Citation: Zhao Li-Dong, Zuo Peng, Yin Bin-Cheng, Hong Chenglin, Ye Bang-Ce. A Cell Membrane-Anchored DNA Tetrahedral Sensor for Real-time Monitoring of Exosome Secretion[J]. Acta Chimica Sinica, ;2020, 78(10): 1076-1081. doi: 10.6023/A20060235 shu

A Cell Membrane-Anchored DNA Tetrahedral Sensor for Real-time Monitoring of Exosome Secretion

Zhao Li-Dong ^a ,
Zuo Peng ^a ,
Yin Bin-Cheng ^a,b,, ,
Hong Chenglin ^b,, ,
Ye Bang-Ce ^a,c,,

a.
Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
b.
Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi, 832000, China
c.
Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310014, China

Corresponding author: Yin Bin-Cheng, binchengyin@ecust.edu.cn Hong Chenglin, hcl_tea@shzu.edu.cn Ye Bang-Ce, bcye@ecust.edu.cn
Received Date: 14 June 2020
Available Online: 25 July 2020
Fund Project: the National Natural Science Foundation of China 21822402Project supported by the National Natural Science Foundation of China (Nos. 21822402, 21675052, 31730004)the National Natural Science Foundation of China 21675052the National Natural Science Foundation of China 31730004

Figures(11)

Exosomes are nanoscale bilayer membrane vesicles actively secreted by cells, which carry abundant cell-specific substances. They can directly reflect the physiological and functional status of the secreting cells and play important roles in intercellular communication, physiological and pathological processes. In this work, we combined membrane modification technique with fluorescence imaging technique and blended CD63 aptamers into a highly stable and universal DNA tetrahedral structure to construct a cell membrane-anchored DNA sensor for real-time monitoring the secretion of exosomes. We designed four functional toes on each vertex of the tetrahedral sensor, respectively. A signal report toe on the top vertex consisted of fluorophore-modified CD63 aptamer, quencher-modified quencher probe(QP) binding part of the CD63 aptamer, and block probe (BP) binding the rest of the CD63 aptamer. The other three extended toes on the vertices were immobilized to the cell membrane by hybridizing with cholesterol-modified anchor probes(AP), which spontaneously incorporated to a lipid bilayer via hydrophobic interaction between the cholesterol moieties and the cellular membrane. In the initial state, the proposed DNA tetrahedral sensor was tethered to membrane with fluorophores quenched by QP and CD63 aptamer blocked by QP and BP. Trigger probes (TP) were add to bind to BP, resulting in the activation of the sensor. Subsequently, CD63 aptamers were specifically bound to the secreted exosomes, leading to the release of QP and concurrent fluorescence restoration of fluorophore. The intensity of the fluorescent signal in cell membrane was proportional to the amount of exosomes captured, thus realizing the real-time monitoring of the exosomes by analysis the changes of the fluorescence intensity. The experimental results showed that the sensor exhibited a good stability and a high capture efficiency for secreted exosomes. This strategy would provide a potentially useful tool for a variety of applications in biomedical research, drug discovery and tissue engineering.
- cell membrane-anchored sensor,
- DNA tetrahedron,
- exosome,
- secretion,
- real-time monitoring,
- aptamer
1.   引言

外泌体是细胞内体膜向内萌芽主动释放的纳米级膜囊泡(30~150 nm), 广泛存在于各种体液中, 例如血液, 脑脊髓液, 尿液, 唾液和母乳等^[1-3]. 1987年, Johnstone等^[4]在研究网状细胞向成熟红细胞转化时发现一种具有膜结构的小囊泡, 命名为“外泌体”.在此后很长时间里, 外泌体仅被视为细胞不需要的成分.然而, 随着对外泌体研究的逐渐深入, 人们发现外泌体中携带细胞特异性蛋白质和核酸, 在细胞间通讯^[5], 包括肿瘤的发生及转移中发挥着重要作用.因此, 实时监测外泌体的分泌对其发生机制, 生物学功能, 疾病相关性等基础和应用研究具有重要意义.目前, 大多数分析方法, 如纳米颗粒跟踪分析^[6], 电化学^[7], 表面等离子体共振^[8]和微流体系统^[9]等, 主要针对细胞已释放的外泌体进行定量分析, 无法对细胞分泌外泌体进行动态实时监测.

膜锚定生物传感器能够实时监测细胞及其微环境的状态, 为监测动态细胞过程提供了强大的工具.目前膜锚定策略主要包括共价络合^[10-12], 静电结合^[13]和疏水性修饰.其中, 疏水性修饰策略具有操作迅速, 普适性高和毒性低等优点, 已在细胞膜表面工程中广泛应用^[14-20].基于胆固醇基团修饰的DNA传感器已应用于细胞外pH^[21], 干扰素^[22], ATP^[23]等的实时监测.然而, 复杂的细胞培养环境和柔软的单链核酸适配体等因素都会对传感器的功能造成一定影响. DNA四面体由Turberfield等^[24]在2004年首次合成, 具有刚性结构, 能够避免探针之间的相互缠绕, 提高了传感器性能, 广泛应用于生物检测^[25-29].

