Preparation method and application of core-shell structure co-assembled photonic crystal bar code

Abstract

The invention provides a preparation method of a core-shell structure co-assembled photonic crystal bar code. The preparation method comprises the following steps: step 1, preparing a carboxylated single-walled carbon nanotube and silicon dioxide mixed solution; 2, preparing a co-assembly photonic crystal bar code; step 3, preparing a core-shell structure co-assembled photonic crystal bar code; and step 4, preparing the aptamer modified core-shell structure co-assembled photonic crystal bar code. The aptamer-modified core-shell structure co-assembled photonic crystal bar code prepared by the invention can be used for simultaneously detecting various myocardial injury markers, such as myoglobin, cardiac troponin I and B-type natriuretic peptide, in a serum sample of a patient in a tiny sample. The prepared co-assembled bar code has stable coding capability and anti-interference capability, can realize accurate identification and capture of a target in a complex serum environment, and is quantified through fluorescence intensity. The method is reasonable in design, low in cost, easy to operate and high in practicability, and a novel reference method is provided for biological detection.

Description

Technical field:

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a preparation method and application of a core-shell structure co-assembled photonic crystal barcode. The barcode can be used in the technical field of biomedical detection. Background technology:

[0002] Acute myocardial infarction, also known as acute myocardial ischemic necrosis, is caused by a sharp reduction or interruption of coronary artery blood supply, resulting in local necrosis of the corresponding myocardium due to persistent ischemia. It is a common cardiovascular disease with a high mortality rate in the clinical field. The identification of biomarkers is an effective method for diagnosing acute myocardial infarction. It can not only detect the occurrence and development of the disease, but also evaluate and track the therapeutic effect of drugs. Studies have reported that during myocardial overload, some protein biomarkers related to acute myocardial infarction are released into the patient's serum and plasma, such as cardiac troponin I and T, cardiac fatty acid binding protein, myoglobin, B-type natriuretic peptide, and C-reactive protein. Among them, myoglobin, cardiac troponin I and B-type natriuretic peptide are important indicators for clinical detection. Commonly used detection methods include enzyme-linked immunosorbent assay, electrochemiluminescence, and surface plasmon resonance. These methods are costly and time-consuming and can only detect one marker at a time. Therefore, a composite high-throughput diagnostic platform is needed to simultaneously detect multiple cardiovascular biomarkers to improve the accuracy and effectiveness of early diagnosis of acute myocardial infarction.

[0003] Multiplexed assays have been used for the detection and quantification of multiple analytes in the fields of clinical diagnosis, gene expression, drug discovery, etc. Most assays are based on the specific recognition or binding of molecules. In order to distinguish different molecules simultaneously, the molecules should be encoded. The most common and versatile methods are planar arrays and suspension arrays. Compared with planar arrays, suspension arrays show the characteristics of high flexibility, fast response speed, and good detection repeatability, and are therefore more widely used. Suspension arrays use coded microspheres as information carriers that specifically bind to analytes. These coded microcarriers are classified based on different encoding technologies, including optical encoding, graphic encoding, magnetic encoding, and shape encoding. In optical encoding, structural color encoding based on photonic crystals has attracted increasing attention. Photonic crystals based on long-range ordered nanoarrays produce photonic band gap effects, and characteristic reflection peaks can be used as multiple encoding elements, which have the advantages of coding stability and no fluorescence interference. At present, photonic crystals have been widely used in wastewater detection, metal ion detection, cancer cell detection, and biomarker detection.

[0004] Therefore, the present invention provides a method for preparing a core-shell structure co-assembled photonic crystal barcode, combining silica photonic crystals and carboxylated single-walled carbon nanotubes together for simultaneous detection of myocardial injury markers myoglobin (Myo), cardiac troponin I (cTnI) and B-type natriuretic peptide (BNP). In order to overcome the problem of silica itself lacking active groups, we creatively combine it with carboxylated single-walled carbon nanotubes by co-assembly. This will not only not affect the reflection peak position and encoding function of the photonic crystal, but also fix the carboxyl group on the silica. The interconnected porous hydrogel shell can enhance the mechanical strength and hydrophilicity of the microcarrier while providing abundant surface area for the functionalization of the aptamer. Summary of the invention:

[0005] The purpose of the present invention is to provide a method for preparing a core-shell structure co-assembled photonic crystal barcode and its application in view of the deficiencies in the prior art.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] (I) The present invention provides a method for preparing a core-shell structure co-assembled photonic crystal barcode, comprising the following steps:

[0008] Step 1: Prepare a mixed solution of carboxylated single-walled carbon nanotubes and silicon dioxide:

[0009] A certain amount of carboxylated single-walled carbon nanotubes is dissolved in anhydrous ethanol, and the carboxylated single-walled carbon nanotubes are uniformly dispersed in the anhydrous ethanol solution by ultrasonication for 2 hours; a certain volume of distilled water is added to the silica nanoparticle solution to obtain a silica nanoparticle dispersion; and the two are mixed to prepare a mixed solution;

[0010] Step 2: preparing a co-assembled photonic crystal barcode: using a droplet template method to prepare the mixed solution into a co-assembled photonic crystal barcode;

[0011] Step 3: Prepare a core-shell co-assembled photonic crystal barcode: immerse the dried co-assembled photonic crystal barcode in a pre-gel solution for half an hour, then irradiate under ultraviolet light to solidify the hydrogel, and then remove the silica positive structure template by etching to obtain a core-shell co-assembled photonic crystal barcode;

[0012] Step 4: Prepare aptamer-modified core-shell co-assembled photonic crystal barcodes: put the core-shell co-assembled photonic crystal barcodes into activation solution for activation, wash them, add aptamer solution and incubate them at 4°C overnight to obtain aptamer-modified core-shell co-assembled photonic crystal barcodes.

[0013] Furthermore, in step 1, the concentration of carboxylated single-walled carbon nanotubes in the carboxylated single-walled carbon nanotube solution is 0.6 mg / mL, and the concentration of silica nanoparticles in the silica nanoparticle dispersion is 20 w / v%; the mixing volume ratio of the two solutions is 1:1.

[0014] Furthermore, in step 2, the droplet template method is as follows: on a microfluidic chip, the mixed liquid and methyl silicone oil (50 cSt) are set as dispersed phase and continuous phase respectively. The two fluids are respectively injected into the microfluidic device through two independent peristaltic pumps, and the inner phase solution is cut into small droplets by the shear force of the outer phase fluid, and collected in a container filled with high viscosity (500 cSt) methyl silicone oil; the collected droplets are dried in an oven overnight; after drying, the residual methyl silicone oil on the surface of the microspheres is thoroughly cleaned with n-hexane, and calcined in a muffle furnace to further improve its mechanical strength.

[0015] Furthermore, in step three, the pre-gel solution is a polyethylene glycol diacrylate aqueous solution, and the pre-gel solution also includes a photoinitiator.

[0016] Furthermore, in step three, in the pre-gel solution, the concentration of polyethylene glycol diacrylate is 20wt%, and the concentration of the photoinitiator is 1v / v%; the photoinitiator is 2-hydroxy-2-methyl-1-phenyl-1-propanone.

[0017] Furthermore, in step three, the etching method is soaking in 4v / v% hydrofluoric acid or 4wt% sodium hydroxide.

[0018] Furthermore, in step 4, the activation solution is a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide; and the activation time is half an hour.

[0019] Furthermore, in step 4, in the aptamer solution, the aptamer concentration is 60 μM; the aptamers are myoglobin (Myo), cardiac troponin I (cTnI) and B-type natriuretic peptide (BNP).

[0020] Furthermore, in order to meet the needs of multiple detection, three kinds of silica nanoparticles with reflection peaks of 660 nm, 563 nm and 457 nm were used to prepare co-assembled photonic crystal barcodes.

[0021] (II) The present invention also provides a core-shell structure co-assembled photonic crystal barcode, which is prepared by the preparation method described above.

[0022] (III) The present invention also provides the application of the core-shell structure co-assembled photonic crystal barcode prepared by the above-mentioned preparation method in the field of biomedical detection technology.

