Nature Nanotechnol.:用于监测脑部细胞外K+含量的超灵敏探针


【引言】

细胞外钾离子浓度([K+]o)变化会影响神经元的膜电位,从而影响神经元活性。同时,[K+]o的改变可能与神经系统疾病有关,例如癫痫和阿尔茨海默氏症等,因此,选择性检测[K+]o可以监测疾病。但是,现有的探针技术不能检测[K+]o的微小变化,特别是在自由移动的动物中。此外,它们容易受到钠离子的干扰。在这里,我们报告了一种超灵敏K+探针,通过配体表界面自组装方法,将“三脚架”配体在负载有荧光染料的介孔硅纳米粒表面自组装形成可以筛选出目标离子的薄膜,从而赋予该探针高灵敏性和高特异性,实现癫痫发作的自由移动小鼠大脑中[K+]o变化的动态监测。

【成果简介】

近日,浙江大学凌代舜教授、陈忠教授以及首尔大学Taeghwan Hyeon教授(共同通讯)在Nature Nanotechnology上发表了一篇题为“A sensitive and specific nanosensor for monitoring extracellular potassium levels in the brain”的文章。这项工作报告了一种通过配体表界面自组装制备超灵敏探针的方法,该离子探针可以监测自由移动小鼠大脑中细胞外离子的动态变化。研究团队通过合成三维的“三脚架”配体,使其在负载有荧光染料的介孔硅纳米粒表面自组装形成对不同离子具有选择性的薄膜,制备得到新型离子探针。其中,利用K+选择性薄膜孔中存在的六个羰基氧原子及孔径与K-O键键长相近的特征,实现了对K+的特异性筛选,随之捕获到的K+在介孔硅纳米粒孔道内富集,增强了该探针检测K+的灵敏度。基于该探针,首次在自由活动的生命体中实现了非侵入性的实时动态的脑部神经活动监测。

【图文导读】

1 K+探针的原子级设计和性能表征

a,K+探针的设计示意图。K+指示剂载入MSN中。MSN表面的K+选择性薄膜仅允许K+进入;

b,K+选择性薄膜中“三脚架”配体的化学结构示意图;

c,捕获K+后,K+选择性薄膜的化学结构示意图:K原子,红色;C原子,灰色;O原子,绿色;N原子,蓝色;

d,K+探针的TEM图;

e,沿d中白线的EDS元素线扫描揭示了元素分布并证实了K+探针的结构;

f,该图显示了K+探针在中性环境下对150 mM [K+]具有高度选择性。然而,在酸性或碱性环境中观察到荧光强度略有下降。添加其他生理阳离子后,荧光强度没有明显变化。ΔF= F-F0,其中,F是给定离子浓度下的荧光强度,F0是0 mM [K+]下的荧光强度;

g,[K+]从0增加到150 mM以及从150减小到0 mM时K+探针的荧光强度变化;

h,在富含K+(150 mM [K+])和不含K+(0 mM [K+])的水溶液中孵育20个循环的K+探针的荧光强度变化。在达到平稳之前的前六个循环中,每次循环荧光强度略有减少;

i,在富含K+和不含K+的水溶液中孵育时,K+探针荧光强度随时间的变化结果。

2 K+探针高选择性和高灵敏度的机理研究

a,在去离子水中K+和Na+水合层的示意图;

b,c,左边,分散在含K+和Na+溶液中的未覆盖K+选择性薄膜的探针(b)和K+探针(c)的TEM图像。右边,沿着TEM图像中白线的EDS元素线扫描结果;

d,在c中用白色虚线框标出区域的EDS元素映射,如白色箭头所示;

e,K+选择性薄膜腔与K+/ Na+之间相互作用的示意图:除去水合层后,K+与孔中的氧原子相互作用,这在能量上有利于K+的通过。但是,Na+无法与孔中的氧原子充分相互作用,这在能量上不利于粒径较小的Na+的通过;

f,K+/Na+通过K+选择性薄膜时,薄膜与K+/Na+之间的结合能计算结果。

3培养的神经元中K+释放的成像

a,在aCSF中对药理调节K+释放的海马神经元进行成像的实验设计图;

