WO2018072745A1 - 基于金-上转换纳米粒子四面体的双信号原位检测胞内microRNA的方法 - Google Patents

基于金-上转换纳米粒子四面体的双信号原位检测胞内microRNA的方法 Download PDF

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WO2018072745A1
WO2018072745A1 PCT/CN2017/107101 CN2017107101W WO2018072745A1 WO 2018072745 A1 WO2018072745 A1 WO 2018072745A1 CN 2017107101 W CN2017107101 W CN 2017107101W WO 2018072745 A1 WO2018072745 A1 WO 2018072745A1
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microrna
dna
tetrahedron
gold
intracellular
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French (fr)
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胥传来
匡华
李斯
徐丽广
马伟
刘丽强
宋珊珊
吴晓玲
孙茂忠
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江南大学
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    • C12Q1/6844—Nucleic acid amplification reactions
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  • a method for in situ detection of intracellular microRNAs based on gold-upconversion nanoparticle tetrahedrons is a field of material chemical biology.
  • MicroRNAs are a class of small-molecule non-coding regulatory genes (19-22 bases) that are widely found in plant, animal, and viral genes. miRNA plays an important role in biological processes such as cell differentiation, proliferation, apoptosis and carcinogenesis. The expression level of miRNA is closely related to human major diseases, especially cancer.
  • Traditional miRNA detection methods are limited to intracellular miRNA imaging and quantitative detection based on fluorescent signals. However, the optical instability, short quantum lifetime, and photobleaching of fluorescent signals greatly limit the application of intracellular in situ detection methods based on fluorescent signals.
  • the nanostructures controlled by nanoparticles are self-assembled with regular geometric shapes, stable structures, and strong optical activity. Based on these properties, self-assembled nanostructures have been widely used in biosensing. However, the detection of intracellular microRNA based on nano-assembled structures has not been reported. In recent years, chiral plasma optical activity has become a hot topic that has attracted widespread attention. A number of ultrasensitive sensory detection methods based on chiral nanoassemblies have been developed to detect nucleic acid fragments and cancer markers in blood. However, the application of living cancer cells in the biological field remains to be explored.
  • the object of the present invention is to provide an ultrasensitive detection method for intracellular microRNA based on chiral gold-upconversion nanoparticle tetrahedron bimodal signal, which is an ultrasensitive and highly specific intracellular microRNA in situ. Detection method.
  • the technical scheme of the present invention is a method for in situ detection of microRNA in a cell based on a luminescence signal of a gold-upconversion nanoparticle tetrahedron and a chiral signal, including design of a nucleic acid for preparing a detection probe, and gold-upconversion nanoparticle tetrahedron detection Preparation of probes, functionalization of tetrahedrons, establishment of intracellular microRNA detection methods; specific steps are:
  • the reaction mixture was slowly stirred at 50 ° C for 30 min, then treated under vacuum at 100 ° C for 10 min to remove methanol, heated to 320 ° C under nitrogen and maintained for 1 h. The mixture was cooled to room temperature and the reaction was completed.
  • the synthesized NaGdF4 particles precipitated in ethanol were collected by centrifugation, washed several times with ethanol, and finally resuspended in THF or cyclohexane solution for subsequent experiments.
  • Phase inversion of up-converting nanoparticles 100 mg of PEG modified with maleimide groups and phosphate groups and 10 mg of oleic acid-coated NaGdF4 particles were mixed in a THF solution and stirred at room temperature overnight.
  • the PEG-coated NaGdF4 particles were precipitated in cyclohexane and washed three times with cyclohexane and dried under vacuum at room temperature to obtain PEG-coated NaGdF4 particles which were soluble in water.
  • the DNA used in the present invention was purchased from Shanghai Shenggong Bioengineering Co., Ltd., and purified by polyacrylamide gel electrophoresis.
  • AuNP gold nanoparticles
  • DNA-Py3, DNA-Py4 molar ratio of 1..3.5 20nM gold nanoparticles (AuNP) were mixed with DNA-Py3, DNA-Py4 molar ratio of 1..3.5, and after 1 hour, NaCl solution was added to dissolve. The degree was 50 mM, the mixture was uniformly mixed, and the reaction was carried out overnight, and the free nucleic acid was removed by centrifugation to obtain AuNP-DNA-Py 3 and AuNP-DNA-Py 4 .
  • up-converting nanoparticles 20 nM up-converting nanoparticles (UCNP) are mixed with DNA-Py1, DNA-Py2 molar ratio of 1..3.5, and after 1 h, NaNO 3 solution is added. The final solubility was 50 mM, the mixture was homogeneous, and the reaction was carried out overnight. The free nucleic acid was removed by centrifugation to obtain UCNP-DNA-Py1, UCNP-DNA-Py 2.
  • UCNP up-converting nanoparticles
  • the obtained AuNP-DNA-Py3 and AuNP-DNA-Py4, UCNP-DNA-Py1 and UCNP-DNA-Py 2 were separately mixed, and NaCl was added to a final concentration of 50 mM, 95 ° C water bath for 5 min, and then incubated at 37 ° C for 8 h. That is, a gold dimer and an upconversion dimer are obtained.
  • the gold dimer and the upconversion dimer were mixed at a molar ratio of 1..1 and incubated at 37 ° C for 24 h to prepare a gold-upconversion nanoparticle tetrahedral detection probe solution for detecting microRNA-21.
