WO2018054391A1 - 一种细胞内atp的圆二色光谱实时检测方法 - Google Patents

一种细胞内atp的圆二色光谱实时检测方法 Download PDF

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WO2018054391A1
WO2018054391A1 PCT/CN2017/108923 CN2017108923W WO2018054391A1 WO 2018054391 A1 WO2018054391 A1 WO 2018054391A1 CN 2017108923 W CN2017108923 W CN 2017108923W WO 2018054391 A1 WO2018054391 A1 WO 2018054391A1
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atp
dimer
gold
aptamer
sequence
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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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    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/19—Dichroism

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  • the invention relates to a real-time detection method of circular dichroism spectrum of intracellular ATP, belonging to the technical field of material chemistry.
  • Adenosine-5'-triphosphate is a major source of energy required for tissue cells in the body, as an energy currency, which is involved in gene duplication, protein or lipid kinase activity, membrane ion channel pumping, immune and neuronal mediated Reaction, drug delivery and other biological activity regulation.
  • changes in the amount of ATP in cells can reflect cell variability and damage, as well as the occurrence of diseases such as anemia, hypoglycemia, cardiovascular disease, and cancer. Therefore, ATP has received extensive attention and research as one of the markers of major diseases.
  • ATP detection methods include: high performance liquid chromatography, capillary electrophoresis, and nuclear magnetic resonance spectroscopy.
  • these methods of analyzing cell lysates have potential degradation of ATP, and the analysis time is long, making real-time detection difficult to achieve.
  • fluorescence analysis, chemiluminescence, and electrochemical tracking techniques have all been used for real-time detection of intracellular ATP.
  • these methods are cumbersome to operate, have large sample consumption, long detection time, unstable performance, and are difficult to miniaturize.
  • Circular dichroism spectroscopy is an analytical method that has been widely used in chemical and biological sensing. Compared to fluorescence detection, due to plasma-plasma coupling between nanoparticles, induced plasma CD Sub-plasma coupling, mixed dipole-dipole coupling and minimum matrix interference allow CDs to collect enough information from the sample and achieve low sensitivity detection levels with greater potential for biomarker analysis . However, real-time detection of intracellular ATP content by self-assembled chiral gold dimer nanosensors has not been reported.
  • the technical solution of the present invention is a method for real-time detection of circular dichroism spectrum of intracellular ATP, and the steps are as follows:
  • Circular dichroic gold dimer sensor constructed by ATP aptamer sequence assembly: 25nm gold nanoparticle GNP synthesized by citrate reduction method, modified thiol ATP aptamer sequence after centrifugation; 15nm gold nanoparticle GNP modification and adaptation
  • the thiol sequence ATP CS1 which is partially complementary to the ligand, is mixed with the GNP-aptamer and GNP-CS1 complexes to obtain a circular dichroic gold dimer aptamer-dimer assembled by ATP aptamer sequence;
  • Circular dichroic gold dimer sensor assembled by ATP mismatch sequence 25nm gold nanoparticle GNP synthesized by citrate reduction method, modified sulfhydryl mismatch DNA sequence after centrifugation; 15nm gold nanoparticle GNP modification and ATP error
  • the GNP-mismatch and GNP-CS2 complexes will be obtained by ligating the partially thiol ATP CS2 sequence. Mixing to obtain a circular dichroic gold dimer mismatch-dimer assembled with ATP mismatch sequence;
  • step (3) to obtain transmembrane peptide modified circular dichroic TAT-aptamer-dimer and different inhibitors and accelerators
  • the cells and the untreated cells were separately incubated.
  • the ATP of the analyte was present, the assembly gradually disaggregated, resulting in a change in the circular dichroic signal, and then detection, establishing a standard curve of intracellular ATP concentration and circular dichroism spectrum.
  • Circular dichroic gold dimer sensor assembled by ATP aptamer sequence 25 nm gold nanoparticles GNP synthesized by citrate reduction method were resuspended in 5 mM PB buffer of pH 7.4, and 100 ⁇ L of 2 nM 25 nm gold was taken. The nanoparticles were mixed with the thiol ATP aptamer sequence at a molar concentration of 1..5; the 15 nm gold nanoparticle GNP synthesized by the citrate reduction method was resuspended in 5 mM PB buffer of pH 7.4, and 100 ⁇ L of 2 nM 15 nm gold was taken.
