Quantum dot magnetic fluorescent encoding microspheres and preparation method thereof
By assembling daughter spheres in multiple layers on the surface of the mother sphere, the problems of limited coding capacity and insufficient fluorescence uniformity of existing fluorescently encoded microspheres are solved, realizing quantum dot magnetic fluorescently encoded microspheres with high coding capacity and good fluorescence intensity, which are suitable for the detection of multiple biological indicators.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HANGZHOU JOINSTAR BIOMEDICAL TECHNOLOGY CO LTD
- Filing Date
- 2023-12-25
- Publication Date
- 2026-06-26
AI Technical Summary
Existing fluorescently encoded microspheres suffer from limited encoding capacity, insufficient fluorescence intensity and uniformity, especially when multiple quantum dots are mixed for encoding, which is prone to fluorescence resonance energy interference and decreased encoding accuracy.
The method of assembling sub-spheres on the surface of a mother sphere is adopted. Each sub-sphere contains quantum dots of different wavelengths and is connected by chemical covalent bonds formed by the reaction of carboxyl and amino groups. The sub-spheres are synthesized using mesoporous or solid silica nanospheres as templates, and then coated with silica after loading quantum dots on their surface to form a multi-layered quantum dot magnetic fluorescently encoded microsphere.
It improves coding capacity and fluorescence intensity, reduces fluorescence variation coefficient, achieves high coding capacity and good coding reproducibility, and is suitable for mass production.
Smart Images
Figure CN117903786B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biological multi-index detection, and in particular to a quantum dot magnetic fluorescently encoded microsphere and its preparation method. Background Technology
[0002] Liquid-phase chip technology is a multiplex detection technique for detecting multiple indicators in a single sample. It offers advantages such as high throughput, small sample volume, simple operation, high sensitivity, wide detection range, good repeatability, high flexibility, and low cost. Protein and nucleic acid detection can be performed on a single platform. Liquid-phase chip technology primarily uses coded microspheres as carriers; therefore, the performance of the coded microspheres significantly affects the detection performance of the liquid-phase chip. This requires the coded microspheres to meet requirements such as uniform size, stable and uniform fluorescence, easy surface functionalization, and clear clustering.
[0003] Traditional fluorescently encoded microspheres primarily employ a swelling method. Taking Luminex as an example, this method involves incorporating two or three different fluorescent dyes into polystyrene-encoded microspheres through swelling, thereby achieving fluorescent encoding. However, dye-based encoding has several drawbacks, such as the broad emission spectrum of dyes limiting their encoding capabilities; unavoidable cross-interference between dye-encoded signals and between the encoded signals and the quantitative labeling signals, necessitating complex and cumbersome fluorescence compensation; and the small Stokes shift of dyes, with each dye having its optimal excitation wavelength, typically requiring different excitation light.
[0004] In comparison, using quantum dots for encoding avoids the defects of organic dyes. However, if multiple quantum dots are mixed for encoding simultaneously, it may cause fluorescence resonance energy differences between quantum dots of different wavelengths, affecting encoding accuracy. Therefore, in previous experiments, the inventors used assembled spheres formed by connecting nanospheres (daughter spheres) to the surface of micron or submicron-sized microspheres (mother spheres) as encoding microspheres. Two different fluorescent materials are encoded separately in the mother sphere and daughter sphere, without interference. However, the inventors found that the encoding capacity of the daughter spheres in this encoding method is limited. If a third color is encoded within the limited surface area of the mother sphere, not only is the fluorescence intensity of the existing fluorescent material sacrificed, but the encoding capacity is also reduced. On the other hand, the inventors also found that the uniformity of the daughter spheres also affects the fluorescence uniformity of the encoding channel, further affecting the encoding capacity. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide a quantum dot magnetic fluorescent encoded microsphere with a small coefficient of variation and a high encoding capacity, and a method for preparing the same, in order to address the shortcomings of the prior art.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] A quantum dot magnetic fluorescent encoded microsphere is characterized by comprising a mother sphere and N layers of daughter spheres located on the surface of the mother sphere; wherein N is an integer greater than 1; each layer of daughter spheres contains only one type of quantum dot, and different layers of daughter spheres contain quantum dot wavelengths of different wavelengths; the daughter spheres are connected to the mother sphere by chemical covalent bonds formed by the reaction of carboxyl and amino groups, and the daughter spheres are connected to adjacent layers of daughter spheres by chemical covalent bonds formed by the reaction of carboxyl and amino groups; the daughter spheres are synthesized by using mesoporous silica nanospheres or solid silica nanospheres as templates, loading quantum dots on their surface and then coating them with silica, each layer of daughter spheres contains the same type of quantum dots, and the N layers of daughter spheres contain m types of quantum dots, where m is less than or equal to N.