  • the thiol-modified PEG (molecular weight: 5000) solution was added to the tetrahedral detection probe solution in a ratio of PEG.. tetrahedral molar ratio of 1000..1, and after 15 minutes, excess PEG molecules were removed by centrifugation. Then, the transmembrane peptide molecule (TAT) was added to the PEG-modified tetrahedral solution according to the TAT.. tetrahedral molar ratio of 1000..1, and reacted at room temperature for 24 hours, and centrifuged to remove excess penetrating peptide which was not attached to the tetrahedron. .
  • TAT transmembrane peptide molecule
  • RNAiMAX transfection reagent lipofectamine RNAiMAX transfection reagent
  • RT-PCR RT-PCR was used to determine the amount of microRNA-21 in the transfected cells.
  • the specific method is as follows: First, a synthetic amplification curve of gradient microRNA-21 is established, and then a linear relationship between the number of cycles and the concentration of microRNA-21 is established.
  • the total RAN in the transfected cells is extracted, and then amplified by RT-PCR to obtain an amplification curve containing microRNA-21, and the number of cycles is obtained according to the amplification curve, and then the microRNA contained in the standard curve is determined according to the number of cycles.
  • -21 then the transfected cells were cultured in a medium containing tetrahedron (1640 + 10% FBS + double antibody), and after 8 hours, the tetrahedron outside the cells was washed away, and the intracellular CD signal and up-conversion fluorescence intensity were measured.
  • a standard curve of CD signal and intracellular microRNA-21 concentration and a standard curve between upconversion fluorescence intensity and intracellular microRNA-21 concentration were then established, respectively.
  • MCF-7, HeLa, and PCS-460-010 cells were cultured in medium containing tetrahedron (1640+10% FBS+double antibody). After 8 hours, the CD signal and up-conversion fluorescence intensity in the cells were detected, and then established separately. A standard curve between the concentration of CD signal and intracellular microRNA-21 and a standard curve between the upconversion fluorescence intensity and the concentration of intracellular microRNA-21 to determine the amount of microRNA-21 in the cells.
  • the present invention uses DNA-driven gold-upconverting nanoparticle-assembled tetrahedrons for real-time detection of intracellular miRNAs.
  • the gold-upconversion nanoparticle tetrahedron has dual optical activity, exhibits a strong plasma circular dichroism signal at 521 nm, and has a significant upconversion fluorescence intensity at 500-600 nm, so this dual signal can be used to detect cells.
  • miRNA In the presence of the target miRNA, the CD signal is attenuated and the fluorescence intensity is increased. The experimental results show that the CD signal has a good linear relationship with the target miRNA concentration in the range of 0.073 to 43.65fmol/10 ⁇ g RNA.
  • the detection limit can reach 0.03fmol/10 ⁇ g RNA, and the linear range of up-conversion fluorescence intensity and target miRNA concentration is 0.16-43.65fmol/10 ⁇ g RNA, the minimum detection limit is 0.12fmol/10 ⁇ g RNA.
  • the invention not only establishes an ultra-sensitive quantitative detection method of intracellular miRNA, but also opens a way for the assembly of plasma chiral biological applications.
  • the invention establishes a method for detecting intracellular microRNA by double signal in situ based on gold-upconversion nanoparticle tetrahedron, and is an ultrasensitive and highly specific in situ detection method of intracellular microRNA.
  • Figure 1 (A) Schematic diagram of microRNAs in gold-upconverting nanoparticle tetrahedron detection cells; (B) tetrahedral nucleic acid backbone, sites 1 and 2 linked to upconverting nanoparticles, sites 3 and 4 Gold nanoparticles are joined.
  • Figure 2 Transmission electron micrograph of the assembled tetrahedron.
  • Figure 3 Yield chart of assembled tetrahedrons.
  • Figure 4 Evaluation of extracellular detection capabilities: (A) Chiral signal maps obtained by adding a series of different concentrations of target microRNAs; (B) Fluorescence signal maps obtained by adding a series of different concentrations of target microRNAs. For each curve in the figure, according to the order of the bottom of each curve, from high to low, it represents the case where the microRNA concentration is 200 pM, 50 pM, 30 pM, 15 pM, 10 pM, 5 pM, 2 pM, 0 pM, respectively.
  • FIG. 5 Evaluation of detection specificity: (C) Chiral signal map obtained by adding different kinds of microRNAs; (D) Fluorescence signal diagram obtained by adding different kinds of microRNAs.
  • Figure 6 CD signal of cells containing different amounts of microRNA21 in Example 6 after incubation with the detection probe for 8 hours.
  • each curve in the figure according to the order of the bottom of each curve, from high to low, it represents 43.65fmol/10 ⁇ g RNA, 15.81fmol/10 ⁇ g RNA, 3.18fmol/10 ⁇ g RNA, 0.48fmol/10 ⁇ g RNA, 0.16fmol/10 ⁇ g, respectively.
  • Figure 7 Fluorescence signal of cells containing different amounts of microRNA21 in Example 6 incubated with the detection probe for 8 hours.
  • Figure 8 is a linear relationship between the intensity of the CD signal at 521 nm in Figure 6 and the amount of microRNA detected by intracellular RT-PCR.
  • Figure 9 is a linear relationship between the up-conversion intensity at 543 nm in Figure 7 and the amount of microRNA detected by intracellular RT-PCR.