  • the thiol ATP CS1 sequence partially complementary to the aptamer was mixed at a molar concentration of 1..5, and respectively added to a final concentration of 50 mM NaCl solution, mixed well, incubated at room temperature overnight, and centrifuged three times to remove the solution.
  • the reacted DNA was resuspended in 100 ⁇ L of 5 mM PB buffer, 100 ⁇ L of GNP-aptamer and 50 ⁇ L of GNP-CS1 complex were mixed, and NaCl solution was added to a final concentration of 50 mM for aging, and incubated at room temperature for 12 h to obtain ATP.
  • the thiol ATP CS2 sequence complementary to the aptamer moiety was mixed at a molar concentration of 1..5, added to a final concentration of 50 mM NaCl solution, mixed well, incubated overnight at room temperature, and centrifuged three times to remove unreacted solution. Resuspend the DNA in 100 ⁇ L of 5 mM PB buffer; mix 100 ⁇ L of GNP-mismatch and 50 ⁇ L of GNP-CS2 complex, add Into the NaCl solution to a final concentration of 50 mM for aging, incubation at room temperature for 12 h, to obtain a circular dichromeric gold dimer mismatch-dimer assembled with ATP mismatch sequence, to be used;
  • Two size gold dimer modified transmembrane peptides the gold dimers obtained in steps (1) and (2) are mixed with SH-PEG5000 and transmembrane peptide TAT at a molar concentration of 1..1000..100, respectively. After homogenization, incubate for 12 h at room temperature, centrifuge at 7500 rpm for 20 min, remove the supernatant, and resuspend the pellet in the cell culture medium to obtain a stable surface-modified penetrant peptide TAT-aptamer-dimer and TAT-mismatch-dimer;
  • the change of the intracellular circular dichroism signal of the two size gold dimer sensors over time the cells were seeded in a 24-well culture plate, so that the number of cells in each well was 104, and the culture solution was removed after 24 hours of culture. 8 wells were added to the final concentration of 5nM transmembrane peptide modified circular dichroic TAT-aptamer-dimer and 104 cells were co-cultured for 0h, 2h, 4h, 6h, 8h, 12h, 16h, 24h; 8 The wells were added to a penetrating peptide modified circular dichroic TAT-mismatch-dimer with a final concentration of 5 nM and co-cultured with 104 cells for 0h, 2h, 4h, 6h, 8h, 12h, 16h, 24h; The cells were each obtained with cell suspensions containing different amounts of round dichromatic aptamer-dimer and mismatch-dimer. The cell suspension was characterized by circular dichroism
  • the cells were digested with 1 mL of trypsin to obtain cell suspensions of circular dichroic aptamer-dimer containing different degrees of dissolving, and the cell suspension was subjected to circular dichroism characterization, and intracellular ATP concentration and circle were established.
  • the standard curve of the two-color signal was established.
  • the ATP aptamer sequence is shown in SEQ ID NO. 1
  • the ATP CS1 sequence is shown in SEQ ID NO. 2
  • the mismatch DNA sequence is shown in SEQ ID NO. 3
  • the ATP CS2 sequence is shown in SEQ ID NO.
  • the TAT polypeptide sequence is shown in SEQ ID NO. 5, and is specifically shown in Table 1.
  • the invention prepares a gold nanoparticle dimer with high assembly yield, stable property and good dispersibility in cells, and provides a method for real-time detection of intracellular ATP content by circular dichroism spectroscopy A standard curve between intracellular ATP concentration and circular dichroic signal intensity is established, which has the advantages of high sensitivity, good selectivity, time and labor saving, and has very good practical application prospects.
  • Figure 1 is a bio-transmission electron micrograph of a gold nanoparticle dimer of the present invention in a cell.
  • 2 is a scatter plot of the two-dimensional gold dimer sensor of the present invention in the intracellular circular dichroism signal over time.
