CN111410951A - A kind of composite quantum dot encoded microsphere based on natural prickly pollen and preparation method thereof - Google Patents
A kind of composite quantum dot encoded microsphere based on natural prickly pollen and preparation method thereof Download PDFInfo
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Abstract
本发明公开了一种基于天然多刺状花粉的复合量子点编码微球及其制备方法,该制备方法以天然多刺状花粉为编码基底,表面逐层修饰特征发射峰位置和强度不同的量子点;花粉基底经过炭化处理消除自发荧光后,多孔和刺状表面结构提供了高孔隙率和较大比表面积,可增加目标分子的吸附量进而显著增强检测荧光信号,提高检测灵敏度;采用聚电解质逐层沉积技术,使花粉基底外表面逐层吸附不同层数、不同种类或不同大小的量子点,简单易行,能够均匀地结合多种类型量子点,编码稳定性好;采用不同种类和不同层数量子点的组合可实现多重特征发射峰和强度的编码形式,扩充编码量,同时可对编码微球荧光编码信息精确控制,满足多指标和高通量生物检测的需要。
The invention discloses a composite quantum dot encoding microsphere based on natural prickly pollen and a preparation method thereof. The preparation method uses the natural prickly pollen as an encoding substrate, and the surface is modified layer by layer with quantum dots with different characteristic emission peak positions and intensities. Point; after the pollen substrate is carbonized to eliminate autofluorescence, the porous and thorn-like surface structure provides high porosity and large specific surface area, which can increase the adsorption of target molecules and significantly enhance the detection fluorescence signal and improve detection sensitivity; polyelectrolyte is used The layer-by-layer deposition technology enables the outer surface of the pollen substrate to adsorb different layers, types or sizes of quantum dots layer by layer. The combination of layers of quantum dots can realize the coding form of multiple characteristic emission peaks and intensities, expand the coding amount, and at the same time can precisely control the fluorescent coding information of the coded microspheres to meet the needs of multi-index and high-throughput biological detection.
Description
技术领域technical field
本发明涉及生物材料技术领域,涉及一种以天然多刺状花粉作为编码基底的量子点编码微球及其制备方法,特别涉及一种基于天然多刺状花粉的复合量子点编码微球及其制备方法。The invention relates to the technical field of biological materials, relates to a quantum dot encoded microsphere using natural prickly pollen as a coding substrate and a preparation method thereof, in particular to a composite quantum dot encoded microsphere based on natural prickly pollen and the same Preparation.
背景技术Background technique
为了研究大量的分子间相互作用,如蛋白与核酸之间、蛋白质与蛋白质之间以及蛋白质与药物之间的作用,高通量的试验平台相继出现,高通量的试验平台要求有高通量的分子载体,即所谓的编码载体,来标识不同的分子以及分子间发生的相互作用,随后通过读取相应编码信息进行结果分析。常见的编码载体中,阵列式生物芯片利用分子固定的不同位置也就是坐标作为编码来区别不同的生物分子,但是由于阵列式生物芯片的坐标只能固定在二维空间内,因此限制了识别反应的速度及其应用。而微球作为一种新型的固相编码载体,已经成为高通量筛选以及组合化学等领域关注的热点。相对于其他形式的固相载体,微球具有显著的优势:第一,微球的比表面积大,能够使表面化学反应在更小的体积内进行;第二,采用微球作为载体可以结合其他辅助手段如搅拌、液体冲刷等实现一种介于固-液反应和液-液反应之间的反应体系,从而加快体系反应速度;第三,微球表面结合的分子在反应完成后可以方便的从溶液中分离出来;第四,在微球表面修饰多样化的功能基团,可以大大扩展微球的用途。以上这些优势使得编码微球在防伪、生物分析、环境监测等领域发挥着作用。In order to study a large number of intermolecular interactions, such as the interaction between proteins and nucleic acids, between proteins and proteins, and between proteins and drugs, high-throughput test platforms have emerged one after another, and high-throughput test platforms require high-throughput The molecular carrier, the so-called coding carrier, is used to identify different molecules and the interactions that occur between them, and then analyze the results by reading the corresponding coding information. Among the common encoding carriers, the array biochip uses the fixed positions of the molecules, that is, the coordinates, as codes to distinguish different biomolecules. However, because the coordinates of the array biochip can only be fixed in a two-dimensional space, the recognition reaction is limited. speed and its applications. As a new type of solid-phase encoding carrier, microspheres have become a hot spot in the fields of high-throughput screening and combinatorial chemistry. Compared with other forms of solid support, microspheres have significant advantages: first, the specific surface area of microspheres is large, which enables surface chemical reactions to be carried out in a smaller volume; second, the use of microspheres as a carrier can combine other Auxiliary means such as stirring and liquid scouring can realize a reaction system between solid-liquid reaction and liquid-liquid reaction, thereby speeding up the reaction speed of the system; third, the molecules bound on the surface of the microspheres can be easily It can be separated from the solution; fourthly, the modification of diverse functional groups on the surface of the microspheres can greatly expand the use of the microspheres. These advantages make encoded microspheres play a role in anti-counterfeiting, biological analysis, environmental monitoring and other fields.
目前编码微球的制造方法主要包括化学合成、模板法、光刻以及微流控技术等。这些方法获得的微球往往呈现出相对简单的球形和平滑的表面结构。复杂表面形貌的缺失,大大降低了在依赖于结构衍生的活性位点上进行的化学反应或光电相互作用的效率,也阻碍了多种物理化学特性和功能与编码载体的整合。此外,这些制造方法大多涉及精密加工技术,设备复杂,费时费力且成本高昂,因而限制了编码微球的大规模生产和广泛使用。At present, the fabrication methods of encoded microspheres mainly include chemical synthesis, template method, photolithography and microfluidic technology. The microspheres obtained by these methods tend to exhibit relatively simple spherical and smooth surface structures. The absence of complex surface topography greatly reduces the efficiency of chemical reactions or optoelectronic interactions at structure-derived active sites, and also hinders the integration of multiple physicochemical properties and functions into encoding vectors. In addition, most of these fabrication methods involve precision machining techniques, complex equipment, time-consuming, labor-intensive, and high-cost, thus limiting the large-scale production and widespread use of encoded microspheres.
自然界为人类提供了丰富的材料,这些材料经过亿万年的进化改造,被赋予了精细复杂的微观结构和非凡的功能,直接利用天然材料省去了人工制备的繁琐过程。花粉是有花植物雄蕊中的生殖细胞,外观呈粉末状,花粉粒是微米级的颗粒,具有高度单分散的粒度分布,其精美的多刺表面结构,即使在苛刻的自然条件下也具有持久的坚固性。此外,天然多刺状花粉数量庞大,极易获取,因此利用花粉作为编码载体的基底材料也能够显著减少生产成本。Nature provides human beings with abundant materials. These materials have been endowed with delicate and complex microstructures and extraordinary functions after billions of years of evolutionary transformation. The direct use of natural materials saves the tedious process of artificial preparation. Pollen is the germ cell in the stamens of flowering plants, powdery in appearance, pollen grains are micron-sized particles with a highly monodisperse particle size distribution, and their exquisite spiny surface structure is durable even under harsh natural conditions sturdiness. In addition, the natural thorny pollen is very abundant and easy to obtain, so the use of pollen as the base material of the encoding carrier can also significantly reduce the production cost.
与传统的有机荧光分子相比,量子点具有明显的优越性:激发光谱宽,发射光谱呈对称分布且宽度窄,颜色可调,光化学稳定性高,不易光解。量子点有连续的激发光谱,而且可以通过调节晶体颗粒的大小以得到连续发射光谱。量子点的发射光谱很窄,仅用一种波长的光就可以同时激发多种大小的量子点,因此用最小的光学带隙就可以得到多种颜色的发射光。基于量子点的编码优势,将不同荧光颜色和荧光强度的半导体纳米晶体发光量子点与天然多刺状花粉基底相结合,可以建立用于多元生物检测的理想编码微载体。Compared with traditional organic fluorescent molecules, quantum dots have obvious advantages: wide excitation spectrum, symmetrical distribution of emission spectrum and narrow width, adjustable color, high photochemical stability, and not easy to photolysis. Quantum dots have a continuous excitation spectrum, and the size of the crystal particles can be adjusted to obtain a continuous emission spectrum. The emission spectrum of quantum dots is very narrow, and only one wavelength of light can simultaneously excite quantum dots of various sizes, so multiple colors of emitted light can be obtained with the smallest optical band gap. Based on the coding advantages of quantum dots, combining semiconductor nanocrystal luminescent quantum dots with different fluorescence colors and fluorescence intensities with natural prickly pollen substrates can establish ideal coding microcarriers for multiplex biological detection.
因此,在本发明中,我们采用天然多刺状花粉作为编码载体,在其上逐层修饰量子点,设计发明了一种可同时测定多靶标的复合量子点编码微球,可用于生物检测分析。Therefore, in the present invention, we use natural prickly pollen as an encoding carrier, and modify quantum dots on it layer by layer, and design and invent a composite quantum dot encoded microsphere that can simultaneously measure multiple targets, which can be used for biological detection and analysis. .
发明内容SUMMARY OF THE INVENTION
本发明所要解决的技术问题是针对上述现有技术的不足,提供一种基于天然多刺状花粉的复合量子点编码微球及其制备方法,该方法采用天然多刺状花粉作为编码载体,在其上逐层修饰量子点,制备出具有独特形态和多重编码组合的微球,解决了传统编码微球结构简单、制备复杂、编码量不足的缺点。The technical problem to be solved by the present invention is to aim at the deficiencies of the above-mentioned prior art, and to provide a composite quantum dot encoded microsphere based on natural prickly pollen and a preparation method thereof. The method adopts the natural prickly pollen as an encoding carrier, and The quantum dots are modified layer by layer on it to prepare microspheres with unique morphology and multiple coding combinations, which solves the shortcomings of the traditional coding microspheres with simple structure, complicated preparation and insufficient coding quantity.