  • Figure 10 CD signal map of microRNAs in different cells obtained by detecting the amount of microRNA21 in different cells using a detection probe.
  • the cells are represented by MCF-7, HeLa, and PCS-460-010, respectively.
  • Figure 11 Up-conversion fluorescence signal of microRNAs in different cells obtained by detecting the amount of microRNA21 in different cells using a detection probe.
  • the reaction mixture was slowly stirred at 50 ° C for 30 min, then treated under vacuum at 100 ° C for 10 min to remove methanol, heated to 320 ° C under nitrogen and maintained for 1 h. The mixture was cooled to room temperature and the reaction was completed.
  • the synthesized NaGdF 4 particles precipitated in ethanol were collected by centrifugation, washed several times with ethanol, and finally resuspended in THF or cyclohexane solution for subsequent experiments.
  • Phase inversion of up-converting nanoparticles 100 mg of PEG modified with maleimide group and phosphate group and 10 mg of oleic acid-coated NaGdF 4 particles were mixed in a THF solution and stirred at room temperature overnight.
  • the PEG-coated NaGdF4 particles were precipitated in cyclohexane and washed three times with cyclohexane and dried under vacuum at room temperature to obtain PEG-coated NaGdF 4 particles which were soluble in water.
  • the DNA used in the present invention was purchased from Shanghai Shenggong Bioengineering Co., Ltd., and purified by polyacrylamide gel electrophoresis.
  • AuNP gold nanoparticles
  • DNA1-Py3, DNA-Py4 molar ratio of 1..3.5 20 nM gold nanoparticles (AuNP) were mixed with DNA1-Py3, DNA-Py4 molar ratio of 1..3.5, and after 1 hour, NaCl solution was added. The final solubility was 50 mM, the mixture was uniformly mixed, and the reaction was carried out overnight. The free nucleic acid was removed by centrifugation to obtain AuNP-DNA-Py3, AuNP-DNA-Py4.
  • preparing a conjugate of the up-converting nanoparticle and the nucleic acid mixing 20 nM up-converting nanoparticles (UCNP) with DNA-Py1, DNA-Py2 molar ratio of 1..3.5, and adding NaNO3 solution after 1 hour of reaction.
  • the final solubility was 50 mM, the mixture was homogeneous, and the reaction was carried out overnight.
  • the free nucleic acid was removed by centrifugation to obtain UCNP-DNA-Py1, UCNP-DNA-Py2.
  • the obtained AuNP-DNA-Py3 and AuNP-DNA-Py4, UCNP-DNA-Py1 and UCNP-DNA-Py2 were separately mixed, and NaCl was added to a final concentration of 50 mM, 95 ° C water bath for 5 min, and then incubated at 37 ° C for 8 h. That is, a gold dimer and an upconversion dimer are obtained.
  • the gold dimer and the upconversion dimer were mixed at a molar ratio of 1..1 and incubated at 37 ° C for 24 h to prepare a gold-upconversion nanoparticle tetrahedral detection probe solution for detecting microRNA-21.
  • 2 is a transmission electron micrograph of a detection probe prepared using gold and up-converting nanoparticles and DNA nucleic acid sequences.
  • Figure 3 is a graph showing the yield statistics of the final assembled tetrahedron by taking TEM photographs of 200 different fields of view.
  • the detection ability of the tetrahedral detection microRNA21 is detected by adding a different concentration of the target microRNA to the detection probe solution, and then testing the CD and fluorescence signals of the solution, and by changing the CD and the fluorescence signal.
  • the results of the assay are shown in Figure 4.
  • the change in target microRNA concentration is significantly reflected in the CD and fluorescence signals.
  • non-target microRNAs or other substances By adding a high concentration of non-target microRNA to the detection probe solution, then testing the CD and fluorescence signals of the solution, detecting the specificity of the tetrahedral probe by the change of CD and fluorescence signal, and confirming whether the signal of the detection probe is affected by Interference from non-target microRNAs or other substances.
  • the CD and fluorescence signal intensity of non-target microRNAs or other substances, and the CD and fluorescence signal intensity of the target microRNA form a very significant difference, non-target microRNA or other substances will not interfere with the target microRNA. Detection.
  • the thiol-modified PEG (molecular weight: 5000) solution was added to the tetrahedral detection probe solution in a ratio of PEG.. tetrahedral molar ratio of 1000..1, and after 15 minutes, excess PEG molecules were removed by centrifugation. Then, the transmembrane peptide molecule (TAT) was added to the PEG-modified tetrahedral solution according to the TAT.. tetrahedral molar ratio of 1000..1, and reacted at room temperature for 24 hours, and centrifuged to remove excess penetrating peptide which was not attached to the tetrahedron. .
  • TAT transmembrane peptide molecule
  • Example 5 Establishment of a method for detecting microRNA-21 in living cells
  • RNAiMAX transfection reagent lipofectamine RNAiMAX transfection reagent
  • RT-PCR was then used to determine the amount of microRNA-21 in the transfected cells.
  • the specific method is as follows: First, a synthetic amplification curve of gradient microRNA-21 is established, and then a linear relationship between the number of cycles and the concentration of microRNA-21 is established.
  • the total RAN in the transfected cells is extracted, and then amplified by RT-PCR to obtain an amplification curve containing microRNA-21, and the number of cycles is obtained according to the amplification curve, and then the microRNA contained in the standard curve is determined according to the number of cycles. -21 amount (concentration).