  • Fig. 3 is a graph showing changes in the circular dichroism signal of the transmembrane peptide-modified circular dichroic TAT-aptamer-dimer of the present invention after incubation with cells and untreated cells supplemented with different inhibitors and promoters, respectively.
  • the curves are the intensity of the circular dichroic signal when the ATP concentrations are 1.5 mM, 2.0 mM, 2.8 mM, 3.5 mM, and 4.2 mM.
  • Figure 4 is a standard curve of intracellular ATP concentration and circular dichroism signal.
  • sequence material in the following examples was purchased from Bioengineering Engineering (Shanghai) Co., Ltd.
  • Circular dichroic gold dimer sensor constructed by ATP aptamer sequence assembly 25nm synthesized by citrate reduction method Gold nanoparticles GNP were resuspended in 5 mM PB buffer, pH 7.4, and 100 ⁇ L of 2 nM 25 nm gold nanoparticles were mixed with the thiol ATP aptamer sequence at a molar concentration of 1..5; 15 nm gold nanoparticles were resuspended by GNP centrifugation.
  • the thiol ATP CS2 sequence was mixed at a molar concentration of 1..5, and added to a final concentration of 50 mM NaCl solution. After thorough mixing, the mixture was incubated overnight at room temperature, and the unreacted DNA in the solution was removed by centrifugation three times and resuspended in 100 ⁇ L. In the 5 mM PB buffer, 100 ⁇ L of GNP-mismatch and 50 ⁇ L of GNP-CS2 complex were mixed, and NaCl solution was added to a final concentration of 50 mM for aging, and incubated at room temperature for 12 h to obtain a circular dichroic gold assembled by ATP mismatch sequence. Dimer mismatch-dimer, ready to use;
  • Two size gold dimer modified transmembrane peptides the gold dimers obtained in steps (1) and (2) are mixed with SH-PEG5000 and transmembrane peptide TAT at a molar concentration of 1..1000..100, respectively. After homogenization, incubate for 12 h at room temperature, centrifuge at 7500 rpm for 20 min, remove the supernatant, and resuspend the pellet in the cell culture medium to obtain a stable surface-modified circular dichroic TAT-aptamer-dimer and TAT-mismatch-dimer with transmembrane peptide.
  • the bio-transmission electron micrograph of the gold nanoparticle dimer in the cell is shown in Fig. 1. As can be seen from the figure, the assembly is still present in the cell in a high yield of dimer.
  • the change of the intracellular circular dichroism signal of the two size gold dimer sensors over time the cells were seeded in a 24-well culture plate, so that the number of cells in each well was 104, and the culture solution was removed after 24 hours of culture. 8 wells were added to the final concentration of 5 nM transmembrane peptide modified circular dichroic TAT-aptamer-dimer and 104 cells were co-cultured for 0h, 2h, 4h, 6h, 8h, 12h, 16h, 24h; The wells were added to a penetrating peptide modified circular dichroic TAT-mismatch-dimer with a final concentration of 5 nM and co-cultured with 104 cells for 0h, 2h, 4h, 6h, 8h, 12h, 16h, 24h; The cells were digested, and different cell suspensions containing circular dichroic aptamer-dimer and mismatch-dimer were obtained. The cell suspension was characterized by circular dichroism
  • the assembly gradually dissipates, causing a change in the circular dichroic signal, and then detecting, and then digesting the cells with trypsin to obtain a circular dichroic aptamer-dimer having different degrees of dissociation in the cells.
  • the cell suspension, transmembrane peptide-modified circular dichroic TAT-aptamer-dimer and the cells with different inhibitors and promoters and untreated cells were separately incubated with the change spectrum of the circular dichroism signal, as shown in Figure 3.
  • the circular dichroism signal is gradually weakened; the cell suspension is characterized by circular dichroism, and the standard curve of intracellular ATP concentration and circular dichroic signal is established, as shown in Fig. 4,
  • the ATP concentration is in the range of 1.5-4.2 mM, and the ATP concentration is inversely proportional to the circular dichroic signal intensity.

