Amphiphilic quantum dot composite microsphere and preparation method thereof

Abstract

The invention belongs to the field of biochemical material preparation, and particularly relates to an amphiphilic quantum dot composite microsphere and a preparation method thereof. The invention provides a one-step preparation method of amphiphilic polystyrene quantum dot composite microspheres, which comprises the following steps: by utilizing the principle that styrene and tetraethoxysilane are similar and soluble, mixing and winding styrene and tetraethoxysilane with each other by adjusting the coexistence conditions of styrene and tetraethoxysilane during polymerization reaction to form composite microspheres with a certain skeleton structure; according to the method disclosed by the invention, not only is the defect of generating the microspheres from a single component improved, but also the links of the preparation process are reduced, and an amphiphilic microsphere carrier and a fluorescence labeling material with lower cost are provided for popularizing the application of the quantum dots in the aspects of biochemical detection and biochemical imaging.

Description

Technical Field

[0001] The invention belongs to the field of biochemical material preparation, and specifically relates to an amphiphilic quantum dot composite microsphere and a preparation method thereof. Background Art

[0002] Quantum dots have the advantages of high fluorescence intensity, narrow and highly symmetrical half-width of emission peak, large Stoke shift, strong resistance to photobleaching, etc. Moreover, the fluorescence emission wavelength of quantum dots can be effectively controlled by material composition and particle size selection, so that fluorescence with multiple emission wavelengths can be obtained simultaneously under the same excitation light. Therefore, in recent years, the use of quantum dot-encoded microspheres for biochemical imaging and high-throughput diagnosis of diseases has become a hot topic in clinical disease diagnosis and basic biomedical research.

[0003] However, high-quality quantum dots are generally synthesized by high-temperature preparation in an organic phase, and are usually extremely hydrophobic, and cannot be directly used in biochemical environments or for labeling large molecular proteins. Therefore, how to change oil-soluble quantum dots into hydrophilic and amphiphilic quantum dots suitable for biochemical reaction environments becomes the key to their application in biological imaging and disease diagnosis.

[0004] To achieve the above goals, the general idea is to first modify the oil-soluble quantum dots into water-soluble quantum dots with functional groups on the surface. How to achieve the water-soluble modification of oil-soluble quantum dots has been widely studied and can be roughly divided into the following three categories: (1) Ligand exchange: that is, using a hydrophilic ligand containing a terminal thiol or amino group at one end to replace the hydrophobic ligand on the surface of the oil-soluble quantum dots, and fixing the hydrophilic ligand molecules on the surface of the quantum dots through coordination bonds, so that the oil-soluble quantum dots become water-soluble.

[0005] The advantages of this method are simple operation, small particle size and large specific surface area of ​​the obtained water-soluble quantum dots, which help to improve the efficiency of subsequent coupling reactions. However, due to the weak binding force of the coordination binding and the susceptibility to the influence of the environmental pH value and ionic strength, the prepared water-soluble quantum dots have poor stability.

[0006] (2) Surface silanization: that is, forming one or more layers of amorphous silica on the surface of oil-soluble quantum dots, thereby giving the quantum dots water solubility. Since the stability of silica in an aqueous environment is not affected by pH and ionic strength, the water-soluble quantum dots modified in this way can not only be stored for a long time in an aqueous environment, but also show higher reactivity when coupled with proteins. However, the surface silanization process of quantum dots is relatively complicated. It is essentially a multi-step chemical reaction. The surfactants, alkaline catalysts, reaction precursors and their hydrolysis products added during the reaction will reduce the quantum yield of the quantum dots to varying degrees, resulting in a loss of fluorescence intensity. These factors will reduce the diagnostic sensitivity and imaging resolution of quantum dot-macromolecule protein conjugates, which is not conducive to their actual biological applications.

