WO2019096069A1 - 一种基于FRET效应的同时检测两种miRNA的方法 - Google Patents

一种基于FRET效应的同时检测两种miRNA的方法 Download PDF

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WO2019096069A1
WO2019096069A1 PCT/CN2018/114795 CN2018114795W WO2019096069A1 WO 2019096069 A1 WO2019096069 A1 WO 2019096069A1 CN 2018114795 W CN2018114795 W CN 2018114795W WO 2019096069 A1 WO2019096069 A1 WO 2019096069A1
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dna
satellite
gold
assembly
upconversion
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匡华
瞿爱华
胥传来
徐丽广
刘丽强
吴晓玲
宋珊珊
胡拥明
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江南大学
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"

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  • the invention relates to a method for simultaneously detecting two miRNAs based on the FRET effect, in particular to a miRNA ultra-sensitive detection platform of a gold rod-upconversion satellite assembly based on the FRET effect, belonging to the field of material chemistry.
  • miRNAs are small RNA regulatory factors widely found in eukaryotic cells. They usually have a length of about 21 bases and play an important role in regulating development. Studies have found that abnormal expression of miRNAs is associated with a variety of cancers, which also makes miRNAs a new biological marker for cancer diagnosis, providing a new means of treatment of human diseases. However, due to its low content in cells, small size and easy degradation, it is very difficult to quantitatively detect miRNAs in cells. miRNA microarray hybridization and real-time quantitative polymerase chain reaction (PCR) are conventional methods for studying the abnormal expression of miRNA, but the equipment is expensive, the process is complex, and the sensitivity is limited, which restricts its development.
  • PCR real-time quantitative polymerase chain reaction
  • the invention establishes a method for simultaneously detecting two miRNAs based on the FRET effect, and is a method for detecting two miRNAs by a gold bar-upconversion satellite assembly based on the FRET effect, which is an ultrasensitive and highly specific intracellular method.
  • the in situ detection method can simultaneously detect intracellular miR-21 and miR-200b levels.
  • the invention firstly provides a gold rod core-upconversion satellite-shaped nano-assembly, the preparation method comprising the following steps:
  • DNA 1 is fully complementary to the miRNA first test, when the measured first encounters miRNA, miRNA and a first test DNA 2 Competition binding DNA 1 .
  • DNA 4. 5 and partially complementary DNA, DNA complementary to DNA portion. 6 and 4, 4 and a second DNA test is fully complementary to miRNA, miRNA when it encounters the second test, a second test DNA miRNA. 5 Competition binding DNA 4 .
  • the first miRNA to be tested is miR-21 and the second miRNA to be tested is miR-200b.
  • the DNA 1 sequence is shown in SEQ ID NO. 1
  • the DNA 2 sequence is shown in SEQ ID NO. 2
  • the DNA 3 sequence is shown in SEQ ID NO. 3
  • DNA 4 The sequence is shown in SEQ ID NO. 4
  • the DNA 5 sequence is shown in SEQ ID NO. 5
  • the DNA 6 sequence is shown in SEQ ID NO.
  • the gold nanorods are resuspended in a 0.005 M CTAB solution to obtain a suspension having a final concentration of 10 nM in a molar ratio of AuNR:DNA 2 of 1: Add DNA 2 to 80-100, react for 12h at room temperature, centrifuge to remove excess DNA 2 , resuspend in 0.001M CTAB solution to obtain a final concentration of 10nM suspension; add DNA 4 at a molar concentration of 1:450-500. DNA 5 , reacted at room temperature for 12 h, centrifuged to remove excess DNA 4 DNA 5 , and resuspended in 0.001 M CTAB solution to give a final concentration of 10 nM.
  • step (2) the up-converting nanoparticles are diluted to 10 nM, 100 ⁇ L each, and DNA 1 and DNA 4 are coupled, respectively, and then the DNA 3 and the end of the terminally modified fluorescent molecule TAMRA are added.
