WO2018054390A1 - 一种用于胞内癌症标志物双重检测的卫星状纳米组装体的制备方法及应用 - Google Patents

一种用于胞内癌症标志物双重检测的卫星状纳米组装体的制备方法及应用 Download PDF

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WO2018054390A1
WO2018054390A1 PCT/CN2017/108922 CN2017108922W WO2018054390A1 WO 2018054390 A1 WO2018054390 A1 WO 2018054390A1 CN 2017108922 W CN2017108922 W CN 2017108922W WO 2018054390 A1 WO2018054390 A1 WO 2018054390A1
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dimer
satellite
gold
gold rod
upconversion
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胥传来
匡华
徐丽广
郝恬甜
马伟
刘丽强
吴晓玲
宋珊珊
孙茂忠
胡拥明
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江南大学
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    • G01N2333/9125—Nucleotidyltransferases (2.7.7) with a definite EC number (2.7.7.-)
    • G01N2333/9128—RNA-directed DNA polymerases, e.g. RT (2.7.7.49)

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  • the invention relates to a preparation method and application of a satellite nano-assembly for dual detection of intracellular cancer markers, and belongs to the technical field of material chemistry.
  • MicroRNAs are a class of 18-25 nucleotide single-stranded endogenous non-coding RNAs that play important regulatory roles in life sciences. It is generally accepted that the level of miRNA expression changes is associated with altered expression and changes in hereditary diseases and immune functions. miRNAs are especially useful in the diagnosis of many solid tumors, and the expression levels of one or more miRNAs are commonly used as important diagnostic and prognostic biomarkers. Things. Traditional technologies, such as gene chips and real-time quantitative PCR, are time consuming, labor intensive, labor intensive, and expensive, which limits their application. Therefore, in situ quantitative detection methods in non-invasive and easy-to-operate living cells have attracted a great deal of attention.
  • telomerase is a ribonucleoprotein complex that uses its own ribonucleic acid component as a template to maintain the length of telomeres. Telomerase is highly expressed in primary tumors and immortalizes in these tumor cells. Therefore, telomerase has an important biomarker that is recognized as an early clinical diagnostic.
  • Traditional polymerase chain reaction (PCR)-based telomere repeat amplification (TRAP) due to the basis of amplification procedures, including length tailoring of telomerase product distribution, false positive results, and primer dimer problems, may be detected Less active telomerase.
  • fluorescent probes prepared with deoxyribonucleic acid in the pores of mesoporous silica nanoparticles have been used to quantify cellular telomerase activity. However, its limited sensitivity and applicability remain an important issue in complex matrices.
  • the technical solution of the present invention is a method for dual detection of intracellular cancer markers by a satellite-shaped nano-assembly, and the steps are as follows:
  • Gold rod dimer nuclear-upconversion satellite-like nano-assembly modified transmembrane peptide gold rod dimer nuclear-upconversion satellite-like structure assembly obtained in step (4) and SH-PEG-5000 and transmembrane The peptide TAT is mixed to obtain a gold rod dimer nuclear-upconversion satellite-like structural assembly having a surface-modified membrane-penetrating peptide;
  • step (6) Intracellular detection and characterization of gold rod dimer nuclear-upconversion satellite-like nanoassemblies: The cells obtained in step (6) were simultaneously subjected to Raman spectroscopy and fluorescence imaging, and a standard curve was established.
  • the satellite nano-assembly is used for the double detection of intracellular cancer markers, and the specific steps are as follows:
  • gold nanorod growth 5mL of 1mM trichloroauric acid trihydrate added to 5mL, 0.2M cetyltrimethylammonium bromide solution, add 4mL of ultrapure water, mix; then 0.125mL 0.01M silver nitrate solution was added to the above mixed system, and mixed; then 70 ⁇ L, 0.1 M ascorbic acid solution was added, stirred vigorously, and the solution became colorless, 2 min. After adding 12 ⁇ L of the seed crystal prepared in step a, stirring for 20 s, placed in a 30 ° C water bath, 2 h;
  • the nucleotide sequence of the thiol DNA1 was mixed at a molar concentration of 1..40; another 100 ⁇ L of the modified PEG-coated gold nanorods and the DNA1 partially complementary thiol DNA2 sequence were mixed at a molar concentration of 1..40;
  • the NaCl solution was mixed at a concentration of 50 mM, and after incubation at room temperature overnight, the unreacted DNA in the solution was removed by centrifugation three times, resuspended in 100 ⁇ L of 5 mM CTAB solution, and the obtained GNR-DNA1 and GNR-DNA2 were combined.