[0007] (3) Coating with amphiphilic polymers: that is, using amphiphilic polymers to encapsulate oil-soluble quantum dots to form micelles with a core-shell structure, thereby giving the quantum dots water solubility. The hydrophobic segments of the amphiphilic polymers will not directly act on the surface of the hydrophobic quantum dots, so the quantum yield of the product can be well maintained. At present, there are many types of amphiphilic polymers (comb-shaped or grafted amphiphilic macromolecules, linear diblock amphiphilic polymers, etc.) used for water-soluble modification of quantum dots; however, the amphiphilic macromolecules prepared by this method are mostly entangled in micelles or loose linear macromolecules. Under mechanical or thermal forces, especially under long-term swelling, they are easily dispersed, and the coated quantum dots are easy to fall off.

[0008] Based on existing research, it can be found that the current mainstream methods for water-soluble modification of oil-soluble quantum dots all have their own shortcomings. However, among these three methods, water-soluble quantum dots prepared by coating with amphiphilic polymers have the greatest development potential, because it can not only maintain the quantum yield of the modified quantum dots, but also maintain high stability in complex aqueous environments, and also has high protein coupling activity.

[0009] Therefore, how to further optimize the structure of micelles or microspheres of polymer-coated oil-soluble quantum dots based on the modification of quantum dots by amphiphilic polymer coating so that they have higher stability after forming a conjugate with protein is the bottleneck restricting the application of quantum dots in clinical disease diagnosis and basic biomedical research. Summary of the invention

[0010] In order to solve the above technical problems, the present invention provides an amphiphilic quantum dot composite microsphere and a preparation method thereof.

[0011] The first aspect of the present invention is to provide an amphiphilic quantum dot composite microsphere, which includes: an outer shell, a three-dimensional skeleton inside the outer shell, a plurality of cavities formed between the three-dimensional skeletons, a hydrophobic polymer encapsulating oil-soluble quantum dots in the cavities, and the outer shell and the internal three-dimensional skeleton are both made of hydrophilic silica gel.

[0012] Preferably, the particle size of the amphiphilic quantum dot composite microspheres is 80-800 nm.

[0013] Preferably, the hydrophobic polymer is selected from any one of polystyrene and polyester.

[0014] Preferably, the oil-soluble quantum dots have a particle size of 2 to 8 nm, and the surface of the oil-soluble quantum dots is covered with any one of hexadecylamine, oleic acid, trioctylphosphine, hexadecyl alcohol, and octylphosphine oxide.

[0015] As further preferred, the oil-soluble quantum dots are selected from: any one of (CdSe)ZnS, (CdSe)CdS, (CdS)ZnS, (CdTe)CdSe, (CdTe)CdS, (CdTe)ZnS, (ZnSe)CdSe, and (InP)ZnS.

[0016] The second aspect of the present invention is to provide a method for preparing the amphiphilic quantum dot composite microspheres.

[0017] The preparation method of amphiphilic quantum dot composite microspheres provided by the present invention is a one-step preparation method, which specifically utilizes the co-solvent polymerization of styrene and tetraethyl orthosilicate in a specific solvent and the polarity difference between polystyrene and silica gel generated during the polymerization process to ultimately form a composite microsphere structure in which most of the hydrophilic silica gel is on the periphery and a small amount of hydrophilic silica gel is formed inside to form a three-dimensional skeleton, which accommodates the hydrophobic polymer polystyrene and the oil-soluble quantum dots in intervals.

[0018] The method for preparing the amphiphilic quantum dot composite microspheres comprises the following steps: S1 Preparation of oil-soluble quantum dots: Evenly mix styrene, tetraethyl orthosilicate, and oil-soluble quantum dots to obtain an oil-soluble quantum dot mixed solution; Preparation of S2 amphiphilic quantum dot composite microspheres: First, polyvinyl pyrrolidone is dissolved in a mixed solvent, then azobisisobutyronitrile and the oil-soluble quantum dots prepared in S1 are mixed evenly and added thereto, and then a mixed solution of water and glacial acetic acid is added, nitrogen is passed through for deoxygenation, stirring is started, and a polymerization reaction is carried out to obtain amphiphilic quantum dot composite microspheres; the mixed solvent is a mixture of ethanol and ethylene glycol methyl ether in a volume ratio of 1:0.1~1.0.