本文以DNA四面体为基础, 结合细胞膜疏水性修饰技术和荧光成像技术, 构建了一种细胞膜表面DNA四面体传感器, 应用于不同细胞类型分泌外泌体的实时监测.如图 1所示, 胆固醇修饰的锚定探针(AP)首先通过疏水作用固定于细胞膜表面, DNA四面体传感器通过25 bp延伸脚趾与AP结合固定于细胞膜表面.为了监测外泌体的分泌, 四面体传感器顶端设计一个信号检测系统, 包括荧光基团修饰的CD63核酸适配体序列, 淬灭探针(QP)和封闭探针(BP). CD63核酸适配体序列被QP及BP封闭, 其携带的荧光基团被QP上的淬灭基团淬灭.该传感器不会与细胞膜上的CD63蛋白结合, 降低了假阳性信号.通过添加与BP完全互补的引发探针(TP)置换BP, 从而激活传感器.当细胞分泌外泌体时, CD63核酸适配体特异性结合外泌体, 从而释放QP, 传感器上的荧光信号恢复, 其荧光信号强度与外泌体量成正相关.因此, 通过监测细胞膜表面荧光的变化, 获得外泌体的分泌情况.

图 1

图 1. DNA四面体传感器的锚定及实时监测外泌体分泌的工作原理示意图

Figure 1. Schematic illustration of DNA tetrahedral sensor anchoring and the working principle for real-time monitoring of exosome secretion

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2.   结果与讨论

2.1   DNA四面体的合成及表征

采用边长为17 bp的DNA四面体来构建传感器.合成DNA四面体结构(TDN-F)的四条链(A-F, B-S, C-S, D-S), QP和BP通过自组装形成传感器(Sensor), 利用非变性聚丙烯酰胺凝胶电泳对传感器进行表征.如图 2所示, 随着A-F到BP的添加, DNA混合物的凝胶迁移率逐渐降低, 表明传感器成功合成.

图 2

图 2. 非变性聚丙烯酰胺凝胶电泳(PAGE)验证TDN-F的形成. M: 8000 bp DNA Ladder Marker; 泳道1: A-F; 泳道2: B-S+C-S; 泳道3: B-S+C-S+D-S; 泳道4: TDN-F; 泳道5: TDN-F+QP; 泳道6: TDN-F+QP+BP(传感器).

Figure 2. PAGE verified the formation of DNA tetrahedron structure. M: 8000 bp DNA Ladder Marker; lane 1: A-F; lane 2: B-S+C-S; lane 3: B-S+C-S+D-S; lane 4: TDN-F; lane 5: TDN-F+QP; lane 6: TDN-F+QP+BP(Sensor).

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2.2   可行性分析

以HepG2细胞作为模型细胞, 研究传感器的固定化及外泌体捕获的可行性.通过共聚焦激光扫描显微镜验证传感器固定化的可行性, 如图 3A所示, 过夜培养的细胞与1 μmol/L AP在37 ℃下孵育20 min, 与0.3 μmol/L TDN-F孵育1 h后, 荧光均匀分布于细胞膜表面, 表明TDN-F能够成功锚定于膜表面.采用已报道的方法^[30]验证TDN-F三个延伸脚趾是否均与AP结合, 我们设计了6条胆固醇修饰的探针, 其中三条锚定探针AP1, AP2, AP3, 除了25 bp的连接部分, 还带有三个不同的7 bp脚趾.另外三条置换探针RP1, RP2, RP3分别与AP1, AP2, AP3完全互补, 能够与AP1, AP2, AP3结合取代TDN-F的延伸脚趾, 将TDN-F从细胞膜上移除(图 3B).如图 3C所示, 当仅添加RP1或同时添加RP1和RP2时, 细胞膜表面显示荧光, 表明TDN-F仍固定于细胞膜表面.而同时添加RP1, RP2, RP3, 细胞膜表面荧光信号消失, 表明TDN-F的三个延伸脚趾均被置换, TDN-F从细胞膜表面移除.以上结果表明, TDN-F的三个延伸脚趾均与AP结合, TDN-F稳定地锚定于细胞膜表面.此外, 显微镜成像(CLSM image)结果显示, 随着RP的添加, 细胞膜表面荧光强度逐渐降低, 我们推测, TDN-F出现一定的脱靶现象, 可能是由于DNA四面体携带有大量负电荷与细胞膜表面负电荷相斥, 且AP上携带的聚乙二醇(PEG)具有亲水性, 使传感器倾向于向溶液端扩散, 而单个胆固醇的疏水相互作用和小窝蛋白对DNA四面体的亲和力不足以将传感器束缚于细胞膜表面, 从而导致传感器部分脱靶.