b,首先将培养的海马神经元与各种传感器预孵育。暴露于Coriaria内酯后,K+探针的荧光强度增加,而未覆盖K+选择性薄膜的探针和游离指示剂未观察到显著变化;

c,在通过微通道连接的两室微腔中培养的被K+探针标记的神经元的CLSM图像,胞体和轴突分离在两个隔间中;

d,从小鼠海马体处记录的EEG数据,虚线所示范围为放大图;

e,在与5 µM海藻酸孵育前后,c中两个选定区域的荧光强度变化结果;

d,当海藻酸的浓度增加至10 µM时,荧光强度变化结果。

4大脑切片中K+释放的成像

a,对急性背侧纹状体脑切片中电刺激诱发的K+释放进行成像的实验设计图;

b,在不同强度的给定电脉冲下细胞外荧光响应结果(右),红点表示信号响应(∆F/F0)大于5%的区域;

c,对b中峰值荧光响应的变化进行量化,结果表明只有K+探针能够灵敏检测到电刺激脑切片中的[K+]o变化;

d,应用K+探针的脑切片荧光图像;

e,通过K+选择性微电极(蓝线)和K+探针光学成像(红线)记录了电刺激时脑切片中的[K+]o变化。

5自由移动小鼠大脑中的动态[K+]o波动监测

a,监测电点燃诱发的癫痫小鼠模型中[K+]o动态变化的体内实验设计图,其中反复电刺激会增加癫痫发作的严重程度;

b,c,同时对不同癫痫发作阶段(b,癫痫发作阶段3;c,癫痫发作阶段5)的小鼠进行神经活动记录和荧光成像。顶部,癫痫发作时EEG数据及其放大图。中间,EEG数据对应的能谱。底部,K+探针对电点燃诱发的癫痫发作的荧光响应;

d,自由移动的正常小鼠注射了K+探针后,没有荧光反应;

e,癫痫小鼠(癫痫发作阶段5)注射了aCSF后,没有荧光反应。顶部,EEG数据。中间,EEG数据对应的能谱。底部,K+探针的荧光响应;

f,g,K+探针荧光信号的振幅(f)和持续时间(g)的变化与刺激次数之间的关系;

h,i,K+探针荧光信号的振幅(h)和持续时间(i)的癫痫发作阶段依赖性变化。

6自由移动小鼠的脑部多位点[K+]o监测

a, 在电点燃诱发的癫痫小鼠的三个不同脑区(海马体,杏仁核和皮质)同时进行EEG记录和光学[K+]o动态成像的实验设计图;

b,c,海马体电点燃刺激导致不同程度的癫痫性发作时,(b,癫痫发作期3; c,癫痫发作期5),EEG结果和荧光成像结果均显示杏仁核和皮层有反应。左图是癫痫发作期间的海马体、杏仁核和皮层中记录的EEG数据及其放大图。中间,EEG数据对应的能谱。右图,在癫痫发作的不同阶段,三个不同脑区的荧光响应;

d,e,在小鼠大脑的三个不同区域,K+探针荧光信号的振幅(d)和持续时间(e)与癫痫发作阶段的关系。

【小结】

研究表明,K+探针能够监测自由移动小鼠大脑中[K+]o变化的动力学过程。与只能用于固定样品中[K+]单点测量的侵入性K+选择微电极相比,此纳米探针是非侵入性的,可以在较大范围内传递[K+]变化的空间信息。此外,还可以进一步开发基于近红外发射的K+探针,用于在全脑成像中精确检测癫痫灶,从而促进癫痫的诊断和治疗,减少不必要的脑部手术。本研究为离子特异性探针的设计和制备提供了一种表界面配体自组装的新思路,为在自由活动的生命体上探究神经元放电活动开辟了实时动态监测离子浓度变化的新方法,进而开创了一种活体脑部多位点无创成像的新策略,为神经退行性疾病机制、神经冲动与行为关联等脑部奥秘的探索带来了曙光。

文献链接:A sensitive and specific nanosensor for monitoring extracellular potassium levels in the brain(Nature Nanotechnology, DOI: 10.1038/s41565-020-0634-4)