为实现上述技术目的,本发明采取的技术方案为:一种基于天然多刺状花粉的复合量子点编码微球的制备方法,包括如下步骤:In order to achieve the above-mentioned technical purpose, the technical solution adopted in the present invention is: a preparation method of composite quantum dot encoded microspheres based on natural prickly pollen, comprising the following steps:
(1)对多刺状花粉进行分散筛选,对筛选的花粉粒进行炭化处理,以获得消除自发荧光后的花粉基底;(1) Disperse and screen the thorny pollen, and carbonize the screened pollen grains to obtain the pollen base after eliminating autofluorescence;
(2)将花粉基底依次浸泡在负聚电解质溶液、正聚电解质溶液中,得到表面带正电的微球基底,利用聚电解质逐层沉积技术,在带正电的微球基底外表面逐层吸附不同层数、不同种类或不同大小的量子点,从而获得以天然多刺状花粉为基底的复合量子点编码微球。(2) The pollen substrate is soaked in the negative polyelectrolyte solution and the positive polyelectrolyte solution in turn to obtain a microsphere substrate with a positively charged surface. Using the polyelectrolyte layer-by-layer deposition technology, the outer surface of the positively charged microsphere substrate is layered layer by layer. Quantum dots with different layers, different types or sizes are adsorbed to obtain composite quantum dot-encoded microspheres based on natural prickly pollen.
进一步地,步骤(1)中,将多刺状花粉浸泡在无水乙醇中震荡,获得花粉粒充分剥离的花粉分散液,对花粉分散液进行干燥处理,以分散筛选出颗粒分明的干燥花粉粒,干燥花粉粒在300℃高温下煅烧并持续通入氮气12小时,使荧光有机物质彻底炭化,以消除自发荧光。Further, in step (1), the thorny pollen is soaked in absolute ethanol and shaken to obtain a pollen dispersion liquid with the pollen grains fully peeled off, and the pollen dispersion liquid is dried to disperse and screen out dry pollen grains with distinct particles. , the dried pollen grains were calcined at a high temperature of 300 °C and nitrogen gas was continuously introduced for 12 hours to completely carbonize the fluorescent organic substances to eliminate autofluorescence.
进一步地,所述的多刺状花粉选自向日葵花粉、豚草花粉、菊花花粉中的一种。Further, the spiny pollen is selected from the group consisting of sunflower pollen, ragweed pollen and chrysanthemum pollen.
进一步地,所述正聚电解质选自聚丙烯胺盐酸盐(PAH)、聚乙烯亚胺(PEI)中的一种,所述负聚电解质选自聚苯乙烯磺酸钠(PSS)、聚丙烯酸(PAA)中的一种。Further, the positive polyelectrolyte is selected from one of polyacrylamine hydrochloride (PAH) and polyethyleneimine (PEI), and the negative polyelectrolyte is selected from polystyrene sulfonate (PSS), polystyrene One of Acrylic Acid (PAA).
进一步地,所述正负聚电解质溶液或负聚电解质溶液的配制方法为:将正聚电解质或负聚电解质固体溶解在0.5mol/L NaCl溶液中,最终获得浓度为1mg/mL的正聚电解质溶液或负聚电解质溶液。Further, the preparation method of the positive and negative polyelectrolyte solution or the negative polyelectrolyte solution is: dissolving the positive polyelectrolyte or the negative polyelectrolyte solid in a 0.5mol/L NaCl solution to finally obtain a positive polyelectrolyte with a concentration of 1 mg/mL solution or negative polyelectrolyte solution.
进一步地,步骤(2)中,通过带正电的微球基底浸泡在带负电的量子点溶液中,该量子点吸附在微球基底的外表面,再通过正-负-正聚电解质层(正聚电解质溶液、负聚电解质溶液、正聚电解质溶液依次吸附形成的正-负-正聚电解质层,该正-负-正聚电解质层将各层量子点相隔开)与带负电量子点之间的静电作用力逐层吸附不同层数、不同种类或不同大小的量子点。Further, in step (2), the positively charged microsphere substrate is immersed in the negatively charged quantum dot solution, the quantum dots are adsorbed on the outer surface of the microsphere substrate, and then the positive-negative-positive polyelectrolyte layer ( The positive-negative-positive polyelectrolyte layer formed by the sequential adsorption of positive polyelectrolyte solution, negative polyelectrolyte solution, and positive polyelectrolyte solution, the positive-negative-positive polyelectrolyte layer separates each layer of quantum dots) and negatively charged quantum dots The electrostatic force between them adsorbs quantum dots of different layers, types or sizes layer by layer.
进一步地,步骤(2)中,所述量子点选自硫化镉(CdS),硒化镉(CdSe),碲化镉(CdTe)、硫化锌包硒化镉(CdSe@ZnS)量子点中的一种。Further, in step (2), the quantum dots are selected from cadmium sulfide (CdS), cadmium selenide (CdSe), cadmium telluride (CdTe), and cadmium selenide on zinc sulfide (CdSe@ZnS) quantum dots. A sort of.
进一步地,步骤(2)中,在最外层的量子点外继续沉积一层正聚电解质,以便于后续检测分子的接枝,所述检测分子选自DNA,RNA,蛋白质中的一种。Further, in step (2), a layer of positive polyelectrolyte is continuously deposited outside the outermost quantum dots to facilitate the grafting of subsequent detection molecules, wherein the detection molecules are selected from one of DNA, RNA and protein.
本发明还提供了一种基于天然多刺状花粉的复合量子点编码微球,采用上述制备方法制得,所述复合量子点编码微球以天然多刺状花粉作为基底,表面逐层吸附不同层数、不同种类或不同大小的量子点。The invention also provides a composite quantum dot encoded microsphere based on natural prickly pollen, which is prepared by the above preparation method. Number of layers, different kinds or different sizes of quantum dots.
本发明还提供了一种基于天然多刺状花粉的混合型复合量子点编码微球,所述混合型复合量子点编码微球由不同特异性的复合量子点编码微球混合而成,所述复合量子点编码微球采用上述制备方法制得,以天然多刺状花粉作为基底,表面逐层吸附不同层数、不同种类或不同大小的量子点。The present invention also provides a hybrid composite quantum dot encoded microsphere based on natural prickly pollen, the hybrid composite quantum dot encoded microsphere is formed by mixing composite quantum dot encoded microspheres with different specificities, and the The composite quantum dot-encoded microsphere is prepared by the above preparation method, and uses natural prickly pollen as a substrate, and the surface adsorbs quantum dots of different layers, types or sizes layer by layer.
与现有技术相比,本发明的有益效果在于:Compared with the prior art, the beneficial effects of the present invention are:
1)本发明采用单分散性良好的天然多刺状花粉作为编码基底,其独特复杂的多孔和刺状表面结构提供了高孔隙率和较大比表面积,可增加目标分子的吸附量进而显著增强检测荧光信号,大大降低检测限,提高检测灵敏度;同时天然多刺状花粉如向日葵花粉来源极其广泛,数量庞大,可用于大规模量产并大大降低成本;1) The present invention uses natural thorny pollen with good monodispersity as the coding substrate, and its unique and complex porous and thorn-like surface structure provides high porosity and large specific surface area, which can increase the adsorption capacity of target molecules and thus significantly enhance the Detecting fluorescent signals, greatly reducing the detection limit and improving detection sensitivity; at the same time, natural prickly pollen, such as sunflower pollen, comes from a wide variety of sources and is available in large quantities, which can be used for large-scale mass production and greatly reduce costs;
2)本发明采用聚电解质逐层吸附的方法,使花粉基底外表面逐层吸附不同层数、不同种类或不同大小的量子点,简单易行,能够均匀地结合多种类型量子点,制备的复合量子点编码微球所用编码稳定性好,量子点激发光谱宽,发射光谱呈对称分布且宽度窄,在应用过程中始终保持稳定的编码,并且容易对所得编码微球的荧光特征峰和强度进行精确控制;2) The present invention adopts the layer-by-layer adsorption method of polyelectrolyte, so that the outer surface of the pollen substrate can adsorb different layers, different types or different sizes of quantum dots layer by layer. The coding stability of the composite quantum dot coding microspheres is good, the excitation spectrum of the quantum dots is wide, the emission spectrum is symmetrically distributed and the width is narrow, and the coding is always stable during the application process, and the fluorescence characteristic peaks and intensity of the obtained coding microspheres are easily determined. precise control;
3)本发明采用不同种类和不同层数量子点的组合来实现多重特征发射峰和强度的编码形式,极大地扩充了编码量,并且制备的复合量子点编码微球解码方法简单,仅用一种波长的光就可以同时激发多种特征发射峰的量子点,解码时可同时获悉靶标分子的种类和浓度信息,检测过程简便快捷,检测时无交叉干扰,可满足同时检测多指标和高通量检测的需要;3) The present invention adopts the combination of different types and different layers of quantum dots to realize the coding form of multiple characteristic emission peaks and intensities, which greatly expands the coding amount, and the decoding method of the prepared composite quantum dot coding microspheres is simple, and only one Light of one wavelength can simultaneously excite quantum dots with various characteristic emission peaks, and the type and concentration information of the target molecule can be obtained at the same time during decoding. the need for quantitative testing;
4)本发明制备的复合量子点编码微球应用范围广,通过吸附聚电解质的方式能够实现多种功能基团的固定,从而提供与下游分子化学偶联的条件。4) The composite quantum dot-encoded microspheres prepared by the invention have a wide range of applications, and can realize the immobilization of various functional groups by adsorbing polyelectrolytes, thereby providing conditions for chemical coupling with downstream molecules.