[0008] Furthermore, the mother sphere is a magnetic microsphere with a particle size of 500 nm to 10 μm and an amino or carboxyl group on its surface. Further, the magnetic microsphere is composed of iron oxide nanoparticles and polymers or silicon dioxide.
[0009] Furthermore, the particle size of the sub-spheres is 100nm to 400nm.
[0010] On the other hand, the present invention also provides a method for preparing quantum dot magnetic fluorescently encoded microspheres, the method comprising the following steps:
[0011] (1) Preparation of multiple quantum spheres loaded with different quantum dots
[0012] Silica nanospheres were dispersed in a mixed solution of ethanol and ammonia, and then aminosilane was added. The reaction was carried out for 4 to 8 hours, and the precipitate was collected by centrifugation to obtain amino-modified silica nanospheres.
[0013] A chloroform or toluene solution of quantum dots was added to amino-modified silica nanospheres, sonicated for 4–8 minutes, and the precipitate was collected by centrifugation to obtain a silica sphere / quantum dot composite, which is a quantum dot silica nanosphere without silica coating.
[0014] The silica sphere / quantum dot composite was dispersed in an aqueous solution of polyethyleneimine and reacted for 0.5–2 hours. After centrifugation to remove the supernatant, the mixture was washed alternately with ethanol and water and then placed back into a mixed solution of ethanol, water, ammonia and tetraethyl orthosilicate and reacted for 10–13 hours to obtain silica-coated quantum dot silica nanospheres.
[0015] Quantum dot silica nanospheres coated with silica were dispersed in a mixed solution of ethanol, ammonia and aminosilane and reacted for 4 to 8 hours. After centrifugation and washing, amino-modified quantum dot silica nanospheres coated with silica were obtained.
[0016] Amino-modified silica-coated quantum dot silica nanospheres were placed in a solution containing succinic anhydride and reacted for 2–6 hours. After centrifugation and washing, carboxyl-modified silica-coated quantum dot silica nanospheres were obtained.
[0017] Repeat step (1) and change the wavelength or amount of quantum dots to prepare various quantum spheres loaded with different quantum dots;
[0018] (2) Connection between the cue ball and the child ball
[0019] The mother sphere is added to an excess of daughter spheres. When the daughter spheres are carboxyl-modified silica-coated quantum dot silica nanospheres, the mother spheres are amino-modified silica-coated quantum dot silica nanospheres; conversely, when the daughter spheres are amino-modified silica-coated quantum dot silica nanospheres, the mother spheres are carboxyl-modified silica-coated quantum dot silica nanospheres. After ultrasonic dispersion, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added. After reacting for 2–4 hours, magnetic separation is performed, the supernatant is discarded, and the precipitate is washed multiple times with purified water to obtain quantum dot magnetic fluorescently encoded microspheres with a single layer of daughter spheres.
[0020] (3) Connection between adjacent sub-spheres
[0021] Depending on whether the second layer of subspheres is modified with amino or carboxyl groups, the quantum dot magnetic fluorescently encoded microspheres with composite 1-layer subspheres prepared in step (2) are modified or not modified. Then, they are added to an excess of the second layer of subspheres, ultrasonically dispersed, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added. After stirring for 2 to 4 hours, magnetic separation is performed, the supernatant is discarded, and the precipitate is washed with purified water to obtain quantum dot magnetic fluorescently encoded microspheres with composite 2-layer subspheres; or, step (3) is repeated to obtain quantum dot magnetic fluorescently encoded microspheres with the required number of subspheres.
[0022] (4) Carboxyl modification of quantum dot magnetic fluorescently encoded microspheres requiring multiple layers of subspheres
[0023] The mother sphere and the quantum dot magnetic fluorescently encoded microspheres that require multiple layers were added to a phosphate buffer solution of polyacrylic acid. After being ultrasonically dispersed, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. After stirring for 2-4 hours, magnetic separation was performed, the supernatant was discarded, and the precipitate was washed three times with purified water to obtain carboxyl-modified quantum dot magnetic fluorescently encoded microspheres that require multiple layers.