图 3

图 3. (A) TDN-F在细胞膜表面锚定的示意图及显微镜成像; (B) RP置换TDN-F延伸脚趾示意图; (C) TDN-F固定化验证的显微镜成像

Figure 3. (A) Schematic illustration and CLSM images of TDN-F anchoring on the cell-surface; (B) Schematic illustration of RP replaced the extended toe of TDN-F; (C) CLSM images of TDN-F immobilization verification

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通过荧光光谱法, 共聚焦显微镜成像和流式细胞仪分析验证传感器捕获外泌体的可行性.首先, 在缓冲溶液中评估探针的功能性及传感器对外泌体的响应情况.如图 4A所示, TDN-F+QP+Exo中荧光信号恢复(绿色曲线), 表明外泌体蛋白成功与适配子结合，将QP从传感器上置换释放; TDN-F+QP+BP+Exo中无荧光信号(蓝色曲线), 表明QP与BP能够有效封闭CD63适配体, 减少传感器固定化过程中的假阳性信号; 在加入TP后传感器荧光信号恢复(粉色曲线), 表明TP能够将传感器上的BP置换下来, 探针具有良好的功能性.检测传感器在不同浓度外泌体中的荧光信号强度评估其捕获外泌体的能力, 如图 4B所示, 荧光信号强度随外泌体浓度增加逐渐增强, 表明传感器对外泌体响应情况良好.我们评估了细胞膜锚定的传感器对外泌体的响应情况.传感器固定到细胞膜表面后, 添加TP及不同浓度的外泌体反应1 h.如图 5A, B所示, 随着外泌体浓度的增加, 细胞膜表面荧光强度显著增强, 表明细胞膜表面锚定的传感器保留了对外泌体的响应能力, 与缓冲液中结果一致.与细胞膜锚定的单链核酸适配体传感器Apt-S (由胆固醇及荧光基团修饰的核酸适配体Apt-F, QP及BP组成)相比, 该传感器捕获外泌体的效率高约100倍(图 5C, D).

图 4

图 4. (A) 缓冲液中探针的功能验证; (B)缓冲液中传感器对外泌体的响应情况

Figure 4. (A) Functional verification of probes in buffer; (B) Response of sensor to exosomes in buffer

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图 5

图 5. 细胞膜表面锚定的传感器与不同浓度外泌体反应的显微镜成像(A)及流式细胞仪分析(B); (C)单链核酸适配体传感器与该传感器捕获外泌体流式细胞仪分析; (D)两种传感器捕获外泌体效率比较.

Figure 5. CLSM images (A) and flow cytometry analysis (B) of cell membrane-anchored sensor reacted with different concentrations of exosomes; (C) Flow cytometry analysis of the single-strand aptamer sensor and this sensor capture exosomes; (D) Differences of the exosomes capture efficiency of two sensors.

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在确定了传感器能够成功锚定于细胞膜上且能够捕获外泌体之后, 我们在缓冲液及细胞体系中对传感器的特异性进行了分析, 对比了该传感器与含随机序列的传感器(R-sensor)对外泌体的响应情况.如图 6A所示, 在添加了引发探针和外泌体的缓冲液中该传感器荧光信号恢复(蓝色曲线), 而含随机序列的传感器中无荧光信号(绿色曲线).在细胞体系中, 将两种传感器分别锚定于细胞膜表面, 添加引发探针激活传感器后, 加入外泌体.如图 6B所示, 该传感器锚定的细胞的荧光信号峰较空白对照有明显偏移, 含随机序列的传感器锚定的细胞荧光信号峰几乎无偏移, 与缓冲液体系的结果一致.表明该传感器能够特异性捕获外泌体.

图 6

图 6. 缓冲液(A)和细胞体系(B)中传感器的特异性验证

Figure 6. Verification of sensor specificity in buffer (A) and cell system (B)

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2.3   条件优化

随后, 我们对AP和TDN-F的固定化条件进行了优化, 以AP-F代替AP进行优化实验.如图 7A所示, 随着AP浓度的升高, 细胞膜表面的平均荧光强度明显增强, 浓度超过2 μmol/L后, 平均荧光强度几乎平稳, 这一结果表明, 2 μmol/L为AP修饰细胞的最佳孵育浓度.如图 7B所示, AP与细胞孵育时间超过30 min后, 细胞膜表面的平均荧光强度几乎平稳, 表明30 min为AP修饰细胞的最佳孵育时间.对于传感器修饰条件的优化, 首先将2 μmol/L AP与过夜培养的细胞孵育30 min, 磷酸盐缓冲液(PBS)洗涤除去游离AP, 加入TDN-F与细胞孵育.如图 7C, D所示, 0.3 μmol/L为TDN-F与AP的最佳结合浓度, 60 min为TDN-F与AP的最佳结合时间.在后续研究中, AP和TDN-F的浓度和孵育时间分别为2 μmol/L, 0.3 μmol/L和30 min, 60 min.