附图说明Description of drawings
图1为本发明的基于天然多刺状花粉的复合量子点编码微球的制备方法流程图,其中,1为天然多刺状花粉,2为消除自发荧光后的花粉基底,3为量子点,4为制备的复合量子点编码微球;Fig. 1 is the flow chart of the preparation method of the composite quantum dot-encoded microspheres based on natural prickly pollen of the present invention, wherein, 1 is the natural prickly pollen, 2 is the pollen substrate after eliminating autofluorescence, and 3 is the quantum dots, 4 is the prepared composite quantum dot encoded microsphere;
图2为天然多刺状花粉和花粉基底的荧光表征,其中,图a为激光共聚焦荧光显微镜明场下拍摄的天然多刺状花粉图像,图b~d依次为紫外光、蓝光和绿光激发下天然多刺状花粉的自发荧光图像,图e为激光共聚焦荧光显微镜明场下拍摄的花粉基底图像,图f~h为天然多刺状花粉炭化处理后的花粉基底依次在紫外光、蓝光和绿光激发下的荧光图像;Figure 2 shows the fluorescence characterization of natural prickly pollen and pollen substrate, in which, Figure a is the image of natural prickly pollen taken in bright field by confocal fluorescence microscope, and Figures b to d are ultraviolet light, blue light and green light in order Autofluorescence images of natural prickly pollen under excitation. Figure e is the image of the pollen substrate taken in bright field by confocal fluorescence microscope. Figures f-h are the pollen substrate after carbonization of natural prickly pollen. Fluorescence images under blue and green excitation;
图3为本发明的基于向日葵花粉的复合量子点编码微球的发射光谱图,其中,图A为实施例1制备的基于向日葵花粉的复合量子点编码微球(微球编码为CdTe 465∶CdTe 513∶CdTe 626= 1:2:3)及其与层数对应的量子点(一层CdTe 465、两层CdTe 513、三层CdTe626)的发射光谱图,a1为实施例1制备的基于向日葵花粉的复合量子点编码微球的荧光发射光谱图,b1为量子点一层CdTe 465的荧光发射光谱图,c1为两层量子点CdTe 513的荧光发射光谱图,d1为三层量子点CdTe 626的荧光发射光谱图,图B为实施例2制备的基于豚草花粉的复合量子点编码微球(微球编码为CdSe 460∶CdSe 525∶CdSe 620= 1:2:1)及其与层数对应的量子点(一层CdSe 460、两层CdSe 525、一层CdSe 620)的发射光谱图,a2为实施例2制备的基于豚草花粉的复合量子点编码微球的荧光发射光谱图,b2为一层量子点CdSe460的荧光发射光谱图,c2为两层量子点CdSe 525的荧光发射光谱图,d2为一层量子点CdSe620的荧光发射光谱图;3 is the emission spectrum of the sunflower pollen-based composite quantum dot-encoded microspheres of the present invention, wherein Figure A is the sunflower pollen-based composite quantum dot-encoded microspheres prepared in Example 1 (the microspheres are coded as CdTe 465:CdTe 513:CdTe 626= 1:2:3) and the quantum dots corresponding to the number of layers (one layer of CdTe 465, two layers of CdTe 513, three layers of CdTe626) emission spectra, a1 is prepared in Example 1 based on sunflower pollen The fluorescence emission spectrum of the composite quantum dot encoded microsphere, b1 is the fluorescence emission spectrum of one layer of quantum dots CdTe 465, c1 is the fluorescence emission spectrum of two layers of quantum dots CdTe 513, d1 is the fluorescence emission spectrum of three layers of quantum dots CdTe 626 Fluorescence emission spectrum, Figure B shows the composite quantum dot-encoded microspheres based on ragweed pollen prepared in Example 2 (the microspheres are coded as CdSe 460:CdSe 525:CdSe 620=1:2:1) and their corresponding layers The emission spectrum of the quantum dots (one layer of
图4为实施例4中干燥向日葵花粉粒和基于向日葵花粉的复合量子点编码微球(编码为CdSe 620=1)的体式显微镜和场发射扫描电子显微镜的表征图像,其中,图a为干燥向日葵花粉粒的体式显微镜下图像,图b、c为干燥向日葵花粉粒的扫描电镜图;图d为基于向日葵花粉的复合量子点编码微球(编码为CdSe 620=1)的体式显微镜下图像,图e、f为基于向日葵花粉的复合量子点编码微球(编码为CdSe 620=1)的扫描电镜图;Figure 4 is the stereoscopic microscope and field emission scanning electron microscope characterization images of dried sunflower pollen grains and sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 620=1) in Example 4, wherein, Figure a is a dried sunflower The stereomicroscope images of pollen grains, Figures b and c are SEM images of dried sunflower pollen grains; Figure d is the stereomicroscope images of sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 620=1), Figure d e and f are SEM images of composite quantum dot-encoded microspheres (coded as CdSe 620=1) based on sunflower pollen;
图5为实施例4中激光扫描共聚焦显微镜扫描的基于向日葵花粉的复合量子点编码微球(编码为CdSe 620=1)不同断层的荧光图像;5 is a fluorescence image of different slices of sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 620=1) scanned by a laser scanning confocal microscope in Example 4;
图6为实施例4中基于向日葵花粉的复合量子点编码微球(编码为CdSe 460=1)、基于向日葵花粉的复合量子点编码微球(编码为CdSe 525=1)、基于向日葵花粉的复合量子点编码微球(编码为CdSe 620=1)在激光扫描共聚焦荧光显微镜下的荧光表征图像,其中,图6a~c为不同倍率下基于向日葵花粉的复合蓝色量子点编码微球(编码为CdSe 460=1)的荧光图像,图6d~f为不同倍率下基于向日葵花粉的复合绿色量子点编码微球(编码为CdSe 525=1)的荧光图像,图6g~i为不同倍率下基于向日葵花粉的复合红色量子点编码微球(编码为CdSe 620=1)的荧光图像;Figure 6 shows the composite quantum dot-encoded microspheres based on sunflower pollen (encoded as CdSe 460=1), the composite quantum dot-encoded microspheres based on sunflower pollen (encoded as
图7为实施例4中紫外光源同时激发下的基于向日葵花粉的混合型复合量子点编码微球(编码为CdSe 620=1,CdSe 525=1,CdSe 460=1)的荧光图像,其中,CdSe 620指代基于向日葵花粉的复合量子点编码微球(编码为CdSe 620=1),CdSe 525指代基于向日葵花粉的复合量子点编码微球(编码为CdSe 525=1),CdSe 460指代基于向日葵花粉的复合量子点编码微球(编码为CdSe 460=1);7 is a fluorescence image of the hybrid composite quantum dot-encoded microspheres based on sunflower pollen (encoded as CdSe 620=1,
图8为实施例5中制备的复合量子点编码微球的生物检测应用过程示意图,其中1为制备得到的复合量子点编码微球,2为探针DNA,3为靶标DNA,4为修饰绿色荧光染料羧酸荧光素(FAM,特征峰位置为520 nm)的标记DNA;8 is a schematic diagram of the biological detection application process of the composite quantum dot-encoded microspheres prepared in Example 5, wherein 1 is the prepared composite quantum dot-encoded microspheres, 2 is probe DNA, 3 is target DNA, and 4 is modified green Labeled DNA with the fluorescent dye fluorescein carboxylate (FAM, characteristic peak position at 520 nm);
图9为实例5中基于向日葵花粉的复合量子点编码微球(编码为CdSe 460=1)和传统生物检测产品玻璃珠(Glass Beads)用于DNA检测时,检测到的FAM的荧光强度与标靶DNA浓度的关系曲线图,图i和ii分别是表面结合有修饰FAM条件下的杂交夹心结构DNA的基于向日葵花粉的复合量子点编码微球(编码为CdSe 460=1)和传统生物检测产品玻璃珠(GlassBeads)在DNA杂交测定实验时的荧光图像;Figure 9 shows the fluorescence intensity of the detected FAM and the standard when the sunflower pollen-based composite quantum dot-encoded microspheres (coded as
图10为实例5中三种基于向日葵花粉的复合量子点编码微球(编码分别为CdSe 460∶CdSe 620=2:1,CdSe 460∶CdSe 620=1:1,CdSe 460∶CdSe 620=1:2)用于多重、同时DNA检测得到的荧光光谱图,其中,图a为标记DNA未修饰FAM条件下无靶标信号只有基于向日葵花粉的复合量子点编码微球(编码为CdSe 460∶CdSe 620=2:1)信号的发射荧光图谱,图b~d为表面结合有修饰FAM条件下的杂交夹心结构DNA的三种基于向日葵花粉的复合量子点编码微球(编码分别为CdSe 460∶CdSe 620=2:1,CdSe 460∶CdSe 620=1:1,CdSe 460∶CdSe 620=1:2)的发射荧光图谱;Figure 10 shows three kinds of composite quantum dot-encoded microspheres based on sunflower pollen in Example 5 (the codes are CdSe 460:CdSe 620=2:1, CdSe 460:CdSe 620=1:1, CdSe 460:CdSe 620=1:1: 2) Fluorescence spectra obtained for multiplexed and simultaneous DNA detection, among which, Figure a is a composite quantum dot-encoded microsphere based on sunflower pollen (encoded as CdSe 460:CdSe 620= 2:1) The emission fluorescence spectrum of the signal, Figures b~d are three kinds of sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 460:CdSe 620= 2:1, CdSe 460:CdSe 620=1:1, CdSe 460:CdSe 620=1:2) emission fluorescence spectrum;
图11为实施例6中天然向日葵花粉和基于向日葵花粉的复合量子点编码微球(编码为CdSe@ZnS 565=1)的荧光发射光谱;Figure 11 is the fluorescence emission spectrum of natural sunflower pollen and sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe@ZnS 565=1) in Example 6;
图12为实施例5中结合DNA后的基于向日葵花粉的复合量子点编码微球(编码为CdSe460=1)的荧光强度随时间变化的曲线图。12 is a graph showing the change of fluorescence intensity with time of the sunflower pollen-based composite quantum dot-encoded microspheres (encoded as CdSe460=1) after DNA binding in Example 5.