  • Body volume mixing hybridization adding NaCl solution to NaCl concentration of 50 mM for aging, incubation at room temperature for 12 h, to obtain gold rod dimer, to be used;
  • Proportionally modified telomerase primer TE primer sequence after overnight incubation, resuspended in 5 mM CTAB solution by centrifugation; 20 nm maleimide-modified water-soluble up-converting nanoparticles were diluted with 10 mM pH 7.4 tris buffer For 5 nM, the thiol mismatch DNA complementary to the linker DNA was modified at a molar concentration of 1..5, and the unbound mismatch DNA was removed by ultrafiltration after overnight incubation, and resuspended in 10 mM tris buffer pH 7.4; 50 ⁇ L TE primer modified gold rod The dimer was mixed with 200 ⁇ L of mismatch DNA-modified up-converting nanoparticles, assembled into a gold rod dimer nuclear-upconversion satellite-like structural assembly by 5 ⁇ L of 10 ⁇ M linker DNA, and the assembly was purified by gradient centrifugation;
  • Gold rod dimer nuclear-upconversion satellite-like structure modified transmembrane peptide gold rod dimer nuclear-upconversion satellite-like structure assembly obtained in step (4): PEG5000.. transmembrane peptide TAT in molar concentration 1 Mix the ratio of .1000..100, incubate for 12h at room temperature, centrifuge at 7500rpm for 10min, remove the supernatant, and resuspend the pellet in the cell culture medium;
  • gold rod dimer nuclear-upconversion satellite-like structural assemblies with surface-modified peptides can be directly introduced into cells for detection.
  • the analyte miRNA21 When the analyte miRNA21 is present, it binds to the microRNA21 antisense nucleotide fragment in the thiol DNA1 sequence, resulting in the disintegration of the gold rod dimer.
  • the Raman signal gradually decreases; when there is an object to be detected
  • TE primer transcribes along the linker DNA and replaces the position where the mismatch DNA is complementary to the linker DNA, resulting in up-conversion nanoparticles.
  • the granules dissociated from the assembly, and the fluorescence signal gradually recovered as the telomerase activity increased.
  • the gold nanorods and the up-converting nanoparticles dissociated from the assembly. Inducing changes in Raman and fluorescence signals, respectively, and then performing detection and characterization;
  • step (6) Intracellular detection and characterization of gold rod dimer nuclear-upconversion satellite-like nanoassemblies: the gold rod dimer nuclear-upconversion satellite-like structure assembly obtained in step (6) was transfected with different amounts of transfection agent After the cells were incubated with the untransfected cells for 8 hours, the cells were digested with 1 mL of trypsin to obtain the cell suspension of the gold rod dimer nuclear-up-conversion satellite structure to detect the concentration of miRNA21, followed by Raman spectroscopy. Establish a standard curve between different miRNA21 concentrations and Raman signal intensities in the cells;
  • the golden rod dimer nuclear-upconversion satellite nano-assembly obtained in step (6) was incubated with different amounts of EGCG for inhibition for 8 hours, and then the gold rod dimer nuclear-upconversion satellite structure was obtained.
  • Cells after detection of telomerase activity were subsequently subjected to fluorescence imaging to establish a standard curve between different telomerase activities and fluorescence intensities in the cells.
  • the DNA1 sequence is shown in SEQ ID NO. 1
  • the DNA2 sequence is shown in SEQ ID NO. 2
  • the TE primer sequence is shown in SEQ ID NO. 3
  • the mismatch DNA sequence is shown in SEQ ID NO. 4, linker DNA.
  • the sequence is shown in SEQ ID NO. 5, and the TAT polypeptide sequence is shown in SEQ ID NO. The details are shown in Table 1.
  • the present invention provides a gold rod dimer nuclear-upconversion satellite-like nanostructure assembly having uniform structure and good biocompatibility, and provides simultaneous detection of intracellular miRNA 21 by Raman and fluorescence dual signals.
  • the advantages of short use time have very good practical application prospects.
  • 1 is a transmission electron micrograph of a gold rod dimer (a), an upconverting nanoparticle (b), and a gold rod dimer core-upconversion satellite nanoassembly (c) of the present invention.
  • FIG. 2 is a Raman spectrum of the intracellular miRNA21 content of the gold rod dimer nuclear-upconversion satellite nano-assembly in the cells transfected with different amounts of transfection agent and untransfected cells in the present invention. (a), and a standard curve (b) of Raman signal and intracellular miRNA21 content.
  • FIG. 3 is a fluorescence imaging diagram (a) of detecting telomerase activity in a cell of a gold rod dimer nuclear-upconversion satellite nanoassembly in the present invention after being inhibited by different amounts of EGCG and in unsuppressed cells, And a standard curve of fluorescence signal and intracellular telomerase activity (b).
