrigerator in the dark for 15 - 24 h to prepare a solution of specific drug encapsulated by nanocapsules; The said surfactant is 8% (w / v) 2 - undecyl - N - carboxymethyl - N - hydroxyethyl imidazoline and 5% (w / v) poloxamer; The said polysaccharide is chitosan or sodium alginate.

2. The method according to claim 1, characterized in that The volume ratio of 8% (w / v) 2 - undecyl - N - carboxymethyl - N - hydroxyethyl imidazoline to 5% (w / v) poloxamer is 1 - 5:

1.

3. The method according to claim 2, characterized in that, The volume ratio of 8% (w / v) 2 - undecyl - N - carboxymethyl - N - hydroxyethyl imidazoline to 5% (w / v) poloxamer is 3:

1.

4. The method according to claim 1, wherein The concentration of the said HPF6 solution is 5% (w / v).

5. The method according to claim 1, wherein The said specific drug is one of tretinoin, arsenic trioxide, and arsenous acid.

6. The method according to claim 5, characterized in that, The said specific drug is tretinoin.

7. A sensor based on photonic crystal, characterized in that, Prepared by the method for preparing a sensor based on photonic crystals described in claim 1.

8. A kit for detecting acute promyelocytic leukemia, characterized in that, A sensor comprising the photonic crystal described in claim 7.

Images