  • the DNA 6 of the fluorescent molecule Cy5.5 was modified and hybridized to obtain up-converting nanoparticles coupled with different fluorescent molecules.
  • step (3) 20 ⁇ L of the up-converting nanoparticles modified with the fluorescent molecule TAMRA and 20 ⁇ L of the modified fluorescent molecule Cy5 are added to 2 ⁇ L of gold nanorods coupled with DNA 2 and DNA 5 . 100 ⁇ L of the up-converted nanoparticles of 5, after overnight incubation, the unbound particles were removed by gradient centrifugation to obtain an assembly of gold rod core-upconversion satellite-like structures.
  • a method for simultaneously detecting two miRNAs using a gold rod core-upconversion satellite-like nanoassembly comprises the following steps:
  • Transmembrane peptide modification the gold rod core-upconversion satellite-like structure assembly: PEG5000: the transmembrane peptide TAT is mixed at a molar concentration of 1:1000:100, and after incubation at room temperature for 12 hours, the supernatant is removed by centrifugation. The resulting precipitate is a transmembrane peptide-modified gold rod core-upconversion satellite nano assembly; the TAT polypeptide sequence is shown in SEQ ID NO.
  • the invention has the advantages that the invention has prepared a gold rod core-upconversion satellite nanostructure assembly with uniform structure and good biocompatibility, and provides a fluorescent signal capable of simultaneously detecting two intracellular miRNA contents by FRET effect.
  • the method establishes a standard curve between the intracellular miRNA concentration and the fluorescence signal intensity, and has the advantages of high sensitivity, good selectivity, low detection limit and short use time, and has a very good practical application prospect.
  • the detection line for miR-21 was 3.2 zmol/ng RNA and the detection line for miR-200b was 10.3 zmol/ng RNA.
  • the gold nanorod core-upconversion satellite-like nanoassemblies were added to the mismatch sequence, and the intracellular interference mass had no significant fluorescence change compared with the assembly, only the miR-21 and miR- were added.
  • FRET occurs and a change in fluorescence occurs.
  • the invention has good selectivity and sensitivity.
  • Figure 1 is a transmission electron micrograph, (a): gold nanorods prepared in Example 1, (b): upconverting nanoparticles, (c): gold nanorod core-upconversion satellite nanoassemblies prepared in Example 1.
  • FIG. 2 is a transmission electron micrograph of the gold nanorod core-upconversion satellite nanoassembly added to miR-21 (a) and miR-200b (b) in the present invention.
  • Figure 3 is an evaluation of extracellular detection ability in the present invention, (a): a fluorescence signal obtained by adding a series of different concentrations of miR-21 and miR-200b, each curve representing 1000 pM miR-21,500 from top to bottom. pM miR-200b, 500pM miR-21, 200pM miR-200b, 100pM miR-21, 100pM miR-200b, 50pM miR-21, 50pM miR-200b, 10pM miR-21, 20pM miR-200b and 5pM miR-21, 10pM miR -200b fluorescence intensity; (b): standard curve of fluorescence signal and miR-21 content; (c): standard curve of fluorescence signal and miR-200b content.
  • Figure 4 is a diagram showing the gold nanorod-core-upconversion satellite nano-assemblies of the present invention into cells transfected with different amounts of transfection agent and untransfected cells, (a) intracellular different miR-21 and miR-200b The fluorescence imaging of the content, from left to right, 1 to 5 columns indicate that the content of miR-21 and miR-200b is gradually increased, and the corresponding fluorescence intensity is also enhanced; (b): fluorescence signal and intracellular miR-21 content Standard curve; (c): standard curve of fluorescence signal and intracellular miR-200b content.
  • Figure 5 is a graph showing the fluorescence signals of a gold nanorod core-upconversion satellite-like nanoassembly incorporating mismatch sequences, intracellular interfering substances, and target miRs in the present invention.
  • the gold nanorods synthesized in the step (1) were centrifuged at 7500 rpm for 15 minutes to remove small molecules such as Vc and AgNO 3 , and concentrated 10 times in a 0.005 M CTAB solution to obtain a suspension having a final concentration of 10 nM, and 50 ⁇ L was taken.