  • biomaterials in the following examples were purchased from Bioengineering (Shanghai) Co., Ltd.
  • the thiol-mismatch DNA complementary to the linker DNA was modified at a molar concentration of 1..5, and the unbound mismatch DNA was removed by ultrafiltration after overnight incubation, and resuspended in 10 mM pH 7.4 tris buffer; 50 ⁇ L TE primer modified gold rod II
  • the polymer was mixed with 200 ⁇ L of mismatch DNA-modified up-converting nanoparticles, assembled into a gold rod dimer core-upconversion satellite-like structural assembly by 5 ⁇ L of 10 ⁇ M linker DNA, and the assembly was purified by gradient centrifugation.
  • the transmission electron micrographs of the obtained gold rod dimer, upconverting nanoparticles, and gold rod dimer core-upconversion satellite nano-assemblies are shown in Fig. 1.
  • the material dispersibility is good, and the obtained assembly yield is high and there are
  • the complete assembly structure, its complete single morphology facilitates the highly sensitive detection of miRNA21 below.
  • Gold rod dimer nuclear-upconversion satellite-like structure modified transmembrane peptide the gold rod dimer nuclear-upconversion satellite-like structure assembly obtained in step (4) and PEG5000 and transmembrane peptide TAT are 1..1000 The molar concentration of .100 was mixed, incubated for 12 h at room temperature, and centrifuged at 7500 rpm for 10 min. The supernatant was removed and the pellet was resuspended in the cell culture medium.
  • gold rod dimer nuclear-upconversion satellite-like structural assemblies with surface-modified peptides can be directly introduced into cells for detection.
  • the analyte miRNA21 When the analyte miRNA21 is present, it binds to the microRNA21 antisense nucleotide fragment in the thiol DNA1 sequence, resulting in the disintegration of the gold rod dimer.
  • the Raman signal gradually decreases; when there is an object to be detected
  • TE primer transcribes along the linker DNA and replaces the position where the mismatch DNA is complementary to the linker DNA, resulting in the dissociation of the up-converting nanoparticles from the assembly.
  • the fluorescent signal gradually recovers;
  • the gold nanorods and the up-converting nanoparticles dissociate from the assembly, causing changes in Raman and fluorescence signals, respectively, and then performing detection and characterization.
  • Intracellular detection and characterization of gold rod dimer nuclear-upconversion satellite-like nanoassemblies cells transfected with different amounts of transfection agent and untransfected cells were subjected to real-time fluorescent PCR for intracellular miRNA21 concentration. Quantification; quantification of intracellular telomerase activity was performed using ELISA standard curves for different amounts of EGCG-inhibited and uninhibited cell lysates. The gold rod dimer nuclear-upconversion satellite nano-assembly obtained in step (6) was incubated with cells transfected with different amounts of transfection agent and untransfected cells for 8 hours, and then digested with 1 mL trypsin.
  • the cell was obtained, and the cell suspension of the gold-band dimer nuclear-upconversion satellite structure was detected to detect the concentration of miRNA21, followed by Raman spectroscopy, and the standard curve between different miRNA21 concentration and Raman signal intensity in the cell was established.
  • the miRNA concentration is linear with the Raman signal intensity;
  • the gold rod dimer nuclear-upconversion satellite structure assembly obtained in step (6) is different
  • the telomerase activity of the gold-rod dimer nuclear-up-conversion satellite structure was detected, followed by fluorescence imaging to establish different telomerase activities in the cells.
  • the standard curve between the fluorescence intensity and the fluorescence intensity is shown in Figure 3.
  • the telomerase activity is linearly related to the fluorescence intensity in the range of 0.6 x 10 -12 to 31 x 10 -12 IU.