图 7

图 7. AP与细胞孵育浓度(A)和时间(B)优化; TDN-F与AP结合浓度(C)和时间(D)优化

Figure 7. Optimization of concentration (A) and time (B) of AP incubated with cells; Optimization of concentration (C) and time (D) of TDN-F hybridized AP

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2.4   内化研究

为了探究传感器在细胞培养条件下的内化情况, 将TDN-F在最优条件下固定, PBS洗涤后加入杜氏改良Eagle培养基(DMEM), 分别继续培养2, 4, 6 h, 显微镜成像分析TDN-F的分布情况.如图 8A所示, 继续培养0~2 h后细胞表面荧光强度无明显变化, 2~6 h荧光强度逐渐减弱, 孵育6 h后在细胞内观察到荧光信号(箭头所指). Image J对TDN-F内化情况的显微镜成像进行分析, 如图 8B所示, 继续培养2 h后约5%的TDN-F被内化, 继续培养4 h后10%左右探针内化, 即使继续培养6 h后仅有不足20%探针被细胞内化.以上结果表明, 该传感器能够较长时间稳定停留在细胞膜表面, 满足后续实验需求.

图 8

图 8. (A) TDN-F在细胞培养条件下内化情况的显微镜成像; (B)细胞膜荧光与总荧光比值(F_membrane/F_total)柱状图

Figure 8. (A) CLSM images of the internalization of TDN-F in cell culture environment; (B) Histogram of cell membrane fluorescence to total fluorescence ratio

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2.5   稳定性研究

为了探究传感器在细胞培养环境下的稳定性，将1 μmol/L传感器与DMEM于37 ℃孵育0, 2, 4, 8, 12 h后, 进行琼脂糖凝胶电泳及Image J分析.如图 9所示, 0~12 h内传感器的降解程度随孵育时间的延长而增加, 而孵育4 h后传感器有约10%降解. 12 h后仅有35%传感器降解, 表明该传感器在细胞培养环境下具有较高的稳定性.可能由于DNA四面体上携带的大量负电荷吸引了部分盐离子聚集于四面体表面，较高的盐离子浓度降低了FBS中蛋白等物质对四面体的影响.而后续实验均在4 h内进行, 该时间内传感器在细胞培养环境下的降解程度较小, 满足实验设计需求.

图 9

图 9. 2.5%琼脂糖凝胶电泳验证传感器在细胞培养环境中的稳定性

Figure 9. 2.5% agarose gel electrophoresis to verify the stability of sensor in cell culture environment

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2.6   细胞分泌外泌体的实时监测

为了进一步分析传感器实时监测外泌体的能力, 将传感器固定化后, 使用不同浓度(0.25, 2.5和25 μmol/L)的外泌体分泌抑制剂GW4869处理细胞, 共聚焦显微镜对处理4 h后的细胞进行成像, 如图 10A所示, 由于抑制剂抑制了外泌体的分泌, 细胞膜表面荧光信号强度与抑制剂浓度呈负相关, 流式细胞仪分析得到了同样的结果(图 10B).进一步分析不同时间点细胞表面荧光强度, 如图 10C所示, 细胞膜表面荧光信号强度随抑制剂浓度增高逐渐减弱, 随监测时间的延长逐渐增强, 以上结果表明该传感器具有良好的外泌体监测能力.

图 10

图 10. 不同浓度GW4869处理细胞的显微镜成像(A)及流式分析(B); (C)不同浓度GW4869处理的细胞在不同时间的荧光值

Figure 10. CLSM images (A) and flow cytometry assay (B) of the cells treated by different concentrations of GW4869; (C) Fluorescence value at different times of cells treated by different concentrations of GW4869

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此外, 将传感器锚定于不同类型细胞膜表面, 进一步表明了其可靠性和通用性.以锚定有传感器且未添加TP的HepG2样品为阴性对照, 如图 11所示, 随着监测时间的延长(1~4 h), 不同细胞类型表面荧光强度均有不同程度的增强, 显示出该传感器良好的外泌体监测能力及适用性.

图 11

图 11. 不同细胞类型分泌外泌体的实时监测

Figure 11. Real-time monitoring of exosomes secretion by different cell types

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3.   结论

本研究中, 我们利用包含核酸适配体序列的DNA四面体结构和疏水性胆固醇修饰核酸探针构建了一种细胞膜表面传感器, 实现了对不同类型细胞分泌外泌体的可视化实时监测.该传感器兼具了核酸适配体的识别特异性、胆固醇基团的膜修饰便捷性和DNA四面体的结构稳定性高等优点.与基于单链核酸适配体直接修饰的细胞膜传感器相比, 该传感器展示了独特的优势.四面体刚性结构避免了单链核酸探针间的相互缠绕, 降低了空间位阻, 为核酸适配体与细胞释放的外泌体提供了更大的结合空间, 因而提高了外泌体的捕获效率.此外, 通过更换四面体支架的顶端功能核酸序列, 该传感器可以拓展应用于不同目标物的实时监测, 在生物医学研究、临床诊断及治疗等方面具有一定的潜在应用价值.