  • PCR tubule molar concentration AuNR DNA 2 1: 80-100 added DNA 2, the reaction at room temperature for 12h after centrifugation to remove superfluous DNA 2, resuspended in 0.001M CTAB solution to a concentration of 10 nM AuNR; molar and then Concentration AuNR: DNA 5 1:450-500 was added to DNA 5 and DNA 4 , and reacted at room temperature for 12 hours, and then the excess DNA 4 and DNA 5 were removed by centrifugation, and resuspended in a 0.001 M CTAB solution to have an AuNR concentration of 10 nM.
  • Fig. 1a and Fig. 1b show gold nanorods with uniform structure and good dispersion.
  • the nanoparticles were converted, and
  • Figure 1c shows a gold rod-up-converting satellite-like nanoassembly obtained by DNA coupling.
  • gold bars TAMRA-coupled upconverting nanoparticles: up to 1:10:50 coupled with Cy5.5
  • upconversion The content is too low to form a complete gold rod core-upconversion satellite structure; the content is too high, too many heterogeneous particles, resulting in a decrease in yield.
  • the miR-21 and miR-200b were added to the gold rod-up-converting satellite-like nano-assembly, respectively, and the assembly structure such as transmission electron micrograph is shown in Fig. 2.
  • Fig. 2a when miR-21 is added, the complementary sequence of the end face is competitive, and the up-converting nanoparticles are dissociated from the end face;
  • Fig. 2b when miR-200b is added, the complementary sequence of the side binding is excited, thereby up-converting The nanoparticles dissociate from the side.
  • Gold rod core-upconversion satellite nano-assembly modified transmembrane peptide Gold rod core-upconversion satellite-like structure assembly obtained in the step (4) of Example 1: PEG5000: transmembrane peptide TAT at a molar concentration of 1:1000 The ratio of 100 was mixed, incubated at room temperature for 12 h, centrifuged at 7000 rpm for 10 min, the supernatant was removed, and the pellet was resuspended in the cell culture medium;
  • the analyte miR-200b When the analyte miR-200b exists in the cell, it binds to the complementary fragment in the sequence DNA 4 , causing the upconversion nanoparticles on the side of the gold rod to be dissipated and excited at 980 nm.
  • the fluorescence signal of the up-converting nanoparticles at 660 nm is weakened, and the Cy5.5 signal of the fluorescent molecule is gradually enhanced; when the analytes miR-21 and miR-200b are simultaneously present in the cells
  • the up-converting nanoparticles on the end face and the side of the gold nanorods are simultaneously dissociated from the assembly, causing changes in the fluorescence signals at 540 nm and 660 nm, respectively, and then performing detection and characterization.
  • the fluorescence intensity of TAMRA green
  • Cy5.5 red
  • the assembly is suitable for quantitative detection of intracellular miRNAs.