[0024] Based on the above technical solution, the solution may also employ the following further technical solutions simultaneously, or a combination of these further technical solutions:
[0025] The aminosilane is selected from 3-aminopropyltrimethoxysilane and 3-aminopropyltriethoxysilane.
[0026] In step (1), the mass ratio of silica nanospheres: ethanol: ammonia: aminosilane is 1:800~1200:10~15:2~10.
[0027] In step (1), the mass ratio of amino-modified silica nanospheres to quantum dots is 1:0.01 to 1:1.
[0028] In step (1), the mass ratio of silica spheres / quantum dot composite to polyethyleneimine is 1:1 to 1:4; the mass ratio of polyethyleneimine-modified silica spheres / quantum dot composite: ethanol: water: ammonia: tetraethyl orthosilicate is 1:800 to 1200: 200 to 300: 10 to 15: 3 to 10.
[0029] In step (1), the mass ratio of quantum dot silica nanospheres coated with silica to ethanol to ammonia to aminosilane is 1:800~1200:10~15:2~10.
[0030] In step (1), the mass ratio of amino-modified silica-coated quantum dot silica nanospheres to succinic anhydride is 1:5 to 1:20.
[0031] In step (3): the mass ratio of polyacrylic acid to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:0.02-0.5:0.2-5; the pH value of the phosphate buffer is 5-7.
[0032] This invention uses silica spheres as templates and quantum dots as encoding elements to synthesize daughter spheres. Multilayer assembly is then performed on the surface of the mother spheres to obtain quantum dot magnetic fluorescent encoded microspheres. The encoded microspheres and their preparation method described in this invention have the following beneficial effects:
[0033] (1) This invention expands upon the original method of assembling a mother ball and a single layer of daughter balls on the surface of the mother ball to obtain encoded microspheres, achieving multi-layer assembly of daughter balls and improving fluorescence intensity and encoding capacity. When the number of encoded colors increases, there is no need to reduce the number of a certain type of daughter ball on the surface of the mother ball to replace it with another type, thus without sacrificing encoding capacity. Furthermore, the quantum dots between different layers of daughter balls do not interfere with each other, making encoding easier.
[0034] (2) The present invention uses the template method to synthesize subspheres, and the obtained subspheres have good uniformity in particle size. Therefore, the fluorescence variation coefficient of the encoded microspheres is small and the encoding capacity is high.
[0035] (3) The present invention uses the template method to synthesize subspheres, which can simply add the required amount of quantum dots to obtain subspheres with different fluorescence intensities. The encoding reproducibility is good and easy to control, so the batch-to-batch difference is small and it is suitable for mass production. Attached Figure Description
[0036] Figure 1 This is a transmission electron microscope (TEM) image of carboxyl-modified quantum dot silica nanospheres obtained using mesoporous silica as a template in Example 1.
[0037] Figure 2 This is a transmission electron microscope (TEM) image of carboxyl-modified quantum dot silica nanospheres obtained using solid silica as a template in Example 2.
[0038] Figure 3 This is a scanning electron microscope image of the quantum dot magnetic fluorescently encoded microspheres with a composite single-layer subsphere in Example 3.
[0039] Figure 4 The results are flow cytometry fluorescence images of the six coded balls in the FITC channel in Example 3.
[0040] Figure 5 This is a scanning electron microscope image of the quantum dot magnetic fluorescently encoded microspheres with composite two-layer subspheres in Example 4.
[0041] Figure 6 This is a flow cytometry spectrum of the quantum dot magnetic fluorescently encoded microsphere composite with two layers of subspheres in Example 4.
[0042] Figure 7 This is a flow cytometry pattern obtained by mixing the quantum dot magnetic fluorescently encoded microspheres with composite two-layer subspheres in Example 4 with the encoded spheres in Example 3.
[0043] Figure 8 This is a flow cytometry diagram of the FITC channel of the quantum dot magnetic fluorescence encoded microspheres of the five composite single-layer subspheres with five different fluorescence types in Example 5.
[0044] Figure 9 This is a flow cytometry diagram of the PE channel of the quantum dot magnetic fluorescence encoded microspheres of the six different fluorescent composite two-layer subspheres obtained in Example 6. Detailed Implementation
[0045] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.
