技术领域Technical Field
本申请涉及生物技术领域，具体涉及一种基于荧光颜色和强度的纳米光学编码技术。The present application relates to the field of biotechnology, and in particular to a nano-optical encoding technology based on fluorescence color and intensity.
背景技术Background technique
目前，慢性病诊断和传染病防控形势日益严峻，针对系统性复杂性疾病的生物检测或成像的需求日益增加。生物标志物是指能客观评价和检测系统、器官、组织、细胞以及亚细胞的结构活功能在常规的生物过程、发病过程或药物反应过程发生改变的生化或分子生物学指标。但是，单一指标、单一因子的生物检测或成像很难准确地实现疾病早期检测、病情监控及预后效果评估。以肿瘤为例，迄今为止，还没有找到灵敏度高、特异性100％的肿瘤标志物。近年来，随着基因组、转录组和蛋白质组等分子生物学技术的不断发展，尤其单细胞分析技术的迅猛发展，生物标志物的种类和数量也变得越来越多。At present, the situation of chronic disease diagnosis and infectious disease prevention and control is becoming increasingly severe, and the demand for biological detection or imaging of systemic complex diseases is increasing. Biomarkers refer to biochemical or molecular biological indicators that can objectively evaluate and detect changes in the structure and function of systems, organs, tissues, cells and subcellular structures in routine biological processes, disease processes or drug response processes. However, biological detection or imaging of a single indicator or a single factor is difficult to accurately achieve early disease detection, disease monitoring and prognosis evaluation. Taking tumors as an example, no tumor markers with high sensitivity and 100% specificity have been found so far. In recent years, with the continuous development of molecular biology technologies such as genomics, transcriptomics and proteomics, especially the rapid development of single-cell analysis technology, the types and numbers of biomarkers have become more and more.
编码是信息通信系统必不可少的组成部分，指信息从一种形式转换为另一种形式的过程典型的通信系统模型主要包括信源、编码器、信道、译码器、信宿。近年来，人们开发出多种信源及信道编码方法以达到提高信息传输的可靠性(准确、不失真)、有效性(成本低、周期短)的目的。基于信息通信原理将核酸、小分子、蛋白质等生物标志物信息(信源)通过生物编码方法转换成可分析的信号(编码)，进而被读取(解码)，实现生物检测及分析目的，越来越引起人们的关注。Coding is an essential component of information communication systems, and refers to the process of converting information from one form to another. The typical communication system model mainly includes the source, encoder, channel, decoder, and destination. In recent years, people have developed a variety of source and channel coding methods to improve the reliability (accuracy, no distortion) and effectiveness (low cost, short cycle) of information transmission. Based on the principle of information communication, the information of biomarkers such as nucleic acids, small molecules, and proteins (source) is converted into analyzable signals (coding) through biological coding methods, and then read (decoding) to achieve the purpose of biological detection and analysis, which has attracted more and more attention.
光学编码的一般策略是探索多个光学维度，以便在单个载体中编码尽可能多的信息。通过组合的方法，能够在单个编码载体上展现不同的光谱特性。目前，常见的光学参数主要包括光谱(颜色)、强度、偏振、寿命、相角。由于光谱和强度编码的简便性和实用性(与大多数光学解码仪器兼容)，在光学编码方法中应用最为广泛。在光谱编码中，一般使用若干种不同颜色(发射波长)的荧光染料作为编码分子。另外，通过控制每种荧光分子在编码载体中的数量或浓度，能够实现荧光强度编码。目前，基于光谱(颜色)/强度的编码策略，已经开发出许多标记生物分子的技术。例如，Yougen Li等人将Alexa Fluor 488(绿色，G)和BODIPY 630/650(红色，R)荧光分子标记树枝状DNA分子，通过控制两种荧光分子的比例——4G1R、2G1R、1G1R、1G2R、1G4R，从而实现荧光强度比值编码。Mingyong Han等人将3种颜色(红、绿、蓝)、不同数量比例的量子点掺入聚合物微球中，实现了生物分析的多色光学编码。2018年，Fanghao Hu等人首次设计了20种具有不同拉曼光谱的polyyne材料(Carbonrainbow)，该材料不仅在活细胞内对多个细胞器具有高度特异性、敏感性和光稳定性，而且通过组合不同polyyne材料，获得了较大的编码容量(3×1013)。虽然能够在活细胞内实现多色成像，但该材料合成较为复杂，实验周期长。The general strategy of optical coding is to explore multiple optical dimensions in order to encode as much information as possible in a single carrier. Through a combination of methods, different spectral characteristics can be displayed on a single coding carrier. At present, common optical parameters mainly include spectrum (color), intensity, polarization, lifetime, and phase angle. Due to the simplicity and practicality of spectral and intensity coding (compatible with most optical decoding instruments), it is the most widely used in optical coding methods. In spectral coding, several fluorescent dyes of different colors (emission wavelengths) are generally used as coding