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Abstract

本发明提供了一种用于胞内癌症标志物双重检测的卫星状纳米组装体的制备方法及应用，包括：一、卫星状纳米组装体的制备：金纳米棒的合成、20nm上转换纳米粒子的准备、金棒二聚体的组装，金棒二聚体上偶联拉曼信标分子DTTC，金棒二聚体核-上转换卫星状结构的组装；二、胞内癌症标志物双重检测：金棒二聚体核-上转换卫星状纳米组装体修饰穿膜肽，金棒二聚体核-上转换卫星状纳米组装体胞内的检测，金棒二聚体核-上转换卫星状纳米组装体胞内检测的表征，建立标准曲线。

Description

一种用于胞内癌症标志物双重检测的卫星状纳米组装体的制备方法及应用 技术领域
本发明涉及一种用于胞内癌症标志物双重检测的卫星状纳米组装体的制备方法及应用，属于材料化学技术领域。
背景技术
MicroRNA是一类18-25核苷酸的单链内源性非编码RNA，在生命科学中发挥重要的调控作用。目前普遍认为miRNA的表达变化水平与改变表达与遗传性疾病和免疫功能变化有关，miRNA尤其在许多实体多元化肿瘤的诊断，和一个或多个miRNAs的表达水平常用来作为重要诊断和预后生物标志物。传统的技术，如基因芯片和实时定量PCR，费时费力，劳动强度大，且价格昂贵，这限制了他们的应用。因此，无创性和容易操作的活细胞中原位定量检测方法已经引起了大量的关注。
端粒酶是一种核糖核蛋白复合物，利用其以自身所具有的核糖核酸组分为模板维持端粒的长度，端粒酶在原发性肿瘤中高表达，并在这些肿瘤细胞中永生。因此，端粒酶具有被公认为早期临床诊断学的重要生物标志物的作用。传统聚合酶链反应(PCR)为基础的端粒重复序列扩增法(TRAP)由于扩增程序的基础，包括长度剪裁端粒酶产物分布，假阳性结果，和引物二聚体问题，可能检测不到活性复杂的端粒酶。最近，在介孔二氧化硅纳米颗粒孔道中用脱氧核糖核酸制备的荧光探针已被用来量化细胞端粒酶的活性。然而，其有限的敏感性和适用性在复矩阵中仍然是一个重要的问题。
因此构建一种结构均一，生物相容性好的金棒二聚体核-上转换卫星状纳米结构，通过拉曼和荧光双重信号同时实现胞内miRNA21的含量和端粒酶的活性的检测很有意义，并且目前还未有报道。
发明内容
本发明的目的是提供一种卫星状纳米组装体用于胞内癌症标志物双重检测的方法，同时检测胞内miRNA21的含量和端粒酶的活性的方法。
本发明的技术方案，一种卫星状纳米组装体用于胞内癌症标志物双重检测的方法，步骤如下：
一、卫星状纳米组装体的制备：
(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800nm的金纳米棒GNR；
(2)金棒二聚体的组装：将步骤(1)得到的金纳米棒GNR修饰巯基聚乙二醇SH-PEG-1000，离心重悬后再分别修饰含有microRNA21反义核苷酸序列的巯基DNA1及与DNA1部分互补的巯基DNA2序列，分别得到GNR-DNA1及GNR-DNA2复合体，再将两种复合物混匀，得到金棒二聚体；
(3)金棒二聚体上偶联拉曼信标分子DTTC：将步骤(2)制备的金棒二聚体与拉曼信标分子3.3’-二乙基硫醛三碳菁化碘DTTC混匀，8h后得到表面修饰有DTTC的金棒二聚体-DTTC复合体；
(4)金棒二聚体核-上转换卫星状结构的组装：将步骤(3)得到的金棒二聚体-DTTC离心重悬后，修饰端粒酶的引物序列TE primer，20nm上转换纳米颗粒上修饰与linker DNA部分互补的巯基mismatch DNA，通过linker DNA组装成金棒二聚体核-上转换卫星状结构组装体，再将组装体通过梯度离心纯化；
二、胞内癌症标志物双重检测：
(5)金棒二聚体核-上转换卫星状纳米组装体修饰穿膜肽：将步骤(4)得到的金棒二聚体核-上转换卫星状结构组装体与SH-PEG-5000和穿膜肽TAT混匀，得到表面修饰有穿膜肽的金棒二聚体核-上转换卫星状结构组装体；
(6)金棒二聚体核-上转换卫星状纳米组装体胞内的检测：将步骤(5)得到的金棒二聚体核-上转换卫星状纳米组装体与细胞共孵育8h，当同时存在待测物miRNA21和端粒酶时，组装体的结构发生改变，金纳米棒和上转换纳米粒子从组装体上解离，分别引起拉曼和荧光信号的变化，进而进行检测；
(7)金棒二聚体核-上转换卫星状纳米组装体胞内检测表征：将步骤(6)得到的细胞同时进行拉曼光谱表征和荧光成像，并建立标准曲线。
所述卫星状纳米组装体用于胞内癌症标志物双重检测的方法，具体步骤如下：
一、卫星状纳米组装体的制备：
(1)金纳米棒的合成：