  • the transfected cells (such as MCF-7) were cultured in a medium containing tetrahedron (1640 + 10% FBS + double antibody). After 8 hours, the tetrahedron outside the cells was washed away, and the intracellular CD signal was measured. Convert fluorescence intensity.
  • MCF-7, HeLa, PCS-460-010 cells were cultured separately with medium containing tetrahedron (1640+10% FBS+double antibody), and CD signal and up-conversion were detected in cells after 8 hours. Fluorescence intensity, then the amount of microRNA-21 in the cells was determined based on a standard curve establishing the concentration of CD signal and intracellular microRNA-21 and a standard curve between the up-conversion fluorescence intensity and the intracellular microRNA-21 concentration.
  • the CD signal was obtained after incubation of cells containing different amounts of microRNA21 with the detection probe for 8 hours. It can be seen that the CD signal can clearly reflect the difference between the microRNA21 content.
  • the abscissa is the content of microRNA in the cell detected by RT-PCR, and the ordinate is the intensity of the CD signal at 521 nm in Fig. 6, which has a good linear relationship between the two; The results of the detection probe detection are in good agreement with the results of the RT-PCR assay.
  • the detection probe was used to detect the amount of microRNA21 in different cells, and the CD signal map of the microRNA in the different cells obtained.
  • the amount of microRNA contained in different cells can be further calculated by the standard curve established in Fig. 8 and the CD signal intensity at 521 nm in the CD spectrum.
  • the amount of microRNA21 in different cells was detected by a detection probe, and the up-converted fluorescent signal map of the microRNA in the different cells obtained was obtained.
  • the amount of microRNA in different cells can be further calculated by the standard curve established in Figure 9 and the intensity of the fluorescent signal at 543 nm in the fluorescence spectrum.

Abstract

本发明提供了基于金-上转换纳米粒子四面体的双信号原位检测胞内microRNA的方法，包括制备检测探针所用核酸的设计，金-上转换纳米粒子四面体检测探针的制备，四面体的功能化，胞内microRNA检测。

Description

基于金-上转换纳米粒子四面体的双信号原位检测胞内microRNA的方法 技术领域
基于金-上转换纳米粒子四面体的双信号原位检测胞内microRNA的方法，属于材料化学生物领域。
背景技术
MicroRNA(miRNA)是一类小分子的非编码调控基因(19～22碱基)，广泛地存在于植物、动物及病毒基因之中。miRNA在细胞分化、增殖、凋亡及癌变等生物过程中都扮演着重要的角色，miRNA的表达水平与人类的重大疾病，尤其癌症，密切相关。传统的miRNA检测方法局限于胞内miRNA成像以及基于荧光信号的定量检测。但是荧光信号的光不稳定性、短的量子寿命、易光漂白等特性大大限制了基于荧光信号的胞内原位检测方法的应用。