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Abstract

公开了一种细胞内ATP的圆二色光谱实时检测方法，属于材料化学技术领域。其包括ATP适配体ATP aptamer序列组装构建的圆二色大小金二聚体传感器，ATP错配序列mismatch DNA组装构建的圆二色大小金二聚体传感器，两种大小金二聚体修饰穿膜肽，两种大小金二聚体传感器在细胞内圆二色信号随时间的变化，基于大小金二聚体的圆二色信号实现胞内ATP实时检测，建立标准曲线等步骤实现。还公开了能够通过圆二色信号实时检测胞内ATP含量的方法以及圆二色性能高、生物相容性好的金纳米粒子二聚体。

Description

一种细胞内ATP的圆二色光谱实时检测方法 技术领域
本发明涉及一种细胞内ATP的圆二色光谱实时检测方法，属于材料化学技术领域。
背景技术
三磷酸腺苷(Adenosine-5’-triphosphate，ATP)是体内组织细胞所需能量的主要来源，作为能量货币，其参与基因复制，蛋白质或脂类的激酶活性，膜离子通道泵，免疫和神经介导的反应，药物传递等生物活性调节。同时，细胞中ATP的含量的变化可以反映细胞的变异和损伤，以及与贫血、低血糖、心血管疾病以及癌症等疾病的发生关系密切。因此ATP作为重大疾病的标志物之一得到了广泛的关注和研究。
传统上ATP检测的方法主要有：高效液相色谱、毛细管电泳和核磁共振波谱分析等，但这些利用细胞裂解液的分析方法存在有ATP潜在的降解，并且分析时间长，使实时检测很难实现；近年来，荧光分析法、化学发光和电化学跟踪技术等都用于胞内ATP的实时检测中，但这些方法操作繁琐，样品消耗量大，检测时间长、性能不稳定，难以微型化。
近几年来，利用自组装纳米材料构建圆二色传感器进行生物传感得到了广泛的关注。圆二色光谱(CD)是一种已被广泛应用于化学和生物传感的分析方法，相比于荧光技术检测，由于纳米粒子之间的等离子体-等离子体耦合、诱导等离子体CD通过激子-等离子体耦合、混合偶极-偶极之间的耦合和最小矩阵干扰，使得CD能够收集样品足够多的信息，并达到灵敏度低的检测水平，具有更大的潜在生物标志物分析应用能力。但是通过自组装的手性大小金二聚体的纳米传感器实现细胞内ATP含量的实时检测还未见报道。
发明内容
本发明的目的是提供一种细胞内ATP的圆二色光谱实时检测方法。
本发明的技术方案，一种细胞内ATP的圆二色光谱实时检测方法，步骤为：
(1)ATP适配体序列组装构建的圆二色大小金二聚体传感器：柠檬酸还原法合成的25nm金纳米粒子GNP离心重悬后修饰巯基ATP aptamer序列；15nm金纳米粒子GNP修饰与适配体部分互补的巯基序列ATP CS1，将得到的GNP-aptamer及GNP-CS1复合体混匀，得到ATP适配体序列组装的圆二色大小金二聚体aptamer-dimer；
(2)ATP错配序列组装构建的圆二色大小金二聚体传感器：柠檬酸还原法合成的25nm金纳米粒子GNP离心重悬后修饰巯基mismatch DNA序列；15nm金纳米粒子GNP修饰与ATP错配序列部分互补的巯基ATP CS2序列，将得到的GNP-mismatch及GNP-CS2复合体 混匀，得到ATP错配序列组装的圆二色大小金二聚体mismatch-dimer；
(3)两种大小金二聚体修饰穿膜肽：将步骤(1)及(2)得到大小金二聚体分别与SH-PEG-5000和穿膜肽TAT混匀，偶联12h，分别得到表面修饰有穿膜肽的圆二色性TAT-aptamer-dimer及TAT-mismatch-dimer；
(4)两种大小金二聚体传感器在细胞内圆二色信号随时间的变化：分别将步骤(3)得到两种穿膜肽修饰的圆二色大小金二聚体与一定数量的细胞共同培养不同时间点后，用胰蛋白酶消化细胞，分别得到细胞内含有不同量的圆二色aptamer-dimer及mismatch-dimer的细胞悬液；将细胞悬液进行圆二色性表征，记录不同时间点的圆二色信号，绘制散点图，得到胞内最佳检测时间。