[0046] Example 1
[0047] This embodiment provides a method for synthesizing quantum dot silica nanospheres with good particle size and fluorescence uniformity based on mesoporous silica spheres;
[0048] (1) Preparation of amino-modified mesoporous silica nanospheres
[0049] 100 mg of mesoporous silica nanospheres with a particle size of 200 nm were dispersed in a mixed solution of 100 g of ethanol and 1.25 g of ammonia. Then, 0.6 g of 3-aminopropyltrimethoxysilane was added, and the mixture was stirred for 6 hours. The precipitate was collected by centrifugation and washed three times with ethanol to obtain amino-modified mesoporous silica nanospheres.
[0050] (2) Preparation of uncoated quantum dot silica nanospheres
[0051] 10 mg of amino-modified mesoporous silica nanospheres were added to 0.5 ml of chloroform solution with a concentration of 10 mg / mL quantum dots (wavelength of 520 nm). After sonication for 5 minutes, the precipitate was collected by centrifugation to obtain uncoated quantum dot silica nanospheres.
[0052] (3) Preparation of quantum dot silica nanospheres coated with silica
[0053] The above-mentioned mesoporous silica sphere / quantum dot composite was ultrasonically dispersed in 0.8 mL of an aqueous solution of polyethyleneimine with a concentration of 50 mg / mL, and reacted for 1 hour. After centrifugation to remove the supernatant, the mixture was washed alternately with ethanol and water, and then redispersed in a mixture of 10 g of ethanol and 2.5 g of water. Finally, 0.125 g of ammonia and 50 mg of tetraethyl orthosilicate were added at once, and the mixture was reacted for 12 hours to obtain silica-coated quantum dot silica nanospheres.
[0054] (4) Preparation of amino-modified silica-coated quantum dot silica nanospheres
[0055] 10 mg of quantum dot silica nanospheres were dispersed in a mixed solution of 10 g of ethanol and 0.125 g of ammonia, and then 0.06 g of 3-aminopropyltrimethoxysilane was added. The mixture was stirred for 6 hours, the precipitate was collected by centrifugation, and washed three times with ethanol to obtain amino-modified silica-coated quantum dot silica nanospheres.
[0056] (5) Preparation of carboxyl-modified silica-coated quantum dot silica nanospheres
[0057] 10 mg of amino-modified quantum dot silica nanospheres were placed in 10 ml of N,N-dimethylformamide solution containing succinic anhydride (10 mg / ml) and stirred for 4 hours. After centrifugation, the nanospheres were washed three times with water to obtain carboxyl-modified silica-coated quantum dot silica nanospheres.
[0058] (6) Repeat steps (1) to (5) above, only changing the concentration of quantum dots in step 2 to 0.2 mg / mL, 0.4 mg / mL, 0.9 mg / mL, 2.2 mg / mL, 5 mg / mL, and 10 mg / mL, to prepare six different green quantum dot silica nanospheres with different fluorescence intensities (wavelength 520 nm). Figure 1 The image shown is a transmission electron microscope (TEM) image of quantum dot silica nanospheres coated with carboxyl groups, obtained by adding 10 mg / mL quantum dot solution to mesoporous silica as a template.
[0059] (7) Repeat steps (1) to (5) above, change the wavelength of the quantum dots in step 2 to 610 nm, and change the concentration of the quantum dots to 0.15 mg / mL, 0.4 mg / mL, 0.9 mg / mL, 2 mg / mL, 4 mg / mL and 10 mg / mL to prepare six kinds of red quantum dot silica nanospheres with different fluorescence intensities (wavelength 610 nm).
[0060] Example 2
[0061] This embodiment provides a method for synthesizing quantum dot silica nanospheres with good particle size and fluorescence uniformity based on solid silica spheres;
[0062] (1) Preparation of amino-modified solid silica nanospheres
[0063] 100 mg of solid silica nanospheres with a particle size of 200 nm were dispersed in a mixed solution of 100 g of ethanol and 1.25 g of ammonia. Then, 0.6 g of 3-aminopropyltrimethoxysilane was added, and the mixture was stirred for 6 hours. The precipitate was collected by centrifugation and washed three times with ethanol to obtain amino-modified solid silica nanospheres.