[0023] Beneficial effects of the present invention:

[0024] (1) The present invention combines carboxylated single-walled carbon nanotubes with silica photonic crystals by co-assembly, thereby solving the problem that silica itself lacks active groups. It not only does not affect the reflection peak position and encoding function of the photonic crystal, but also can fix the carboxyl groups on silica.

[0025] (2) The interconnected porous hydrogel shell not only enhances the mechanical strength and hydrophilicity of the microcarrier, but also provides abundant surface area for the functionalization of aptamers.

[0026] (3) Core-shell co-assembled photonic crystal barcodes can simultaneously detect multiple biomarkers in trace samples, showing great application potential in clinical testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the preparation process of the core-shell structure co-assembled photonic crystal barcode of the present invention.

[0028] Figure 2 This is a scanning electron microscope image of the core-shell structure co-assembled photonic crystal barcode prepared in Example 1, wherein a is the overall morphology of the co-assembled photonic crystal barcode, b is the surface nanostructure of the co-assembled photonic crystal barcode, c is the structure of the hydrogel shell, and d is the morphology of the single-walled carbon nanotubes inside the barcode.

[0029] Figure 3 The bright field, fluorescence images and quantitative analysis diagrams of the single biomarker detection obtained by simultaneously detecting multiple myocardial injury markers in Example 4, wherein in Figure a, Ⅰ is the optical image after incubation of the barcode with Myo, Ⅱ is the optical image after incubation of the barcode with cTnI, Ⅲ is the optical image after incubation of the barcode with BNP, Ⅳ is the fluorescence image after incubation of the barcode with Myo, Ⅴ is the fluorescence image after incubation of the barcode with cTnI, Ⅵ is the fluorescence image after incubation of the barcode with BNP; Figure b is a quantitative analysis of the results of Figure a.

[0030] Figure 4 The bright field, fluorescence images and quantitative analysis diagrams of the simultaneous detection of multiple biomarkers obtained by simultaneously detecting multiple myocardial injury markers in Example 4, in Figure a, Ⅰ is the optical image after the barcode is incubated with Myo and cTnI, Ⅱ is the optical image after the barcode is incubated with cTnI and BNP, Ⅲ is the optical image after the barcode is incubated with Myo and BNP, Ⅳ is the optical image after the barcode is incubated with Myo, cTnI and BNP, Ⅴ is the fluorescence image after the barcode is incubated with Myo and cTnI, Ⅵ is the fluorescence image after the barcode is incubated with cTnI and BNP, Ⅶ is the fluorescence image after the barcode is incubated with Myo and BNP, and VIII is the fluorescence image after the barcode is incubated with Myo, cTnI and BNP; Figure b is a quantitative analysis of the results of Figure a. Specific implementation method:

[0031] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly and completely described below in conjunction with the relevant drawings. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The implementation conditions not specified in the embodiments are usually the conditions in routine experiments.

[0032] Example 1

[0033] This embodiment provides a core-shell structure co-assembled photonic crystal barcode, and the preparation method includes the following steps:

[0034] (1) Preparing a mixed solution of carboxylated single-walled carbon nanotubes and silicon dioxide:

[0035] 6 mg of carboxylated single-walled carbon nanotubes were dissolved in 10 mL of anhydrous ethanol, and the carboxylated single-walled carbon nanotubes were uniformly dispersed in the anhydrous ethanol solution by ultrasonication for 2 hours. A certain volume of distilled water was added to the silica nanoparticle solution (reflection peak at 660 nm) to make its concentration 20%. Then, the two were mixed in equal volumes to prepare a mixed solution.

[0036] (2) Preparation of co-assembled photonic crystal barcodes:

[0037] On the microfluidic chip, the mixed liquid and methyl silicone oil (50cSt) were set as the dispersed phase and the continuous phase, respectively. The two fluids were injected into the microfluidic chip by two independent peristaltic pumps, and the corresponding flow rates were set to 2.0mL / h and 3.0mL / h. The inner phase solution (dispersed phase) was cut into small droplets by the shear force of the continuous phase fluid and collected in a disposable lunch box filled with high viscosity (500cSt) methyl silicone oil. The collected droplets were dried in an oven overnight. After drying, the residual methyl silicone oil on the surface of the microspheres was thoroughly cleaned with n-hexane, and calcined at 800℃ in a muffle furnace for 4 hours to further improve its mechanical strength.