molecules. In addition, by controlling the number or concentration of each fluorescent molecule in the coding carrier, fluorescence intensity coding can be achieved. At present, many technologies for labeling biomolecules have been developed based on the spectral (color)/intensity coding strategy. For example, Yougen Li et al. labeled dendritic DNA molecules with Alexa Fluor 488 (green, G) and BODIPY 630/650 (red, R) fluorescent molecules, and achieved fluorescence intensity ratio coding by controlling the ratio of the two fluorescent molecules-4G1R, 2G1R, 1G1R, 1G2R, 1G4R. Mingyong Han et al. incorporated quantum dots of three colors (red, green, and blue) and different numbers and proportions into polymer microspheres to achieve multicolor optical coding for biological analysis. In 2018, Fanghao Hu et al. designed 20 polyyne materials (Carbonrainbow) with different Raman spectra for the first time. This material not only has high specificity, sensitivity, and photostability for multiple organelles in living cells, but also obtains a large coding capacity (3×1013 ) by combining different polyyne materials. Although multicolor imaging can be achieved in living cells, the synthesis of this material is relatively complex and the experimental cycle is long.
之前我们利用DNA四面体作为编码载体，首次构建了基于FRET效应构建的荧光强度条形码。虽然以上编码方案具有一定意义，但由于仅利用荧光强度这一维度进行编码，无法实现更大的编码能力。Previously, we used DNA tetrahedron as a coding carrier to construct the first fluorescence intensity barcode based on the FRET effect. Although the above coding scheme has certain significance, it cannot achieve greater coding capabilities because it only uses the fluorescence intensity dimension for coding.
发明内容Summary of the invention
鉴于以上所述现有技术的缺点，本申请的目的在于提供一种基于荧光颜色和强度的纳米光学编码技术，用于解决现有技术中的问题。In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a nano-optical encoding technology based on fluorescence color and intensity to solve the problems in the prior art.
为实现上述目的及其他相关目的，本申请第一方面提供一种编码载体，所述编码载体包括由四条DNA单链组装形成的DNA四面体构型，所述DNA四面体构型的顶点组装有荧光分子，所述荧光分子选自四种Alexa Flour染料；所述编码载体的条形码信息包括四种荧光分子的种类和/或数量。To achieve the above-mentioned purpose and other related purposes, the first aspect of the present application provides a coding vector, which includes a DNA tetrahedron configuration formed by assembling four DNA single strands, and the vertices of the DNA tetrahedron configuration are assembled with fluorescent molecules, and the fluorescent molecules are selected from four Alexa Flour dyes; the barcode information of the coding vector includes the types and/or quantities of the four fluorescent molecules.
在本申请的任意实施例中，所述四种荧光分子包括Alexa Flour 405、AlexaFlour488、Alexa Flour 546和Alexa Flour 647中的一种或多种。In any embodiment of the present application, the four fluorescent molecules include one or more of Alexa Flour 405, Alexa Flour 488, Alexa Flour 546 and Alexa Flour 647.
在本申请的任意实施例中，所述条形码包括ABCD，其中A、B、C、D分别代表不同的荧光分子的数量。In any embodiment of the present application, the barcode includes ABCD, wherein A, B, C, and D respectively represent the number of different fluorescent molecules.
在本申请的任意实施例中，所述条形码的种类不低于33。In any embodiment of the present application, the types of the barcodes are no less than 33.
在本申请的任意实施例中，所述荧光分子修饰于所述DNA单链的5’端或3’端。In any embodiment of the present application, the fluorescent molecule is modified at the 5' end or 3' end of the DNA single strand.
在本申请的任意实施例中，所述DNA单链的边长为10～17bp。In any embodiment of the present application, the side length of the single-stranded DNA is 10 to 17 bp.
在本申请的任意实施例中，所述DNA四面体构型为正四面体构型。In any embodiment of the present application, the DNA tetrahedral configuration is a regular tetrahedral configuration.
本申请第二方面提供所述的编码载体在细胞成像中的用途。The second aspect of the present application provides the use of the encoding vector in cell imaging.
本申请第三方面提供所述的编码载体在制备检测产品中的用途。The third aspect of the present application provides the use of the encoding carrier in the preparation of a detection product.