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Abstract

一种基于FRET效应的同时检测两种miRNA的方法，属于材料化学领域。该方法合成了金棒核—上转换卫星状结构组装体，并建立了标准曲线，最终制备出了结构均一、生物相容性好的金棒核—上转换卫星状纳米组装体，提供了一种能够通过FRET效应实现胞内两种miRNA的定量检测的平台。

Description

一种基于FRET效应的同时检测两种miRNA的方法 技术领域
本发明涉及一种基于FRET效应的同时检测两种miRNA的方法，尤其是一种基于FRET效应的金棒-上转换卫星组装体的miRNA超灵敏检测平台，属于材料化学领域。
背景技术
MicroRNA(miRNA)是广泛存在于真核生物细胞中的小分子RNA调控因子，通常具有21个碱基左右的长度，在调控发育过程中发挥重要作用。研究发现，miRNA的异常表达与多种癌症相关，这也使miRNA成为癌症诊断的新的生物学标记，给人类疾病的治疗提供一种新的手段。但是由于其在细胞中的含量低、尺寸小和易降解等特点，定量检测细胞内的miRNA是非常困难的。miRNA微阵列杂交和实时定量聚合酶链反应(PCR)是用来研究miRNA的异常表达的常规方法，但是设备昂贵、工艺复杂、灵敏度有限，制约了其发展。
近年来，基于DNA的纳米粒子可控自主装技术已经被广泛的应用于生物传感，其有规则的几何形状，稳定的结构，很强的光学活性。除此以外，发展了许多基于FRET效应的检测方法来检测金属粒子、癌症标志物等，已经成为一个热点话题引起了广泛的关注，但是，主要集中在检测一种miRNA，且有机染料光学信号不稳定、寿命短、背景信号强等缺点都限制了该技术在胞内同时检测多种miRNA中的应用。
发明内容
本发明建立了一种基于FRET效应的同时检测两种miRNA的方法，是基于FRET效应的金棒-上转换卫星组装体的检测两种miRNA的方法，是一种超灵敏，高特异性的胞内的原位检测方法，可同时检测胞内的miR-21和miR-200b含量。
本发明首先提供一种金棒核一上转换卫星状纳米组装体，其制备方法包括如下步骤：
(1)合成金纳米棒，并偶联DNA2、DNA5；
(2)将上转换纳米粒子分别与DNA 1、DNA 4偶联，其中，DNA 2与DNA 1部分互补，DNA 5与DNA 4部分互补；荧光染料分子TAMRA偶联DNA 3，荧光染料分子Cy5.5偶联DNA 6；其中DNA 3与DNA 1部分互补，DNA 6与DNA 4部分互补；
(3)将偶联了DNA 2、DNA 5的金纳米棒与步骤(2)得到的荧光染料修饰后的上转换纳米粒子组装。
其中，DNA 2与DNA 1部分互补，DNA 3与DNA 1部分互补，DNA 1与第一待测miRNA完全互补，当遇到第一待测miRNA时，第一待测miRNA与DNA 2竞争结合DNA 1。同样，DNA 5 与DNA 4部分互补，DNA 6与DNA 4部分互补，DNA 4与第二待测miRNA完全互补，当遇到第二待测miRNA时，第二待测miRNA与DNA 5竞争结合DNA 4。在本发明的一种实施方式中，所述第一待测miRNA是miR-21，所述第二待测miRNA是miR-200b。
在本发明的一种实施方式中，所述DNA 1序列如SEQ ID NO.1所示，DNA 2序列如SEQ ID NO.2所示，DNA 3序列如SEQ ID NO.3所示，DNA 4序列如SEQ ID NO.4所示，DNA 5序列如SEQ ID NO.5所示，DNA 6序列如SEQ ID NO.6所示。