[0019] In the above preparation method, preferably, the mass ratio of styrene to tetraethyl orthosilicate described in S1 is 1:0.5~1.6, and the mass volume ratio of styrene to oil-soluble quantum dots is 1 g: (20~50) μL.

[0020] Preferably, in S2, the mass volume ratio of styrene to the mixed solvent is 1 g: (2.5-5) mL, and the added mass of the other components is as follows, based on the mass of styrene: Polyvinylpyrrolidone 5%~12%, azobisisobutyronitrile 0.5%~1.5%, water 10%~30%, glacial acetic acid 5%~25%.

[0021] Preferably, in S2, stirring is started for 10 min and then the temperature is raised to start polymerization, pre-initiated at 40-50° C. for 20-50 min, then the temperature is raised to 60-80° C. and the reaction is carried out for 12-16 h.

[0022] In the present invention, the particle size of the composite microspheres is regulated to be between 80 and 800 nm by adjusting the ratio of styrene to tetraethyl orthosilicate, reaction temperature, reaction time, stirring speed and other conditions in the preparation process of amphiphilic quantum dot composite microspheres.

[0023] In addition, the polarity of the mixed solvent during the reaction has a significant effect on the structure and stability of the microspheres. This is because the polarity of the product has changed compared to the reactants before polymerization. During the reaction, the non-polar hydrophobic polymer polystyrene and oil-soluble quantum dots move toward the inside of the microspheres, and the hydrophilic silica moves toward the solvent, causing most of the hydrophilic silica to be distributed on the surface of the microspheres, and the hydrophobic polymer polystyrene and the quantum dots encapsulated therein to be distributed inside the microspheres. In this way, the quantum dots encapsulated inside the microspheres not only maintain the original optical properties and quantum yield, but also enhance the stability of the quantum dot microspheres with a three-dimensional skeleton structure, and the oil-soluble quantum dots are not easy to fall off. In addition, the surface silica layer has many silanol groups, which increases the hydrophilicity of the microspheres.

[0024] The one-step preparation method of the composite microspheres of the present invention is not only simple to operate, but also realizes the modification of oil-soluble quantum dots. On this basis, carboxyl groups, sulfhydryl groups and amino groups can be further introduced to couple protein macromolecules, ultimately achieving efficient coupling and stable labeling of quantum dots to biomacromolecules.

[0025] The beneficial effects of the present invention are: (1) Compared with the water-soluble quantum dots prepared by the ligand exchange method, the amphiphilic quantum dot composite microspheres of the present invention have a three-dimensional skeleton-type silica gel structure, the surface is mainly hydrophilic silica gel, and the interior is a cavity formed by discontinuous silica gel to accommodate the hydrophobic polymer polystyrene and the oil-soluble quantum dots. The polystyrene does not react with the quantum dots, but only wraps the quantum dots through hydrophobic interactions, providing a hydrophobic environment for the hydrophobic quantum dots, thereby maintaining the quantum yield and stability of the oil-soluble quantum dots. At the same time, the hydrophilic silica gel provides a skeleton support, enhances the stability of the microspheres, and makes it difficult for the quantum dots wrapped by the internal polystyrene to move relative to each other and fall off; in addition, most of the hydrophilic silica gel is distributed on the surface of the microspheres to ensure the hydrophilicity of the microspheres, and realizes the stable dispersion of the amphiphilic quantum dot microspheres in the aqueous environment. Therefore, the amphiphilic quantum dot composite microspheres of the present invention have higher stability than the water-soluble quantum dots prepared by the ligand exchange method, and are not affected by the polarity and ionic strength in the aqueous environment, and are more suitable for biological imaging and protein coupling reactions under different conditions.