[0064] (2) Preparation of solid silica sphere / quantum dot composite
[0065] 10 mg of amino-modified solid silica nanospheres were added to 0.5 ml of chloroform solution with a concentration of 2 mg / mL quantum dots (wavelength of 520 nm). After sonication for 5 minutes, the precipitate was collected by centrifugation to obtain uncoated solid silica nanospheres of quantum dots.
[0066] (3) Preparation of quantum dot silica nanospheres coated with silica
[0067] The solid silica sphere / quantum dot composite was ultrasonically dispersed in 0.4 mL of an aqueous solution of polyethyleneimine with a concentration of 50 mg / mL, and reacted for 1 hour. The supernatant was removed by centrifugation, and the mixture was washed alternately with ethanol and water, then redispersed in a mixture of 10 g ethanol and 2.5 g water. Finally, 0.125 g ammonia and 20 mg tetraethyl orthosilicate were added, and the mixture was stirred for 12 hours to obtain silica-coated quantum dot silica nanospheres.
[0068] (4) Preparation of amino-modified quantum dot silica nanospheres
[0069] 10 mg of quantum dot silica nanospheres were dispersed in a mixed solution of 10 g of ethanol and 0.125 g of ammonia, and then 0.06 g of 3-aminopropyltrimethoxysilane was added. The mixture was stirred for 6 hours, the precipitate was collected by centrifugation, and washed three times with ethanol to obtain amino-modified quantum dot silica nanospheres.
[0070] (5) Preparation of carboxyl-modified quantum dot silica nanospheres
[0071] 10 mg of amino-modified quantum dot silica nanospheres were placed in 10 ml of N,N-dimethylformamide solution containing succinic anhydride (10 mg / ml), stirred for 4 hours, centrifuged, and washed three times with water to obtain carboxyl-modified silica-coated quantum dot silica nanospheres. Figure 2 The image shown is a transmission electron microscope (TEM) image of quantum dot silica nanospheres with carboxyl-modified silica coating obtained using solid silica as a template.
[0072] Example 3: Connection between the cue ball and the daughter ball.
[0073] First, 5 mg of amino-modified 6 μm magnetic spheres and 10 mg of carboxyl-modified daughter spheres were ultrasonically dispersed in 0.5 mL of 10 mM MES (2-(N-morphoyl)ethanesulfonic acid) solution (containing 0.05 wt% Tween 20). Then, the dispersed mother spheres were added to the daughter spheres, and sonication continued for 1 minute. Next, 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mg of N-hydroxysuccinimide were added, and the mixture was stirred for 3 hours. After magnetic separation, the supernatant was discarded, and the precipitate was washed three times with purified water to obtain quantum dot magnetic fluorescently encoded microspheres with a single layer of daughter spheres. Figure 3 The image shown is a scanning electron microscope (SEM) image of a quantum dot magnetic fluorescently encoded microsphere composed of a composite single-layer subsphere.
[0074] The six carboxyl-modified silica-coated quantum dot silica nanospheres with different green fluorescence intensities obtained in Example 1 were linked with 6 μm amino-modified magnetic microspheres according to the above steps to obtain six FITC channel-coded spheres. Figure 4Table 1 shows the flow cytometry results of the FITC channels for the six types of encoded spheres. The results indicate that the encoded spheres prepared by this method have a small coefficient of variation, and the fluorescence intensity can be controlled by the amount of quantum dots added.
[0075] Table 1
[0076] serial number fluorescence intensity Coefficient of variation (CV) 1 12377 6.93％ 2 30408 5.83％ 3 56056 5.8％ 4 116234 5.38％ 5 229168 4.17％ 6 482694 6.76％
[0077] Example 4: Connection of two adjacent sub-spheres.
[0078] (1) 5 mg of the complex of the mother ball with the highest fluorescence intensity in Example 3 and the 1-layer daughter ball was added to 1 ml of an aqueous solution of polyethyleneimine with a concentration of 20 mg / ml. After ultrasonic dispersion, 5 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. After stirring for 2 hours, magnetic separation was performed, the supernatant was discarded, and the precipitate was washed 3 times with purified water to obtain the quantum dot magnetic fluorescently encoded microspheres of the amino-modified composite 1-layer daughter ball.