本文由水手供稿。

【团队介绍】

凌代舜,2013年于首尔大学获得博士学位,之后在IBS担任资深研究员,于2015年12月在浙江大学成立独立课题组,担任PI。经过五年多的团队发展,已搭建了完善的分子探针和纳米药物研究平台。团队充分利用药学和化学、材料学、医学以及生物医学工程等多学科交叉的优势,致力于通过材料表界面配体改性和诱导自组装来可控合成高灵敏/特异性的分子探针和纳米药物,进一步结合靶向递送、分子影像可视化和疾病微环境响应性诊疗功能放大等先进技术,在神经退行性疾病和肿瘤等重大疾病的早期诊断和高效治疗方面取得了系列化显著成果。相关成果在Nature Nanotechnol.Nature Mater.Nature Biomed. Eng.J. Am. Chem. Soc.Adv. Mater.Angew. Chem.Adv. Funct. Mater.ACS NanoACS Cent. Sci.Nano TodayNano Lett.Mater. Horiz.等国际学术刊物发表论文80余篇。

团队在该领域部分代表性工作汇总

  • Jianan Liu, Fangyuan Li, Yi Wang, Limin Pan, Peihua Lin, Bo Zhang, Yanrong Zheng, Yingwei Xu, Hongwei Liao, Giho Ko, Fan Fei, Cenglin Xu, Yang Du, Kwangsoo Shin, Dokyoon Kim, Sung-Soo Jang, Hee Jung Chung, He Tian, Qi Wang, Wei Guo, Jwa-Min Nam, Zhong Chen*, Taeghwan Hyeon*,Daishun Ling*, A Sensitive and Specific Nanosensor for Monitoring Extracellular Potassium Levels in the Brain,Nature Nanotechnology,2020, DOI: 10.1038/s41565-020-0634-4
  • Qiyue Wang, Xibo Ma, Hongwei Liao, Zeyu Liang, Fangyuan Li*, Jie Tian*,Daishun Ling*, Artificially Engineered Cubic Iron Oxide Nanoparticle as a High-Performance Magnetic Particle Imaging Tracer for Stem Cell Tracking,ACS Nano,2020, DOI: 10.1021/acsnano.9b08660
  • Wei Shao, Qiaolin Wei, Shuaifei Wang, Fangyuan Li*, Jiahe Wu, Jiafeng Ren, Fangyi Cao, Hongwei Liao, Jian-Qing Gao, Min Zhou*,Daishun Ling*, Molecular engineering of D-A-D conjugated small molecule nanoparticles for high performance NIR-II photothermal therapy,Materials Horizons,2020, DOI: 10.1039/C9MH00660E
  • Xi Hu, Fangyuan Li, Fan Xia, Xia Guo, Nan Wang, Lili Liang, Bo Yang, Kelong Fan, Xiyun Yan,Daishun Ling*, Biodegradation-Mediated Enzymatic Activity-Tunable Molybdenum Oxide Nanourchins for Tumor-Specific Cascade Catalytic Therapy,Journal of the American Chemical Society,2019, 142, 1636
  • Jiahe Wu, Fangyuan Li, Xi Hu, Jingxiong Lu, Xiaolian Sun, Jianqing Gao*,Daishun Ling*, Responsive Assembly of Silver Nanoclusters with a Biofilm Locally Amplified Bactericidal Effect to Enhance Treatments against Multi-Drug-Resistant Bacterial Infections,ACS Central Science,2019, 5, 1366
  • Qinjie Weng, Xi Hu, Jiahuan Zheng, Fan Xia, Nan Wang, Hongwei Liao, Ying Liu, Dokyoon Kim, Jianan Liu, Fangyuan Li, Qiaojun He, Bo Yang, Chunying Chen, Taeghwan Hyeon,Daishun Ling*, Toxicological Risk Assessments of Iron Oxide Nanocluster- and Gadolinium-Based T1 MRI Contrast Agents in Renal Failure Rats,ACS Nano,2019, 13, 6801
  • Fangyuan Li, Zeyu Liang, Jianan Liu, Jihong Sun, Xi Hu, Meng Zhao, Jiaxin Liu, Ruiliang Bai, Dokyoon Kim, Xiaolian Sun, Taeghwan Hyeon,Daishun Ling*, Dynamically Reversible Iron Oxide Nanoparticle Assemblies for Targeted Amplification of T1-Weighted Magnetic Resonance Imaging of Tumors,Nano Letters,2019, 19, 4213