(5)基于大小金二聚体的圆二色信号实现胞内ATP实时检测：将步骤(3)得到穿膜肽修饰的圆二色性TAT-aptamer-dimer与添加不同抑制剂和促进剂的细胞及未处理的细胞分别共同孵育，当存在待测物ATP时，组装体逐渐解散，导致圆二色信号的变化，进而进行检测，建立胞内ATP浓度与圆二色光谱的标准曲线。
具体步骤如下：
(1)ATP适配体序列组装构建的圆二色大小金二聚体传感器：柠檬酸还原法合成的25nm金纳米粒子GNP离心重悬于5mM pH 7.4的PB缓冲液中，取100μL 2nM 25nm金纳米粒子将其与巯基ATP aptamer序列以摩尔浓度1︰5的比例混匀；将柠檬酸还原法合成的15nm金纳米粒子GNP离心重悬于5mM pH 7.4的PB缓冲液中，取100μL 2nM 15nm金纳米粒子与适配体部分互补的巯基ATP CS1序列以摩尔浓度1︰5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 PB缓冲液中，取100μL GNP-aptamer及50μL GNP-CS1复合体混匀，加入NaCl溶液至终浓度为50mM进行老化，室温下孵育12h，得到ATP适配体序列组装的圆二色大小金二聚体aptamer-dimer，待用；
(2)ATP错配序列组装构建的圆二色大小金二聚体传感器：柠檬酸还原法合成的25nm金纳米粒子GNP离心重悬于5mM PH 7.4的pB缓冲液，取100μL 2nM 25nm金纳米粒子将其与巯基mismatch DNA序列以摩尔浓度1︰5的比例混匀；将柠檬酸还原法合成的15nm金纳米粒子GNP离心重悬于5mM pH 7.4的PB缓冲液中，取100μL 2nM 15nm金纳米粒子与适配体部分互补的巯基ATP CS2序列以摩尔浓度1︰5的比例混匀，分别加入终浓度为50mM的NaCl溶液中，充分混合后，室温孵育过夜，离心3次除去溶液中未反应的DNA，分别重悬于100μL的5mM PB缓冲液中；取100μL GNP-mismatch及50μL GNP-CS2复合体混匀，加 入NaCl溶液至终浓度为50mM进行老化，室温下孵育12h，得到ATP错配序列组装的圆二色大小金二聚体mismatch-dimer，待用；
(3)两种大小金二聚体修饰穿膜肽：将步骤(1)及(2)得到大小金二聚体分别与SH-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20min，去除上清液，沉淀重悬于细胞培养液中，得到稳定的表面修饰有穿膜肽的圆二色TAT-aptamer-dimer及TAT-mismatch-dimer；
(4)两种大小金二聚体传感器在细胞内圆二色信号随时间的变化：将细胞接种于24孔培养板中，使每孔中的细胞数量为104个，培养24h后去掉培养液，取8个孔加入终浓度为5nM的穿膜肽修饰的圆二色TAT-aptamer-dimer分别与104个细胞共同培养0h、2h、4h、6h、8h、12h、16h、24h；取8个孔加入终浓度为5nM的穿膜肽修饰的圆二色性TAT-mismatch-dimer分别与104个细胞共同培养0h、2h、4h、6h、8h、12h、16h、24h；之后分别用胰蛋白酶消化细胞，分别得到细胞内含有不同量的圆二色aptamer-dimer及mismatch-dimer的细胞悬液。将细胞悬液进行圆二色性表征，记录不同时间点的圆二色信号，绘制散点图，得到胞内最佳检测时间；
(5)基于大小金二聚体的圆二色信号实现胞内ATP实时检测：在将步骤(3)得到穿膜肽修饰的圆二色性TAT-aptamer-dimer与不同量寡霉素A抑制细胞中ATP浓度、不同量依托泊苷促进细胞中ATP浓度及未经处理的细胞分别共孵育6h后，当存在待测物ATP时，组装体逐渐解散，导致圆二色信号的变化，进而进行检测，然后用1mL胰蛋白酶消化细胞，分别得到细胞内含有不同解散程度的圆二色性aptamer-dimer的细胞悬液，将细胞悬液进行圆二色性表征，并建立胞内ATP浓度与圆二色信号的标准曲线。