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的十六烷基三甲基溴化铵溶液中，溶液颜色由无色变成黄褐色，然后加入0.12mL新配制的0.01M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十六烷基三甲基溴化铵溶液中，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)金棒二聚体的组装：采用晶种生长法合成的金纳米棒，将其离心浓缩，重悬于5mM十六烷基三甲基溴化铵(CTAB)溶液中，使金纳米棒的浓度为5nM；将金纳米棒与巯基聚乙二醇PEG-1000以摩尔浓度1︰100的比例混匀，静置12h，之后取100μL修饰过PEG的金纳米棒将其与含有microRNA21反义核苷酸序列的巯基DNA1以摩尔浓度1︰40的比例混匀；另取100μL修饰过PEG的金纳米棒与DNA1部分互补的巯基DNA2序列以摩尔浓度1︰40的比例混匀；分别加入终浓度为50mM的NaCl溶液，充分混合后，室温孵育过夜后，离心3次除去溶液中未反应的DNA，分别重悬于100μL的5mM CTAB溶液中，再将得到的GNR-DNA1及GNR-DNA2复合体等体积混合杂交，加入NaCl溶液至NaCl浓度为50mM进行老化，室温下孵育12h，得到金棒二聚体，待用；
(3)金棒二聚体上偶联拉曼信标分子DTTC：将1mM的拉曼信标分子3.3’-二乙基硫醛三碳菁化碘(DTTC)加入到上述步骤(2)制备的金棒二聚体溶液中，混合均匀，保持DTTC终浓度为10μM，室温孵育8h；
(4)金棒二聚体核-上转换卫星状结构的组装：将步骤(3)修饰好拉曼信标分子的金棒二聚体离心重悬于5mM CTAB溶液中，以摩尔浓度1︰500的比例修饰端粒酶的引物TE primer序列，孵育过夜后，离心重悬于5mM CTAB溶液中；20nm的马来酰亚胺修饰的水溶性上转换纳米颗粒用10mM pH 7.4的tris缓冲液将浓度稀释为5nM，以摩尔浓度1︰5修饰与linkerDNA部分互补的巯基mismatch DNA，孵育过夜后超滤去除未结合的mismatch DNA，用10mM pH 7.4的tris缓冲液重悬待用；50μL TE primer修饰的金棒二聚体与200μL mismatch DNA修饰的上转换纳米颗粒混合，通过5μL 10μM linker DNA组装成金棒二聚体核-上转换卫星状结构组装体，再将组装体通过梯度离心纯化；
二、胞内癌症标志物双重检测：
(5)金棒二聚体核-上转换卫星状结构修饰穿膜肽：将步骤(4)得到的金棒二聚体核-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，7500rpm离心10min，去除上清液，沉淀重悬于细胞培养液中；
(6)金棒二聚体核-上转换卫星状纳米组装体胞内的检测：表面修饰有穿膜肽的金棒二聚体核-上转换卫星状结构组装体可直接进入细胞中进行检测，当存在待测物miRNA21时，其与巯基DNA1序列中的microRNA21反义核苷酸片段结合，导致的金棒二聚体解散，随着miRNA21浓度的增加，拉曼信号逐渐减弱；当存在待测物端粒酶和dNTP时，TE primer会沿着linkerDNA转录，并取代mismatchDNA与linkerDNA互补的位置，导致上转换纳米颗 粒从组装体上解离，随着端粒酶活性的增加，荧光信号逐渐恢复；当同时存在待测物miRNA21和端粒酶时，金纳米棒和上转换纳米粒子从组装体上解离，分别引起拉曼和荧光信号的变化，进而进行检测表征；
(7)金棒二聚体核-上转换卫星状纳米组装体胞内检测表征：将步骤(6)得到的金棒二聚体核-上转换卫星状结构组装体与经不同量转染剂转染后细胞及未经转染后的细胞分别共孵育8h后，用1mL胰蛋白酶消化细胞，得到金棒二聚体核-上转换卫星状结构检测miRNA21浓度后的细胞悬液，随后进行拉曼光谱表征，建立细胞中不同miRNA21浓度与拉曼信号强度两者之间的标准曲线；
将步骤(6)得到的金棒二聚体核-上转换卫星状纳米组装体与不同量EGCG抑制后和未经抑制的细胞分别共孵育8h后，得到金棒二聚体核-上转换卫星状结构检测端粒酶活性后的细胞，随后进行荧光成像，建立细胞中不同端粒酶活性与荧光强度两者之间的标准曲线。
所述DNA1序列如SEQ ID NO.1所示，DNA2序列如SEQ ID NO.2所示，TE primer序列如SEQ ID NO.3所示，mismatch DNA序列如SEQ ID NO.4所示，linker DNA序列如SEQ ID NO.5所示，TAT多肽序列如SEQ ID NO.6所示。具体如表1所示。
表1
Figure PCTCN2017108922-appb-000001