4.   实验部分

4.1   传感器的合成及表征

传感器的合成及表征参照已报道的方法^[31], 实验所用探针序列见表S1.将等摩尔量的A-F, B-S, C-S, D-S, QP, BP在TM缓冲液(20 mmol/L Tris, 50 mmol/L MgCl₂, pH 8.0)中混合均匀, 置于PCR仪中加热至95 ℃持续10 min, 放入冰盒迅速冷却10 min, 即得到传感器.采用10%非变性聚丙烯酰胺凝胶电泳表征传感器的形成, 将A-F, B-S+C-S, B-S+C-S+D-S, TDN-F, TDN-F+QP, TDN-F+QP+BP(传感器)样品加样到胶孔中, 在TBE(89 mmol/L Tris-Borate, 2 mmol/L EDTA, pH8.3)缓冲液中80 V电压下电泳3 h, 电泳结束后, 置于1×SYBR Gold染色液中避光震荡染色30 min, ddH₂O清洗2次, 在DNA凝胶成像系统中成像.

4.2   荧光检测

荧光检测用于研究探针的功能性及传感器对外泌体的响应情况.将传感器与不同浓度外泌体在1.5 mL离心管中混合, 37 ℃孵育30 min后, 将混合物转移到光路长度为1.0 cm的荧光微池中, 在激发波长为490 nm, 带通量为5 nm的条件下, 记录500 nm至700 nm的荧光发射光谱.

4.3   细胞培养

宫颈癌细胞系(HeLa), 肝癌细胞系(HepG2), 乳腺癌细胞系(MCF-7)在含有CO₂ (φ＝5%)的37 ℃培养箱中培养, 细胞培养时所用培养基为添加10% (V/V) FBS, 青霉素(100 U/mL)和链霉素(100 μg/mL)的DMEM, 细胞修饰实验中所用培养基为Opti-MEM.将长满细胞的T25方瓶中的培养基吸出, PBS洗一次, 加入1 mL胰蛋白酶, 在37 ℃培养箱中消化4 min, 加入1 mL DMEM终止消化, 将消化液转移至15 mL离心管中, 800 g离心1 min, 吸出上清丢弃, 加入3 mL DMEM重悬为种子液, 用于之后的传代与接种.传代:吸取200~300 μL种子液至T25方瓶中, 补加5 mL DMEM, 混匀置于37 ℃培养箱中培养; 接种:将种子液计数, 以8×10⁴/孔的密度接种于24孔板中, 过夜培养使其贴壁, 用于后续实验.

4.4   琼脂糖凝胶电泳

将1 μmol/L的A-F, B-S, C-S, D-S, QP, BP在TM缓冲液中混合均匀, 置于PCR仪中加热至95 ℃持续10 min, 放入冰盒迅速冷却10 min, 得到传感器.然后与含10% (V/V) FBS的DMEM于37 ℃孵育0, 2, 4, 8, 12 h后, 分别加样到2.5%琼脂糖凝胶胶孔中, 在TAE缓冲液(40 mmol/L Tris-acetate, 1 mmol/L EDTA, pH 8.0)中170 V电压下电泳10 min, 电泳结束后, 在DNA凝胶成像系统中成像.

4.5   共聚焦显微镜成像及流式细胞仪分析

空白对照是未添加任何探针的细胞.在细胞修饰实验, 探针固定化验证实验, 探针特异性分析实验, 探针固定化条件优化实验以及外泌体监测实验中, 反应结束后进行显微镜成像和流式细胞仪分析.显微镜成像测量以40倍物镜进行.在使用流式细胞仪进行检测之前, 将细胞用胰蛋白酶消化并收集, PBS洗涤3次, 采用波长561 nm的激发光检测细胞的荧光强度.

