技术领域technical field
本发明涉及pH检测技术领域，特别是指一种pH敏感的荧光纳米传感器及其制备方法。The invention relates to the technical field of pH detection, in particular to a pH-sensitive fluorescent nanometer sensor and a preparation method thereof.
背景技术Background technique
近年来，生物医学的快速发展对分析检测技术提出了新的挑战，要求对单个活细胞等微观环境进行实时、无损、动态过程监测。氢离子对生物体的生命活动起着重要的调控作用，一些生命现象的发生常伴随着氢离子浓度的变化，因此生物组织内微观水平的pH值测量具有非常重要的意义。在生物组织pH检测方面，传统宏观尺寸的光、电生物化学传感器存在较多限制，尺寸较大容易导致细胞的生物学损伤并产生相关的生化影响。因此，设计可以实现无创、实时及动态检测微观环境的pH传感探针已经成为人们的一个研究热点。In recent years, the rapid development of biomedicine has posed new challenges to analytical and detection technologies, requiring real-time, nondestructive, and dynamic process monitoring of microenvironments such as single living cells. Hydrogen ions play an important role in regulating the life activities of organisms. The occurrence of some life phenomena is often accompanied by changes in the concentration of hydrogen ions. Therefore, the measurement of pH at the microscopic level in biological tissues is of great significance. In terms of pH detection in biological tissues, traditional optical and electrical biochemical sensors with macroscopic dimensions have many limitations. Larger sizes are likely to cause biological damage to cells and produce related biochemical effects. Therefore, the design of pH sensing probes that can realize non-invasive, real-time and dynamic detection of micro-environment has become a research hotspot.
随着纳米科学技术的发展，基于纳米材料的传感技术引起了广泛的关注。目前通过纳米载体负载荧光pH敏感探针分子制备得到的pH纳米传感器，还存在生物兼容性差的问题。With the development of nanoscience and technology, sensing technology based on nanomaterials has attracted extensive attention. At present, pH nanosensors prepared by loading fluorescent pH-sensitive probe molecules on nanocarriers still have the problem of poor biocompatibility.
发明内容Contents of the invention
有鉴于此，本发明的目的在于提出一种具有良好生物兼容性的pH敏感的荧光纳米传感器及其制备方法，能够方便进入细胞内部进行pH检测。In view of this, the object of the present invention is to propose a pH-sensitive fluorescent nanosensor with good biocompatibility and a preparation method thereof, which can conveniently enter cells for pH detection.
基于上述目的本发明提供的一种pH敏感的荧光纳米传感器，所述荧光纳米传感器包括内核以及包覆所述内核的外壳，所述内核包括二氧化硅表层以及分散在内部的异硫氰酸荧光素、疏水性聚合物和长链烷基三甲氧基硅烷，所述外壳为多聚赖氨酸层。Based on the above purpose, the present invention provides a pH-sensitive fluorescent nanosensor. The fluorescent nanosensor includes an inner core and a shell covering the inner core. The inner core includes a silicon dioxide surface layer and isothiocyanate fluorescence dispersed inside. element, hydrophobic polymer and long-chain alkyltrimethoxysilane, and the shell is a polylysine layer.
进一步的，所述疏水性聚合物选自聚苯乙烯或聚9,9-二辛基芴中的至少一者。Further, the hydrophobic polymer is at least one selected from polystyrene or poly-9,9-dioctylfluorene.
进一步的，所述荧光纳米传感器的粒径为100～160nm。Further, the particle diameter of the fluorescent nanosensor is 100-160 nm.
本发明实施例的第二个方面，提供了一种pH敏感的荧光纳米传感器的制备方法，包括：The second aspect of the embodiments of the present invention provides a method for preparing a pH-sensitive fluorescent nanosensor, including:
将异硫氰酸荧光素、疏水性聚合物和长链烷基三甲氧基硅烷按比例加入至四氢呋喃中，振荡混合得到混合溶液；Add fluorescein isothiocyanate, hydrophobic polymer and long-chain alkyltrimethoxysilane to tetrahydrofuran in proportion, shake and mix to obtain a mixed solution;
在超声条件下，将所述混合溶液加入到多聚赖氨酸的水溶液中即可得到荧光纳米传感器分散液，透析，得到荧光纳米传感器。Under ultrasonic conditions, the mixed solution is added to the aqueous solution of polylysine to obtain the fluorescent nanometer sensor dispersion liquid, which is dialyzed to obtain the fluorescent nanometer sensor.
进一步的，所述疏水性聚合物选自聚苯乙烯或聚9,9-二辛基芴中的至少一者。Further, the hydrophobic polymer is at least one selected from polystyrene or poly-9,9-dioctylfluorene.
进一步的，所述长链烷基三甲氧基硅烷选自十六烷基三甲氧基硅烷或十二烷基三甲氧基硅烷中的至少一者。Further, the long-chain alkyltrimethoxysilane is selected from at least one of hexadecyltrimethoxysilane or dodecyltrimethoxysilane.