在本申请的任意实施例中，所述检测包括疾病早期检测、病情监控及预后效果评估。In any embodiment of the present application, the detection includes early disease detection, condition monitoring and prognosis effect assessment.
与现有技术相比，本申请的有益效果为：Compared with the prior art, the beneficial effects of this application are:
1、本发明将不同数量和不同种类的荧光分子装载于DNA四面体上，解决了之前工作中编码要素单一，编码容量小的问题，进一步扩大了多重生物分析的能力。1. The present invention loads different numbers and types of fluorescent molecules onto DNA tetrahedrons, solving the problem of single coding elements and small coding capacity in previous work, and further expanding the capability of multiple biological analysis.
2、相较于其他类型的染料，本发明所用Alexa Fluor系列的染料在生物光学成像中具有难以比拟的优势，有利于细胞内生物靶标的高亮度成像。2. Compared with other types of dyes, the Alexa Fluor series of dyes used in the present invention have incomparable advantages in bio-optical imaging, which is conducive to high-brightness imaging of intracellular biological targets.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1显示为本发明所使用的编码载体——四面体DNA纳米结构示意图。FIG. 1 shows a schematic diagram of a tetrahedral DNA nanostructure, which is an encoding vector used in the present invention.
图2显示为本发明编码策略示意图。FIG. 2 is a schematic diagram showing the encoding strategy of the present invention.
图3A显示为10bp-TDN相关链的凝胶电泳表征。FIG. 3A shows the gel electrophoresis characterization of the 10 bp-TDN related chain.
图3B显示为17bp-TDN相关链的凝胶电泳表征。FIG. 3B shows the gel electrophoresis characterization of the 17 bp-TDN related chains.
图4A显示为以10bp-TDN作为编码载体，使用Alexa Fluor 488尝试强度编码的荧光发射光谱。FIG4A shows the fluorescence emission spectrum of intensity encoding attempted using Alexa Fluor 488 using 10 bp-TDN as the encoding vector.
图4B显示为以10bp-TDN作为编码载体，使用Alexa Fluor 488尝试强度编码的荧光最大发射值散点拟合图。FIG4B shows a scatter plot of the maximum fluorescence emission value obtained by intensity encoding with Alexa Fluor 488 using 10 bp-TDN as the encoding vector.
图5A显示为以17bp-TDN作为编码载体，使用Alexa Fluor 488尝试强度编码的荧光发射光谱。FIG5A shows the fluorescence emission spectrum of intensity encoding attempted using Alexa Fluor 488 using 17 bp-TDN as the encoding vector.
图5B显示为以17bp-TDN作为编码载体，使用Alexa Fluor 488尝试强度编码的荧光最大发射值散点拟合图。FIG5B shows a scatter plot of the maximum fluorescence emission value obtained by intensity encoding with Alexa Fluor 488 using 17 bp-TDN as the encoding vector.
图6A显示为10bp-TDN作为编码载体，所有颜色条形码的荧光发射光谱。FIG6A shows the fluorescence emission spectra of all color barcodes for 10 bp-TDN as the encoding vector.
图6B显示为17bp-TDN作为编码载体，所有颜色条形码的荧光发射光谱。FIG6B shows the fluorescence emission spectra of all color barcodes for 17 bp-TDN as the encoding vector.
图7A显示为供体的最大发射值。FIG7A shows the emission maximum of the donor.
图7B显示为受体的最大发射值。FIG7B shows the maximum emission values of the receptor.
图8显示为MCF-7细胞毒性实验的存活率。FIG. 8 shows the survival rate of MCF-7 cell cytotoxicity assay.
具体实施方式Detailed ways
为了使本申请的发明目的、技术方案和有益效果更加清晰，下面结合实施例对本申请作进一步说明。应理解，所述实施例只用于解释本申请，并非用于限定申请的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法，熟悉此技术的人士可由本说明所揭露的内容容易地了解本申请的其他优点及功效。In order to make the invention purpose, technical scheme and beneficial effect of the present application clearer, the present application is further described below in conjunction with the examples. It should be understood that the examples are only used to explain the present application and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified, and those familiar with the technology can easily understand other advantages and effects of the present application from the contents disclosed in this description.
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明。Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related conventional techniques in the art. These techniques have been fully described in existing literature.