References
1. [1]
  Kowal, J.; Tkach, M.; Théry, C. Curr. Opin. Cell Biol. 2014, 29, 116. doi: 10.1016/j.ceb.2014.05.004
2. [2]
  Shao, H.; Im, H.; Castro, C. M.; Breakefield, X.; Weissleder, R.; Lee, H. Chem. Rev. 2018, 118, 1917. doi: 10.1021/acs.chemrev.7b00534
3. [3]
  Record, M.; Subra, C.; Silventepoirot, S.; Poirot, M. Biochem. Pharmacol. 2011, 81, 1171. doi: 10.1016/j.bcp.2011.02.011
4. [4]
  Johnstone, R. M.; Adam, M.; Hammond, J. R.; Orr, L.; Turbide, C. J. Biol. Chem. 1987, 262, 9412.
5. [5]
  Imjeti, N. S.; Menck, K.; Egea-Jimenez, A. L.; Lecointre, C.; Lembo, F.; Bouguenina, H.; Badache, A.; Ghossoub, R.; David, G.; Roche, S.; Zimmermann, P. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, 12495. doi: 10.1073/pnas.1713433114
6. [6]
  Gallego-Urrea, J. A.; Tuoriniemi, J.; Hassell v, M. TrAC, Trends Anal. Chem. 2011, 30, 473. doi: 10.1016/j.trac.2011.01.005
7. [7]
  Wang, S.; Zhang, L.; Wan, S.; Cansiz, S.; Cui, C.; Liu, Y.; Cai, R.; Hong, C.; Teng, I. T.; Shi, M.; Wu, Y.; Dong, Y.; Tan, W. ACS Nano. 2017, 11, 3943. doi: 10.1021/acsnano.7b00373
8. [8]
  Rupert, D. L.; L sser, C.; Eldh, M.; Block, S.; Zhdanov, V. P.; Lotvall, J. O.; Bally, M.; H k, F. Anal. Chem. 2014, 86, 5929. doi: 10.1021/ac500931f
9. [9]
  Xu, H.; Liao, C.; Zuo, P.; Liu, Z.; Ye, B. C. Anal. Chem. 2018, 90, 13451. doi: 10.1021/acs.analchem.8b03272
10. [10]
  Germain, M.; Balaguer, P.; Nicolas, J. C.; Lopez, F.; Esteve, J. P.; Sukhorukov, G. B.; Winterhalter, M.; Richard-Foy, H.; Fournier, D. Biosens. Bioelectron. 2006, 21, 1566. doi: 10.1016/j.bios.2005.07.011
11. [11]
  Teramura, Y.; Kaneda, Y.; Totani, T.; Iwata, H. Biomaterials. 2008, 29, 1345. doi: 10.1016/j.biomaterials.2007.11.048
12. [12]
  Kim, H.; Shin, K.; Park, O. K.; Choi, D.; Kim, H. D.; Baik, S.; Lee, S. H.; Kwon, S. H.; Yarema, K. J.; Hong, J.; Hyeon, T.; Hwang, N. S. J. Am. Chem. Soc. 2018, 140, 1199. doi: 10.1021/jacs.7b08440
13. [13]
  Chanana, M.; Gliozzi, A.; Diaspro, A.; Chodnevskaja, I.; Huewel, S.; Moskalenko, V.; Ulrichs, K.; Galla, H. J.; Krol, S. Nano Letters. 2005, 5, 2605. doi: 10.1021/nl0521219
14. [14]
  Zhou, W. -Y.; Yang, P. -T.; Yang, R. -H.; Zheng, J. J. Instrum. Anal. 2019, 038, 154 (in Chinese).
15. [15]
  Chen, X.; Zhang, X.; Wang, H. Y.; Chen, Z.; Wu, F. G. Langmuir. 2016, 32, 10126. doi: 10.1021/acs.langmuir.6b02288
16. [16]
  Matsuda, M.; Hatanaka, W.; Takeo, M.; Kim, C. W.; Niidome, T.; Yamamoto, T.; Kishimura, A.; Mori, T.; Katayama, Y. Bioconjug. Chem. 2014, 25, 2134. doi: 10.1021/bc500465j
17. [17]
  Altman, M. O.; Chang, Y. M.; Xiong, X.; Tan, W. Sci. Rep. 2013, 3, 3343. doi: 10.1038/srep03343
18. [18]
  Qiu, L.; Wimmers, F.; Weiden, J.; Heus, H. A.; Tel, J.; Figdor, C. G. Chem. Commun. 2017, 53, 8066. doi: 10.1039/C7CC03576D
19. [19]
  Li, J.; Xun, K.; Pei, K.; Liu, X.; Peng, X.; Du, Y.; Qiu, L.; Tan, W. J. Am. Chem. Soc. 2019, 141, 18013. doi: 10.1021/jacs.9b04725
20. [20]
  Świtalska, A.; Anna, D.; Agnieszka, F. W.; Juskowiak, B. Sensors 2018, 18, 2201. doi: 10.3390/s18072201
21. [21]
  Zeng, S.; Liu, D.; Li, C.; Yu, F.; Fan, L.; Lei, C.; Huang, Y.; Nie, Z.; Yao, S. Anal. Chem. 2018, 90, 13459. doi: 10.1021/acs.analchem.8b03299
22. [22]
  Qiu, L.; Wimmers, F.; Weiden, J.; Heus, H. A.; Tel, J.; Figdor, C. G. Chem. Commun. 2017, 53, 8066. doi: 10.1039/C7CC03576D
23. [23]