进一步的，按质量份计，所述异硫氰酸荧光素：所述疏水性聚合物：所述长链烷基三甲氧基硅烷为5～15:35～45:45～60。Further, in parts by mass, the ratio of the fluorescein isothiocyanate: the hydrophobic polymer: the long-chain alkyltrimethoxysilane is 5-15:35-45:45-60.
进一步的，所述混合溶液中的异硫氰酸荧光素、疏水性聚合物和长链烷基三甲氧基硅烷的总浓度为200～800ppm。Further, the total concentration of fluorescein isothiocyanate, hydrophobic polymer and long-chain alkyltrimethoxysilane in the mixed solution is 200-800ppm.
进一步的，所述多聚赖氨酸的水溶液pH＝9。Further, the aqueous solution of poly-lysine has pH=9.
进一步的，所述多聚赖氨酸的分子量在30KDa到50KDa之间。Further, the molecular weight of the polylysine is between 30KDa and 50KDa.
从上面所述可以看出，本发明实施例提供的pH敏感的荧光纳米传感器，具有多聚赖氨酸形成的外壳，所述荧光纳米传感器具有良好的生物兼容性，容易被细胞吞噬，能够实现对细胞内部pH的检测。It can be seen from the above that the pH-sensitive fluorescent nanosensor provided by the embodiment of the present invention has a shell formed by polylysine. The fluorescent nanosensor has good biocompatibility, is easily phagocytized by cells, and can realize Detection of pH inside the cell.
本发明实施例提供的pH敏感的荧光纳米传感器的制备方法，利用共沉淀-包覆法制备荧光纳米传感器，简单方便易操作。The preparation method of the pH-sensitive fluorescent nanosensor provided in the embodiment of the present invention uses the co-precipitation-coating method to prepare the fluorescent nanosensor, which is simple, convenient and easy to operate.
附图说明Description of drawings
图1为本发明实施例提供的pH敏感的荧光纳米传感器的截面结构示意图；Fig. 1 is a schematic cross-sectional structure diagram of a pH-sensitive fluorescent nanosensor provided by an embodiment of the present invention;
图2为本发明实施例提供的pH敏感的荧光纳米传感器的TEM电镜照片图；Fig. 2 is the TEM photomicrograph of the pH-sensitive fluorescent nanosensor provided by the embodiment of the present invention;
图3为本发明实施例提供的pH敏感的荧光纳米传感器的动态光散射粒径分布图；Fig. 3 is the dynamic light scattering particle size distribution diagram of the pH-sensitive fluorescent nanosensor provided by the embodiment of the present invention;
图4为本发明实施例提供的pH敏感的荧光纳米传感器的吸收光谱图；Fig. 4 is the absorption spectrum diagram of the pH-sensitive fluorescent nanosensor provided by the embodiment of the present invention;
图5A为本发明实施例提供的pH敏感的荧光纳米传感器在不同pH环境下的发射光谱图；FIG. 5A is an emission spectrum diagram of a pH-sensitive fluorescent nanosensor provided in an embodiment of the present invention under different pH environments;
图5B为图5A中发光峰值的pH拟合曲线。Fig. 5B is a pH fitting curve of the luminescence peak in Fig. 5A.
图6为本发明实施例提供的pH敏感的荧光纳米传感器的pH检测可逆性测试图；Fig. 6 is the pH detection reversibility test diagram of the pH-sensitive fluorescent nanosensor provided by the embodiment of the present invention;
图7为本发明实施例提供的pH敏感的荧光纳米传感器的响应灵敏度测试图；Fig. 7 is the response sensitivity test diagram of the pH-sensitive fluorescent nanosensor provided by the embodiment of the present invention;
图8A为本发明实施例提供的pH敏感的荧光纳米传感器的HepG2细胞的明场成像图；Fig. 8A is a bright field imaging diagram of HepG2 cells of the pH-sensitive fluorescent nanosensor provided by the embodiment of the present invention;
图8B为本发明实施例提供的pH敏感的荧光纳米传感器的HepG2细胞的共聚焦成像图；Fig. 8B is a confocal imaging diagram of HepG2 cells of the pH-sensitive fluorescent nanosensor provided by the embodiment of the present invention;
图8C为图8A和图8B的叠加成像图。Fig. 8C is a superimposed image of Fig. 8A and Fig. 8B.
具体实施方式Detailed ways
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本发明进一步详细说明。In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be described in further detail below in conjunction with specific embodiments and with reference to the accompanying drawings.
需要说明的是，本发明实施例中所有使用“第一”和“第二”的表述均是为了区分两个相同名称非相同的实体或者非相同的参量，可见“第一”“第二”仅为了表述的方便，不应理解为对本发明实施例的限定，后续实施例对此不再一一说明。It should be noted that all expressions using "first" and "second" in the embodiments of the present invention are to distinguish two entities with the same name but different parameters or parameters that are not the same, see "first" and "second" It is only for the convenience of expression, and should not be construed as a limitation on the embodiments of the present invention, which will not be described one by one in the subsequent embodiments.