本申请的发明人经过大量探索研究，分析发现Alexa染料具有极高的FRET效率，且该染料满足选择FRET对量子产率和R0的要求。接下来，考虑光谱重叠积分J(λ)，研究表明，Cy3、Cy3.5、Cy5或Alexa/>488、Cy3、Cy5或Cy3、Cy3.5、Alexa/>647或的光谱重叠积分较大。其中，Alexa/>555染料的光谱与Cy3染料的光谱完全匹配，而且Alexa555染料的共轭物比Cy3染料的共轭物具有更强的荧光和更高的光稳定性，为图像捕获提供了更多时间。Alexa/>647染料的光谱与Cy5染料的光谱基本匹配。一方面，AlexaFlour 647与核酸分子结合后的荧光强度远远强于Cy5染料共轭物的荧光强度。另一方面，与Cy5染料不同，当Alexa Flour 647与大多数蛋白质或核酸分子结合后，吸收光谱或荧光光谱的变化很小。因此，可在相同结合程度下产生更加显著的荧光。After extensive research and analysis, the inventor of this application found that Alexa The dye has a very high FRET efficiency and meets the requirements for FRET quantum yield and R0. Next, considering the spectral overlap integral J(λ), studies have shown that Cy3, Cy3.5, Cy5 or Alexa/> 488, Cy3, Cy5 or Cy3, Cy3.5, Alexa/> 647 or 647 have a large spectral overlap integral. Among them, Alexa/> The spectrum of 555 dye matches that of Cy3 dye exactly, and Alexa The 555 dye conjugate has stronger fluorescence and higher photostability than the Cy3 dye conjugate, providing more time for image capture. The spectrum of 647 dye is basically matched with that of Cy5 dye. On the one hand, the fluorescence intensity of AlexaFlour 647 after binding to nucleic acid molecules is much stronger than that of Cy5 dye conjugate. On the other hand, unlike Cy5 dye, when AlexaFlour 647 binds to most proteins or nucleic acid molecules, the change in absorption spectrum or fluorescence spectrum is very small. Therefore, more significant fluorescence can be produced at the same degree of binding.
基于上述分析，发明人认为可以使用Alexa染料替代/>染料。利用荧光分子的光谱(颜色)特征，使用DNA四面体和4种有机荧光分子构建了一种新型编码策略，通过将不同数量的荧光分子装载于DNA四面体上，从而实现细胞内的多色成像。Based on the above analysis, the inventor believes that Alexa can be used Dye replacement/> Dyes. Using the spectral (color) characteristics of fluorescent molecules, a new encoding strategy was constructed using DNA tetrahedrons and four organic fluorescent molecules. By loading different numbers of fluorescent molecules on DNA tetrahedrons, multicolor imaging in cells was achieved.
本申请一方面提供一种编码载体，编码载体包括由四条DNA单链组装形成的DNA四面体构型，DNA四面体构型的顶点组装有荧光分子，荧光分子选自四种不同的Alexa Flour染料；编码载体的条形码信息包括四种荧光分子的种类和/或数量作为条形码。不同的条形码由于荧光分子的种类和数量不相同，不同条形码之间具有不同的FRET路径和效率，从而产生荧光颜色和强度的差异。On one hand, the present application provides a coding vector, which includes a DNA tetrahedron structure formed by assembling four DNA single strands, and the vertices of the DNA tetrahedron structure are assembled with fluorescent molecules, and the fluorescent molecules are selected from four different Alexa Flour dyes; the barcode information of the coding vector includes the types and/or quantities of the four fluorescent molecules as barcodes. Different barcodes have different FRET paths and efficiencies due to the different types and quantities of fluorescent molecules, thereby generating differences in fluorescence color and intensity.
本申请所提供的编码载体中，四种荧光分子包括Alexa Flour 405、Alexa Flour488、Alexa Flour 546和Alexa Flour 647中的一种或多种。Alexa染料是带负电荷且亲水的荧光染料系列，该系列染料囊括范围较广，且经常用于荧光显微镜技术之中。其中，Alexa Flour 405由405nm激光激发，产生蓝光。Alexa Flour 488由488nm激光激发，产生绿光。Alexa Flour546由546nm激光激发，产生橙光。Alexa Flour 647由647nm激光激发，产生红光。In the encoding vector provided in the present application, the four fluorescent molecules include one or more of Alexa Flour 405, Alexa Flour 488, Alexa Flour 546 and Alexa Flour 647. Dyes are a series of negatively charged and hydrophilic fluorescent dyes. This series of dyes covers a wide range and is often used in fluorescence microscopy. Among them, Alexa Flour 405 is excited by a 405nm laser and produces blue light. Alexa Flour 488 is excited by a 488nm laser and produces green light. Alexa Flour 546 is excited by a 546nm laser and produces orange light. Alexa Flour 647 is excited by a 647nm laser and produces red light.