由纳米粒子可控自组装的纳米结构有规则的几何形状，稳定的结构，很强的光学活性。基于这些性质，自组装的纳米结构已经被广泛的应用于生物传感。但是基于纳米组装结构的胞内microRNA的检测还没有报导。近年来，手性等离子光学活性已经成为一个热点话题引起了广泛的关注。发展了很多基于手性纳米组装体的超灵敏的传感检测方法来检测核酸片段和血液中的癌症标志物。但是，在生物领域活的癌细胞中的应用仍然有待探索。
发明内容
本发明的目的是提供一种基于手性金-上转换纳米粒子四面体的双模态信号的胞内microRNA的超灵敏检测方法，是一种超灵敏，高特异性的胞内microRNA的原位检测方法。
本发明的技术方案，基于金-上转换纳米粒子四面体的发光信号和手性信号原位检测细胞中microRNA的方法，包括制备检测探针所用核酸的设计，金-上转换纳米粒子四面体检测探针的制备，四面体的功能化，胞内microRNA检测方法的建立；具体步骤为:
(1)20nm金纳米粒子的制备
洁净的三口烧瓶中加入97.5mL超纯水，加入2.5mL 0.4％氯金酸溶液，搅拌并加热至沸腾，7-8min后加入1.6mL 1％柠檬酸三钠溶液，溶液从无色变为红色后，停止加热，继续搅拌15min，自然冷却后放置在4℃冰箱备用。
(2)19nm上转换纳米粒子(NaGdF4:Yb,Er)的制备
在含有14mL OA(配体十八烯酸)和16mL ODE(非配位有机溶剂十八烯)的混合液中加入0.80mmol的GdCl3·6H2O，0.18mmol的YbCl3·6H2O和0.02mmol的ErCl3·6H2O，溶解。在氮气的保护下加热到150℃形成均相的溶液。降至室温后，含有0.100g的NaOH和 0.148g的NH4F的10mL甲醇溶液慢慢加入。反应混合液在50℃下慢慢搅拌30min，然后在100℃在真空下处理10min除去甲醇，在氮气的保护下加热到320℃，并且保持1h。将混合物降至室温，反应结束。乙醇中沉淀的合成的NaGdF4粒子用离心收集，并用乙醇洗涤几次，最后重悬在THF或者环己烷溶液中供后续实验使用。上转换纳米粒子的转相：100mg用马来酰亚胺基团和磷酸基团修饰的PEG和10mg油酸包裹的NaGdF4粒子混合在THF溶液里面，在室温下搅拌过夜。PEG包裹的NaGdF4粒子在环己烷中沉淀，并用环己烷洗涤三次，在室温真空的条件下干燥，得到了可以在水中溶解的PEG包裹的NaGdF4粒子。
(3)检测用的核酸探针的设计与合成
表1. microRNA检测用DNA序列设计
Figure PCTCN2017107101-appb-000001
注：本发明所使用的DNA均购自中国上海生工生物工程有限公司，并通过聚丙烯酰胺凝胶电泳进行纯化。
(4)金-上转换纳米粒子四面体检测探针的制备
首先制备金纳米粒子和核酸的偶联物：把20nM金纳米粒子(AuNP)分别和DNA-Py3， DNA-Py4按摩尔比1︰3.5的比例混合在一起，反应1h后加入NaCl溶液至终溶度为50mM，混合均匀，反应过夜，离心去除游离的核酸，即得到AuNP-DNA-Py 3，AuNP-DNA-Py 4。然后制备上转换纳米粒子和核酸的偶联物：把20nM上转换纳米粒子(UCNP)分别和DNA-Py1，DNA-Py2按摩尔比1︰3.5比例混合在一起，反应1h后加入NaNO3溶液至终溶度为50mM，混合均匀，反应过夜，离心去除游离的核酸，即得到UCNP-DNA-Py1，UCNP-DNA-Py 2。接下来把得到的AuNP-DNA-Py3和AuNP-DNA-Py4，UCNP-DNA-Py1和UCNP-DNA-Py 2分别混合，加入NaCl至终浓度为50mM，95℃水浴5min，然后37℃孵育8h，即得到金二聚体及上转换二聚体。把金二聚体和上转换二聚体按照摩尔比1︰1的比例混合，37℃孵育24h，即制备得到检测microRNA-21用的金-上转换纳米粒子四面体检测探针溶液。
(5)四面体的功能化
将巯基修饰的PEG(分子量：5000)溶液按照PEG︰四面体摩尔比1000︰1的比例加入到四面体检测探针溶液中，15min后离心除去多余的PEG分子。然后把穿膜肽分子(TAT)按照TAT︰四面体摩尔比1000︰1的比例加入到PEG修饰过的四面体溶液中，室温反应24h，离心除去多余的未连在四面体上的穿膜肽。
(6)活细胞内microRNA-21检测方法的建立
首先用商业转染试剂(lipofectamine RNAiMAX transfection reagent)和不同量的microRNA-21来增加细胞中microRNA-21的量，或者用microRNA-21的反义序列来降低细胞中的microRNA-21的量，然后用RT-PCR来确定转染后细胞中的microRNA-21的量。具体方法如下：首先建立人工合成的有梯度的microRNA-21的扩增曲线，然后建立循环数和microRNA-21的浓度之间的线性关系。然后提取转染后的细胞中的总RAN，然后用RT-PCR扩增得到里面含有microRNA-21的扩增曲线，根据扩增曲线得到循环数，然后根据循环数从标准曲线中确定含有的microRNA-21，然后用含有四面体的培养基(1640+10％FBS+双抗)来培养转染后的细胞，8h后洗去细胞外面的四面体，测定细胞内的CD信号及上转换荧光强度，然后分别建立CD信号和细胞内microRNA-21的浓度的标准曲线以及上转换荧光强度与细胞内microRNA-21的浓度之间的标准曲线。
(7)不同细胞中microRNA-21的检测
用含有四面体的培养基(1640+10％FBS+双抗)分别培养MCF-7，HeLa,PCS-460-010细胞，8h后，检测细胞中的CD信号及上转换荧光强度，然后根据分别建立CD信号和细胞内microRNA-21的浓度的标准曲线以及上转换荧光强度与细胞内microRNA-21的浓度之间的标准曲线来确定细胞中microRNA-21的含量。