基于上述目的，本发明实施例的第一个方面，提出了一种pH敏感的荧光纳米传感器的一个实施例。如图1所示，为本发明提供的pH敏感的荧光纳米传感器的一个实施例的截面结构示意图。所述荧光纳米传感器包括内核2以及包覆所述内核的外壳3，所述内核2包括二氧化硅表层(图中未标示)以及分散在内部的异硫氰酸荧光素1、疏水性聚合物和长链烷基三甲氧基硅烷，所述外壳3为多聚赖氨酸层。Based on the above purpose, the first aspect of the embodiments of the present invention proposes an embodiment of a pH-sensitive fluorescent nanosensor. As shown in FIG. 1 , it is a schematic cross-sectional structure diagram of an embodiment of the pH-sensitive fluorescent nanosensor provided by the present invention. The fluorescent nanosensor includes an inner core 2 and a shell 3 covering the inner core. The inner core 2 includes a silicon dioxide surface layer (not shown in the figure) and fluorescein isothiocyanate 1 and a hydrophobic polymer dispersed inside. and long-chain alkyltrimethoxysilane, and the shell 3 is a polylysine layer.
本发明实施例提供的pH敏感的荧光纳米传感器，多聚赖氨酸层修饰的荧光纳米传感器具有良好的水溶性和生物兼容性，使得荧光纳米传感器具有高的细胞吞噬效率，能够方便的进入细胞内，实现对细胞内pH的精准检测。The pH-sensitive fluorescent nanosensor provided by the embodiment of the present invention, the fluorescent nanosensor modified by the polylysine layer has good water solubility and biocompatibility, so that the fluorescent nanosensor has high cell phagocytosis efficiency and can easily enter cells within, to achieve precise detection of intracellular pH.
本发明实施例提供的pH敏感的荧光纳米传感器，选择异硫氰酸荧光素作为pH敏感荧光探针且异硫氰酸荧光素均匀分散于荧光纳米传感器的内核部分，当将荧光纳米传感器置于不同pH值缓冲溶液后，氢离子能够自由的通过具有网状正电势的多聚赖氨酸层以及二氧化硅表层，与均匀分散的异硫氰酸荧光素接触引起异硫氰酸荧光素的结构变化，进而通过检测异硫氰酸荧光素的荧光强度的变化，实现对待测环境的pH值的检测。这样的pH检测方式，具有响应速度快，灵敏度高的优势。In the pH-sensitive fluorescent nanosensor provided by the embodiments of the present invention, fluorescein isothiocyanate is selected as the pH-sensitive fluorescent probe and fluorescein isothiocyanate is uniformly dispersed in the core part of the fluorescent nanosensor. When the fluorescent nanosensor is placed After buffering solutions with different pH values, hydrogen ions can freely pass through the polylysine layer and the silica surface layer with a network positive potential, and contact with the uniformly dispersed fluorescein isothiocyanate to cause the formation of fluorescein isothiocyanate Structural changes, and then by detecting changes in the fluorescence intensity of fluorescein isothiocyanate, the detection of the pH value of the environment to be tested is realized. Such a pH detection method has the advantages of fast response and high sensitivity.
本发明实施例提供的pH敏感的荧光纳米传感器，借助二氧化硅表层将异硫氰酸荧光素1、疏水性聚合物和长链烷基三甲氧基硅烷封装成荧光纳米传感器的内核，疏水性聚合物具有较大的分子量，能够有效避免异硫氰酸荧光素由二氧化硅表层泄露，提高荧光纳米传感器的稳定性，不但能够有效阻止异硫氰酸荧光素对生物的毒性伤害，而且能够保证荧光纳米传感器性能的精准稳定性，其原理在于：当异硫氰酸荧光素泄露后，将引起荧光强度的改变，而这将和pH变化引起的荧光强度的变化发生干扰，引起pH检测的误差。此外，多聚赖氨酸形成的外壳能够增强二氧化硅表层的封装效果，减小异硫氰酸荧光素的泄露。In the pH-sensitive fluorescent nanosensor provided by the embodiment of the present invention, fluorescein isothiocyanate 1, hydrophobic polymer and long-chain alkyltrimethoxysilane are encapsulated into the core of the fluorescent nanosensor by means of a silica surface layer, and the hydrophobicity The polymer has a large molecular weight, which can effectively prevent the leakage of fluorescein isothiocyanate from the surface layer of silica, improve the stability of the fluorescent nanosensor, not only can effectively prevent the toxicity of fluorescein isothiocyanate to organisms, but also can To ensure the precise and stable performance of fluorescent nanosensors, the principle is that when fluorescein isothiocyanate leaks, it will cause a change in fluorescence intensity, which will interfere with the change in fluorescence intensity caused by pH changes, causing pH detection. error. In addition, the shell formed by polylysine can enhance the encapsulation effect of the silica surface and reduce the leakage of fluorescein isothiocyanate.