本申请所提供的编码载体中，条形码包括ABCD，其中A、B、C、D分别代表不同的荧光分子的数量，在本申请的具体实施方式中，ABCD例如可以依次表示Alexa Flour 405、AlexaFlour 488、Alexa Flour 546、Alexa Flour 647的数量。通过用不同种类的荧光染料实现颜色编码，用不同数量的荧光染料实现强度编码。基于颜色编码和强度编码的复合编码策略，使得条形码的种类不低于33，具体如表1所示。In the coding carrier provided by the present application, the barcode includes ABCD, wherein A, B, C, and D represent the number of different fluorescent molecules, respectively. In the specific embodiment of the present application, ABCD can, for example, represent the number of Alexa Flour 405, Alexa Flour 488, Alexa Flour 546, and Alexa Flour 647 in sequence. Color coding is achieved by using different types of fluorescent dyes, and intensity coding is achieved by using different numbers of fluorescent dyes. Based on the composite coding strategy of color coding and intensity coding, the number of barcode types is not less than 33, as shown in Table 1.
表1组装33种条形码的DNA链信息Table 1 DNA chain information of 33 barcodes
在颜色编码中，通过将不同种类的荧光染料与DNA四面体结合，实现颜色编码。In color coding, color coding is achieved by binding different kinds of fluorescent dyes to DNA tetrahedrons.
颜色编码分为4种情况：①仅结合一种染料，包括条形码1000、0100、0010、0001；②结合两种染料，包括条形码1100、1010、1001、0110、0101、0011。除了1001外，其他条形码均可通过染料之间的FRET效应产生不同颜色；③结合三种染料，包括条形码1110、1011、1101、0111，以上条形码均可通过染料之间的FRET效应产生不同的颜色；④结合四种染料，即条形码1111，通过4种染料之间的FRET效应实现颜色编码。Color coding is divided into 4 cases: ① Combining only one dye, including barcodes 1000, 0100, 0010, 0001; ② Combining two dyes, including barcodes 1100, 1010, 1001, 0110, 0101, 0011. Except for 1001, other barcodes can produce different colors through the FRET effect between dyes; ③ Combining three dyes, including barcodes 1110, 1011, 1101, 0111, all of the above barcodes can produce different colors through the FRET effect between dyes; ④ Combining four dyes, that is, barcode 1111, color coding is achieved through the FRET effect between the four dyes.
在强度编码中，通过将不同数量的荧光染料与DNA四面体结合，实现强度编码。Intensity encoding is achieved by binding different numbers of fluorescent dyes to DNA tetrahedrons.
以Alexa Flour 405一个荧光染料为例，强度编码也分为4种情况：①仅结合一个染料，即条形码1000；②结合两个染料，即条形码2000；③结合三个染料，即条形码3000；④结合四个染料，即条形码4000。通过结合1、2、3、4个荧光分子，可以分别得到Ⅰ、Ⅱ、Ⅲ、Ⅳ水平的荧光强度，从而为荧光分析提供了更多的可能。Taking Alexa Flour 405 as an example, intensity coding can be divided into four cases: ① Combining only one dye, i.e. barcode 1000; ② Combining two dyes, i.e. barcode 2000; ③ Combining three dyes, i.e. barcode 3000; ④ Combining four dyes, i.e. barcode 4000. By combining 1, 2, 3, and 4 fluorescent molecules, the fluorescence intensity of levels I, II, III, and IV can be obtained respectively, thus providing more possibilities for fluorescence analysis.
在强度编码中，若条形码的载体中含有剩余结合位点，则成倍增加原先装载的染料数量，从而实现强度编码。例如，当条形码为1000时，组装有一个Alexa Flour 405，还有三个剩余结合位点，则以此条形码为基础，还可以再组装两个Alexa Flour 405形成2000，或者组装三个Alexa Flour 405形成3000，或者组装四个Alexa Flour 405形成4000。再例如，当条形码为1100时，组装有一个Alexa Flour 405和一个Alexa Flour 488，还有两个剩余结合位点，则以此条形码为基础，还可以再组装两个Alexa Flour 405和两个AlexaFlour 488形成2000。In intensity coding, if the barcode carrier contains remaining binding sites, the number of dyes originally loaded is multiplied to achieve intensity coding. For example, when the barcode is 1000, one Alexa Flour 405 is assembled, and there are three remaining binding sites. Based on this barcode, two more Alexa Flour 405s can be assembled to form 2000, or three Alexa Flour 405s can be assembled to form 3000, or four Alexa Flour 405s can be assembled to form 4000. For another example, when the barcode is 1100, one Alexa Flour 405 and one Alexa Flour 488 are assembled, and there are two remaining binding sites. Based on this barcode, two more Alexa Flour 405s and two Alexa Flour 488s can be assembled to form 2000.