所述ATP aptamer序列如SEQ ID NO.1所示，ATP CS1序列如SEQ ID NO.2所示，mismatch DNA序列如SEQ ID NO.3所示，ATP CS2序列如SEQ ID NO.4所示，TAT多肽序列如SEQ ID NO.5所示，具体如表1所示。
表1
Figure PCTCN2017108923-appb-000001
本发明的有益效果：本发明制备出了组装产率高，性质稳定，在细胞中分散性好的的金纳米粒子二聚体，提供了能够通过圆二色光谱实时检测胞内ATP含量的方法，建立了细胞内ATP浓度与圆二色信号强度两者之间的标准曲线，具有灵敏度高、选择性好，省时省力的优点，具有非常好的实际应用前景。
附图说明
图1是本发明的金纳米粒子二聚体在细胞中的生物透射电镜图。
图2是本发明中两种大小金二聚体传感器在细胞内圆二色信号随时间的变化，绘制散点图。
图3是本发明中穿膜肽修饰的圆二色性TAT-aptamer-dimer与添加不同抑制剂和促进剂的细胞及未处理的细胞分别共同孵育后圆二色信号的变化谱图。图中，按照曲线谷底的由高到低，各曲线依次为ATP浓度为1.5mM、2.0mM、2.8mM、3.5mM、4.2mM时圆二色信号的强度。
图4是胞内ATP浓度与圆二色信号的标准曲线。
具体实施方式
以下实施例中序列材料购自生工生物工程(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)ATP适配体序列组装构建的圆二色大小金二聚体传感器：柠檬酸还原法合成的25nm 金纳米粒子GNP离心重悬于5mM pH 7.4的PB缓冲液中，取100μL 2nM 25nm金纳米粒子将其与巯基ATP aptamer序列以摩尔浓度1︰5的比例混匀；15nm金纳米粒子GNP离心重悬于5mM pH 7.4的PB缓冲液中，取100μL 2nM 15nm金纳米粒子与适配体部分互补的巯基序列ATP CS1以摩尔浓度1︰5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 PB缓冲液中，取100μL GNP-aptamer及50μL GNP-CS1复合体混匀，加入NaCl溶液至终浓度为50mM进行老化，室温下孵育12h，得到ATP适配体序列组装的圆二色大小金二聚体aptamer-dimer，待用；
(2)ATP错配序列组装构建的圆二色大小金二聚体传感器：柠檬酸还原法合成的25nm金纳米粒子GNP离心重悬于5mMpH 7.4的PB缓冲液，取100μL 2nM 25nm金纳米粒子将其与巯基mismatch DNA序列以摩尔浓度1︰5的比例混匀；15nm金纳米粒子GNP离心重悬于5mM pH 7.4的PB缓冲液中，取100μL 2nM 15nm金纳米粒子与ATP适配体部分互补的巯基ATP CS2序列以摩尔浓度1︰5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 PB缓冲液中，取100μL GNP-mismatch及50μL GNP-CS2复合体混匀，加入NaCl溶液至终浓度为50mM进行老化，室温下孵育12h，得到ATP错配序列组装的圆二色大小金二聚体mismatch-dimer，待用；
(3)两种大小金二聚体修饰穿膜肽：将步骤(1)及(2)得到大小金二聚体分别与SH-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20min，去除上清液，沉淀重悬于细胞培养液中，得到稳定的表面修饰有穿膜肽的圆二色性TAT-aptamer-dimer及TAT-mismatch-dimer，金纳米粒子二聚体在细胞中的生物透射电镜图如图1所示从图中可以看到，该组装体在细胞内，仍以高产率的二聚体形式存在。