在本发明的一些实施例中，所述疏水性聚合物选自聚苯乙烯或聚9,9-二辛基芴中的至少一者。聚苯乙烯或聚9,9-二辛基芴作为优选的疏水性聚合物，包含多个苯环结构且具有较大的分子量，能够有效避免异硫氰酸荧光素这样的小分子从二氧化硅表层的缝隙中泄露。In some embodiments of the present invention, the hydrophobic polymer is at least one selected from polystyrene or poly-9,9-dioctylfluorene. Polystyrene or poly-9,9-dioctylfluorene is a preferred hydrophobic polymer, which contains multiple benzene ring structures and has a relatively large molecular weight, which can effectively prevent small molecules such as fluorescein isothiocyanate from being oxidized Leakage from crevices in the silicon surface.
在本发明的一些实施例中，所述荧光纳米传感器的粒径为100～160nm。这样的较小的粒径，易于对细胞或生物组织等进行非侵入性地微观pH检测。可选的，所述荧光纳米传感器的粒径为100～155nm、100～150nm、100～145nm、105～155nm、105～150nm、110～150nm、115～145nm、120～140nm。In some embodiments of the present invention, the particle size of the fluorescent nanosensor is 100-160 nm. Such a small particle size facilitates non-invasive microscopic pH detection of cells or biological tissues. Optionally, the particle size of the fluorescent nanosensor is 100-155nm, 100-150nm, 100-145nm, 105-155nm, 105-150nm, 110-150nm, 115-145nm, 120-140nm.
现有技术中的荧光纳米传感器的合成过程较为复杂，为了保障生物兼容性，通常先制备获得纳米颗粒，然后在对纳米颗粒表面进行修饰以增加其生物兼容性，导致步骤繁琐耗时长。The synthesis process of fluorescent nanosensors in the prior art is relatively complicated. In order to ensure biocompatibility, nanoparticles are usually prepared first, and then the surface of nanoparticles is modified to increase their biocompatibility, resulting in cumbersome and time-consuming steps.
由此，本发明实施例的第二个方面，提出了一种pH敏感的荧光纳米传感器的制备方法的一个实施例，包括：Therefore, in the second aspect of the embodiments of the present invention, an embodiment of a method for preparing a pH-sensitive fluorescent nanosensor is proposed, including:
将异硫氰酸荧光素、疏水性聚合物和长链烷基三甲氧基硅烷按比例加入至四氢呋喃中，振荡混合得到混合溶液；Add fluorescein isothiocyanate, hydrophobic polymer and long-chain alkyltrimethoxysilane to tetrahydrofuran in proportion, shake and mix to obtain a mixed solution;
在超声条件下，将所述混合溶液加入到多聚赖氨酸的水溶液中即可得到荧光纳米传感器分散液，透析，得到荧光纳米传感器。Under ultrasonic conditions, the mixed solution is added to the aqueous solution of polylysine to obtain the fluorescent nanometer sensor dispersion liquid, which is dialyzed to obtain the fluorescent nanometer sensor.
本发明实施例提供的一种pH敏感的荧光纳米传感器的制备方法，通过简单灵活的共沉淀-包覆法，利用一步操作同时完成荧光纳米颗粒的形成以及纳米颗粒表面的修饰，简单方便，容易实现。具体的，通过将一定比例的异硫氰酸荧光素、疏水性聚合物和长链烷基三甲氧基硅烷的混合溶液，在超声条件下注入到含有多聚赖氨酸的水溶液中，由于环境极性的快速改变，疏水性物质(疏水性聚合物和长链烷基三甲氧基硅烷)遇水缩聚形成纳米颗粒，异硫氰酸荧光素分子同时被随机的掺杂于疏水纳米颗粒中；同时，长链烷基三甲氧基硅烷发生凝结和水解过程形成带负电的二氧化硅表层实现对纳米颗粒的封装，由此带负电荷的内核形成。随后由于多聚赖氨酸的氨基和硅烷醇基团的静电吸附作用，多聚赖氨酸分子吸附到纳米颗粒的表面，完成表面修饰形成包覆内核的多聚赖氨酸层，由此得到pH敏感的荧光纳米传感器。可见，本发明实施例提供的制备方法具有步骤简单灵活、容易实现的优势。The preparation method of a pH-sensitive fluorescent nanosensor provided by the embodiment of the present invention uses a simple and flexible co-precipitation-coating method to simultaneously complete the formation of fluorescent nanoparticles and the modification of the surface of the nanoparticles, which is simple, convenient, and easy. accomplish. Specifically, by injecting a mixed solution of a certain proportion of fluorescein isothiocyanate, hydrophobic polymer and long-chain alkyl trimethoxysilane into the aqueous solution containing polylysine under ultrasonic conditions, due to the environmental Rapid change of polarity, hydrophobic substances (hydrophobic polymers and long-chain alkyltrimethoxysilane) polycondensate to form nanoparticles, and fluorescein isothiocyanate molecules are randomly doped in the hydrophobic nanoparticles at the same time; At the same time, the condensation and hydrolysis of the long-chain alkyltrimethoxysilane forms a negatively charged silica surface layer to encapsulate the nanoparticles, thereby forming a negatively charged core. Subsequently, due to the electrostatic adsorption of the amino groups and silanol groups of polylysine, polylysine molecules are adsorbed to the surface of nanoparticles, and the surface modification is completed to form a polylysine layer covering the inner core, thus obtaining pH-sensitive fluorescent nanosensors. It can be seen that the preparation method provided by the embodiment of the present invention has the advantages of simple and flexible steps and easy implementation.