具体实施方式Detailed ways
为了使本领域技术领域人员更好地理解本发明的技术方案,下面结合附图对本发明的实施例作进一步详细描述。In order to make those skilled in the art better understand the technical solutions of the present invention, the embodiments of the present invention are described in further detail below with reference to the accompanying drawings.
下述实施例中所使用的实验方法,如无特殊说明,均为常规方法,所用的试剂、方法和设备,如无特殊说明,均为本技术领域常规试剂、方法和设备。The experimental methods used in the following examples, unless otherwise specified, are conventional methods, and the used reagents, methods and equipment, unless otherwise specified, are conventional reagents, methods and equipment in the technical field.
本发明提供了基于天然多刺状花粉的复合量子点编码微球的制备方法,工艺流程如图1所示,包括如下步骤:The present invention provides a method for preparing composite quantum dot-encoded microspheres based on natural prickly pollen. The process flow is shown in Figure 1, and includes the following steps:
(1)将多刺状花粉浸泡在无水乙醇中震荡,获得花粉粒充分剥离的花粉分散液,对花粉分散液进行干燥处理,以分散筛选出颗粒分明的干燥花粉粒,将干燥花粉粒在300℃高温下煅烧并持续通入氮气12小时,使荧光有机物质彻底炭化,以消除自发荧光;(1) Soak the thorny pollen in absolute ethanol and shake to obtain a pollen dispersion with fully peeled pollen grains. The pollen dispersion is dried to disperse and screen out dry pollen grains with distinct particles. Calcined at a high temperature of 300 °C and continuously passed nitrogen for 12 hours to completely carbonize the fluorescent organic substances to eliminate autofluorescence;
(2)将花粉基底依次浸泡在负聚电解质溶液、正聚电解质溶液中,得到表面带正电的微球基底,利用聚电解质逐层沉积技术,在带正电的微球基底外表面逐层吸附不同层数、不同种类或不同大小的量子点,从而获得以天然多刺状花粉为基底的复合量子点编码微球。(2) The pollen substrate is soaked in the negative polyelectrolyte solution and the positive polyelectrolyte solution in turn to obtain a microsphere substrate with a positively charged surface. Using the polyelectrolyte layer-by-layer deposition technology, the outer surface of the positively charged microsphere substrate is layered layer by layer. Quantum dots with different layers, different types or sizes are adsorbed to obtain composite quantum dot-encoded microspheres based on natural prickly pollen.
其中,多刺状花粉选自向日葵花粉、豚草花粉、菊花花粉中的一种。Wherein, the spiny pollen is selected from the group consisting of sunflower pollen, ragweed pollen and chrysanthemum pollen.
其中,正聚电解质选自聚丙烯胺盐酸盐(PAH)、聚乙烯亚胺(PEI)中的一种。Wherein, the positive polyelectrolyte is selected from one of polyacrylamine hydrochloride (PAH) and polyethyleneimine (PEI).
其中,负聚电解质选自聚苯乙烯磺酸钠(PSS)、聚丙烯酸(PAA)中的一种。Wherein, the negative polyelectrolyte is selected from one of polystyrene sulfonate (PSS) and polyacrylic acid (PAA).
进一步地,正负聚电解质溶液或负聚电解质溶液的配制方法为:将正聚电解质或负聚电解质固体溶解在0.5mol/L NaCl溶液中,最终获得浓度为1mg/mL的正聚电解质溶液或负聚电解质溶液。Further, the preparation method of the positive and negative polyelectrolyte solution or the negative polyelectrolyte solution is: dissolving the positive polyelectrolyte or the negative polyelectrolyte solid in a 0.5mol/L NaCl solution to finally obtain a positive polyelectrolyte solution with a concentration of 1 mg/mL or Negative polyelectrolyte solution.
进一步地,步骤(2)中,通过带正电的微球基底浸泡在带负电的量子点溶液中,该量子点吸附在微球基底的外表面,再通过正-负-正聚电解质层(正聚电解质溶液、负聚电解质溶液、正聚电解质溶液依次吸附形成的聚电解质层,该聚电解质层将各层量子点相隔开)与带负电量子点之间的静电作用力逐层吸附不同层数、不同种类或不同大小的量子点。Further, in step (2), the positively charged microsphere substrate is immersed in the negatively charged quantum dot solution, the quantum dots are adsorbed on the outer surface of the microsphere substrate, and then the positive-negative-positive polyelectrolyte layer ( The polyelectrolyte layer formed by the sequential adsorption of positive polyelectrolyte solution, negative polyelectrolyte solution and positive polyelectrolyte solution, the polyelectrolyte layer separates each layer of quantum dots) and the electrostatic force between the negatively charged quantum dots are different layer by layer adsorption Number of layers, different kinds or different sizes of quantum dots.
进一步地,量子点选自硫化镉(CdS),硒化镉(CdSe),碲化镉(CdTe)、硫化锌包硒化镉(CdSe@ZnS)量子点中的一种。Further, the quantum dots are selected from one of cadmium sulfide (CdS), cadmium selenide (CdSe), cadmium telluride (CdTe), and cadmium selenide in zinc sulfide (CdSe@ZnS) quantum dots.
进一步地,步骤(2)中,在最外层的量子点外继续沉积一层正聚电解质,以便于后续检测分子的接枝,所述后续检测分子选自DNA,RNA,蛋白质中的一种。Further, in step (2), a layer of positive polyelectrolyte is continued to be deposited outside the outermost quantum dots, so as to facilitate the grafting of subsequent detection molecules, and the subsequent detection molecules are selected from one of DNA, RNA, and protein. .
本发明提供的基于天然多刺状花粉的复合量子点编码微球的制备方法以天然多刺状花粉作为编码基底,材料来源广泛,可用于大规模量产并大大降低成本,采用聚电解质逐层吸附的方法,使花粉基底外表面逐层吸附不同层数、不同种类或不同大小的量子点,简单易行,能够均匀地结合多种类型量子点,制备的复合量子点编码微球所用编码稳定性好,采用不同种类和不同层数量子点的组合来实现多重特征发射峰和强度的编码形式,极大地扩充了编码量。The preparation method of composite quantum dot encoded microspheres based on natural prickly pollen provided by the present invention uses natural prickly pollen as an encoding substrate, has a wide range of material sources, can be used for large-scale mass production and greatly reduces costs, and adopts polyelectrolyte layer by layer. The adsorption method enables the outer surface of the pollen substrate to adsorb quantum dots of different layers, types or sizes layer by layer, which is simple and easy to implement, and can evenly combine various types of quantum dots. It has good performance, and adopts the combination of different types and different layers of quantum points to realize the encoding form of multiple characteristic emission peaks and intensities, which greatly expands the amount of encoding.
采用上述制备方法可制备得到基于天然多刺状花粉的复合量子点编码微球,该编码微球以天然多刺状花粉作为基底,表面逐层吸附不同层数、不同种类或不同大小的量子点。Using the above preparation method, composite quantum dot encoded microspheres based on natural prickly pollen can be prepared. The encoded microspheres use natural prickly pollen as a base, and the surface adsorbs quantum dots of different layers, types or sizes layer by layer. .
采用上述制备方法还可制备得到基于天然多刺状花粉的混合型复合量子点编码微球,其由不同特异性的复合编码微球混合而成,复合编码微球以天然多刺状花粉作为基底,表面逐层吸附不同层数、不同种类或不同大小的量子点。The above preparation method can also be used to prepare hybrid composite quantum dot-encoded microspheres based on natural prickly pollen, which are formed by mixing composite encoded microspheres with different specificities, and the composite encoded microspheres use natural prickly pollen as a substrate , the surface adsorbs quantum dots of different layers, types or sizes layer by layer.
上述基于天然多刺状花粉的复合量子点编码微球或混合型复合量子点编码微球以多刺状花粉作为编码基底,其多孔和刺状表面结构具有荧光增强效益,提高检测灵敏度,花粉基底表面逐层吸附的量子点采用不同种类和不同层数量子点的组合,极大地扩充了编码量,且所用编码稳定性好,复合量子点编码微球解码方法简单,仅用一种波长的光就可以同时激发多种特征发射峰的量子点,解码时可同时获悉靶标分子的种类和浓度信息,检测过程简便快捷,检测时无交叉干扰,可满足同时检测多指标和高通量检测的需要。The above-mentioned composite quantum dot-encoded microspheres or hybrid composite quantum dot-encoded microspheres based on natural prickly pollen use prickly pollen as an encoding substrate, and its porous and prickly surface structure has the benefit of fluorescence enhancement, improving detection sensitivity, and the pollen substrate The quantum dots adsorbed layer by layer on the surface use a combination of different types and layers of quantum dots, which greatly expands the amount of coding, and the coding stability used is good. The decoding method of composite quantum dot-coded microspheres is simple, and only one wavelength of light is used. The quantum dots with a variety of characteristic emission peaks can be excited at the same time, and the type and concentration information of the target molecule can be obtained at the same time when decoding. .