  Yuan, J.; Deng, Z.; Liu, H.; Li, X.; Li, J.; He, Y.; Qing, Z.; Yang, Y.; Zhong, S. ACS Sens. 2019, 4, 1648. doi: 10.1021/acssensors.9b00482
24. [24]
  Goodman, R. P.; Berry, R. M.; Turberfield, A. J. Chem. Commun. 2004, 12, 1372.
25. [25]
  Schlapak, R.; Danzberger, J.; Armitage, D.; Morgan, D.; Ebner, A.; Hinterdorfer, P.; Pollheimer, P.; Gruber, H. J.; Sch ffler, F.; Howorka, S. Small 2012, 8, 89. doi: 10.1002/smll.201101576
26. [26]
  Ye, D.-K.; Zuo, X.-L.; Fan, C.-H. Prog. Chem. 2017, 01, 36 (in Chinese).
27. [27]
  Chen, X.; Zhou, G.; Song, Ping.; Wang, J.; Gao, J.; Lu, J.; Fan, C.; Zuo, X. Anal. Chem. 2014, 86, 7337. doi: 10.1021/ac500054x
28. [28]
  Zhu, F.-L.; Bian, X.-J.; Tian, R.; Li, L.; Yan, J.; Liu, G. Chin. J. Anal. Chem. 2020, 4, 473 (in Chinese).
29. [29]
  He, L.; Lu, D.; Liang, H.; Xie, S.; Zhang, X.; Liu, Q.; Yuan, Q.; Tan, W. J. Am. Chem. Soc. 2018, 140, 258. doi: 10.1021/jacs.7b09789
30. [30]
  Conway, J. W.; Madwar, C.; Edwardson, T. G.; McLaughlin, C. K.; Fahkoury, J.; Lennox, R. B.; Sleiman, H. F. J. Am. Chem. Soc. 2014, 136, 12987. doi: 10.1021/ja506095n
31. [31]
  Lin, M.; Wang, J.; Zhou, G.; Wang, J.; Wu, N.; Lu, J.; Gao, J.; Chen, X.; Shi, J.; Zuo, X.; Fan, C. Angew. Chem. Int. Ed. 2015, 54, 2151. doi: 10.1002/anie.201410720
1. [1]
  Kowal, J.; Tkach, M.; Théry, C. Curr. Opin. Cell Biol. 2014, 29, 116. doi: 10.1016/j.ceb.2014.05.004
2. [2]
  Shao, H.; Im, H.; Castro, C. M.; Breakefield, X.; Weissleder, R.; Lee, H. Chem. Rev. 2018, 118, 1917. doi: 10.1021/acs.chemrev.7b00534
3. [3]
  Record, M.; Subra, C.; Silventepoirot, S.; Poirot, M. Biochem. Pharmacol. 2011, 81, 1171. doi: 10.1016/j.bcp.2011.02.011
4. [4]
  Johnstone, R. M.; Adam, M.; Hammond, J. R.; Orr, L.; Turbide, C. J. Biol. Chem. 1987, 262, 9412.
5. [5]
  Imjeti, N. S.; Menck, K.; Egea-Jimenez, A. L.; Lecointre, C.; Lembo, F.; Bouguenina, H.; Badache, A.; Ghossoub, R.; David, G.; Roche, S.; Zimmermann, P. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, 12495. doi: 10.1073/pnas.1713433114
6. [6]
  Gallego-Urrea, J. A.; Tuoriniemi, J.; Hassell v, M. TrAC, Trends Anal. Chem. 2011, 30, 473. doi: 10.1016/j.trac.2011.01.005
7. [7]
  Wang, S.; Zhang, L.; Wan, S.; Cansiz, S.; Cui, C.; Liu, Y.; Cai, R.; Hong, C.; Teng, I. T.; Shi, M.; Wu, Y.; Dong, Y.; Tan, W. ACS Nano. 2017, 11, 3943. doi: 10.1021/acsnano.7b00373
8. [8]
  Rupert, D. L.; L sser, C.; Eldh, M.; Block, S.; Zhdanov, V. P.; Lotvall, J. O.; Bally, M.; H k, F. Anal. Chem. 2014, 86, 5929. doi: 10.1021/ac500931f
9. [9]
  Xu, H.; Liao, C.; Zuo, P.; Liu, Z.; Ye, B. C. Anal. Chem. 2018, 90, 13451. doi: 10.1021/acs.analchem.8b03272
10. [10]
  Germain, M.; Balaguer, P.; Nicolas, J. C.; Lopez, F.; Esteve, J. P.; Sukhorukov, G. B.; Winterhalter, M.; Richard-Foy, H.; Fournier, D. Biosens. Bioelectron. 2006, 21, 1566. doi: 10.1016/j.bios.2005.07.011
11. [11]
  Teramura, Y.; Kaneda, Y.; Totani, T.; Iwata, H. Biomaterials. 2008, 29, 1345. doi: 10.1016/j.biomaterials.2007.11.048
12. [12]
  Kim, H.; Shin, K.; Park, O. K.; Choi, D.; Kim, H. D.; Baik, S.; Lee, S. H.; Kwon, S. H.; Yarema, K. J.; Hong, J.; Hyeon, T.; Hwang, N. S. J. Am. Chem. Soc. 2018, 140, 1199. doi: 10.1021/jacs.7b08440
13. [13]
  Chanana, M.; Gliozzi, A.; Diaspro, A.; Chodnevskaja, I.; Huewel, S.; Moskalenko, V.; Ulrichs, K.; Galla, H. J.; Krol, S. Nano Letters. 2005, 5, 2605. doi: 10.1021/nl0521219
14. [14]
  Zhou, W. -Y.; Yang, P. -T.; Yang, R. -H.; Zheng, J. J. Instrum. Anal. 2019, 038, 154 (in Chinese).