[0079] (2) The 5 mg of amino-modified composite 1-layer subsphere quantum dot magnetic fluorescently encoded microspheres and 10 mg of the green fluorescent carboxyl-modified subspheres with the highest fluorescence intensity obtained in Example 1 were ultrasonically dispersed in 0.5 mL of 10 mM MES (2-(N-morpholino)ethanesulfonic acid) solution (containing 0.05 wt% Tween 20). Then, the dispersed mother sphere and the composite of 1 or more subspheres were added to the subspheres, and ultrasonication was continued for 1 minute. Then, 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mg of N-hydroxysuccinimide were added. After stirring for 3 hours, magnetic separation was performed, the supernatant was discarded, and the precipitate was washed 3 times with purified water to obtain quantum dot magnetic fluorescently encoded microspheres with 2-layer subspheres.
[0080] (4) Add 5 mg of the above-mentioned mother sphere and 2-layer daughter sphere complex to 0.5 ml of phosphate buffer of polyacrylic acid (pH = 6) with a concentration of 1 mg / ml. After ultrasonic dispersion, add 5 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride. After stirring for 2 hours, perform magnetic separation, discard the supernatant, and wash the precipitate 3 times with purified water to obtain quantum dot magnetic fluorescently encoded microspheres of carboxyl-modified composite 2-layer daughter spheres. Figure 5 The image shown is a scanning electron microscope (SEM) image of a complex of a carboxyl-modified mother sphere and two layers of daughter spheres. Figure 6 This is a flow cytometry fluorescence image of this type of coded sphere. Figure 7 The flow cytometry fluorescence pattern is obtained by mixing this type of coding ball with the coding ball in Example 3. Figure 6 The results show that, compared to a single-layer subsphere, a two-layer subsphere can further improve the fluorescence intensity of the coding sphere. Figure 4 and Figure 7The results show that two-layer subspheres can increase the coding multiplicity of a single fluorescent coding sphere.
[0081] Example 5: Synthesis of a single-layer dual-fluorescent coding sphere
[0082] First, 5 mg of 5 of the 6 green quantum dot silica nanospheres with higher fluorescence intensity obtained in Example 1 (quantum dot concentrations of 0.4 mg / mL, 0.9 mg / mL, 2.2 mg / mL, 5 mg / mL, and 10 mg / mL, respectively) were selected and uniformly mixed with 5 mg of red quantum dot silica nanospheres (quantum dot concentration of 10 mg / mL) to obtain 10 mg of each of the 5 different fluorescence quantum dot silica nanosphere mixtures. Then, 5 mg of amino-modified 6 μm mother spheres and 10 mg of carboxyl-modified nanospheres were ultrasonically dispersed in 0.5 mL of 10 mM MES (2-(N-morphoyl)ethanesulfonic acid) solution (containing 0.05 wt% Tween 20). The dispersed mother spheres were then added to the daughter spheres, and ultrasonication was continued for 1 minute. Then, 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mg of N-hydroxysuccinimide were added. After stirring for 3 hours, magnetic separation was performed, the supernatant was discarded, and the precipitate was washed 3 times with purified water to obtain quantum dot magnetic fluorescently encoded microspheres with 5 different fluorescence composite single-layer daughter spheres. Figure 8 This is a flow cytometry spectrum of the FITC channels of quantum dot magnetic fluorescence-encoded microspheres of five composite single-layer subspheres with five different fluorescence characteristics. (Comparison) Figure 4 and Figure 8 Tables 1 and 2 show that when half of the space on the surface of the mother sphere is occupied by red fluorescent nanospheres, the fluorescence intensity of the FITC channel of the remaining green fluorescent nanospheres decreases during encoding, resulting in a reduction in the encoding multiplicity. At the same time, the coefficient of variation increases, and adjacent two types of encoded nanospheres become closer during clustering, which is not conducive to clustering and encoding.
[0083] Table 2
[0084] serial number fluorescence intensity Coefficient of variation (CV) 1 13162 8.18％ 2 27088 7.00％ 3 55128 6.82％ 4 114647 6.42％ 5 235737 6.01％
[0085] Example 6: Synthesis of a double-layered, double-fluorescent encoded sphere.
[0086] (1) The 5 mg of quantum dot magnetic fluorescently encoded microspheres with the highest fluorescence intensity in Example 3 were added to 1 ml of 20 mg / ml aqueous solution of polyethyleneimine. After being ultrasonically dispersed evenly, 5 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. After stirring for 2 hours, magnetic separation was performed, the supernatant was discarded, and the precipitate was washed 3 times with purified water to obtain the amino-modified quantum dot magnetic fluorescently encoded microspheres with the composite layer.