在本发明的一些实施例中，振荡混合的时间为20～40分钟，以保证溶液混合均匀。可选的，振荡混合时间为20分钟、30分钟或40分钟。In some embodiments of the present invention, the time for shaking and mixing is 20-40 minutes to ensure that the solution is evenly mixed. Optionally, the shaking mixing time is 20 minutes, 30 minutes or 40 minutes.
在本发明的一些实施例中，透析在去离子水中进行，透析时间由透析效果决定。可选的，透析时间可以是10～16小时，具体可以是10小时、12小时、13小时或16小时。In some embodiments of the present invention, dialysis is performed in deionized water, and the dialysis time is determined by the dialysis effect. Optionally, the dialysis time may be 10-16 hours, specifically 10 hours, 12 hours, 13 hours or 16 hours.
在本发明的一些实施例中，所述疏水性聚合物选自聚苯乙烯或聚9,9-二辛基芴中的至少一者。聚苯乙烯或聚9,9-二辛基芴作为优选的疏水性聚合物，包含多个苯环结构且具有较大的分子量，在水/四氢呋喃比例的突然增加时，有利于疏水纳米颗粒的形成，能够提高纳米颗粒的稳定性。In some embodiments of the present invention, the hydrophobic polymer is at least one selected from polystyrene or poly-9,9-dioctylfluorene. Polystyrene or poly-9,9-dioctylfluorene, as the preferred hydrophobic polymer, contains multiple benzene ring structures and has a large molecular weight, which is beneficial to the formation of hydrophobic nanoparticles when the water/THF ratio suddenly increases. Formation can improve the stability of nanoparticles.
可选的，所述聚苯乙烯的分子量为80,000～120,000；聚9,9-二辛基芴的分子量为50000～70000。Optionally, the polystyrene has a molecular weight of 80,000-120,000; the poly-9,9-dioctylfluorene has a molecular weight of 50,000-70,000.
在本发明的一些实施例中，所述长链烷基三甲氧基硅烷选自十六烷基三甲氧基硅烷或十二烷基三甲氧基硅烷中的至少一者。长链烷基三甲氧基硅烷不仅作为疏水性物质性参与纳米颗粒的形成，同时利用长链烷基三甲氧基硅烷的水解的特点形成二氧化硅表层实现对纳米颗粒的封装。十六烷基三甲氧基硅烷或十二烷基三甲氧基硅烷在这两方面性能优异，利于纳米颗粒的形成和封装。In some embodiments of the present invention, the long-chain alkyltrimethoxysilane is selected from at least one of hexadecyltrimethoxysilane or dodecyltrimethoxysilane. Long-chain alkyltrimethoxysilane not only participates in the formation of nanoparticles as a hydrophobic substance, but also uses the hydrolysis characteristics of long-chain alkyltrimethoxysilane to form a silica surface layer to encapsulate nanoparticles. Hexadecyltrimethoxysilane or dodecyltrimethoxysilane excels in both respects and facilitates the formation and encapsulation of nanoparticles.
在本发明的一些实施例中，按质量份计，所述异硫氰酸荧光素：所述疏水性聚合物：所述长链烷基三甲氧基硅烷为5～15:35～45:45～60。这一范围内的异硫氰酸荧光素既能保证用于检测的荧光强度，又不会因含量过高引起荧光淬灭。这一范围内的疏水性聚合物和长链烷基三甲氧基硅烷适宜形成纳米颗粒，特别是，该范围的长链烷基三甲氧基硅烷能够保证适宜的二氧化硅表层，以便于吸引多聚赖氨酸形成壳层。In some embodiments of the present invention, in parts by mass, the ratio of the fluorescein isothiocyanate: the hydrophobic polymer: the long-chain alkyltrimethoxysilane is 5-15:35-45:45 ~60. The fluorescein isothiocyanate within this range can not only ensure the fluorescence intensity used for detection, but also not cause fluorescence quenching due to too high content. Hydrophobic polymers and long-chain alkyltrimethoxysilanes in this range are suitable for forming nanoparticles. In particular, long-chain alkyltrimethoxysilanes in this range can ensure a suitable silica surface layer for attracting multiple Polylysine forms the shell.
可选的，所述异硫氰酸荧光素：所述疏水性聚合物：所述长链烷基三甲氧基硅烷可以是7～12:36～42:48～56、10～15:40～45:50～60、12～15:40～43:50～55或10～12:38～41:50～60等。Optionally, the ratio of the fluorescein isothiocyanate: the hydrophobic polymer: the long-chain alkyltrimethoxysilane can be 7～12:36～42:48～56, 10～15:40～ 45:50-60, 12-15:40-43:50-55 or 10-12:38-41:50-60, etc.