[0038] (3) Preparation of core-shell structure co-assembled photonic crystal barcode:

[0039] 200 μL of polyethylene glycol diacrylate was dissolved in 1 mL of deionized water, and then 10 μL of 2-hydroxy-2-methyl-1-phenyl-1-propanone was added as a photoinitiator to prepare a hydrogel prepolymer solution. The prepolymer solution was added to the dried co-assembled photonic crystal barcode and infiltrated for 0.5 hours, and then irradiated under a UV lamp for ten seconds to solidify the hydrogel. After removing the excess hydrogel on the surface, the surface was etched with 4% HF to remove the silica positive structure template to obtain a core-shell structure microcarrier encapsulated by a porous hydrogel. Scanning electron microscopy showed that Figure 2 As shown, the core-shell co-assembled photonic crystal barcode has good sphericity ( Figure 2 a), consisting of a core of tightly packed silica nanoparticles ( Figure 2 b) and porous hydrogel shell ( Figure 2 c), carboxylated single-walled carbon nanotubes co-assembled around silica nanoparticles ( Figure 2 d).

[0040] (4) Preparation of aptamer-modified core-shell structure co-assembled photonic crystal barcode:

[0041] The core-shell structure co-assembled photonic crystal barcode was placed in an activation solution composed of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (the concentrations of the two were 35 mg / mL and 55 mg / mL, respectively) for activation for half an hour. After washing, 10 μL of 60 μM myoglobin (Myo) aptamer solution was added and incubated overnight in a 4°C refrigerator to obtain the aptamer-modified core-shell structure co-assembled photonic crystal barcode.

[0042] Example 2

[0043] This embodiment provides a core-shell structure co-assembled photonic crystal barcode, and its preparation process is the same as that of Example 1, except that: this embodiment uses silica nanoparticles with a reflection peak of 563nm, and the aptamer in the aptamer solution is cardiac troponin I (cTnI).

[0044] Example 3

[0045] This embodiment provides a core-shell structure co-assembled photonic crystal barcode, and its preparation process is the same as that of Example 1, except that: this embodiment uses silica nanoparticles with a reflection peak of 457nm, and the aptamer in the aptamer solution is B-type natriuretic peptide (BNP).

[0046] Example 4

[0047] Taking the core-shell structure co-assembled photonic crystal barcodes prepared in Examples 1 to 3 as an example, the effect of the core-shell structure co-assembled photonic crystal barcodes in detecting one or more myocardial injury markers was characterized as follows:

[0048] (1) Single biomarker detection:

[0049] The core-shell structure co-assembled photonic crystal barcode prepared in Examples 1 to 3 was placed in an activation solution composed of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide for activation for half an hour, and then washed and added to three test tubes, 10 μL of 60 μM Myo, cTnI and BNP aptamer solutions were added to the three test tubes, and incubated overnight in a 4°C refrigerator to obtain three aptamer-modified core-shell structure co-assembled photonic crystal barcodes. The excess carboxyl groups were blocked with 10 μL of 10% bovine serum albumin solution, and then one of the proteins was added and incubated at 37°C for 2 hours. Finally, 10 μL of 10 μM FAM-labeled aptamer was added, and the corresponding bright field and fluorescence images were taken after incubation at 37°C for 20 minutes, and the results were statistically analyzed.

[0050] The statistical results are shown in Figure 3 ,from Figure 3 The results show that in mixed samples, the core-shell structure co-assembled photonic crystal barcode modified by aptamers can accurately identify and capture the target protein without interference from other proteins, with a high level of specificity and anti-interference ability. After each step of the reaction, the barcode was washed with PBS buffer.