[0026] (2) Compared with surface silanized water-soluble quantum dots, the amphiphilic quantum dot composite microspheres of the present invention can highly maintain the original quantum yield of the quantum dots. This is mainly due to the following three characteristics in the preparation process of the amphiphilic quantum dot composite microspheres of the present invention: First, the reagents and reaction conditions involved in the preparation process will not have any effect on the quantum dots; second, the amphiphilic quantum dot composite microspheres are only bound by the hydrophobic interaction between the internal hydrophobic polystyrene and the hydrophobic ligands on the surface of the oil-soluble quantum dots, and then as the polarity of the polymer silica gel changes, the hydrophobic substance migrates into the interior of the microspheres to form an amphiphilic structure. Therefore, the amphiphilic polymer does not directly contact the surface of the quantum dots and will not affect the fluorescence properties of the quantum dots; third, the hydrolysis and condensation of tetraethyl orthosilicate mainly occurs in an aqueous environment, while the quantum dots have been wrapped in polystyrene, migrated inward and separated from the aqueous environment, and the silica on the surface of the microspheres has very little absorption in the visible light region and basically does not affect the fluorescence properties of the quantum dots, so it will not affect the quantum dots in the hydrophobic core.

[0027] Based on the above reasons, the amphiphilic quantum dot microspheres of the present invention will not have a potential impact on the quantum yield of oil-soluble quantum dots, and thus can show higher biological detection sensitivity.

[0028] (3) Compared with water-soluble quantum dots prepared by coating with comb-shaped amphiphilic polymers or grafted amphiphilic macromolecules and water-soluble quantum dot micelles prepared by coating with diblock amphiphilic linear polymers, the amphiphilic quantum dot composite microspheres of the present invention have a three-dimensional skeleton silica gel structure entangled with linear polystyrene macromolecules inside and a hydrophilic silica layer aggregated outside. Therefore, the structure is more stable and not easy to loosen and cause the quantum dots to fall off. Moreover, the amphiphilic quantum dot composite microspheres of the present invention can be prepared by a one-step reaction, and the preparation process is simpler and the cost is lower. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A schematic diagram of a method for preparing amphiphilic quantum dot composite microspheres provided by the present invention; Figure 2 This is a microscopic structure diagram of the amphiphilic quantum dot composite microspheres prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0031] Example 1 An amphiphilic quantum dot composite microsphere is prepared by the following steps: S1 Preparation of oil-soluble quantum dots: Oil-soluble quantum dots (CdSe)ZnS were purchased from Shanghai Kundao Biotechnology Co., Ltd.

[0032] The particle size of the oil-soluble quantum dots is between 4 and 5 nm, the surface is covered with trioctylphosphine oxide, showing strong hydrophobicity, and the fluorescence emission wavelength is 605 nm.

[0033] Weigh 13.5 g styrene, 8 g ethyl orthosilicate, and 500 uL oil-soluble quantum dots, mix well and set aside; Preparation of S2 amphiphilic quantum dot composite microspheres: First, 1 g of polyvinyl pyrrolidone (PVP) was weighed and dissolved in 50 mL of a mixed solvent of anhydrous ethanol and ethylene glycol methyl ether, with a volume ratio of anhydrous ethanol to ethylene glycol methyl ether of 1:0.5. Then the mixed solution was added into a 100 mL three-necked flask with stirring as a reaction system. Next, weigh 0.15 g of azobisisobutyronitrile (AIBN) and dissolve it in the quantum dot mixture prepared in S1, mix well and then add it to the above reaction system, then weigh 3 g of water and 2 g of glacial acetic acid, mix well and then add them to the reaction system; Nitrogen was introduced into the above reaction system to deoxygenate, stirring was started, and the temperature was raised after 10 minutes to start polymerization. Pre-initiation was carried out at 45°C for 30 minutes, and then the temperature was raised to 70°C and the reaction was carried out for 12 hours.

[0034] After the polymerization reaction in S2 is completed, the mixture is naturally cooled to obtain a white emulsion product. The white emulsion product obtained by the reaction is centrifuged at a speed of 6000 rpm. After sedimentation, the supernatant is poured off, and the precipitate is washed with anhydrous ethanol for 5 times. Finally, the obtained amphiphilic quantum dot composite microspheres are dispersed in an ethanol aqueous solution with a volume fraction of 25%.