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二聚体核-上转换卫星状纳米结构组装体，提供了能够通过拉曼和荧光双重信号同时检测胞内miRNA21的含量和端粒酶的活性的方法，建立了细胞内miRNA21浓度与拉曼信号强度，及细胞中端粒酶活性与荧光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。
附图说明
图1是本发明的金棒二聚体(a)、上转换纳米颗粒(b)、金棒二聚体核-上转换卫星状纳米组装体(c)的透射电镜照片。
图2是本发明中金棒二聚体核-上转换卫星状纳米组装体进入经不同量转染剂转染后细胞及未经转染的细胞中，检测胞内不同miRNA21含量的拉曼光谱图(a)，以及拉曼信号与胞内miRNA21含量的标准曲线(b)。
图3是本发明中金棒二聚体核-上转换卫星状纳米组装体进入经不同量EGCG抑制后及未经抑制的细胞中，检测胞内不同端粒酶活性的荧光成像图(a)，以及荧光信号与胞内端粒酶活性的标准曲线(b)。
具体实施方式
以下实施例中生物材料均购自生工生物工程(上海)股份有限公司。
实施例1
所有的玻璃仪器都用王水浸泡24h，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：
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的十六烷基三甲基溴化铵溶液中，溶液颜色由无色变成黄褐色，然后加入0.12mL新配制的0.01M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十六烷基三甲基溴化铵溶液中，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抗坏血酸溶液加入，剧烈搅拌，溶液变为无色，2min后，加入12μL的晶种，搅拌20s，放入30℃水浴，2h；
(2)金棒二聚体的组装：采用晶种生长法合成的金纳米棒，将其离心浓缩，重悬于5mM CTAB溶液中，使金纳米棒浓度为5nM；将金纳米棒与巯基聚乙二醇PEG-1000以摩尔浓度1︰100的比例混匀，静置12h，之后取100μL修饰过PEG的金纳米棒将其与含有microRNA21反义核苷酸序列的巯基DNA1以摩尔浓度1︰40的比例混匀；另取100μL修饰PEG的金纳米棒与DNA1部分互补的巯基DNA2序列以摩尔浓度1︰40的比例混匀，分别加入50mM的NaCl溶液，充分混合后，室温孵育过夜后，离心3次除去溶液中未反应的DNA，分别重悬于100μL的5mM CTAB溶液中，再将得到的GNR-DNA1及GNR-DNA2复合体等体积混合杂交，加入NaCl溶液至其终浓度为50mM进行老化，室温下孵育12h，得到金棒二聚体， 待用。
(3)金棒二聚体上偶联拉曼信标分子DTTC：将1mM的拉曼信标分子3.3’-二乙基硫醛三碳菁化碘DTTC加入到上述步骤(2)制备的金棒二聚体溶液中，混合均匀，保持其终浓度为10μM，室温孵育8h。
(4)金棒二聚体核-上转换卫星状结构的组装：将步骤(3)修饰好拉曼信标分子的金棒二聚体离心重悬于5mM CTAB溶液中，以摩尔浓度1︰500的比例修饰上端粒酶的引物TE primer序列，孵育过夜后，离心重悬于5mM CTAB溶液中；20nm的马来酰亚胺修饰的水溶性上转换纳米颗粒用10mM pH 7.4的tris缓冲液稀释为5nM，以摩尔浓度1︰5修饰上与linker DNA部分互补的巯基mismatch DNA，孵育过夜后超滤去除未结合的mismatch DNA，用10mMpH 7.4的tris缓冲液重悬待用；50μL TE primer修饰的金棒二聚体与200μLmismatch DNA修饰的上转换纳米颗粒混合，通过5μL 10μM linker DNA组装成金棒二聚体核-上转换卫星状结构组装体，再将组装体通过梯度离心纯化。得到的金棒二聚体、上转换纳米颗粒、金棒二聚体核-上转换卫星状纳米组装体的透射电镜照片如图1所示，材料分散性良好，得到的组装体产率较高且有完整的组装结构，其完整单一的形貌有利于下文中miRNA21的高灵敏检测。
(5)金棒二聚体核-上转换卫星状结构修饰穿膜肽：将步骤(4)得到的金棒二聚体核-上转换卫星状结构组装体与PEG5000和穿膜肽TAT以1︰1000︰100的摩尔浓度混匀，室温孵育12h后，7500rpm离心10min，去除上清液，沉淀重悬于细胞培养液中。