15. [15]
  Chen, X.; Zhang, X.; Wang, H. Y.; Chen, Z.; Wu, F. G. Langmuir. 2016, 32, 10126. doi: 10.1021/acs.langmuir.6b02288
16. [16]
  Matsuda, M.; Hatanaka, W.; Takeo, M.; Kim, C. W.; Niidome, T.; Yamamoto, T.; Kishimura, A.; Mori, T.; Katayama, Y. Bioconjug. Chem. 2014, 25, 2134. doi: 10.1021/bc500465j
17. [17]
  Altman, M. O.; Chang, Y. M.; Xiong, X.; Tan, W. Sci. Rep. 2013, 3, 3343. doi: 10.1038/srep03343
18. [18]
  Qiu, L.; Wimmers, F.; Weiden, J.; Heus, H. A.; Tel, J.; Figdor, C. G. Chem. Commun. 2017, 53, 8066. doi: 10.1039/C7CC03576D
19. [19]
  Li, J.; Xun, K.; Pei, K.; Liu, X.; Peng, X.; Du, Y.; Qiu, L.; Tan, W. J. Am. Chem. Soc. 2019, 141, 18013. doi: 10.1021/jacs.9b04725
20. [20]
  Świtalska, A.; Anna, D.; Agnieszka, F. W.; Juskowiak, B. Sensors 2018, 18, 2201. doi: 10.3390/s18072201
21. [21]
  Zeng, S.; Liu, D.; Li, C.; Yu, F.; Fan, L.; Lei, C.; Huang, Y.; Nie, Z.; Yao, S. Anal. Chem. 2018, 90, 13459. doi: 10.1021/acs.analchem.8b03299
22. [22]
  Qiu, L.; Wimmers, F.; Weiden, J.; Heus, H. A.; Tel, J.; Figdor, C. G. Chem. Commun. 2017, 53, 8066. doi: 10.1039/C7CC03576D
23. [23]
  Yuan, J.; Deng, Z.; Liu, H.; Li, X.; Li, J.; He, Y.; Qing, Z.; Yang, Y.; Zhong, S. ACS Sens. 2019, 4, 1648. doi: 10.1021/acssensors.9b00482
24. [24]
  Goodman, R. P.; Berry, R. M.; Turberfield, A. J. Chem. Commun. 2004, 12, 1372.
25. [25]
  Schlapak, R.; Danzberger, J.; Armitage, D.; Morgan, D.; Ebner, A.; Hinterdorfer, P.; Pollheimer, P.; Gruber, H. J.; Sch ffler, F.; Howorka, S. Small 2012, 8, 89. doi: 10.1002/smll.201101576
26. [26]
  Ye, D.-K.; Zuo, X.-L.; Fan, C.-H. Prog. Chem. 2017, 01, 36 (in Chinese).
27. [27]
  Chen, X.; Zhou, G.; Song, Ping.; Wang, J.; Gao, J.; Lu, J.; Fan, C.; Zuo, X. Anal. Chem. 2014, 86, 7337. doi: 10.1021/ac500054x
28. [28]
  Zhu, F.-L.; Bian, X.-J.; Tian, R.; Li, L.; Yan, J.; Liu, G. Chin. J. Anal. Chem. 2020, 4, 473 (in Chinese).
29. [29]
  He, L.; Lu, D.; Liang, H.; Xie, S.; Zhang, X.; Liu, Q.; Yuan, Q.; Tan, W. J. Am. Chem. Soc. 2018, 140, 258. doi: 10.1021/jacs.7b09789
30. [30]
  Conway, J. W.; Madwar, C.; Edwardson, T. G.; McLaughlin, C. K.; Fahkoury, J.; Lennox, R. B.; Sleiman, H. F. J. Am. Chem. Soc. 2014, 136, 12987. doi: 10.1021/ja506095n
31. [31]
  Lin, M.; Wang, J.; Zhou, G.; Wang, J.; Wu, N.; Lu, J.; Gao, J.; Chen, X.; Shi, J.; Zuo, X.; Fan, C. Angew. Chem. Int. Ed. 2015, 54, 2151. doi: 10.1002/anie.201410720
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18. [18]
  Xiping Luo , Xing Wang , Shengxiang Yang , Jianzhong Guo , Yuxuan Wang , Xuejuan Yang . Innovative “One Body, Dual Wings” Embedded Talent Cultivation Model: Practice in the Construction of Applied Chemistry Major at Zhejiang Agriculture and Forestry University. University Chemistry, 2024, 39(3): 205-209. doi: 10.3866/PKU.DXHX202309058
19. [19]
  Lijun Huo , Mingcun Wang , Tianyi Zhao , Mingjie Liu . Exploration of Undergraduate and Graduate Integrated Teaching in Polymer Chemistry with Aerospace Characteristics. University Chemistry, 2024, 39(6): 103-111. doi: 10.3866/PKU.DXHX202312059
20. [20]
  Li Zhou , Dongyan Tang , Yunchen Du . Focusing on the Cultivation of Outstanding Talents: A “Five in One” Approach to Promoting the Construction of Chemical Experimental and Practical Teaching System. University Chemistry, 2024, 39(7): 121-128. doi: 10.12461/PKU.DXHX202405037