在本发明的一些实施例中，所述混合溶液中的异硫氰酸荧光素、疏水性聚合物和长链烷基三甲氧基硅烷的总浓度为200～800ppm。可选的，所述总浓度为200ppm、250ppm、300ppm、370ppm、400ppm、450ppm、480ppm、520ppm、600ppm、700ppm或800ppm。In some embodiments of the present invention, the total concentration of fluorescein isothiocyanate, hydrophobic polymer and long-chain alkyltrimethoxysilane in the mixed solution is 200-800 ppm. Optionally, the total concentration is 200ppm, 250ppm, 300ppm, 370ppm, 400ppm, 450ppm, 480ppm, 520ppm, 600ppm, 700ppm or 800ppm.
在本发明的一些实施例中，所述多聚赖氨酸的水溶液pH＝9。可选的，所述多聚赖氨酸的水溶液由多聚赖氨酸溶解于去离子水中形成。In some embodiments of the present invention, the aqueous solution of poly-lysine has pH=9. Optionally, the aqueous solution of polylysine is formed by dissolving polylysine in deionized water.
在本发明的一些实施例中，所述多聚赖氨酸的水溶液的浓度为0.02mg/mL。In some embodiments of the present invention, the concentration of the aqueous solution of polylysine is 0.02 mg/mL.
在本发明的一些实施例中，所述多聚赖氨酸的分子量在30KDa到50KDa之间。In some embodiments of the present invention, the molecular weight of the polylysine is between 30KDa and 50KDa.
为了进一步理解本发明，下面结合实施例对本发明提供的技术方案进行详细的说明，本发明的保护范围不受以下实施例的限制。In order to further understand the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the examples, and the protection scope of the present invention is not limited by the following examples.
实施例1Example 1
将异硫氰酸荧光素分子、聚苯乙烯(分子量100,000)和十六烷基三甲氧基硅烷按照10:40:50的质量比溶解于四氢呋喃中，使其在溶液中的总浓度为400ppm，振荡混合30分钟；Dissolve fluorescein isothiocyanate molecule, polystyrene (molecular weight 100,000) and hexadecyltrimethoxysilane in tetrahydrofuran according to the mass ratio of 10:40:50, so that the total concentration in the solution is 400ppm, Shake and mix for 30 minutes;
取上述混合溶液0.5mL，在超声条件下迅速注入到8mL溶有0.16mg多聚赖氨酸(分子量为30KDa～50KDa)的去离子水中(pH＝9)，最后将其在去离子水中透析12小时左右，即得到pH敏感的荧光纳米传感器。Take 0.5 mL of the above mixed solution, inject it into 8 mL of deionized water (pH=9) dissolved with 0.16 mg polylysine (molecular weight: 30KDa～50KDa) under ultrasonic conditions, and finally dialyze it in deionized water for 12 In about an hour, a pH-sensitive fluorescent nanosensor is obtained.
图2是本实施例得到的荧光纳米传感器的透射电子显微镜照片。观察其形貌大小可知，荧光纳米传感器为球形颗粒，粒径大约为100～160nm，具有良好的分散性。图3是荧光纳米传感器的动态光散射粒径分布图，从图中可以看出，荧光纳米传感器粒径大约为120nm左右，与透射电子显微镜成像统计的结果较一致。Fig. 2 is a transmission electron micrograph of the fluorescent nanosensor obtained in this example. Observing its shape and size, it can be seen that the fluorescent nanosensor is a spherical particle with a particle size of about 100-160 nm, and has good dispersion. Figure 3 is a dynamic light scattering particle size distribution diagram of the fluorescent nanosensor. It can be seen from the figure that the particle size of the fluorescent nanosensor is about 120nm, which is consistent with the statistical results of transmission electron microscope imaging.
实施例2Example 2
本实施例与实施例1的不同之处在于异硫氰酸荧光素、聚苯乙烯和十六烷基三甲氧基硅烷质量比为5:45:50。The difference between this example and Example 1 is that the mass ratio of fluorescein isothiocyanate, polystyrene and hexadecyltrimethoxysilane is 5:45:50.
实施例3Example 3
本实施例与实施例1的不同之处用十二烷基三甲氧基硅烷替换十六烷基三甲氧基硅烷，同时异硫氰酸荧光素分子、聚苯乙烯和十二烷基三甲氧基硅烷质量比为15:35:50。The difference between this example and Example 1 is that hexadecyltrimethoxysilane is replaced by dodecyltrimethoxysilane, while fluorescein isothiocyanate molecule, polystyrene and dodecyltrimethoxy The mass ratio of silane is 15:35:50.
以实施例1得到的pH敏感的荧光纳米传感器为例，说明本发明提供的pH敏感的荧光纳米传感器优异性能。Taking the pH-sensitive fluorescent nanosensor obtained in Example 1 as an example, the excellent performance of the pH-sensitive fluorescent nanosensor provided by the present invention is illustrated.
pH敏感特性pH Sensitive Properties
荧光纳米传感器的吸收光谱由日本岛津公司生产的型号为UV21011PC的紫外可见(UV-Vis)吸收光谱分析仪测得。结果如图4所示，显示荧光纳米传感器在490nm左右具有很强的吸收峰，证明异硫氰酸荧光素分子被有效地掺杂于纳米颗粒中。The absorption spectrum of the fluorescent nanosensor was measured by an ultraviolet-visible (UV-Vis) absorption spectrum analyzer model UV21011PC produced by Shimadzu Corporation of Japan. The results are shown in Figure 4, showing that the fluorescent nanosensor has a strong absorption peak around 490 nm, which proves that the FITC molecules are effectively doped in the nanoparticles.