[0051] (2) Simultaneous detection of multiple biomarkers:

[0052] The core-shell structure co-assembled photonic crystal barcode was activated in an activation solution composed of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide for half an hour. After washing, 10 μL of 60 μM Myo, cTnI and BNP aptamer solutions were added to three test tubes respectively, and incubated overnight in a 4°C refrigerator to obtain aptamer-modified core-shell structure co-assembled photonic crystal barcodes. The excess carboxyl groups were blocked with 10 μL of 10% bovine serum albumin solution, and then two or three of the proteins were added and incubated at 37°C for 2 hours. Finally, 10 μL of 10 μM FAM-labeled aptamer was added, and the corresponding bright field and fluorescence images were taken after incubation at 37°C for 20 minutes, and the results were statistically analyzed.

[0053] The statistical results are shown in Figure 4 ,from Figure 4 The results show that the aptamer-modified core-shell structure co-assembled photonic crystal barcode can simultaneously detect two or three target proteins. After each step of the reaction, the barcode is washed with PBS buffer.

[0054] The above are only preferred implementations of the present invention. The protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a core-shell structure co-assembled photonic crystal barcode, characterized in that: The following steps are involved: Step 1: Prepare a mixed solution of carboxylated single-walled carbon nanotubes and silicon dioxide: preparing carboxylated single-walled carbon nanotubes and silicon dioxide nanoparticles into solutions respectively, and then mixing the two into a mixed solution; Step 2: Preparation of co-assembled photonic crystal barcodes: The mixed solution was prepared into a co-assembled photonic crystal barcode using a droplet template method; Step 3: Prepare core-shell structure to co-assemble photonic crystal barcode: The dried co-assembled photonic crystal barcode is immersed in a pre-gel solution and solidified, and the silicon dioxide positive structure template is removed by etching to obtain a core-shell structure co-assembled photonic crystal barcode; Step 4: Preparation of aptamer-modified core-shell structure co-assembled photonic crystal barcode: The core-shell co-assembled photonic crystal barcode was placed in an activation solution for activation, and then an aptamer solution was added for overnight incubation at 4°C to obtain an aptamer-modified core-shell co-assembled photonic crystal barcode.

2. The method for preparing a core-shell structure co-assembled photonic crystal barcode according to claim 1, characterized in that: In step 1, the concentration of carboxylated single-walled carbon nanotubes in the carboxylated single-walled carbon nanotube solution is 0.6 mg / mL, and the concentration of silica nanoparticles in the silica nanoparticle solution is 20 w / v%; The mixing volume ratio of the two solutions was 1:

1.

3. The method for preparing a core-shell structure co-assembled photonic crystal barcode according to claim 1, characterized in that: In step 2, the droplet template method uses the mixed liquid as the dispersed phase and the silicone oil as the continuous phase. The dispersed phase is cut into small droplets by the shear force of the fluid and collected in a container. After cleaning, the droplets are calcined to obtain a co-assembled photonic crystal barcode.

4. The method for preparing a core-shell structure co-assembled photonic crystal barcode according to claim 1, characterized in that: In step three, the pre-gel solution is a polyethylene glycol diacrylate aqueous solution, and the pre-gel solution also includes a photoinitiator.

5. The method for preparing a core-shell structure co-assembled photonic crystal barcode according to claim 4, characterized in that: In step three, in the pre-gel solution, the concentration of polyethylene glycol diacrylate is 20wt%, and the concentration of the photoinitiator is 1v / v%; the photoinitiator is 2-hydroxy-2-methyl-1-phenyl-1-propanone.

6. The method for preparing a core-shell structure co-assembled photonic crystal barcode according to claim 1, characterized in that: In step three, the etching method is to immerse in 4v / v% hydrofluoric acid or 4wt% sodium hydroxide.

7. The method for preparing a core-shell structure co-assembled photonic crystal barcode according to claim 1, characterized in that: In step 4, the activation solution is a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide; and the activation time is half an hour.

8. The method for preparing a core-shell structure co-assembled photonic crystal barcode according to claim 1, characterized in that: In step 4, in the aptamer solution, the aptamer concentration is 60 μM; the aptamers are myoglobin, cardiac troponin I and B-type natriuretic peptide.

9. The core-shell structure co-assembled photonic crystal barcode prepared by the preparation method according to any one of claims 1 to 9.

10. Application of the core-shell structure co-assembled photonic crystal barcode prepared by the preparation method according to any one of claims 1 to 9 in the field of biomedical detection technology.

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