本发明的有益效果：本发明将DNA驱使的金-上转换纳米粒子组装的四面体用于胞内miRNA的实时检测。金-上转换纳米粒子四面体具有双重的光学活性，在521nm呈现了很强的等离子圆二色谱信号，并在500-600nm有显著的上转换荧光强度，因此可以利用这双重信号来检测细胞中的miRNA。在目标miRNA的存在下，CD信号会减弱，荧光强度会增强。实验结果显示，CD信号与目标miRNA浓度在0.073到43.65fmol/10μg RNA范围内有很好的线性关系，检测限可以达到0.03fmol/10μg RNA，而上转换荧光强度与目标miRNA浓度的线性范围是0.16-43.65fmol/10μg RNA，最低检测限是0.12fmol/10μg RNA。这些结果说明了CD信号对miRNA的浓度变化比荧光强度有更高的灵敏度。强的CD信号来自于光子自旋角动量的相互作用以及四面体中DNA分子对手性的增强作用。
本发明不仅建立了胞内miRNA的超灵敏定量检测方法，同时也为组装体等离子手性的生物应用开辟了道路。本发明建立了一种基于金-上转换纳米粒子四面体的双信号原位检测胞内microRNA的检测方法，是一种超灵敏，高特异性的胞内microRNA的原位检测方法。
附图说明
图1：(A)金-上转换纳米粒子四面体检测细胞中microRNA的示意图；(B)四面体的核酸骨架，1和2的位点与上转换纳米粒子联接，3和4的位点与金纳米粒子联接。
图2：组装的四面体的透射电镜图。
图3：组装的四面体的产率统计图。
图4：细胞外检测能力的评价：(A)添加一系列不同浓度目标microRNA得到的手性信号图；(B)添加一系列不同浓度目标microRNA得到的荧光信号图。对于图中的各条曲线，按照各曲线谷底的高低顺序，从高到底，分别代表microRNA浓度为200pM、50pM、30pM、15pM、10pM、5pM、2pM、0pM的情形。
图5：检测特异性的评价：(C)添加不同种类的microRNA得到的手性信号图；(D)添加不同种类的microRNA得到的荧光信号图。
图6：实施例6中含有不同量microRNA21的细胞与检测探针孵育8小时后的CD信号。对于图中的各条曲线，按照各曲线谷底的高低顺序，从高到底，分别代表43.65fmol/10μg RNA、15.81fmol/10μg RNA、3.18fmol/10μg RNA、0.48fmol/10μg RNA、0.16fmol/10μg RNA、0.073fmol/10μg RNA、空白对照的情形。
图7：实施例6中含有不同量microRNA21的细胞与检测探针孵育8小时的荧光信号。
图8为图6中521nm处的CD信号的强度与细胞内用RT-PCR检测得到的microRNA的量之间的线性关系。
图9为图7中543nm处的上转换强度与细胞内用RT-PCR检测得到的microRNA的量之间的线性关系。
图10：用检测探针检测不同细胞中microRNA21的量，所得到不同细胞中的microRNA的CD信号图。对于图中的各条曲线，按照各曲线谷底的高低顺序，从高到底，分别代表细胞为MCF-7、HeLa、PCS-460-010的情形。
图11：用检测探针检测不同细胞中microRNA21的量，所得到不同细胞中的microRNA的上转换荧光信号图。
具体实施方式
实施例1 金纳米粒子和上转换纳米粒子(NaGdF4:Yb,Er)的制备
(1)20nm金纳米粒子的制备
洁净的三口烧瓶中加入97.5mL超纯水，加入2.5mL 0.4％氯金酸溶液，搅拌并加热至沸腾，7-8min后加入1.6mL 1％柠檬酸三钠溶液，溶液从无色变为红色后，停止加热，继续搅拌15min。自然冷却后放置在4℃冰箱备用。
(2)19nm上转换纳米粒子(NaGdF4:Yb,Er)的制备
在含有14mL OA(配体十八烯酸)和16mL ODE(非配位有机溶剂十八烯)的混合液中加入0.80mmol的GdCl3·6H2O，0.18mmol的YbCl3·6H2O和0.02mmol的ErCl3·6H2O，溶解。在氮气的保护下加热到150℃形成均相的溶液。降至室温后，含有0.100g的NaOH和0.148g的NH4F的10mL甲醇溶液慢慢加入。反应混合液在50℃下慢慢搅拌30min，然后在100℃在真空下处理10min除去甲醇，在氮气的保护下加热到320℃，并且保持1h。将混合物降至室温，反应结束。乙醇中沉淀的合成的NaGdF4粒子用离心收集，并用乙醇洗涤几次，最后重悬在THF或者环己烷溶液中供后续实验使用。上转换纳米粒子的转相：100mg用马来先亚胺基团和磷酸基团修饰的PEG和10mg油酸包裹的NaGdF4粒子混合在THF溶液里面，在室温下搅拌过夜。PEG包裹的NaGdF4粒子在环己烷中沉淀，并用环己烷洗涤三次，在室温真空的条件下干燥，得到了可以在水中溶解的PEG包裹的NaGdF4粒子。
实施例2 microRNA检测用DNA序列设计
Figure PCTCN2017107101-appb-000002
Figure PCTCN2017107101-appb-000003
注：本发明所使用的DNA均购自中国上海生工生物工程有限公司，并通过聚丙烯酰胺凝胶电泳进行纯化。
实施例3 检测探针的制备
首先，制备金纳米粒子和核酸的偶联物：把20nM的金纳米粒子(AuNP)分别和DNA1-Py3，DNA-Py4按摩尔比1︰3.5的比例混合在一起，反应1h后加入NaCl溶液至终溶度为50mM，混合均匀，反应过夜，离心去除游离的核酸，即得到AuNP-DNA-Py3，AuNP-DNA-Py4。
然后，制备上转换纳米粒子和核酸的偶联物：把20nM上转换纳米粒子(UCNP)分别和DNA-Py1，DNA-Py2按摩尔比1︰3.5的比例混合在一起，反应1h后加入NaNO3溶液至终溶度为50mM，混合均匀，反应过夜，离心去除游离的核酸，即得到UCNP-DNA-Py1，UCNP-DNA-Py2。