[0035] The schematic diagram of the preparation of amphiphilic quantum dot composite microspheres in this embodiment is shown in the attached figure. Figure 1 As shown, the particle size of the obtained amphiphilic quantum dot composite microspheres is 520 nm.

[0036] The obtained amphiphilic quantum dot composite microspheres were dispersed in 0.2 M PBS buffer (pH=7.4) with a concentration of 0.3 mg / mL. They were then placed at 4°C, 25°C, 37.5°C, and 45°C, respectively, and their dispersion states were observed every 2 days for 5 consecutive weeks, and the changes in their fluorescence intensity were measured.

[0037] The results showed that the amphiphilic quantum dot composite microspheres were insensitive to temperature and could maintain a stably dispersed state in PBS buffer at different temperatures, and the fluorescence intensity remained unchanged.

[0038] The micrograph of the amphiphilic quantum dot composite microspheres obtained in this example is shown in the attached Figure 2 Shown (magnification 1000x).

[0039] Attached Figure 2 It can be seen that the prepared amphiphilic quantum dot composite microspheres have an obvious spherical structure, including an outer shell formed by hydrophilic silica gel and a three-dimensional skeleton inside the outer shell. A plurality of cavities are formed between the three-dimensional skeletons, and oil-soluble quantum dots are dispersed in the cavities.

[0040] Example 2 An amphiphilic quantum dot composite microsphere is prepared by the following steps: S1 Preparation of oil-soluble quantum dots: Oil-soluble quantum dots (CdSe)ZnS were purchased from Shanghai Kundao Biotechnology Co., Ltd.

[0041] The particle size of the oil-soluble quantum dots is between 4 and 8 nm. The surface is covered with trioctylphosphine oxide, which is highly hydrophobic. The fluorescence emission wavelengths of the two quantum dots are 605 nm and 660 nm.

[0042] Weigh 13.5 g of styrene, 5 g of tetraethyl orthosilicate, 300 uL of oil-soluble quantum dots with a fluorescence wavelength of 605 nm and 300 uL of oil-soluble quantum dots with a fluorescence wavelength of 660 nm, mix them evenly and set aside; Preparation of S2 amphiphilic quantum dot composite microspheres: The difference from Example 1 is that: In S2, the amount of AIBN added was 0.2 g, water was 2 g, and glacial acetic acid was 1 g, and the reaction time was 15 h.

[0043] The particle size of the amphiphilic quantum dot composite microspheres prepared by the method of this example is 580 nm.

[0044] The amphiphilic quantum dot composite microspheres were dispersed in 0.2 M PBS buffer (pH=7.4) at a certain concentration (0.1 mg / mL) and placed in a shaker at 25°C at different speeds of 0 rpm, 50 rpm, 150 rpm, and 220 rpm. The dispersion state was observed every 2 days for 2 weeks, and the changes in the fluorescence intensity and quantum dot content of each sample were measured.

[0045] The results showed that the composite microspheres were insensitive to mechanical vibration and could maintain a relatively stable dispersion state in PBS buffer at different vibration speeds, with unchanged fluorescence intensity.

[0046] Example 3 Different from Example 1, in the mixed solvent used in S2, the volume ratio of anhydrous ethanol to ethylene glycol methyl ether is 1:0.2.

[0047] The composite microspheres obtained in this example were dispersed in 0.2 M PBS buffer (pH=7.4) to verify the performance of the microspheres. The results showed that although the composite microspheres had good dispersibility, the mechanical strength of the composite microspheres was reduced, and the fluorescence intensity of the composite microspheres was slightly reduced after 5 weeks.

[0048] Example 4 Different from Example 1, in the mixed solvent used in S2, the volume ratio of anhydrous ethanol to ethylene glycol methyl ether is 1:0.8.

[0049] The composite microspheres obtained in this example were dispersed in 0.2 M PBS buffer (pH=7.4) to verify the performance of the microspheres. The results showed that the dispersibility of the composite microspheres in the solution was slightly reduced, some microspheres tended to agglomerate, and the stability was reduced.