(4)两种大小金二聚体传感器在细胞内圆二色信号随时间的变化：将细胞接种于24孔培养板中，使每孔中的细胞数量为104个，培养24h后去掉培养液，取8个孔加入终浓度为5nM的穿膜肽修饰的圆二色性TAT-aptamer-dimer分别与104个细胞共同培养0h、2h、4h、6h、8h、12h、16h、24h；取8个孔加入终浓度为5nM的穿膜肽修饰的圆二色性TAT-mismatch-dimer分别与104个细胞共同培养0h、2h、4h、6h、8h、12h、16h、24h；之后分别用胰蛋白酶消化细胞，分别得到细胞内含有不同量的圆二色性aptamer-dimer及mismatch-dimer的细胞悬液；将细胞悬液进行圆二色性表征，记录不同时间点的圆二色信号，绘制散点图如图2所示，得到胞内最佳检测时间为6h。
(5)基于大小金二聚体的圆二色信号实现胞内ATP实时检测：将不同量寡霉素A抑制后、不同量依托泊苷促进后及未经抑制的细胞裂解液用ELISA标准曲线进行胞内ATP的定量。在将步骤(3)得到穿膜肽修饰的圆二色性TAT-aptamer-dimer与不同量寡霉素A抑制后、不同量依托泊苷促进后及未经抑制的细胞分别共孵育6h后，当存在待测物ATP时，组装体逐渐解散，导致圆二色信号的变化，进而进行检测，然后用胰蛋白酶消化细胞，分别得到细胞内含有不同解离程度的圆二色性aptamer-dimer的细胞悬液，穿膜肽修饰的圆二色性TAT-aptamer-dimer与添加不同抑制剂和促进剂的细胞及未处理的细胞分别共同孵育后圆二色信号的变化谱图，如图3所示随着ATP浓度的升高，圆二色信号逐渐减弱；对细胞悬液进行圆二色性表征，并建立胞内ATP浓度与圆二色信号的标准曲线，具体如图4所示，在ATP浓度为1.5-4.2mM范围内，ATP浓度与圆二色信号强度成反比。
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 PCTCN2017108923-appb-000002
Figure PCTCN2017108923-appb-000003

Claims (3)

  1. 一种细胞内ATP的圆二色光谱实时检测方法，其特征在于步骤为：
    (1)ATP适配体序列组装构建的圆二色大小金二聚体传感器：柠檬酸还原法合成的25nm金纳米粒子GNP离心重悬后修饰巯基ATP aptamer序列；15nm金纳米粒子GNP修饰与适配体部分互补的巯基ATP CS1序列；将得到的GNP-aptamer及GNP-CS1复合体混匀，得到ATP适配体序列组装的圆二色大小金二聚体aptamer-dimer；
    (2)ATP错配序列组装构建的圆二色大小金二聚体传感器：柠檬酸还原法合成的25nm金纳米粒子GNP离心重悬后修饰巯基mismatch DNA序列；15nm金纳米粒子GNP修饰与ATP错配序列部分互补的巯基ATP CS2序列；将得到的GNP-mismatch及GNP-CS2复合体混匀，得到ATP错配序列组装的圆二色大小金二聚体mismatch-dimer；
    (3)两种大小金二聚体修饰穿膜肽：将步骤(1)及(2)得到大小金二聚体分别与SH-PEG-5000和穿膜肽TAT混匀，偶联12h，分别得到表面修饰有穿膜肽的圆二色性TAT-aptamer-dimer及TAT-mismatch-dimer；
    (4)两种大小金二聚体传感器在细胞内圆二色信号随时间的变化：分别将步骤(3)得到两种穿膜肽修饰的圆二色大小金二聚体与一定数量的细胞共同培养不同时间点后，用胰蛋白酶消化细胞，分别得到细胞内含有不同量的圆二色aptamer-dimer及mismatch-dimer的细胞悬液；将细胞悬液进行圆二色性表征，记录不同时间点的圆二色信号，绘制散点图，得到胞内最佳检测时间；
    (5)基于大小金二聚体的圆二色信号实现胞内ATP实时检测：将步骤(3)得到穿膜肽修饰的圆二色性TAT-aptamer-dimer与添加不同抑制剂和促进剂的细胞及未处理的细胞分别共同孵育，当存在待测物ATP时，组装体逐渐解散，导致圆二色信号的变化，进而进行检测，建立胞内ATP浓度与圆二色光谱的标准曲线。
  2. 根据权利要求1所述细胞内ATP的圆二色光谱实时检测方法，其特征在于具体步骤如下：