以下为实施例:The following are examples:
实施例1Example 1
一种基于向日葵花粉的复合量子点编码微球,通过以下步骤制备:A composite quantum dot-encoded microsphere based on sunflower pollen is prepared by the following steps:
(1)炭化处理制备消除自发荧光后的向日葵花粉基底(1) Preparation of sunflower pollen substrate after eliminating autofluorescence by carbonization
将向日葵花粉浸泡在无水乙醇中保持震荡20分钟,获得花粉粒充分剥离的分散液,将分散液置于热台上100℃加热至乙醇完全挥发,花粉处于干燥状态,筛选出颗粒分明的干燥向日葵花粉粒;将干燥向日葵花粉粒置于马弗炉中,300℃高温下煅烧并持续通入氮气12小时,得到荧光有机物质彻底炭化后的带正电向日葵花粉基底,如图1所示;Soak the sunflower pollen in absolute ethanol and keep shaking for 20 minutes to obtain a dispersion with fully peeled pollen grains. The dispersion is placed on a hot table and heated at 100 ° C until the ethanol is completely volatilized, and the pollen is in a dry state. Sunflower pollen grains; the dried sunflower pollen grains are placed in a muffle furnace, calcined at a high temperature of 300°C and continuously fed with nitrogen for 12 hours, to obtain a positively charged sunflower pollen substrate after complete carbonization of fluorescent organic substances, as shown in Figure 1;
对天然向日葵花粉和经炭化处理后的向日葵花粉基底使用激光扫描共聚焦荧光显微镜进行荧光表征,图2为天然向日葵花粉和向日葵花粉基底的荧光表征,其中,图a为激光共聚焦荧光显微镜明场下拍摄的天然多刺状花粉图像,图b~d依次为紫外光、蓝光和绿光激发下天然多刺状花粉的自发荧光图像,图e为激光共聚焦荧光显微镜明场下拍摄的花粉基底图像,图f~h为天然多刺状花粉炭化处理后的花粉基底依次在紫外光、蓝光和绿光激发下的荧光图像,对比天然向日葵花粉和向日葵花粉基底的荧光图像可见,经炭化处理后的向日葵花粉基底刺状结构存留度较好,且自发荧光已全部去除;应用动态光散射仪测量向日葵花粉基底的Zeta电位,结果显示制备的向日葵花粉基底表面带正电荷,电位均值为+27.1mV;The natural sunflower pollen and the carbonized sunflower pollen substrate were characterized by laser scanning confocal fluorescence microscopy. Figure 2 shows the fluorescence characterization of the natural sunflower pollen and the sunflower pollen substrate. Figure a is the bright field of the laser confocal fluorescence microscope. The images of natural spiny pollen taken under the following conditions, Figures b-d are the autofluorescence images of natural spiny pollen under the excitation of ultraviolet light, blue light and green light in turn, and Figure e is the pollen substrate taken under the bright field of confocal fluorescence microscope. Images, Figures f-h are the fluorescence images of the pollen substrate after carbonization treatment of natural thorny pollen under the excitation of ultraviolet light, blue light and green light in turn. Comparing the fluorescence images of natural sunflower pollen and sunflower pollen substrate, it can be seen that after carbonization treatment The thorn-like structure of the sunflower pollen substrate was well preserved, and the autofluorescence had been completely removed; the Zeta potential of the sunflower pollen substrate was measured by dynamic light scattering, and the results showed that the surface of the prepared sunflower pollen substrate was positively charged, with an average potential of +27.1mV. ;
(2)采用聚电解质逐层吸附法制备基于向日葵花粉的复合量子点编码微球(2) Preparation of composite quantum dot-encoded microspheres based on sunflower pollen by layer-by-layer adsorption of polyelectrolytes
将PAH、PSS固体颗粒分别溶解0.5mol/L NaCl溶液中,配制得到浓度为1 mg/mL的PAH溶液和PSS溶液;将上述消除自发荧光后的向日葵花粉基底浸泡在PSS溶液中,静置吸附20分钟,离心,用超纯水冲洗三次,以洗掉多余的PSS溶液,获得表面带负电的花粉微球,将带负电花粉微球再浸泡在PAH溶液中,同样静置吸附20分钟后,离心,用超纯水冲洗三次,获得表面带正电的向日葵花粉基底,将该花粉基底浸泡在巯基乙酸修饰的蓝色量子点CdTe 465(特征发射峰在465nm位置) 水溶液中,静置吸附60分钟后离心,再用超纯水洗涤三次;参照上述聚电解质吸附步骤,在蓝色量子点CdTe 465外表面吸附正-负-正聚电解质层(依次吸附PAH溶液、PSS溶液、PAH溶液),然后在正-负-正聚电解质层外表面吸附第一层绿色量子点CdTe 513,重复上述操作,继续吸附第二层绿色量子点CdTe 513,通过聚电解质逐层沉积技术再吸附三层红色量子点CdTe 626,从而制备得到基于向日葵花粉的复合量子点编码微球;该编码微球采用不同颜色(特征发射峰)及不同强度(层数)的量子点进行复合编码,最终得到的编码为CdTe 465∶CdTe 513∶CdTe 626= 1:2:3;The PAH and PSS solid particles were dissolved in 0.5mol/L NaCl solution to prepare PAH solution and PSS solution with a concentration of 1 mg/mL; the sunflower pollen substrate after eliminating autofluorescence was immersed in PSS solution, and allowed to stand for adsorption. 20 minutes, centrifuge, rinse three times with ultrapure water to wash off excess PSS solution, and obtain negatively charged pollen microspheres. The negatively charged pollen microspheres are then immersed in PAH solution, and also after standing for adsorption for 20 minutes, Centrifuge, rinse with ultrapure water three times to obtain a sunflower pollen substrate with a positively charged surface, soak the pollen substrate in an aqueous solution of thioglycolic acid-modified blue quantum dots CdTe 465 (characteristic emission peak at 465 nm), and stand to absorb 60 Centrifuge after 2 minutes, and then wash with ultrapure water three times; referring to the above-mentioned polyelectrolyte adsorption steps, adsorb positive-negative-positive polyelectrolyte layer on the outer surface of blue quantum dot CdTe 465 (sequentially adsorb PAH solution, PSS solution, PAH solution), Then adsorb the first layer of green quantum dots CdTe 513 on the outer surface of the positive-negative-positive polyelectrolyte layer, repeat the above operation, continue to adsorb the second layer of green quantum dots CdTe 513, and then adsorb three layers of red quantum dots through the polyelectrolyte layer-by-layer deposition technology. dot CdTe 626 to prepare composite quantum dot encoded microspheres based on sunflower pollen; the encoded microspheres use quantum dots of different colors (characteristic emission peaks) and different intensities (layers) for composite encoding, and the final code is CdTe 465: CdTe 513: CdTe 626 = 1:2:3;
对制备的基于向日葵花粉的复合量子点编码微球(编码为CdTe 465∶CdTe 513∶CdTe626= 1:2:3)及其与层数对应的量子点(一层CdTe 465,两层CdTe 513,三层CdTe 626)在同一紫外激发光下进行发射光谱的测定,使用光纤光谱仪测得它们的荧光发射光谱图,如图3A所示,其中a1为本实施例制备的基于向日葵花粉的复合量子点编码微球的荧光发射光谱图,b1为一层量子点CdTe 465的荧光发射光谱图,c1为两层量子点CdTe 513的荧光发射光谱图,d1为三层量子点CdTe 626的荧光发射光谱图,由图可见,量子点被修饰在花粉基底上之后,其荧光强度并没有明显的降低。For the prepared sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdTe 465:CdTe 513:CdTe626 = 1:2:3) and the quantum dots corresponding to the number of layers (one layer of CdTe 465, two layers of CdTe 513, Three layers of CdTe 626) were measured under the same ultraviolet excitation light, and their fluorescence emission spectra were measured using a fiber optic spectrometer, as shown in Figure 3A, where a1 was the sunflower pollen-based composite quantum dots prepared in this example. The fluorescence emission spectrum of the encoded microspheres, b1 is the fluorescence emission spectrum of one-layer quantum dots CdTe 465, c1 is the fluorescence emission spectrum of two-layer quantum dots CdTe 513, d1 is the fluorescence emission spectrum of three-layer quantum dots CdTe 626 , it can be seen from the figure that after the quantum dots are modified on the pollen substrate, the fluorescence intensity does not decrease significantly.