如图2所示，本申请将不同数量和不同种类的荧光分子装载于DNA四面体上，实现了最大编码33种不同的条形码，解决了之前工作中编码要素单一，编码容量小的问题，进一步扩大了多重生物分析的能力。As shown in FIG2 , the present application loads different numbers and types of fluorescent molecules onto DNA tetrahedrons, achieving the encoding of a maximum of 33 different barcodes, solving the problem of single encoding elements and small encoding capacity in previous work, and further expanding the capabilities of multiple biological analysis.
本申请所提供的编码载体中，荧光分子修饰于所述DNA单链的5’端或3’端。DNA四面体的4个端点总体上最多可以修饰4个荧光分子。In the encoding vector provided in the present application, the fluorescent molecule is modified at the 5' end or 3' end of the DNA single strand. The four endpoints of the DNA tetrahedron can be modified with up to four fluorescent molecules in total.
本申请所提供的编码载体中，DNA单链的边长为10～17bp；优选地，为10bp。In the encoding vector provided in the present application, the side length of the single-stranded DNA is 10 to 17 bp; preferably, it is 10 bp.
本申请所提供的编码载体中，如图1所示，DNA四面体构型为正四面体构型。DNA四面体的4个顶点之间的距离均相等。因此，不同的荧光修饰方式可能产生相同的样品，最终产生33种不同的样品。In the encoding vector provided by the present application, as shown in Figure 1, the DNA tetrahedron configuration is a regular tetrahedron configuration. The distances between the four vertices of the DNA tetrahedron are equal. Therefore, different fluorescent modification methods may produce the same sample, and ultimately produce 33 different samples.
本申请所提供的编码载体中，其制备方法包括将各所述DNA单链等比例混合，置于聚合酶链式反应仪进行退火杂交，制备获得。在本申请的一具体实施例中，所述聚合酶链式反应仪的程序为加热至95℃，时间为10min；然后冷却至4℃。In the coding vector provided in the present application, the preparation method thereof comprises mixing the single-stranded DNA in equal proportions, placing the single-stranded DNA in a polymerase chain reaction apparatus for annealing and hybridization, and preparing the coding vector. In a specific embodiment of the present application, the program of the polymerase chain reaction apparatus is to heat the single-stranded DNA to 95°C for 10 minutes, and then cool the single-stranded DNA to 4°C.
本申请另一方面提供编码载体在细胞成像中的用途。通过修饰不同种类和不同数量的荧光分子，可以实现在细胞种的多色成像，且本发明所用Alexa Fluor系列的染料在生物光学成像中具有难以比拟的优势，有利于细胞内生物靶标的高亮度成像。On the other hand, the present application provides the use of the encoding vector in cell imaging. By modifying different types and different numbers of fluorescent molecules, multi-color imaging in cell species can be achieved, and the Alexa Fluor series of dyes used in the present invention have incomparable advantages in bio-optical imaging, which is conducive to high-brightness imaging of intracellular biological targets.
本申请另一方面提供编码载体在制备检测产品中的用途。检测包括疾病早期检测、病情监控及预后效果评估。编码载体种的单链例如可以是核酸、小分子、蛋白质等生物标志物，而生物标志物可以作为客观评价和检测系统、器官、组织、细胞以及亚细胞的结构活功能在常规的生物过程、发病过程或药物反应过程发生改变的生化或分子生物学指标，因此可以通过生物编码方法将生物标志物转换成可分析的信号，进而被读取，实现生物检测及分析。On the other hand, the present application provides the use of coding vectors in the preparation of detection products. Detection includes early disease detection, disease monitoring and prognosis effect evaluation. The single strand of the coding vector can be, for example, a biomarker such as a nucleic acid, a small molecule, or a protein, and the biomarker can be used as a biochemical or molecular biological indicator for objectively evaluating and detecting changes in the structure and function of systems, organs, tissues, cells, and subcellular structures and functions in conventional biological processes, pathogenesis, or drug response processes. Therefore, the biomarker can be converted into an analyzable signal through a biological coding method, and then read to achieve biological detection and analysis.
下面通过实施例对本申请予以进一步说明，但并不因此而限制本申请的范围。The present application is further described below by way of examples, but the scope of the present application is not limited thereby.