(6)金棒二聚体核-上转换卫星状纳米组装体胞内的检测:表面修饰有穿膜肽的金棒二聚体核-上转换卫星状结构组装体可直接进入细胞中进行检测，当存在待测物miRNA21时，其与巯基DNA1序列中的microRNA21反义核苷酸片段结合，导致的金棒二聚体解散，随着miRNA21浓度的增加，拉曼信号逐渐减弱；当存在待测物端粒酶和dNTP时，TE primer会沿着linkerDNA转录，并取代mismatchDNA与linkerDNA互补的位置，导致上转换纳米颗粒从组装体上解离，随着端粒酶活性的增加，荧光信号逐渐恢复；当同时存在待测物miRNA21和端粒酶时，金纳米棒和上转换纳米粒子从组装体上解离，分别引起拉曼和荧光信号的变化，进而进行检测表征。
(7)金棒二聚体核-上转换卫星状纳米组装体胞内检测表征：将经不同量转染剂转染后细胞及未经转染后的细胞用实时荧光PCR进行胞内miRNA21浓度的定量；将不同量EGCG抑制后及未经抑制的细胞裂解液用ELISA标准曲线进行胞内端粒酶活性的定量。将步骤(6)得到的金棒二聚体核-上转换卫星状纳米组装体与经不同量转染剂转染后细胞及未经转染后的细胞分别共孵育8h后，用1mL胰蛋白酶消化细胞，得到金棒二聚体核-上转换卫星状结构 检测miRNA21浓度后的细胞悬液，随后进行拉曼光谱表征，建立细胞中不同miRNA21浓度与拉曼信号强度两者之间的标准曲线如图2所示，在0.011am/ngRNA-22.36amol/ngRNA范围内，miRNA浓度与拉曼信号强度成线性关系；将步骤(6)得到的金棒二聚体核-上转换卫星状结构组装体与不同量EGCG抑制后及未经抑制的细胞分别共孵育8h后，得到金棒二聚体核-上转换卫星状结构检测端粒酶活性后的细胞，随后进行荧光成像，建立细胞中不同端粒酶活性与荧光强度两者之间的标准曲线如图3所示，在0.6×10-12～31×10-12IU范围内端粒酶活性与荧光强度成线性关系。
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。
Figure PCTCN2017108922-appb-000002
Figure PCTCN2017108922-appb-000003
Figure PCTCN2017108922-appb-000004

Claims (3)

  1. 一种卫星状纳米组装体用于胞内癌症标志物双重检测的方法，其特征在于步骤如下：
    (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800nm的金纳米棒GNR；
    (2)金棒二聚体的组装：将步骤(1)得到的金纳米棒GNR修饰巯基聚乙二醇SH-PEG-1000，离心重悬后再分别修饰含有microRNA21反义核苷酸序列的巯基DNA1及与DNA1部分互补的巯基DNA2序列，分别得到GNR-DNA1及GNR-DNA2复合体，再将两种复合物混匀，得到金棒二聚体；
    (3)金棒二聚体上偶联拉曼信标分子DTTC：将步骤(2)制备的金棒二聚体与拉曼信标分子3.3’-二乙基硫醛三碳菁化碘DTTC混匀，8h后得到表面修饰有DTTC的金棒二聚体-DTTC复合体；
    (4)金棒二聚体核-上转换卫星状结构的组装：将步骤(3)得到的金棒二聚体-DTTC离心重悬后，修饰端粒酶的引物TE primer序列，20nm上转换纳米颗粒上修饰与linker DNA部分互补的巯基mismatch DNA，通过linker DNA组装成金棒二聚体核-上转换卫星状结构组装体，再将组装体通过梯度离心纯化；
    (5)金棒二聚体核-上转换卫星状纳米组装体修饰穿膜肽：将步骤(4)得到的金棒二聚体核-上转换卫星状结构组装体与SH-PEG-5000和穿膜肽TAT混匀，得到表面修饰有穿膜肽的金棒二聚体核-上转换卫星状结构组装体；
    (6)金棒二聚体核-上转换卫星状纳米组装体胞内的检测：将步骤(5)得到的金棒二聚体核-上转换卫星状纳米组装体与细胞共孵育8h，当同时存在待测物miRNA21和端粒酶时，组装体的结构发生改变，金纳米棒和上转换纳米粒子从组装体上解离，分别引起拉曼和荧光信号的变化，进而进行检测；
    (7)金棒二聚体核-上转换卫星状纳米组装体胞内检测表征：将步骤(6)得到的细胞同时进行拉曼光谱表征和荧光成像，并建立标准曲线。
  2. 根据权利要求1所述卫星状纳米组装体用于胞内癌症标志物双重检测的方法，其特征在于具体步骤如下：
    (1)金纳米棒的合成：