通过日立公司生产的型号为F4600的光谱仪测量荧光纳米传感器对pH的敏感特性。具体方法是：样品池为1.0cm×l.0cm的四面透光的石英比色皿，将0.2mL纳米传感器悬浮液置于不同的pH值(4、5、6、7、8和9)缓冲液中，待二者充分反应5分钟后，通过荧光光谱仪对不同pH缓冲液中的荧光纳米传感器的发射光谱进行表征，激发波长为490nm，监测波长为500～600nm。The sensitivity of fluorescent nanosensors to pH was measured by a spectrometer model F4600 produced by Hitachi. The specific method is: the sample cell is a four-sided light-transmitting quartz cuvette of 1.0 cm × 1.0 cm, and 0.2 mL of nanosensor suspension is placed in buffers of different pH values (4, 5, 6, 7, 8 and 9). After the two fully reacted for 5 minutes, the emission spectra of the fluorescent nanosensors in different pH buffers were characterized by a fluorescence spectrometer, the excitation wavelength was 490nm, and the monitoring wavelength was 500-600nm.
测量结果表明，本发明实施例提供的pH敏感的荧光纳米传感器的发光强度对pH极为敏感。图5A表示pH敏感的荧光纳米传感器的发光强度随pH值的变化情况，可以观察到，当pH值从4增加到5时，发光强度随着pH值的上升而逐渐上升；当pH值从5增加到7时，发光强度随着pH值的上升而迅速增加；当pH值从8增加到9时，发光强度增幅减缓，在pH值4至9范围内荧光强度的变化率达到1154％，证明该荧光纳米传感器的发光强度具有极高的pH灵敏度。在518nm处的发光强度随着pH值的变化趋势可以通过对数函数进行拟合，得到如图5B所示的拟合曲线，通过该拟合曲线可以看出，pH＝5至pH＝7的范围内，纳米传感器的荧光强度与pH具有较好的线性关系，通过该pH纳米传感器可灵活、精确检测该区间内的pH变化。The measurement results show that the luminous intensity of the pH-sensitive fluorescent nanosensor provided by the embodiment of the present invention is extremely sensitive to pH. Figure 5A shows the variation of the luminous intensity of the pH-sensitive fluorescent nanosensor with the pH value. It can be observed that when the pH value increases from 4 to 5, the luminous intensity gradually increases with the increase of the pH value; When the pH value increases to 7, the luminescence intensity increases rapidly with the rise of the pH value; when the pH value increases from 8 to 9, the increase rate of the luminescence intensity slows down, and the change rate of the fluorescence intensity reaches 1154% in the range of pH value 4 to 9, proving that The luminous intensity of the fluorescent nanosensor has extremely high pH sensitivity. The variation trend of the luminous intensity at 518nm with the pH value can be fitted by a logarithmic function to obtain a fitting curve as shown in Figure 5B. It can be seen from the fitting curve that pH=5 to pH=7 Within the range, the fluorescence intensity of the nanosensor has a good linear relationship with the pH, and the pH nanosensor can flexibly and accurately detect the pH change within this range.
荧光纳米传感器的可逆性研究Study on the reversibility of fluorescent nanosensors
利用荧光纳米传感器进行pH值检测时，其重复使用的稳定性可以通过可逆性测试来验证。具体的，通过F4600光谱仪测定pH敏感的荧光纳米传感器的pH检测可逆性，将荧光纳米颗粒分散液交替置于pH值为4和9的缓冲溶液中，随后检测纳米传感器的发光强度变化，结果如图6所示，表明pH敏感的荧光纳米传感器具有良好的可逆性，反复改变pH值后发光强度仍可快速恢复，可重复用于pH值检测，监测细胞内的pH变化情况，具有良好的稳定性和检测准确性，可降低使用成本。When fluorescent nanosensors are used for pH detection, their repeated use stability can be verified by reversibility tests. Specifically, the pH detection reversibility of the pH-sensitive fluorescent nanosensor was measured by the F4600 spectrometer, and the fluorescent nanoparticle dispersion was alternately placed in the buffer solution with a pH value of 4 and 9, and then the change in the luminous intensity of the nanosensor was detected. The results are as follows As shown in Figure 6, it shows that the pH-sensitive fluorescent nanosensor has good reversibility, and the luminous intensity can still recover quickly after changing the pH value repeatedly. High performance and detection accuracy, which can reduce the cost of use.