[0087] (2) The 5 mg of amino-modified composite 1-layer subsphere quantum dot magnetic fluorescently encoded microspheres and 10 mg of the 6 different fluorescence intensities of red quantum dot silica nanospheres (wavelength 610 nm) obtained in Example 1 were ultrasonically dispersed in 0.5 mL of 10 mM MES (2-(N-morpholino)ethanesulfonic acid) solution (containing 0.05 wt% Tween 20). Then, the dispersed 5 mg of amino-modified composite 1-layer subsphere quantum dot magnetic fluorescently encoded microspheres were added to 10 mg of red quantum dot silica nanospheres and ultrasonicated for 1 minute. Then, 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mg of N-hydroxysuccinimide were added respectively. After stirring for 3 hours, magnetic separation was performed, the supernatant was discarded, and the precipitate was washed 3 times with purified water to obtain 6 different fluorescence composite 2-layer subsphere quantum dot magnetic fluorescently encoded microspheres.
[0088] (4) The six different fluorescent composite two-layer subspheres were added to 0.5 ml of phosphate buffer of polyacrylic acid (pH=6) with a concentration of 1 mg / ml. After ultrasonic dispersion, 5 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. After stirring for 2 hours, magnetic separation was performed, the supernatant was discarded, and the precipitate was washed 3 times with purified water to obtain carboxyl-modified quantum dot magnetic fluorescently encoded microspheres. Figure 9 The images show flow cytometry fluorescence spectra of the PE channels in the quantum dot magnetic fluorescence-encoded microspheres of the composite two-layer subspheres with six different fluorescence characteristics. Figure 9 This indicates that the present invention can achieve precise encoding on the second layer of sub-spheres.
[0089] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.
Claims
1. A quantum dot magnetic fluorescently encoded microsphere, characterized in that... The system comprises a mother sphere and N layers of daughter spheres located on the surface of the mother sphere; where N is an integer greater than 1; each layer of daughter spheres contains only one type of quantum dot, and different layers of daughter spheres contain quantum dot wavelengths of different wavelengths; the daughter spheres are connected to the mother sphere by chemical covalent bonds formed by the reaction of carboxyl and amino groups, and the daughter spheres are connected to the daughter spheres of adjacent layers by chemical covalent bonds formed by the reaction of carboxyl and amino groups; the daughter spheres are synthesized by using mesoporous silica nanospheres or solid silica nanospheres as templates, loading quantum dots on their surface, and then coating them with silica; each layer of daughter spheres contains the same quantum dots, and the N layers of daughter spheres contain m types of quantum dots, where m equals N; The mother ball is a magnetic microsphere.
2. The quantum dot magnetic fluorescently encoded microsphere as described in claim 1, characterized in that... The mother spheres have a particle size of 500 nm to 10 μm and have amino or carboxyl groups on their surface.
3. The quantum dot magnetic fluorescently encoded microsphere as described in claim 2, characterized in that... Magnetic microspheres are composed of iron oxide nanoparticles and polymers or silicon dioxide.
4. The quantum dot magnetic fluorescently encoded microsphere as described in claim 1, characterized in that... The particle size of the sub-spheres ranges from 100 nm to 400 nm.