实施例2Example 2
一种基于豚草花粉的复合量子点编码微球,通过以下步骤制备:A composite quantum dot-encoded microsphere based on ragweed pollen is prepared by the following steps:
(1)炭化处理制备消除自发荧光后的豚草花粉花粉基底(1) Preparation of ragweed pollen substrate after autofluorescence elimination by carbonization
将豚草花粉浸泡在无水乙醇中保持震荡20分钟,获得花粉粒充分剥离的分散液,将分散液置于热台上100℃加热至乙醇完全挥发,花粉处于干燥状态,筛选出颗粒分明的干燥豚草花粉粒;将干燥豚草花粉粒置于马弗炉中,300℃高温下煅烧并持续通入氮气12小时,得到荧光有机物质彻底炭化后的带正电豚草花粉基底;Soak the ragweed pollen in absolute ethanol and keep shaking for 20 minutes to obtain a dispersion with fully peeled pollen grains. The dispersion is placed on a hot table and heated at 100 ° C until the ethanol is completely volatilized, and the pollen is in a dry state. Drying the ragweed pollen grains; placing the dried ragweed pollen grains in a muffle furnace, calcining at a high temperature of 300° C. and continuously feeding nitrogen for 12 hours, to obtain a positively charged ragweed pollen substrate after the fluorescent organic substances are completely carbonized;
(2)采用聚电解质逐层吸附法制备基于豚草花粉的复合量子点编码微球(2) Preparation of composite quantum dot-encoded microspheres based on ragweed pollen by polyelectrolyte layer-by-layer adsorption
将PAH、PAA固体颗粒分别溶解0.5mol/L NaCl溶液中,配制得到浓度为1 mg/mL的PAH溶液和PAA溶液;将上述消除自发荧光后的豚草花粉基底浸泡在PAA溶液中,静置吸附20分钟,离心,用超纯水冲洗三次,以洗掉多余的PAA溶液,再将花粉微球浸泡在PAH溶液中,同样静置吸附20分钟后,离心,用超纯水冲洗三次,获得表面带正电的豚草花粉基底,参照实施例1的聚电解质逐层吸附法,通过聚电解质隔离层(依次吸附PAH溶液、PAA溶液、PAH溶液)逐层吸附巯基乙酸修饰的蓝色量子点CdSe 460、第一层绿色量子点CdSe 525、第二层绿色量子点CdSe 525和红色量子点CdSe 620,从而制备得到基于豚草花粉的复合量子点编码微球;该编码微球采用不同颜色(特征发射峰)及不同强度(层数)的量子点进行复合编码,最终得到的编码为CdSe 460∶CdSe 525∶CdSe 620= 1:2:1;The PAH and PAA solid particles were dissolved in 0.5mol/L NaCl solution, respectively, to prepare a PAH solution and a PAA solution with a concentration of 1 mg/mL; the ragweed pollen substrate after eliminating autofluorescence was immersed in the PAA solution, and allowed to stand. Adsorbed for 20 minutes, centrifuged, rinsed three times with ultrapure water to wash off excess PAA solution, then immersed the pollen microspheres in PAH solution, also stood for adsorption for 20 minutes, centrifuged, and rinsed three times with ultrapure water to obtain The positively charged ragweed pollen substrate, referring to the polyelectrolyte layer-by-layer adsorption method in Example 1, adsorbed thioglycolic acid-modified blue quantum dots layer by layer through a polyelectrolyte isolation layer (sequentially adsorbing PAH solution, PAA solution, and PAH solution).
对制备的基于豚草花粉的复合量子点编码微球(编码为CdSe 460∶CdSe 525∶CdSe 620= 1:2:1)及其与层数对应的量子点(一层CdSe 460,两层CdSe 525,一层CdSe 620)在同一紫外激发光下进行发射光谱的测定,使用光纤光谱仪测得它们的荧光发射光谱图如图3B所示,其中a2为本实施例制备的基于向日葵花粉的复合量子点编码微球的荧光发射光谱图,b2为量子点一层CdSe 460的荧光发射光谱图,c2为两层量子点CdSe 525的荧光发射光谱图,d2为量子点一层CdSe 620的荧光发射光谱图,由图可见,量子点被修饰在花粉基底上之后,其荧光强度并没有明显的降低。The prepared ragweed pollen-based composite quantum dot-encoded microspheres (coded as CdSe 460:CdSe 525:CdSe 620 = 1:2:1) and their corresponding quantum dots (one layer of
实施例3Example 3
一种基于菊花花粉的混合型复合量子点编码微球,通过以下步骤制备:A hybrid composite quantum dot-encoded microsphere based on chrysanthemum pollen is prepared by the following steps:
(1)将菊花花粉浸泡在无水乙醇中保持震荡20分钟,获得花粉粒充分剥离的分散液,将分散液置于热台上100℃加热至乙醇完全挥发,花粉处于干燥状态,筛选出颗粒分明的干燥豚草花粉粒;将干燥菊花花粉粒置于马弗炉中,300℃高温下煅烧并持续通入氮气12小时,得到荧光有机物质彻底炭化后的带正电菊花花粉基底;(1) Soak chrysanthemum pollen in anhydrous ethanol and keep shaking for 20 minutes to obtain a dispersion with fully peeled pollen particles. Heat the dispersion on a hot table at 100°C until the ethanol is completely volatilized and the pollen is in a dry state. Screen out the particles Distinct dry ragweed pollen grains; the dry chrysanthemum pollen grains are placed in a muffle furnace, calcined at a high temperature of 300°C and continuously fed with nitrogen for 12 hours to obtain a positively charged chrysanthemum pollen substrate after the fluorescent organic substances are completely carbonized;
(2)采用聚电解质逐层吸附法制备基于菊花花粉的混合型复合量子点编码微球(2) Hybrid composite quantum dot-encoded microspheres based on chrysanthemum pollen were prepared by layer-by-layer adsorption of polyelectrolytes
将PEI、PSS固体颗粒分别溶解0.5mol/L NaCl溶液中,配制得到浓度为1 mg/mL的PEI溶液和PSS溶液;将上述消除自发荧光后的菊花花粉基底浸泡在PSS溶液中,静置吸附20分钟,离心,用超纯水冲洗三次,以洗掉多余的PSS溶液,再将花粉微球浸泡在PEI溶液中,同样静置吸附20分钟后,离心,用超纯水冲洗三次,获得表面带正电的菊花花粉基底,参照实施例1的聚电解质逐层吸附法,通过聚电解质隔离层(依次吸附PEI溶液、PSS溶液、PEI溶液)逐层吸附第一层蓝色量子点CdS 540、第二层蓝色量子点CdS 540、第三层蓝色量子点CdS 540,从而制备得到编码为CdS 540= 3的基于菊花花粉的复合量子点编码微球;参照上述聚电解质逐层吸附法,在菊花花粉基底外表面逐层吸附量子点CdS 540、第一层量子点CdS 580、第二层量子点CdS 580、第三层量子点CdS 580,制备得到编码为CdS 540∶CdS 580= 1∶3的基于菊花花粉的复合量子点编码微球;参照上述聚电解质逐层吸附法,在菊花花粉基底外表面逐层吸附量子点CdS 540、量子点CdS 580、量子点CdS 620,制备得到编码为CdS 540∶CdS580∶CdS 620= 1∶1∶1的基于菊花花粉的复合量子点编码微球;将以上三种编码微球混合,得到三种不同特异性的基于菊花花粉的混合型复合量子点编码微球,即量子点编码分别为CdS 540= 3,CdS 540∶CdS 580= 1∶3,CdS 540∶CdS 580∶CdS 620= 1∶1∶1的复合量子点编码微球的组合;The PEI and PSS solid particles were dissolved in 0.5mol/L NaCl solution, respectively, to prepare a PEI solution and a PSS solution with a concentration of 1 mg/mL; the chrysanthemum pollen substrate after eliminating autofluorescence was immersed in the PSS solution, and allowed to stand for adsorption. 20 minutes, centrifuge, rinse three times with ultrapure water to wash off excess PSS solution, then soak the pollen microspheres in PEI solution, also stand for adsorption for 20 minutes, centrifuge, rinse three times with ultrapure water to obtain the surface The positively charged chrysanthemum pollen substrate, referring to the polyelectrolyte layer-by-layer adsorption method in Example 1, adsorbed the first layer of blue quantum dots CdS 540, The second layer of blue quantum dots CdS 540 and the third layer of blue quantum dots CdS 540 are prepared to obtain a chrysanthemum pollen-based composite quantum dot encoded microsphere encoded as CdS 540=3; with reference to the above-mentioned polyelectrolyte layer-by-layer adsorption method, Quantum dots CdS 540, the first layer of quantum dots CdS 580, the second layer of quantum dots CdS 580, and the third layer of quantum dots CdS 580 were adsorbed layer by layer on the outer surface of the chrysanthemum pollen substrate, and the prepared code was CdS 540:CdS 580= 1: The composite quantum dot coding microsphere based on chrysanthemum pollen according to 3; with reference to the above-mentioned polyelectrolyte layer-by-layer adsorption method, quantum dots CdS 540, quantum dots CdS 580 and quantum dots CdS 620 are adsorbed layer by layer on the outer surface of the chrysanthemum pollen substrate, and the prepared code is CdS 540:CdS580:CdS 620=1:1:1 chrysanthemum pollen-based composite quantum dot-encoded microspheres; mixing the above three encoded microspheres to obtain three different specific chrysanthemum pollen-based hybrid quantum dots Coding microspheres, that is, the combination of quantum dot coding microspheres with CdS 540=3, CdS 540: CdS 580= 1: 3, CdS 540: CdS 580: CdS 620= 1: 1: 1 respectively;
实施例中,在各复合量子点编码微球最外层量子点外侧再沉积一层PEI溶液,可用于多种待测分子的检验,所述分子选自DNA,RNA,蛋白质中的一种。In the embodiment, a layer of PEI solution is deposited outside the outermost quantum dots of each composite quantum dot encoded microsphere, which can be used for the detection of various molecules to be tested, and the molecules are selected from one of DNA, RNA and protein.