接下来，把得到的AuNP-DNA-Py3和AuNP-DNA-Py4，UCNP-DNA-Py1和UCNP-DNA-Py2分别混合，加入NaCl至终浓度为50mM，95℃水浴5min，然后37℃孵育8h，即得到金二聚体及上转换二聚体。
把金二聚体和上转换二聚体按照摩尔比1︰1的比例混合，37℃孵育24h，即制备得到检测microRNA-21用的金-上转换纳米粒子四面体检测探针溶液。图2为用金和上转换纳米粒子以及DNA核酸序列制备的检测探针的透射电镜图。图3为通过拍摄200张不同视野的TEM照片，对最终组装的四面体进行产率统计。
通过在检测探针溶液中添加不同浓度的目标microRNA，然后测试溶液的CD和荧光信号，通过CD和荧光信号的变化来检测四面体检测microRNA21的检测能力。检测结果如图4所示，目标microRNA浓度的变化，能在CD和荧光信号上得到显著的体现。
通过向检测探针溶液中添加高浓度的非目标microRNA，然后测试溶液的CD和荧光信号，通过CD和荧光信号的变化来检测四面体探针的特异性，确认检测探针的信号是否会受非目标microRNA或者其他物质的干扰。检测结果如图5所示，非目标microRNA或者其他物质的CD和荧光信号强度，与目标microRNA的CD和荧光信号强度，形成了非常显著的差异，非目标microRNA或者其他物质不会干扰目标microRNA的检测。
实施例4 四面体的功能化
将巯基修饰的PEG(分子量：5000)溶液按照PEG︰四面体摩尔比1000︰1的比例加入到四面体检测探针溶液中，15min后离心除去多余的PEG分子。然后把穿膜肽分子(TAT)按照TAT︰四面体摩尔比1000︰1的比例加入到PEG修饰过的四面体溶液中，室温反应24h，离心除去多余的未连在四面体上的穿膜肽。
实施例5 活细胞内microRNA-21检测方法的建立
首先，用商业转染试剂(lipofectamine RNAiMAX transfection reagent)和不同量的microRNA-21来增加细胞中microRNA-21的量，或者用microRNA-21的反义序列来降低细胞中的microRNA-21的量，然后用RT-PCR来确定转染后细胞中的microRNA-21的量。具体方法如下：首先建立人工合成的有梯度的microRNA-21的扩增曲线，然后建立循环数和microRNA-21的浓度之间的线性关系。然后提取转染后的细胞中的总RAN，然后用RT-PCR扩增得到里面含有microRNA-21的扩增曲线，根据扩增曲线得到循环数，然后根据循环数从标准曲线中确定含有的microRNA-21的量(浓度)。
然后，用含有四面体的培养基(1640+10％FBS+双抗)来培养转染后的细胞(如MCF-7)，8h后洗去细胞外面的四面体，测定细胞内的CD信号和上转换荧光强度。
然后，分别建立CD信号和细胞内microRNA-21的浓度的标准曲线以及上转换荧光强度与细胞内microRNA-21的浓度之间的标准曲线。
实施例6 不同细胞中microRNA-21的检测
参照实施例5中的方法，用含有四面体的培养基(1640+10％FBS+双抗)分别培养MCF-7，HeLa，PCS-460-010细胞，8h后检测细胞中的CD信号及上转换荧光强度，然后根据建立CD信号和细胞内microRNA-21的浓度的标准曲线以及上转换荧光强度与细胞内microRNA-21的浓度之间的标准曲线来确定细胞中microRNA-21的含量。
如图6所示，含有不同量microRNA21的细胞与检测探针孵育8小时后，得到的CD信号。可以看出，CD信号能够明显体现出microRNA21含量间的差异。如图8所示，横坐标为以RT-PCR检测细胞内的microRNA的含量，纵坐标为图6中521nm处的CD信号的强度，两者之间具有很好的线性关系；说明采用本发明的检测探针检测的结果与采用RT-PCR检测的结果非常吻合。
如图7所示，含有不同量microRNA21的细胞与检测探针孵育8小时后，得到了荧光信号。可以看出，荧光信号能够明显体现出microRNA21含量间的差异。如图9所示，横坐标为以RT-PCR检测细胞内的microRNA的含量，纵坐标为图7中543nm处的上转换强度，两者之间具有很好的线性关系；说明采用本发明的检测探针检测的结果与采用RT-PCR检测的结果非常吻合。
如图10所示，用检测探针检测不同细胞中microRNA21的量，所得到的不同细胞中的microRNA的CD信号图。可进一步通过图8所建立的标准曲线以及CD光谱中521nm处的CD信号强度来计算不同细胞内含有的microRNA的量。
如图11所示，用检测探针检测不同细胞中microRNA21的量，所得到的不同细胞中的microRNA的上转换荧光信号图。可进一步通过图9所建立的标准曲线以及荧光光谱中543nm处的荧光信号强度来计算不同细胞中的microRNA的量。
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 PCTCN2017107101-appb-000004
Figure PCTCN2017107101-appb-000005
Figure PCTCN2017107101-appb-000006
Figure PCTCN2017107101-appb-000007

Claims (6)

  1. 一种基于金-上转换纳米粒子四面体的双信号原位检测胞内microRNA的方法，其特征在于，包括制备检测探针所用核酸的设计、金-上转换纳米粒子四面体检测探针的制备、四面体的功能化、胞内microRNA-21检测方法的建立；步骤为：
    (1)所用DNA序列设计；
    (2)金-上转换纳米粒子四面体检测探针的制备；
    (3)四面体的功能化；
    (4)胞内microRNA-21检测方法的建立；
    (5)不同细胞中microRNA-21的检测。