[0050] In addition, the present invention also adjusts the parameters such as reaction temperature, molar ratio of styrene to tetraethyl orthosilicate in the process of preparing microspheres. Some experiments are as follows: Example 5 Different from Example 1, the polymerization temperature in S2 is 60°C.

[0051] The particle size of the obtained composite microspheres is slightly larger than that of Example 1, and the overall particle size of the microspheres is about 750 nm.

[0052] Example 6 Different from Example 1, the polymerization temperature in S2 is 80°C.

[0053] The spherical regularity of the obtained composite microspheres is reduced, the particle size uniformity is poor, the particle size difference is large, the polymer is formed too quickly, the average molecular weight is small, and the mechanical strength of the microspheres is also reduced.

[0054] Example 7 Different from Example 1, the molar ratio of styrene to tetraethyl orthosilicate is 1:0.8, and the hydrophobicity of the obtained composite microspheres is improved, and the stability thereof is slightly reduced.

Claims

1. An amphiphilic quantum dot composite microsphere, characterized in that: The amphiphilic quantum dot composite microspheres include: an outer shell, a three-dimensional skeleton inside the outer shell, a plurality of cavities formed between the three-dimensional skeletons, a hydrophobic polymer encapsulating the oil-soluble quantum dots in the cavities, and the outer shell and the inner three-dimensional skeleton are both composed of hydrophilic silica gel.

2. The amphiphilic quantum dot composite microsphere according to claim 1, characterized in that: The particle size of the amphiphilic quantum dot composite microspheres is 80-800 nm.

3. The amphiphilic quantum dot composite microsphere according to claim 1, characterized in that: The hydrophobic polymer is selected from any one of polystyrene and polyester.

4. The amphiphilic quantum dot composite microsphere according to claim 1, characterized in that: The oil-soluble quantum dots have a particle size of 2-8 nm, and the surface of the oil-soluble quantum dots is covered by any one of hexadecylamine, oleic acid, trioctylphosphine, hexadecyl alcohol, and octylphosphine oxide.

5. The amphiphilic quantum dot composite microsphere according to claim 4, characterized in that: The oil-soluble quantum dots are selected from any one of (CdSe)ZnS, (CdSe)CdS, (CdS)ZnS, (CdTe)CdSe, (CdTe)CdS, (CdTe)ZnS, (ZnSe)CdSe, and (InP)ZnS.

6. A method for preparing the amphiphilic quantum dot composite microspheres according to claim 1, characterized in that: The steps include: S1 Preparation of oil-soluble quantum dots: Evenly mix styrene, tetraethyl orthosilicate, and oil-soluble quantum dots to obtain an oil-soluble quantum dot mixed solution; Preparation of S2 amphiphilic quantum dot composite microspheres: First, polyvinyl pyrrolidone is dissolved in a mixed solvent, then azobisisobutyronitrile and the oil-soluble quantum dots prepared in S1 are mixed evenly and added thereto, and then a mixed solution of water and glacial acetic acid is added, nitrogen is passed through to deoxygenate, stirring is started, and a polymerization reaction is carried out to obtain amphiphilic quantum dot composite microspheres; The mixed solvent is prepared by mixing ethanol and ethylene glycol methyl ether in a volume ratio of 1:0.1-1.

0.

7. The preparation method according to claim 6, characterized in that: The mass ratio of styrene to tetraethyl orthosilicate described in S1 is 1:0.5~1.6, and the mass volume ratio of styrene to oil-soluble quantum dots is 1 g:(20~50) μL.

8. The preparation method according to claim 6, characterized in that: In S2, the mass volume ratio of styrene to mixed solvent is 1 g: (2.5~5) mL. Based on the mass of styrene, the added masses of other components are as follows: Polyvinylpyrrolidone 5%~12%, azobisisobutyronitrile 0.5%~1.5%, water 10%~30%, glacial acetic acid 5%~25%.

9. The preparation method according to claim 6, characterized in that: In S2, the polymerization was started after stirring for 10 min and the temperature was raised. The polymerization was pre-initiated at 40-50°C for 20-50 min, and then the temperature was raised to 60-80°C and the reaction was carried out for 12-16 h.

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