在本发明的一种实施方式中，步骤(1)，将金纳米棒重悬在0.005M的CTAB溶液中，得到终浓度为10nM的悬液，以摩尔浓度AuNR:DNA 2的比例为1:80-100加入DNA 2，室温下反应12h后离心去除多余的DNA 2，重悬在0.001M的CTAB溶液中，得到终浓度为10nM的悬液；再以摩尔浓度1:450-500加入DNA 4DNA 5，室温下反应12h后离心去除多余的DNA 4DNA 5，重悬在0.001M的CTAB溶液中，得到终浓度为10nM的悬液。
在本发明的一种实施方式中，步骤(2)中，将上转换纳米粒子稀释为10nM，各取100μL，分别偶联DNA 1和DNA 4，然后加入末端修饰荧光分子TAMRA的DNA 3和末端修饰荧光分子Cy5.5的DNA 6，进行杂交得到偶联不同荧光分子的上转换纳米粒子。
在本发明的一种实施方式中，步骤(3)，向偶联了DNA 2、DNA 5的金纳米棒2μL中，加入修饰了荧光分子TAMRA的上转换纳米粒子20μL和修饰了荧光分子Cy5.5的上转换纳米粒子100μL，孵育过夜后梯度离心去除未结合的粒子，得到金棒核一上转换卫星状结构的组装体。
应用金棒核一上转换卫星状纳米组装体同时检测两种miRNA的方法，包括如下步骤：
(1)制备金棒核一上转换卫星状纳米组装体；
(2)制作细胞中不同浓度的miRNA与荧光强度之间的标准曲线：将金棒核一上转换卫星状结构组装体与经不同量转染剂转染后的待检测目标细胞及未经转染后的待检测目标细胞，分别共孵育一段时间，随后进行荧光成像，建立不同浓度的miRNA与荧光强度之间的标准曲线；
(3)穿膜肽修饰：将金棒核一上转换卫星状结构组装体：PEG5000：穿膜肽TAT以摩尔浓度1:1000:100的比例混匀，室温孵育12h后，离心去除上清液，所得沉淀即为穿膜肽修饰的金棒核一上转换卫星状纳米组装体；TAT多肽序列如SEQ ID NO.7所示；
(4)将膜肽修饰修饰后的金棒核一上转换卫星状结构组装体转入待测细胞，培养8小时后，细胞用PBS洗三遍，去除未进入细胞的组装体后，进行上转换荧光共聚焦呈像，在980nm激发下，得到荧光图像，将荧光强度代入标准曲线，计算得到胞内miRNA的含量。
本发明的有益效果：本发明制备出了结构均一,生物相容性好的金棒核一上转换卫星状纳米结构组装体，提供了能够通过FRET效应的荧光信号同时检测胞内的两种miRNA含量的方法，建立了细胞内miRNA浓度与荧光信号强度之间的标准曲线，具有灵敏度高、选择性好，检测限低、用时短的优点，具有非常好的实际应用前景。对于灵敏度，如图4所示，miR-21的检测线为3.2zmol/ngRNA，miR-200b的检测线为10.3zmol/ngRNA。对于选择性，如图5所示，金纳米棒核一上转换卫星状纳米组装体加入错配序列、细胞内干扰质与组装体相比没有明显的荧光变化，只有加入miR-21和miR-200b时，才会发生FRET，产生荧光变化。说明本发明具有良好的选择性和灵敏度。
附图说明
图1透射电镜照片，(a)：实施例1制备的金纳米棒，(b)：上转换纳米颗粒，(c)：实施例1制备的金纳米棒核一上转换卫星状纳米组装体。
图2是本发明中金纳米棒核一上转换卫星状纳米组装体加入miR-21(a)和miR-200b(b)的透射电镜照片。
图3是本发明中胞外检测能力的评价，(a)：添加一系列不同浓度的miR-21和miR-200b得到的荧光信号图，各曲线从上到下依次分别代表添加1000pM miR-21,500pM miR-200b、500pM miR-21,200pM miR-200b、100pM miR-21,100pM miR-200b、50pM miR-21,50pM miR-200b、10pM miR-21,20pM miR-200b和5pM miR-21,10pM miR-200b的荧光强度；(b)：荧光信号与miR-21含量的标准曲线；(c)：荧光信号与miR-200b含量的标准曲线。