荧光纳米传感器的响应特性研究Study on Response Characteristics of Fluorescent Nanosensors
荧光纳米传感器的响应特性通过F4600光谱仪进行表征。具体的，将纳米传感器分散液分别置于pH值为4、7、9的缓冲溶液中，随即检测荧光纳米传感器的发光强度随时间的变化趋势，测量时间间隔为1分钟。如图7所示，荧光纳米传感器具有较快的响应特性，反应1分钟后，纳米传感器的发光强度已接近稳定值，表明本发明实施例提供的荧光纳米传感器具有很高的响应灵敏度，能够有效提高检测效率。The response characteristics of fluorescent nanosensors were characterized by F4600 spectrometer. Specifically, the nanosensor dispersion was placed in buffer solutions with pH values of 4, 7, and 9, respectively, and then the luminous intensity of the fluorescent nanosensor was detected with a time interval of 1 minute. As shown in Figure 7, the fluorescent nanosensor has a faster response characteristic. After 1 minute of reaction, the luminous intensity of the nanosensor is close to a stable value, indicating that the fluorescent nanosensor provided by the embodiment of the present invention has a high response sensitivity and can effectively Improve detection efficiency.
荧光纳米传感器的生物兼容性Biocompatibility of fluorescent nanosensors
将HepG2细胞在共聚焦培养皿中培养24小时，然后加入荧光纳米传感器分散液(30mg/L)共培养12小时，使用PBS冲洗细胞以去除死细胞和黏贴到细胞表面的荧光纳米传感器，再通过共聚焦显微镜观察其在HepG2细胞中的荧光成像，其中异硫氰酸荧光素的绿色荧光由488nm的光激发，发射收集波段为500～560nm。从图8A～8C中可以看出，该荧光纳米传感器具有良好的生物兼容性，能够被细胞有效吞噬。HepG2 cells were cultured in a confocal culture dish for 24 hours, then co-cultured with fluorescent nanosensor dispersion (30 mg/L) for 12 hours, and washed with PBS to remove dead cells and fluorescent nanosensors adhered to the cell surface, and then The fluorescence imaging of FITC in HepG2 cells was observed by confocal microscope, in which the green fluorescence of FITC was excited by 488nm light, and the emission collection band was 500-560nm. It can be seen from Figures 8A-8C that the fluorescent nanosensor has good biocompatibility and can be effectively phagocytized by cells.
所属领域的普通技术人员应当理解：以上任何实施例的讨论仅为示例性的，并非旨在暗示本公开的范围(包括权利要求)被限于这些例子；在本发明的思路下，以上实施例或者不同实施例中的技术特征之间也可以进行组合，步骤可以以任意顺序实现，并存在如上所述的本发明的不同方面的许多其它变化，为了简明它们没有在细节中提供。Those of ordinary skill in the art should understand that: the discussion of any of the above embodiments is exemplary only, and is not intended to imply that the scope of the present disclosure (including claims) is limited to these examples; under the idea of the present invention, the above embodiments or Combinations between technical features in different embodiments are also possible, steps may be carried out in any order, and there are many other variations of the different aspects of the invention as described above, which are not presented in detail for the sake of brevity.
另外，为简化说明和讨论，并且为了不会使本发明难以理解，在所提供的附图中可以示出或可以不示出与集成电路(IC)芯片和其它部件的公知的电源/接地连接。此外，可以以框图的形式示出装置，以便避免使本发明难以理解，并且这也考虑了以下事实，即关于这些框图装置的实施方式的细节是高度取决于将要实施本发明的平台的(即，这些细节应当完全处于本领域技术人员的理解范围内)。在阐述了具体细节(例如，电路)以描述本发明的示例性实施例的情况下，对本领域技术人员来说显而易见的是，可以在没有这些具体细节的情况下或者这些具体细节有变化的情况下实施本发明。因此，这些描述应被认为是说明性的而不是限制性的。In addition, well-known power/ground connections to integrated circuit (IC) chips and other components may or may not be shown in the provided figures, for simplicity of illustration and discussion, and so as not to obscure the present invention. . Furthermore, devices may be shown in block diagram form in order to avoid obscuring the invention, and this also takes into account the fact that details regarding the implementation of these block diagram devices are highly dependent on the platform on which the invention is to be implemented (i.e. , these details should be well within the understanding of those skilled in the art). Where specific details (eg, circuits) have been set forth to describe example embodiments of the invention, it will be apparent to those skilled in the art that other embodiments may be implemented without or with variations from these specific details. Implement the present invention down. Accordingly, these descriptions should be regarded as illustrative rather than restrictive.
尽管已经结合了本发明的具体实施例对本发明进行了描述，但是根据前面的描述，这些实施例的很多替换、修改和变型对本领域普通技术人员来说将是显而易见的。例如，其它存储器架构(例如，动态RAM(DRAM))可以使用所讨论的实施例。Although the invention has been described in conjunction with specific embodiments of the invention, many alternatives, modifications and variations of those embodiments will be apparent to those of ordinary skill in the art from the foregoing description. For example, other memory architectures such as dynamic RAM (DRAM) may use the discussed embodiments.
本发明的实施例旨在涵盖落入所附权利要求的宽泛范围之内的所有这样的替换、修改和变型。因此，凡在本发明的精神和原则之内，所做的任何省略、修改、等同替换、改进等，均应包含在本发明的保护范围之内。Embodiments of the present invention are intended to embrace all such alterations, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent replacements, improvements, etc. within the spirit and principles of the present invention shall be included within the protection scope of the present invention.
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