  2. 根据权利要求1所述基于金-上转换纳米粒子四面体的双信号原位检测胞内microRNA的方法，其特征在于，所用DNA序列设计：
    DNA-Py1：5’-SH-TTTTCAACAT CAGTCTGATA AGCTACTGTT CTTTCCCTCA ACATCAGTCT GATAAGCTAC GGTTTTCAAC ATCAGTCTGATAAGCTACTT GAC-3’，下划线标注的碱基用硫代磷酸修饰；
    DNA-Py2：5’-SH-TTTCCGTAGC TTATCAGTTA AGCTAGGGTT TCATTAATCA ACATCAGTTC GATAAGCTAT TTTCAACATC AGTCTGATAA GCTACAACTT-3’，下划线标注的碱基用硫代磷酸修饰；
    DNA-Py3：5’-SH-TTTACTAAGC GATTACTGAT GTTGATTAAT GTTTACAACA TCAGTCTGATAAGCTAACTGATTTTGAACAGTAGCTTATC AGGACTCGTG GTC-3’，下划线标注的碱基用硫代磷酸修饰；
    DNA-Py4：5’-SH-TTTAAGTTGT AGCTTATCAG CTGACTACAT TTTTGTCAAG TAGCTTATCA GTTACAGCTC GCTTTATCAG TTAGCTTATC AGAGATCGTA GCT-3’，下划线标注的碱基用硫代磷酸修饰；
    miR-21：5’-UAGCUUAUCA GACUGAUGUU GA-3’，
    control1：5’-AAGCUUAUCU GACUGAUGUU GU-3’，
    control2：5’-UACCUUAUCA GACUGAUGCU GA-3’，
    miR-200b：5’-UAAUACUGCC UGGUAAUGAU GA-3’，
    let-7d：5’-AGAGGUAGUA GGUUGCAUAG UU-3’。
  3. 根据权利要求1或2所述基于金-上转换纳米粒子四面体的双信号原位检测胞内microRNA的方法，其特征在于，金-上转换纳米粒子四面体检测探针的制备：
    (1)首先制备金纳米粒子和核酸的偶联物：把20nM的金纳米粒子AuNP分别和DNA-Py3，DNA-Py4按摩尔比1︰3.5比例混合在一起，反应1h后加入NaCl溶液至终浓度 为50mM，混合均匀，反应过夜，离心去除游离的核酸，即得到AuNP-DNA-Py 3，AuNP-DNA-Py 4；
    (2)然后制备上转换纳米粒子和核酸的偶联物：把20nM的上转换纳米粒子UCNP分别和DNA-Py1，DNA-Py2按摩尔比1︰3.5比例混合在一起，反应1h后加入NaNO3溶液至终浓度为50mM，混合均匀，反应过夜，离心去除游离的核酸，即得到UCNP-DNA-Py 1，UCNP-DNA-Py 2；
    (3)把步骤(1)、(2)得到的AuNP-DNA-Py3和AuNP-DNA-Py4，UCNP-DNA-Py1和UCNP-DNA-Py 2分别混合，加入NaCl至终浓度为50mM，95℃水浴5min，然后37℃孵育8h，即得到金二聚体及上转换二聚体；把金二聚体和上转换二聚体按照摩尔比1︰1的比例混合，37℃孵育24h，即制备得到检测microRNA-21用的金-上转换纳米粒子四面体检测探针溶液。
  4. 根据权利要求1所述基于金-上转换纳米粒子四面体的双信号原位检测胞内microRNA的方法，其特征在于四面体的功能化：将巯基修饰的PEG-5000溶液按照PEG︰四面体摩尔比1000︰1的比例加入到四面体检测探针溶液中，15min后离心除去多余的PEG分子，然后把穿膜肽分子TAT按照TAT︰四面体摩尔比1000︰1的比例加入到PEG修饰过的四面体溶液中，室温反应24h，离心除去多余的未连在四面体上的穿膜肽。
  5. 根据权利要求1所述基于金-上转换纳米粒子四面体的双信号原位检测胞内microRNA的方法，其特征在于，胞内microRNA-21检测方法的建立：
    首先用商业转染试剂lipofectamine RNAiMAX transfection reagent和不同量的microRNA-21来增加细胞中microRNA-21的量，或者用microRNA-21的反义序列来降低细胞中的microRNA-21的量，然后用RT-PCR来确定转染后细胞中的microRNA-21的量；具体方法如下：
    (1)首先建立人工合成的有梯度的microRNA-21的扩增曲线，然后建立循环数和microRNA-21的浓度之间的线性关系；
    (2)然后提取转染后的细胞中的总RAN，用RT-PCR扩增得到microRNA-21的扩增曲线，根据扩增曲线得到循环数，根据循环数从标准曲线中确定含有的microRNA-21，用含有四面体的培养基1640+10％FBS+双抗来培养转染后的细胞，8h后洗去细胞外面的四面体，测定细胞内的CD信号及上转换荧光强度，分别建立CD信号和细胞内microRNA-21的浓度的标准曲线以及上转换荧光强度与细胞内microRNA-21的浓度之间的标准曲线。
  6. 根据权利要求1所述基于金-上转换纳米粒子四面体的双信号原位检测胞内microRNA的方法，其特征在于，不同细胞中microRNA-21的检测：
    用含有四面体的培养基1640+10％FBS+双抗分别培养MCF-7，HeLa，PCS-460-010细胞，8h后，检测细胞中的CD信号及上转换荧光强度，然后根据分别建立CD信号和细胞内microRNA-21的浓度的标准曲线以及上转换荧光强度与细胞内microRNA-21的浓度的标准曲线来确定细胞中microRNA-21的含量。
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