图4是本发明中金纳米棒核一上转换卫星状纳米组装体进入经不同量转染剂转染后及未经转染的细胞中，(a)胞内不同miR-21和miR-200b含量时的荧光成像图，从左向右1到5列表示miR-21和miR-200b的含量逐渐增加，对应的荧光强度也随之增强；(b)：荧光信号与胞内miR-21含量的标准曲线；(c)：荧光信号与胞内miR-200b含量的标准曲线。
图5是本发明中金纳米棒核一上转换卫星状纳米组装体加入错配序列、细胞内干扰质和目标miRs的荧光信号图。
具体实施方式
实施例1金棒核一上转换卫星状纳米组装体的制备
所有的玻璃仪器都用王水浸泡，并用双蒸水清洗，晾干备用。实验中使用的水均为18.2MΩ的Milli-Q超纯水。
(1)金纳米棒的合成：采用晶种生长法合成横向等离子吸收峰控制在780nm的金纳米棒；
a、晶种合成：室温下，将0.05mL浓度为10mM的三水合四氯金酸，加入到1mL的0.2M的十六烷基三甲基嗅化铵溶液中，溶液颜色由无色变成黄褐色，然后加入0.12mL新配制的硼氢化钠溶液，快速搅拌2min，溶液颜色即由黄褐色变为浅棕色；
b、金纳米棒生长：5mL的1mM的三水合四氯金酸加入到5mL、0.2M十六烷基三甲基嗅化铵溶液中，加入4mL的超纯水，混匀；再将0.125mL的0.01M硝酸银溶液加入到上述混合体系中，混匀；随后将70μL、0.1M的抗坏血酸溶液加入，剧烈搅拌，溶液变为无色，2min后，加入12μL步骤a制备的晶种，搅拌20s，放入30℃水浴，2h；
(2)上转换纳米粒子的准备：由北京万德高科技发展有限公司购买；上转换纳米粒子基本组成单位是NaGdF 4，掺杂Yb 3+,Er 3+，大小为20±3nm，具有良好的耐光性和化学稳定性，同时毒性小和信噪比高，适用于生物检测。
(3)上转换纳米粒子上偶联荧光染料分子：将购买的上转换纳米粒子稀释为10nM，各取100μL于PCR中，分别偶联DNA 1和DNA 4，然后分别加入末端修饰荧光分子TAMRA的DNA 3(与DNA 1部分互补)和末端修饰荧光分子Cy5.5.的DNA 6(与DNA 4部分互补)，进行杂交得到偶联了不同荧光分子的上转换纳米粒子。
表1
Figure PCTCN2018114795-appb-000001
(4)金棒核一上转换卫星状结构的组装：
将步骤(1)中合成的金纳米棒7500rpm离心15分钟，去除Vc、AgNO 3等小分子物质，浓缩10倍重悬在0.005M的CTAB溶液中得到终浓度为10nM的悬液，取50μL于PCR小管中，以摩尔浓度AuNR：DNA 21:80-100加入DNA 2，室温下反应12h后离心去除多余的 DNA 2，重悬在0.001M的CTAB溶液中使AuNR浓度为10nM；再以摩尔浓度AuNR：DNA 5 1:450-500加入DNA 5、DNA 4，室温下反应12h后离心去除多余的DNA 4、DNA 5，重悬在0.001M的CTAB溶液中使AuNR浓度为10nM。然后，取上述偶联DNA的金纳米棒2μL，加入步骤(3)中修饰荧光分子TAMRA的上转换纳米粒子20μL和修饰荧光分子Cy5.5的上转换纳米粒子100μL，孵育过夜后梯度离心去除未结合的粒子，得到金棒核一上转换卫星状结构的组装体。
得到的金纳米棒、上转换纳米颗粒、金棒核一上转换卫星状纳米组装体的透射电镜照片如图1所示，图1a、图1b展示了结构均一、分散性良好的金纳米棒和上转换纳米颗粒，图1c表示通过DNA偶联得到的金棒核-上转换卫星状纳米组装体。
在金棒偶联DNA过程中，由于端面和侧面分别修饰不同的DNA序列，所以控制CTAB的浓度以及DNA的浓度是关键。金棒重悬在高浓度的CTAB时，DNA 2优先与端面结合，所以要控制好DNA 2的浓度，浓度太高导致过量的DNA 2与侧面结合；这时，在较低浓度的CTAB时，加入适量的DNA 4与侧面结合。除此之外，控制金棒与上转换纳米粒子的比例(金棒：偶联TAMRA的上转换纳米粒子：偶联Cy5.5的上转换纳米粒子大概为1:10:50)也十分关键，上转换含量过低，形成不了完整的金棒核一上转换卫星结构；含量过高，杂粒子太多，导致产率降低。