实施例4:基于天然多刺状花粉的复合量子点编码微球的表征实验Example 4: Characterization experiment of composite quantum dot-encoded microspheres based on natural prickly pollen
4.1)制备干燥向日葵花粉粒、基于向日葵花粉的复合量子点编码微球(编码为CdSe620=1)、基于向日葵花粉的复合量子点编码微球(编码为CdSe 460=1)、基于向日葵花粉的复合量子点编码微球(编码为CdSe 525=1);4.1) Preparation of dry sunflower pollen grains, sunflower pollen-based composite quantum dot-encoded microspheres (encoded as CdSe620=1), sunflower pollen-based composite quantum dot-encoded microspheres (encoded as
参照实施例1,将向日葵花粉浸泡在无水乙醇中保持震荡20分钟,获得花粉粒充分剥离的分散液,将分散液置于热台上100℃加热至乙醇完全挥发,花粉处于干燥状态,获得颗粒分明的干燥向日葵花粉粒;取部分干燥向日葵花粉粒干燥向日葵花粉粒置于马弗炉中,300℃高温下煅烧并持续通入氮气12小时,获得消除自发荧光后的向日葵花粉花粉基底,将向日葵花粉花粉基底依次浸泡在浓度为1 mg/mL的PSS溶液和PAH溶液中,再将表面带正电的向日葵花粉基底浸泡在量子点CdSe 620中,获得基于天然向日葵花粉的表面修饰一层红色量子点CdSe 620的复合量子点编码微球,即基于向日葵花粉的复合量子点编码微球(编码为CdSe 620=1);类似地,将表面带正电的向日葵花粉基底浸泡在量子点CdSe 525中,获得基于天然向日葵花粉的表面修饰一层绿色量子点CdSe 525的复合量子点编码微球,即基于向日葵花粉的复合量子点编码微球(编码为CdSe 525=1);将表面带正电的向日葵花粉基底浸泡在量子点CdSe 460中,获得基于天然向日葵花粉的表面修饰一层蓝色量子点CdSe 460的复合量子点编码微球,即基于向日葵花粉的复合量子点编码微球(编码为CdSe 460=1);With reference to Example 1, sunflower pollen was soaked in absolute ethanol and kept oscillating for 20 minutes to obtain a dispersion with fully peeled pollen grains. Dry sunflower pollen grains with distinct particles; take part of the dried sunflower pollen grains and dry sunflower pollen grains in a muffle furnace, calcine at a high temperature of 300 ° C and continuously pass nitrogen for 12 hours to obtain the sunflower pollen pollen base after eliminating autofluorescence. The sunflower pollen substrate was immersed in PSS solution and PAH solution with a concentration of 1 mg/mL in turn, and then the positively charged sunflower pollen substrate was immersed in quantum dot CdSe 620 to obtain a surface-modified layer of red based on natural sunflower pollen. Composite quantum dot-encoded microspheres of quantum dots CdSe 620, namely sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 620=1); similarly, the surface-positively charged sunflower pollen substrate was immersed in
4.2)对步骤4.1)制备的干燥向日葵花粉粒和基于向日葵花粉的复合量子点编码微球(编码为CdSe 620=1)进行体式显微镜和场发射扫描电子显微镜的表征,结果如图4所示;由图4d-f可见,基于向日葵花粉的复合量子点编码微球(编码为CdSe 620=1)的直径在20-30μm范围内,比天然向日葵花粉(图4a-c)的尺寸缩小了约10μm,这是300℃高温煅烧的结果,虽然复合量子点编码微球直径变小,但花粉刺状结构存留度较好,同时可以明显观察到量子点CdSe 620在花粉基底表面均匀分布,表明以天然多刺状花粉为编码基底,其独特复杂的多孔和刺状表面结构提供了高孔隙率和较大比表面积,采用聚电解质逐层吸附的方法能够均匀地结合多种类型量子点;4.2) The dried sunflower pollen grains prepared in step 4.1) and the sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 620=1) were characterized by stereo microscopy and field emission scanning electron microscopy, and the results are shown in Figure 4; It can be seen from Fig. 4d–f that the diameter of the sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 620=1) is in the range of 20–30 μm, which is about 10 μm smaller than that of natural sunflower pollen (Fig. 4a–c). , this is the result of high temperature calcination at 300 °C. Although the diameter of the composite quantum dot-encoded microspheres becomes smaller, the retention of the pollen-like structure is better. At the same time, it can be clearly observed that the quantum dot CdSe 620 is uniformly distributed on the surface of the pollen substrate, indicating that the natural Spiny pollen is the coding substrate. Its unique and complex porous and thorn-like surface structure provides high porosity and large specific surface area. The layer-by-layer adsorption method of polyelectrolyte can uniformly bind various types of quantum dots;
4.3)对步骤4.1)制备的基于向日葵花粉的复合量子点编码微球(编码为CdSe 620=1)进行激光扫描共聚焦显微镜下扫描的不同断层的荧光图像,如图5所示,红色量子点CdSe620(图中亮白色部分)覆盖整个花粉表面且仅仅分布在表面,表明采用单分散性良好的多刺状花粉作为编码基底,其独特复杂的多孔和刺状表面结构提供了高孔隙率和较大比表面积,通过聚电解质逐层吸附的方法可获得有效的量子点涂层,进而可增加目标分子的吸附量进而显著增强检测荧光信号,大大降低检测限,提高检测灵敏度;4.3) Fluorescence images of different slices scanned under a laser scanning confocal microscope on the sunflower pollen-based composite quantum dot-encoded microspheres (coded as CdSe 620=1) prepared in step 4.1), as shown in Figure 5, red quantum dots CdSe620 (bright white part in the figure) covers the entire pollen surface and is only distributed on the surface, indicating the use of spiny pollen with good monodispersity as the coding substrate, and its unique complex porous and spiny surface structure provides high porosity and relatively Large specific surface area, effective quantum dot coating can be obtained by layer-by-layer adsorption of polyelectrolyte, which can increase the adsorption amount of target molecules and significantly enhance the detection fluorescence signal, greatly reduce the detection limit, and improve the detection sensitivity;
4.4)对步骤4.1)制备的基于向日葵花粉的复合量子点编码微球(编码为CdSe 460=1)、基于向日葵花粉的复合量子点编码微球(编码为CdSe 525=1)、基于向日葵花粉的复合量子点编码微球(编码为CdSe 620=1)进行激光扫描共聚焦荧光显微镜下荧光表征,结果如图6所示,其中,图6a-c为基于向日葵花粉的复合量子点编码微球(编码为CdSe 460=1)的荧光图像,蓝色量子点CdSe 460(图中亮色部分)覆盖整个花粉表面且仅仅分布在表面,图b, c分别为不同放大倍率下的图a,图6d-f为基于向日葵花粉的复合量子点编码微球(编码为CdSe 525=1)的荧光图像,绿色量子点CdSe 525(图中亮色部分)覆盖整个花粉表面且仅仅分布在表面,图e, f分别为不同放大倍率下的图d,图6g-i为基于向日葵花粉的复合量子点编码微球(编码为CdSe 620=1)的荧光图像,红色量子点CdSe 620(图中亮色部分)覆盖整个花粉表面且仅仅分布在表面,图h, i分别为不同放大倍率下的图g,同时可以观察到在花粉表面尖刺的顶部具有更强的荧光;4.4) For the sunflower pollen-based composite quantum dot-encoded microspheres (coded as
将基于向日葵花粉的复合量子点编码微球(编码为CdSe 460=1)、基于向日葵花粉的复合量子点编码微球(编码为CdSe 525=1)、基于向日葵花粉的复合量子点编码微球(编码为CdSe 620=1)混合,得到三种不同特异性的基于向日葵花粉的混合型复合量子点编码微球,即量子点编码分别为CdSe 460=1,CdSe 525=1,CdSe 620=1的复合量子点编码微球的组合;将基于向日葵花粉的混合型复合量子点编码微球(编码分别为CdSe 460=1,CdSe 525=1,CdSe 620=1)进行激光扫描共聚焦荧光显微镜下荧光表征,如图7所示,在同一紫外光源的激发下,可以同时激发出多种特征发射峰的量子点,可同时检测到红(编码为CdSe 620=1)、绿(编码为CdSe 525=1)、蓝(编码为CdSe 460=1)三种颜色的编码微球,表明本发明制备的基于天然多刺状花粉的复合量子点编码微球或混合型复合量子点编码微球解码方法简单,仅用一种波长的光就可以同时激发多种特征发射峰的量子点。The composite quantum dot-encoded microspheres based on sunflower pollen (coded as
实施例5:DNA杂交测定实验Example 5: DNA Hybridization Assay Experiment
(1)以实施例4制备的基于向日葵花粉的复合量子点编码微球(编码为CdSe 460=1)为例,将探针DNA固定在复合量子点编码微球上,具体的实现过程是:首先探针DNA在5'端进行胺官能化,并在标记DNA的5'端用绿色荧光染料羧酸荧光素(FAM,特征峰位置为520 nm)标记;杂交夹心结构如图8所示,将制备的带有羧基的复合量子点编码微球添加到50 mL现配的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐EDC(4.2 mg / mL)溶液中,同时,活性中间体用50 mL N-羟基琥珀酰亚胺NHS(5 mg / mL)溶液进行稳定;然后将1 mL胺官能化的探针DNA溶液添加到上述混合物中,并将样品在室温避光条件下,于4℃持续振荡,孵育2小时;由于复合量子点编码微球带有的羧基能够与胺官能化后DNA带有的氨基形成酰胺键,因此探针DNA能被固定在复合量子点编码微球上,离心后用是磷酸缓冲盐溶液在洗涤三次去除未结合的多余探针DNA;接着分别依次以等摩尔比添加5’端标记FAM的标记DNA和靶标DNA混合液,并将样品在室温避光条件下,于4℃持续振荡,孵育6小时;事先设计好的靶标DNA、探针DNA和标记DNA序列能够通过杂交在基于向日葵花粉的复合量子点编码微球(编码为CdSe460=1)表面形成杂交夹心结构,通过激光扫描共聚焦显微镜和光纤光谱仪对表面结合有杂交夹心结构DNA的基于向日葵花粉的复合量子点编码微球(编码为CdSe 460=1)进行荧光图像和发射光谱的表征。(1) Taking the sunflower pollen-based composite quantum dot-encoded microspheres (coded as