    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的十六烷基三甲基溴化铵溶液中，溶液颜色由无色变成黄褐色，然后加入0.12mL新配制的0.01M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 溴化铵溶液中，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)金棒二聚体的组装：采用晶种生长法合成的金纳米棒，将其离心浓缩，重悬于5mM十六烷基三甲基溴化铵CTAB溶液中，使金纳米棒的浓度为5nM；将金纳米棒与巯基聚乙二醇PEG-1000以摩尔浓度1︰100的比例混匀，静置12h，之后取100μL修饰PEG的金纳米棒将其与含有microRNA21反义核苷酸序列的巯基DNA1以摩尔浓度1︰40的比例混匀；另取100μL修饰PEG的金纳米棒与DNA1部分互补的巯基DNA2序列以摩尔浓度1︰40的比例混匀；分别加入终浓度为50mM的NaCl溶液，充分混合后，室温孵育过夜后，离心3次除去溶液中未反应的DNA，分别重悬于100μL的5mM CTAB溶液中，再将得到的GNR-DNA1及GNR-DNA2复合体等体积混合杂交，加入NaCl溶液至NaCl浓度为50mM进行老化，室温下孵育12h，得到金棒二聚体，待用；
    (3)金棒二聚体上偶联拉曼信标分子DTTC：将1mM的拉曼信标分子3.3’-二乙基硫醛三碳菁化碘DTTC加入到上述步骤(2)制备的金棒二聚体溶液中，混合均匀，保持DTTC终浓度为10μM，室温孵育8h；
    (4)金棒二聚体核-上转换卫星状结构的组装：将步骤(3)修饰拉曼信标分子的金棒二聚体离心重悬于5mM CTAB溶液中，以摩尔浓度1︰500的比例修饰端粒酶的引物TE primer序列，孵育过夜后，离心重悬于5mM CTAB溶液中；20nm的马来酰亚胺修饰的水溶性上转换纳米颗粒用10mM pH 7.4的tris缓冲液将浓度稀释为5nM，以摩尔浓度1︰5修饰与linkerDNA部分互补的巯基mismatch DNA，孵育过夜后超滤去除未结合的mismatch DNA，用10mM pH 7.4的tris缓冲液重悬待用；50μL TE primer修饰的金棒二聚体与200μL mismatch DNA修饰的上转换纳米颗粒混合，通过5μL 10μM linker DNA组装成金棒二聚体核-上转换卫星状结构组装体，再将组装体通过梯度离心纯化；
    (5)金棒二聚体核-上转换卫星状结构修饰穿膜肽：将步骤(4)得到的金棒二聚体核-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，7500rpm离心10min，去除上清液，沉淀重悬于细胞培养液中；
    (6)金棒二聚体核-上转换卫星状纳米组装体胞内的检测：表面修饰有穿膜肽的金棒二聚体核-上转换卫星状结构组装体直接进入细胞中进行检测，当存在待测物miRNA21时，其与巯基DNA1序列中的microRNA21反义核苷酸片段结合，导致的金棒二聚体解散，随着miRNA21浓度的增加，拉曼信号逐渐减弱；当存在待测物端粒酶和dNTP时，TE primer会沿着linkerDNA转录，并取代mismatchDNA与linkerDNA互补的位置，导致上转换纳米颗 粒从组装体上解离，随着端粒酶活性的增加，荧光信号逐渐恢复；当同时存在待测物miRNA21和端粒酶时，金纳米棒和上转换纳米粒子从组装体上解离，分别引起拉曼和荧光信号的变化，进而进行检测表征；
    (7)金棒二聚体核-上转换卫星状纳米组装体胞内检测表征：将步骤(6)得到的金棒二聚体核-上转换卫星状结构组装体与经不同量转染剂转染后细胞及未经转染后的细胞分别共孵育8h后，用1mL胰蛋白酶消化细胞，得到金棒二聚体核-上转换卫星状结构检测miRNA21浓度后的细胞悬液，随后进行拉曼光谱表征，建立细胞中不同miRNA21浓度与拉曼信号强度两者之间的标准曲线；
    将步骤(6)得到的金棒二聚体核-上转换卫星状纳米组装体与不同量EGCG抑制后及未经抑制的细胞分别共孵育8h后，得到金棒二聚体核-上转换卫星状结构检测端粒酶活性后的细胞，随后进行荧光成像，建立细胞中不同端粒酶活性与荧光强度两者之间的标准曲线。
  3. 根据权利要求1所述卫星状纳米组装体用于胞内癌症标志物双重检测的方法，其特征在于：所述DNA1序列如SEQ ID NO.1所示，DNA2序列如SEQ ID NO.2所示，TE primer序列如SEQ ID NO.3所示，mismatch DNA序列如SEQ ID NO.4所示，linker DNA序列如SEQ ID NO.5所示，TAT多肽序列如SEQ ID NO.6所示。
PCT/CN2017/108922 2016-09-20 2017-11-01 一种用于胞内癌症标志物双重检测的卫星状纳米组装体的制备方法及应用 WO2018054390A1 (zh)

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