向金棒核-上转换卫星状纳米组装体分别中加入miR-21和miR-200b，组装体结构如透射电镜照片如图2所示。如图2a所示，加入miR-21时，竞争结合端面的互补序列，上转化纳米粒子从端面解离；如图2b所示，加入miR-200b时，竞争结合侧面的互补序列，从而上转换纳米粒子从侧面解离。
实施例2胞内癌症标志物的检测
(1)金棒核一上转换卫星纳米组装体修饰穿膜肽：将实施例1步骤(4)得到的金棒核一上转换卫星状结构组装体：PEG5000：穿膜肽TAT以摩尔浓度1:1000:100的比例混匀，室温孵育12h后，7000rpm离心10min，去除上清液，沉淀重悬于细胞培养液中；
(2)金棒核一上转换卫星纳米组装体胞内检测两种miRNA：表面修饰有穿膜肽的金棒核一上转换卫星状结构组装体直接进入细胞中进行检测，当细胞内存在待测物miR-21时，其与序列DNA 1中的互补片段结合，导致的金棒端面的上转换纳米粒子解散，在980nm的激发下，产生FRET效应，随着miR-21浓度的增加，上转换纳米粒子在540nm的荧光信号减弱，荧光分子TAMRA信号逐渐增强；当细胞内存在待测物miR-200b时，与序列DNA 4中的互补片段结合，导致金棒侧面的上转换纳米粒子解散，在980nm的激发下，产生FRET效应，随 着miR-200b浓度的增加，上转换纳米粒子在660nm的荧光信号减弱，荧光分子Cy5.5信号逐渐增强；当细胞内同时存在待测物miR-21和miR-200b时，金纳米棒端面和侧面的上转换纳米粒子同时从组装体上解离，分别引起540nm和660nm处的荧光信号变化，进而进行检测表征。
(3)金棒核一上转换卫星纳米组装体胞内检测的表征，建立标准曲线：将实施例1制备得到的金棒核一上转换卫星状结构组装体与经不同量转染剂转染后的Hela细胞及未经转染后的细胞分别共孵育8h后，得到金棒核一上转换卫星状结构检测miRNA的细胞，随后进行荧光成像，建立细胞中不同浓度的miRNA与荧光强度两者之间的标准曲线。
如图4所示，随着Hela细胞内miR-21和miR-200b含量的增加，TAMRA(绿色)和Cy5.5(红色)的荧光强度随之增加，并呈现良好的线性关系。所以，该组装体适用于细胞内miRNA的定量检测。
实施例3胞外癌症标志物的检测
在体外，向金棒核一上转换卫星纳米组装体中加入不同浓度的miR-21和miR-200b。结果如图3所示，随着miR-21和miR-200b浓度的增加，端面和侧面的上转化纳米粒子解离程度增加，在980nm激发下，产生FRET，分别将能量转移给荧光分子TAMRA和Cy5.5，导致荧光增加。同时，miR-21、miR-200b的浓度分别与TAMRA和Cy5.5的荧光强度呈现良好的线性关系。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

  1. 一种金棒核一上转换卫星状纳米组装体，其特征在于，其制备方法包括如下步骤：
    (1)合成金纳米棒，并偶联DNA 2、DNA 5；
    (2)将上转换纳米粒子分别与DNA 1、DNA 4偶联，其中，DNA 2与DNA 1部分互补，DNA 5与DNA 4部分互补；荧光染料分子TAMRA偶联DNA 3，荧光染料分子Cy5.5偶联DNA 6；其中DNA 3与DNA 1部分互补，DNA 6与DNA 4部分互补；
    (3)将偶联了DNA 2、DNA 5的金纳米棒与步骤(2)得到的荧光染料修饰后的上转换纳米粒子组装。