参照上述步骤,使用传统生物检测产品玻璃珠(Glass Beads)作为对照,在其他实验参数条件完成相同的情况下,对比基于向日葵花粉的复合量子点编码微球(编码为CdSe460=1)和Glass Beads在DNA杂交测定实验中的表现。通过激光扫描共聚焦显微镜和光纤光谱仪对表面结合有修饰FAM条件下的杂交夹心结构DNA的Glass Beads进行荧光图像和发射光谱的表征。如图9i, 9ii所示,荧光图像可检测到标记染料FAM的绿色荧光,且基于向日葵花粉的复合量子点编码微球(编码为CdSe 460=1)发射的荧光强度远高于Glass Beads。如图9所示,荧光曲线证明了基于向日葵花粉的复合量子点编码微球(编码为CdSe 460=1)可以检测到比Glass Beads更宽的DNA浓度范围,进一步测得Glass Beads的检测限为1090pM,而本发明制备得到的量子点编码微球的检测限仅为9.7 pM,可见以多刺状花粉作为编码基底,其独特复杂的多孔和刺状表面结构提供了高孔隙率和较大比表面积,增加了目标分子的吸附量进而显著增强检测荧光信号,大大降低了检测限,提高了检测灵敏度。Referring to the above steps, using glass beads (Glass Beads), a traditional biological detection product, as a control, and under the same conditions as other experimental parameters, compare the composite quantum dot-encoded microspheres based on sunflower pollen (coded as CdSe460=1) and Glass Beads Performance in DNA hybridization assay experiments. Fluorescence images and emission spectra of Glass Beads with hybridized sandwich DNA under modified FAM conditions were characterized by laser scanning confocal microscopy and fiber optic spectrometer. As shown in Fig. 9i, 9ii, the green fluorescence of the labeled dye FAM can be detected in the fluorescence images, and the fluorescence intensity emitted by the sunflower pollen-based composite quantum dot-encoded microspheres (coded as
(2)多重DNA靶标序列的测定(2) Determination of multiple DNA target sequences
参照实施例3的制备方法,制备基于向日葵花粉的混合型复合量子点编码微球(编码为CdSe 460∶CdSe 620=2:1,1:1,1:2),参照本实施例的上述步骤(1),将不同的探针DNA(1nmol /L)分别固定在三种基于向日葵花粉的复合量子点编码微球(编码分别为CdSe 460∶CdSe 620=2:1,CdSe 460∶CdSe 620=1:1,CdSe 460∶CdSe 620=1:2)上,在室温下混合不同的载体-探针结合物,并以等摩尔比添加四种具有不同序列的靶标DNA分子,将混合物在37℃避光条件下连续振荡孵育6小时,得到表面结合有杂交夹心结构DNA的混合型复合量子点编码微球(编码为CdSe 460∶CdSe 620=2:1,1:1,1:2);然后用光谱仪检测不同复合量子点编码微球上反射的光谱信号(在荧光测量之前,将编码微球用缓冲液洗涤3次)。结果如图10所示,图a为标记DNA未修饰FAM条件下无靶标信号只有基于向日葵花粉的复合量子点编码微球(编码为CdSe 460∶CdSe 620=2:1)信号的发射荧光图谱,图b~d依次为表面结合有修饰FAM条件下的杂交夹心结构DNA的三种基于向日葵花粉的复合量子点编码微球(编码分别为CdSe 460∶CdSe 620=2:1,CdSe 460∶CdSe 620=1:1,CdSe 460∶CdSe 620=1:2)的发射荧光图谱,在波长520 nm位置的特征峰为检测到的靶标信号的发射峰;综合图10所示检测结果可见,混合型复合量子点编码微球检测时无交叉干扰,能够清晰地反映靶标分子的种类,定量化检测分子的浓度信息;表明本发明采用不同种类和不同层数量子点的组合来实现多重特征发射峰和强度的编码形式,极大地扩充了编码量,解码时可同时获悉靶标分子的种类和浓度信息,并且检测时无交叉干扰,可满足同时检测多指标和高通量检测的需要。Referring to the preparation method of Example 3, prepare hybrid composite quantum dot-encoded microspheres based on sunflower pollen (encoded as CdSe 460:CdSe 620=2:1, 1:1, 1:2), refer to the above steps of this example (1), different probe DNAs (1 nmol/L) were immobilized on three kinds of sunflower pollen-based composite quantum dot-encoded microspheres (encoded as CdSe 460:CdSe 620=2:1, CdSe 460:CdSe 620= 1:1, CdSe 460:CdSe 620=1:2), mix different carrier-probe conjugates at room temperature, and add four target DNA molecules with different sequences in an equimolar ratio, and put the mixture at 37 °C. Incubate with continuous shaking for 6 hours in the dark to obtain hybrid composite quantum dot-encoded microspheres (coded as CdSe 460:CdSe 620=2:1, 1:1, 1:2) with hybrid sandwich DNA bound on the surface; then Spectral signals reflected on different composite quantum dot-encoded microspheres were detected with a spectrometer (encoded microspheres were washed 3 times with buffer before fluorescence measurement). The results are shown in Figure 10. Figure a is the emission fluorescence spectrum of the composite quantum dot-encoded microspheres based on sunflower pollen (encoded as CdSe 460:CdSe 620=2:1) without target signal under the condition of labeled DNA unmodified FAM. Figures b to d show three kinds of composite quantum dot-encoded microspheres based on sunflower pollen with hybrid sandwich structure DNA bound on the surface in turn under the condition of modified FAM (the codes are CdSe 460:CdSe 620=2:1, CdSe 460:CdSe 620 =1:1, CdSe 460:CdSe 620=1:2) emission fluorescence spectrum, the characteristic peak at the wavelength of 520 nm is the emission peak of the detected target signal; from the detection results shown in Figure 10, it can be seen that the mixed composite There is no cross-interference in the detection of quantum dot-encoded microspheres, which can clearly reflect the types of target molecules and quantitatively detect the concentration information of the molecules; it shows that the present invention adopts the combination of different types and different layers of quantum dots to achieve multiple characteristic emission peaks and intensities The coding format of TM greatly expands the coding amount, and the type and concentration information of the target molecule can be obtained at the same time during decoding, and there is no cross-interference during detection, which can meet the needs of simultaneous detection of multiple indicators and high-throughput detection.
实施例6:量子点编码的发射光谱及稳定性表征Example 6: Emission Spectra and Stability Characterization of Quantum Dot Encoding
参照是实施例1所述的复合量子点编码微球的制备方法,以天然向日葵花粉为基底,通过聚电解质逐层沉积技术,在向日葵花粉基底表面修饰一层CdSe@ZnS565量子点,从而制备得到基于向日葵花粉的复合量子点编码微球(编码为CdSe@ZnS565=1),对天然向日葵花粉和基于向日葵花粉的复合量子点编码微球(编码为CdSe@ZnS565=1)通过荧光分光光度计进行激发光谱和发射光谱的测定,结果如图11所示,可以观察到基于向日葵花粉的复合量子点编码微球(编码为CdSe@ZnS565=1)(图示中记作复合量子点编码微球)激发光谱宽,发射光谱呈对称分布且宽度窄,相比天然向日葵花粉,本复合量子点编码微球的荧光具有极强的光学编码优势;The reference is the preparation method of the composite quantum dot-encoded microspheres described in Example 1. Using natural sunflower pollen as a substrate, a layer of CdSe@ZnS565 quantum dots is modified on the surface of the sunflower pollen substrate by polyelectrolyte layer-by-layer deposition technology, thereby preparing Sunflower pollen-based composite quantum dot-encoded microspheres (encoded as CdSe@ZnS565=1), natural sunflower pollen and sunflower pollen-based composite quantum dot-encoded microspheres (encoded as CdSe@ZnS565=1) were analyzed by fluorescence spectrophotometer The measurement of excitation spectrum and emission spectrum is shown in Figure 11. It can be observed that the composite quantum dot-encoded microspheres based on sunflower pollen (coded as CdSe@ZnS565=1) (referred to as composite quantum dot-encoded microspheres in the figure) The excitation spectrum is wide, and the emission spectrum is symmetrically distributed and narrow in width. Compared with natural sunflower pollen, the fluorescence of the composite quantum dot-encoded microspheres has a strong optical encoding advantage;
在实施例5的DNA杂交测定实验中,通过光纤光谱仪检测结合DNA后的基于向日葵花粉的复合量子点编码微球(编码为CdSe 460=1)的荧光强度的变化,结果如图12所示,可见较长时间内,量子点荧光强度降低幅度较小;表明,本发明制备的基于天然多刺状花粉的复合编码微球在应用过程中,编码稳定性较好。In the DNA hybridization assay experiment in Example 5, the fluorescence intensity of the sunflower pollen-based composite quantum dot-encoded microspheres (coded as
以上仅是本发明的优选实施方式,本发明的保护范围并不仅局限于上述实施例,凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理前提下的若干改进和润饰,应视为本发明的保护范围。The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions that belong to the idea of the present invention belong to the protection scope of the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.
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