    (1)ATP适配体序列组装构建的圆二色大小金二聚体传感器：柠檬酸还原法合成的25nm金纳米粒子GNP离心重悬于5mM pH 7.4的PB缓冲液中，取100μL 2nM 25nm金纳米粒子将其与巯基ATP aptamer序列以摩尔浓度1︰5的比例混匀；将柠檬酸还原法合成的15nm金纳米粒子GNP离心重悬于5mM pH 7.4的PB缓冲液中，取100μL 2nM 15nm金纳米粒子与适配体部分互补的巯基ATP CS1序列以摩尔浓度1︰5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 PB缓冲液中；取100μL GNP-aptamer及50μL GNP-CS1复合体混匀，加 入NaCl溶液至终浓度为50mM进行老化，室温下孵育12h，得到ATP适配体序列组装的圆二色大小金二聚体aptamer-dimer，待用；
    (2)ATP错配序列组装构建的圆二色大小金二聚体传感器：柠檬酸还原法合成的25nm金纳米粒子GNP离心重悬于5mM pH 7.4的PB缓冲液，取100μL 2nM 25nm金纳米粒子将其与巯基mismatch DNA序列以摩尔浓度1︰5的比例混匀；将柠檬酸还原法合成的15nm金纳米粒子GNP离心重悬于5mM pH 7.4的PB缓冲液中，取100μL 2nM 15nm金纳米粒子与适配体部分互补的巯基ATP CS2序列以摩尔浓度1︰5的比例混匀，分别加入终浓度为50mM的NaCl溶液中，充分混合后，室温孵育过夜，离心3次除去溶液中未反应的DNA，分别重悬于100μL的5mM PB缓冲液中；取100μL GNP-mismatch及50μL GNP-CS2复合体混匀，加入NaCl溶液至终浓度为50mM进行老化，室温下孵育12h，得到ATP错配序列组装的圆二色大小金二聚体mismatch-dimer，待用；
    (3)两种大小金二聚体修饰穿膜肽：将步骤(1)及(2)得到大小金二聚体分别与SH-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20min，去除上清液，沉淀重悬于细胞培养液中，得到稳定的表面修饰有穿膜肽的圆二色TAT-aptamer-dimer及TAT-mismatch-dimer；
    (4)两种大小金二聚体传感器在细胞内圆二色信号随时间的变化：将细胞接种于24孔培养板中，使每孔中的细胞数量为104个，培养24h后去掉培养液，取8个孔加入终浓度为5nM的穿膜肽修饰的圆二色TAT-aptamer-dimer分别与104个细胞共同培养0h、2h、4h、6h、8h、12h、16h、24h；取8个孔加入终浓度为5nM的穿膜肽修饰的圆二色性TAT-mismatch-dimer分别与104个细胞共同培养0h、2h、4h、6h、8h、12h、16h、24h；之后分别用胰蛋白酶消化细胞，分别得到细胞内含有不同量的圆二色aptamer-dimer及mismatch-dimer的细胞悬液；将细胞悬液进行圆二色性表征，记录不同时间点的圆二色信号，绘制散点图，得到胞内最佳检测时间；
    (5)基于大小金二聚体的圆二色信号实现胞内ATP实时检测：在将步骤(3)得到穿膜肽修饰的圆二色性TAT-aptamer-dimer与不同量寡霉素A抑制细胞中ATP浓度、不同量依托泊苷促进细胞中ATP浓度及未经处理的细胞分别共孵育6h后，当存在待测物ATP时，组装体逐渐解散，导致圆二色信号的变化，进而进行检测，然后用1mL胰蛋白酶消化细胞，分别得到细胞内含有不同解散程度的圆二色性aptamer-dimer的细胞悬液，将细胞悬液进行圆二色性表征，并建立胞内ATP浓度与圆二色信号的标准曲线。
  3. 根据权利要求1所述细胞内ATP的圆二色光谱实时检测方法，其特征在于：所述ATP aptamer序列如SEQ ID NO.1所示，ATP CS1序列如SEQ ID NO.2所示，mismatch DNA序 列如SEQ ID NO.3所示，ATP CS2序列如SEQ ID NO.4所示，TAT多肽序列如SEQ ID NO.5所示。
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