5. A method for preparing quantum dot magnetic fluorescently encoded microspheres as described in any one of claims 1-4, characterized in that... Includes the following steps: (1) Preparation of multiple quantum spheres loaded with different quantum dots Silica nanospheres were dispersed in a mixed solution of ethanol and ammonia, and then aminosilane was added. The reaction was carried out for 4 to 8 hours, and the precipitate was collected by centrifugation to obtain amino-modified silica nanospheres. A chloroform or toluene solution of quantum dots was added to amino-modified silica nanospheres, sonicated for 4-8 minutes, and the precipitate was collected by centrifugation to obtain a silica sphere / quantum dot composite, which is a quantum dot silica nanosphere without silica coating. The silica sphere / quantum dot composite was dispersed in an aqueous solution of polyethyleneimine and reacted for 0.5 to 2 hours. After centrifugation to remove the supernatant, the mixture was washed alternately with ethanol and water and then placed back into a mixed solution of ethanol, water, ammonia and tetraethyl orthosilicate and reacted for 10 to 13 hours to obtain silica-coated quantum dot silica nanospheres. Quantum dot silica nanospheres coated with silica were dispersed in a mixed solution of ethanol, ammonia and aminosilane and reacted for 4-8 hours. After centrifugation and washing, amino-modified quantum dot silica nanospheres coated with silica were obtained. Amino-modified silica-coated quantum dot silica nanospheres were placed in a solution containing succinic anhydride and reacted for 2-6 hours. After centrifugation and washing, carboxyl-modified silica-coated quantum dot silica nanospheres were obtained. Repeat step (1) and change the wavelength or amount of quantum dots to prepare various quantum spheres loaded with different quantum dots; (2) Connection between the cue ball and the child ball The mother sphere is added to an excess of daughter spheres. When the daughter spheres are carboxyl-modified silica-coated quantum dot silica nanospheres, the mother spheres are amino-modified magnetic microspheres. If the daughter spheres are amino-modified silica-coated quantum dot silica nanospheres, the mother spheres are carboxyl-modified magnetic microspheres. After ultrasonic dispersion, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added. After reacting for 2-4 hours, magnetic separation is performed, the supernatant is discarded, and the precipitate is washed multiple times with purified water to obtain quantum dot magnetic fluorescently encoded microspheres with a composite of one layer of daughter spheres. (3) Connection between two adjacent sub-spheres Depending on whether the second layer of subspheres is modified with amino or carboxyl groups, the quantum dot magnetic fluorescently encoded microspheres with a composite 1-layer subsphere prepared in step (2) can be modified or left unmodified. Then, they are added to an excess of the second layer of subspheres, ultrasonically dispersed, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added. After stirring for 2-4 hours, magnetic separation is performed, the supernatant is discarded, and the precipitate is washed with purified water to obtain quantum dot magnetic fluorescently encoded microspheres with a composite 2-layer subsphere. Alternatively, step (3) can be repeated to obtain quantum dot magnetic fluorescently encoded microspheres with the required number of subspheres. (4) Carboxyl modification of quantum dot magnetic fluorescently encoded microspheres obtained in step (3) The quantum dot magnetic fluorescently encoded microspheres obtained in step (3) were added to the phosphate buffer of polyacrylic acid and ultrasonically dispersed. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added and stirred for 2-4 hours. After magnetic separation, the supernatant was discarded and the precipitate was washed three times with purified water to obtain the carboxyl-modified composite quantum dot magnetic fluorescently encoded microspheres with multiple layers.
6. The method for preparing quantum dot magnetic fluorescently encoded microspheres according to claim 5, characterized in that... The aminosilane is selected from 3-aminopropyltrimethoxysilane and 3-aminopropyltriethoxysilane.
7. The method for preparing quantum dot magnetic fluorescently encoded microspheres according to claim 5, characterized in that... In step (1), the mass ratio of silica nanospheres: ethanol: ammonia: aminosilane is 1:800~1200:10~15:2~10.
8. The method for preparing quantum dot magnetic fluorescently encoded microspheres according to claim 5, characterized in that... In step (1), the mass ratio of amino-modified silica nanospheres to quantum dots is 1:0.01 to 1:
1.
9. The method for preparing quantum dot magnetic fluorescently encoded microspheres according to claim 5, characterized in that... In step (1), the mass ratio of silica spheres / quantum dot composite to polyethyleneimine is 1:1 to 1:4; the mass ratio of polyethyleneimine-modified silica spheres / quantum dot composite: ethanol: water: ammonia: tetraethyl orthosilicate is 1:800 to 1200: 200 to 300: 10 to 15: 3 to 10.
10. The method for preparing quantum dot magnetic fluorescently encoded microspheres according to claim 5, characterized in that... In step (1), the mass ratio of quantum dot silica nanospheres coated with silica to ethanol to ammonia to aminosilane is 1:800~1200:10~15:2~10.
11. The method for preparing quantum dot magnetic fluorescently encoded microspheres according to claim 5, characterized in that... In step (1), the mass ratio of amino-modified silica-coated quantum dot silica nanospheres to succinic anhydride is 1:5 to 1:
20.
12. The method for preparing quantum dot magnetic fluorescently encoded microspheres according to claim 5, characterized in that... In step (4), the mass ratio of the composite quantum dot magnetic fluorescently encoded microspheres requiring multiple layers is 1:0.02~0.5:0.2~5 for polyacrylic acid and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; the pH of the phosphate buffer solution is 5~7.