本申请在下述具体的实施方式中所采用的材料及设备如下：The materials and equipment used in the following specific implementations of this application are as follows:
1×TM buffer,1×TE buffer,20×TBS buffer,1×TE buffer(low EDTA,pH8.0),5×TBE buffer等试剂均购自生工生物工程(上海)股份有限公司；实验所用水为Millipore-Q水(18.2MΩ·cm-1)。1×TM buffer, 1×TE buffer, 20×TBS buffer, 1×TE buffer (low EDTA, pH 8.0), 5×TBE buffer and other reagents were purchased from Sangon Biotech (Shanghai) Co., Ltd.; the water used in the experiment was Millipore-Q water (18.2 MΩ·cm-1).
NanoDrop微量分光光度计(BIO-RAD，PowerPac Basic)、PCR仪(BIO-RAD，T100Thermal Cycler)、荧光分光光度计(日立，F-7000)、酶标仪(BioTek Gen5)等。NanoDrop micro-volume spectrophotometer (BIO-RAD, PowerPac Basic), PCR instrument (BIO-RAD, T100 Thermal Cycler), fluorescence spectrophotometer (Hitachi, F-7000), microplate reader (BioTek Gen5), etc.
DNA链购于生工生物工程(上海)股份有限公司，使用高效液相色谱纯化。DNA strands were purchased from Sangon Biotech (Shanghai) Co., Ltd. and purified using high performance liquid chromatography.
实施例1Example 1
(1)条形码的合成(1) Barcode synthesis
构建10bp-TDN和17bp-TDN的DNA四面体，取四条单链各(ssDNA)0.5μL，共2μL，加入48μL 1×TM Buffer中，置于PCR仪中，设置合成程序：加热至95℃，时间为10min；然后冷却至4℃。制备成终体积为50μL，终浓度为500nM的条形码。最后将合成的条形码样品置于4℃冷藏储存。单链序列见表2。To construct DNA tetrahedrons of 10bp-TDN and 17bp-TDN, 0.5μL of each of the four single strands (ssDNA) was taken, a total of 2μL, added to 48μL 1×TM Buffer, placed in a PCR instrument, and set the synthesis program: heating to 95°C for 10min; then cooling to 4°C. Prepare a barcode with a final volume of 50μL and a final concentration of 500nM. Finally, the synthesized barcode sample was stored in a refrigerated container at 4°C. The single strand sequence is shown in Table 2.
表2序列信息Table 2 Sequence information
(2)SDS-PAGE跑胶(2) SDS-PAGE gel
配制A、B液(储存液)：A液由6mL 5×TBE、3.75mL 100mM MgCl2(0.076g，终浓度为12.5mM)、20.25mL H2O组成；B液由6mL 5×TBE、3.75mL 100mM MgCl2(0.076g，终浓度为12.5mM)、16mL 30％聚丙烯酰胺、4.25mL H2O组成。Prepare solution A and solution B (stock solution): solution A consists of 6 mL 5×TBE, 3.75 mL 100 mM MgCl2 (0.076 g, final concentration is 12.5 mM), and 20.25 mL H2 O; solution B consists of 6 mL 5×TBE, 3.75 mL 100 mM MgCl2 (0.076 g, final concentration is 12.5 mM), 16 mL 30% polyacrylamide, and 4.25 mL H2 O.
配制胶液：取A、B液各4mL，加入80μL APS(10％过硫酸铵)和8μL TEMED(四甲基乙二胺)，迅速混合均匀。Prepare glue solution: Take 4 mL of solution A and solution B, add 80 μL APS (10% ammonium persulfate) and 8 μL TEMED (tetramethylethylenediamine), and mix quickly.
将上述配制好的胶液均匀加入至玻璃片的夹层中，并插入电泳梳。待1小时后成胶即可使用。将(1)构建得到的10bp-TDN相关链和17bp-TDN相关链进行电泳，电泳条件分别为：电压100V、时间100min；电压100V、时间120min。Add the prepared gel solution evenly to the sandwich of glass slides and insert the electrophoresis comb. After 1 hour, the gel can be used. The 10 bp-TDN related chain and 17 bp-TDN related chain constructed in (1) were electrophoresed under the following conditions: voltage 100 V, time 100 min; voltage 100 V, time 120 min.
结果如图3所示，证明成功构建DNA四面体的条形码样品。The results are shown in FIG3 , demonstrating that the barcoded sample of DNA tetrahedron was successfully constructed.