  2. 根据权利要求1所述的一种金棒核一上转换卫星状纳米组装体，其特征在于，其中，DNA 2与DNA 1部分互补，DNA 3与DNA 1部分互补，DNA 1与第一待测miRNA完全互补，当遇到第一待测miRNA时，第一待测miRNA与DNA 2竞争结合DNA 1；DNA 5与DNA 4部分互补，DNA 6与DNA 4部分互补，DNA 4与第二待测miRNA完全互补，当遇到第二待测miRNA时，第二待测miRNA与DNA 5竞争结合DNA 4。
  3. 根据权利要求1或2所述的一种金棒核一上转换卫星状纳米组装体，其特征在于，所述第一待测miRNA是miR-21，所述第二待测miRNA是miR-200b。
  4. 根据权利要求1或2所述的一种金棒核一上转换卫星状纳米组装体，其特征在于，所述DNA 1序列如SEQ ID NO.1所示，DNA 2序列如SEQ ID NO.2所示，DNA 3序列如SEQ ID NO.3所示，DNA 4序列如SEQ ID NO.4所示，DNA 5序列如SEQ ID NO.5所示，DNA 6序列如SEQ ID NO.6所示。
  5. 根据权利要求1所述的一种金棒核一上转换卫星状纳米组装体，其特征在于，步骤(1)，将金纳米棒重悬在0.005M的CTAB溶液中，得到终浓度为10nM的悬液，以摩尔浓度AuNR:DNA 2的比例为1:80-100加入DNA 2，室温下反应12h后离心去除多余的DNA 2，重悬在0.001M的CTAB溶液中，得到终浓度为10nM的悬液；再以摩尔浓度1:450-500加入DNA 4DNA 5，室温下反应12h后离心去除多余的DNA 4DNA 5，重悬在0.001M的CTAB溶液中，得到终浓度为10nM的悬液。
  6. 根据权利要求1所述的一种金棒核一上转换卫星状纳米组装体，其特征在于，步骤(2)中，将上转换纳米粒子稀释为10nM，各取100μL，分别偶联DNA 1和DNA 4，然后加入末端修饰荧光分子TAMRA的DNA 3和末端修饰荧光分子Cy5.5的DNA 6，进行杂交得到偶联不同荧光分子的上转换纳米粒子。
  7. 根据权利要求1所述的一种金棒核一上转换卫星状纳米组装体，其特征在于，步骤(3)，向偶联了DNA 2、DNA 5的金纳米棒2μL中，加入修饰了荧光分子TAMRA的上转换纳米粒子20μL和修饰了荧光分子Cy5.5的上转换纳米粒子100μL，孵育过夜后梯度离心去除未结 合的粒子，得到金棒核一上转换卫星状结构的组装体。
  8. 一种应用权利要求1～7任一所述金棒核一上转换卫星状纳米组装体同时检测两种miRNA的方法，其特征在于，包括如下步骤：
    (1)制备金棒核一上转换卫星状纳米组装体；
    (2)制作细胞中不同浓度的miRNA与荧光强度之间的标准曲线：将金棒核一上转换卫星状结构组装体与经不同量转染剂转染后的待检测目标细胞及未经转染后的待检测目标细胞，分别共孵育一段时间，随后进行荧光成像，建立不同浓度的miRNA与荧光强度之间的标准曲线；
    (3)穿膜肽修饰：将金棒核一上转换卫星状结构组装体：PEG5000：穿膜肽TAT以摩尔浓度1:1000:100的比例混匀，室温孵育12h后，离心去除上清液，所得沉淀即为穿膜肽修饰的金棒核一上转换卫星状纳米组装体；TAT多肽序列如SEQ ID NO.7所示；
    (4)将膜肽修饰修饰后的金棒核一上转换卫星状结构组装体转入待测细胞，培养8小时后，细胞用PBS洗三遍，去除未进入细胞的组装体后，进行上转换荧光共聚焦呈像，在980nm激发下，得到荧光图像，将荧光强度代入标准曲线，计算得到胞内miRNA的含量。
  9. 一种用于检测胞内miRNA的试剂盒，其特征在于，含有权利要求1～7任一所述金棒核一上转换卫星状纳米组装体。
  10. 权利要求9所述试剂盒在检测miRNA中的应用。
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