(3)检测荧光光谱(3) Detection of fluorescence spectrum
取(2)中验证成功的终浓度为500nM的条形码样品，置于微量荧光比色皿中。检测每种条形码的最大发射光谱。其中，使用400.0nm作为Alexa 405的激发波长，发射波长范围为405.0nm-900.0nm；使用485.0nm作为Alexa 488的激发波长，发射波长范围为490.0nm-900.0nm；使用545.0nm作为Alexa 546的激发波长，发射波长范围为550.0nm-900.0nm；使用635.0nm作为Alexa 647的激发波长，发射波长范围为640.0nm-900.0nm。Take the barcode sample with a final concentration of 500nM successfully verified in (2) and place it in a micro-fluorescence cuvette. Detect the maximum emission spectrum of each barcode. Among them, 400.0nm is used as the excitation wavelength of Alexa 405, and the emission wavelength range is 405.0nm-900.0nm; 485.0nm is used as the excitation wavelength of Alexa 488, and the emission wavelength range is 490.0nm-900.0nm; 545.0nm is used as the excitation wavelength of Alexa 546, and the emission wavelength range is 550.0nm-900.0nm; 635.0nm is used as the excitation wavelength of Alexa 647, and the emission wavelength range is 640.0nm-900.0nm.
结果如图4和图5所示，当不同尺寸的TDN载体与不同数量的Alexa Fluor 488组装时，随着染料数量的增加，Alexa 488的特征峰对应的荧光强度呈现递增趋势，且均表现为良好的线性关系(10bp-TDN和17bp-TDN的R2分别为0.99391和0.99363)，结果证明了两种尺寸的载体均能实现强度编码。The results are shown in Figures 4 and 5. When TDN vectors of different sizes were assembled with different amounts of Alexa Fluor 488, the fluorescence intensity corresponding to the characteristic peak of Alexa 488 showed an increasing trend with the increase in the amount of dye, and both showed a good linear relationship (R2 of 10bp-TDN and 17bp-TDN were 0.99391 and 0.99363, respectively). The results proved that both sizes of vectors can achieve intensity encoding.
如图6所示，两种编码载体构建的颜色条形码具有相同的荧光发射峰，但在强度方面略有差别。如图7所示，相较于边长为17bp的四面体，边长10bp四面体作为载体时，供体的荧光强度略低，且受体的荧光强度相近或略高，说明10bp-TDN的FRET效率较高，更适合作为编码载体。As shown in Figure 6, the color barcodes constructed by the two encoding vectors have the same fluorescence emission peak, but slightly different in intensity. As shown in Figure 7, compared with the tetrahedron with a side length of 17 bp, when the tetrahedron with a side length of 10 bp is used as a vector, the fluorescence intensity of the donor is slightly lower, and the fluorescence intensity of the acceptor is similar or slightly higher, indicating that the FRET efficiency of 10 bp-TDN is higher and it is more suitable as an encoding vector.
(4)细胞毒性实验(4) Cytotoxicity assay
设置调零组(仅含DMEM)、对照组(DMEM+细胞)和实验组(DMEM+细胞+TDN载体)。将密度为5×104个/mL的MCF-7细胞悬液加入96孔板中，每孔100μL。培养24小时后，将(2)中验证成功的100nM 10bp-TDN和17bp-TDN分别与MCF-7细胞共孵育2、6、12、24、36和48小时。共孵育完成后，每孔加入10μL CCK-8溶液(Sangon Biotech，E606335)，反应0.5小时。使用酶标仪检测450nm处的吸光值。计算存活率，存活率＝实验组/(对照组-调零组)×100％。A zeroing group (containing only DMEM), a control group (DMEM + cells), and an experimental group (DMEM + cells + TDN vector) were set up. A MCF-7 cell suspension with a density of 5×104 cells/mL was added to a 96-well plate, 100 μL per well. After 24 hours of culture, the 100nM 10bp-TDN and 17bp-TDN successfully verified in (2) were incubated with MCF-7 cells for 2, 6, 12, 24, 36, and 48 hours, respectively. After the co-incubation was completed, 10 μL of CCK-8 solution (Sangon Biotech, E606335) was added to each well and reacted for 0.5 hours. The absorbance at 450 nm was detected using an ELISA reader. The survival rate was calculated, and the survival rate = experimental group/(control group-zeroing group) × 100%.
结果如图8所示，存活率一直维持高水平，说明10bp-TDN和17bp-TDN均能在细胞内能够长期稳定存在。The results are shown in FIG8 . The survival rate has been maintained at a high level, indicating that both 10bp-TDN and 17bp-TDN can exist stably in cells for a long time.
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本申请。任何熟悉此技术的人士皆可在不违背本申请的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本申请的权利要求所涵盖。The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present application. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present application.
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