  • Haibin Wu, Fangyuan Li, Wei Shao, Jianqing Gao*,Daishun Ling*, Promoting Angiogenesis in Oxidative Diabetic Wound Microenvironment Using a Nanozyme-Reinforced Self-Protecting Hydrogel,ACS Central Science,2019, 5, 477
  • Tianyuan Zhang, Fangyuan Li, Qianhao Xu, Qiyue Wang, Xinchi Jiang, Zeyu Liang, Hongwei Liao, Xianglei Kong, Jianan Liu, Honghui Wu, Danping Zhang, Changhua An, Liang Dong, Yang Lu, Hongcui Cao, Dokyoon Kim, Jihong Sun, Taeghwan Hyeon, Jianqing Gao*,Daishun Ling*, Ferrimagnetic Nanochains-Based Mesenchymal Stem Cell Engineering for Highly Efficient Post-Stroke Recovery,Advanced Functional Materials,2019, 29, 1900603
  • Hongwei Liao, Zeyu Liang, Nan Wang, Min Wei, Ying Chen, Fangyuan Li*,Daishun Ling*, Dynamic Supraparticles for the Treatment of Age-Related Diseases,Science Bulletin,2019, 64, 1850
  • Shuaifei Wang, Hongwei Liao, Fangyuan Li*,Daishun Ling*, A Mini-review and Perspective on Ferroptosis-inducing Strategies in Cancer Therapy,Chinese Chemical Letters,2019, 30, 847
  • Yonghong Song, Zhujun Sheng, Yunjun Xu, Liang Dong, Weiping Xu, Fangyuan Li, Jing Wang, Zeyu Wu, Yi Yang, Yang Su, Xiaolian Sun,Daishun Ling*, Yang Lu*, Magnetic liposomal emodin composite with enhanced killing efficiency against breast cancer,Biomaterials Science,2019, 7, 867
  • Qiyue Wang, Shuying Wang, Xi Hu, Fangyuan Li,Daishun Ling*, Controlled Synthesis and Assembly of Ultra-small Nanoclusters for Biomedical Applications,Biomaterials Science,2019, 7, 480
  • Baoping Tian, Fangyuan Li, Ruiqing Li, Xi Hu, Tianwen Lai, Jingxiong Lu, Yun Zhao, Yang Du, Zeyu Liang, Chen Zhu, Wei Shao, Wen Li, Zhi-hua Chen, Xiaolian Sun, Xiaoyuan Chen, Songmin Ying*,Daishun Ling*, Huahao Shen*, Nanoformulated ABT-199 to Effectively Target Bcl-2 at Mitochondrial Membrane Alleviates Airway Inflammation by Inducing Apoptosis,Biomaterials,2019, 192, 429
  • Fangyuan Li, Yang Du, Jianan Liu, Heng Sun, Jin Wang, Ruiqing Li, Dokyoon Kim, Taeghwan Hyeon,Daishun Ling*, Responsive Assembly of Upconversion Nanoparticles for pH-Activated and Near-Infrared-Triggered Photodynamic Therapy of Deep Tumors,Advanced Materials,2018, 30, 1802808
  • Jingxiong Lu, Jihong Sun, Fangyuan Li, Jin Wang, Jianan Liu, Dokyoon Kim, Chunhai Fan, Taeghwan Hyeon,Daishun Ling*, Highly Sensitive Diagnosis of Small Hepatocellular Carcinoma Using pH-Responsive Iron Oxide Nanocluster Assemblies,Journal of the American Chemical Society,2018, 140, 10071
  • Shuaifei Wang, Fangyuan Li, Ruirui Qiao, Xi Hu, Hongwei Liao, Lumin Chen, Jiahe Wu, Haibin Wu, Meng Zhao, Jianan Liu, Rui Chen, Xibo Ma, Dokyoon Kim, Jihong Sun, Thomas P. Davis, Chunying Chen, Jie Tian, Taeghwan Hyeon,Daishun Ling*, Arginine-Rich Manganese Silicate Nanobubbles as a Ferroptosis-Inducing Agent for Tumor-Targeted Theranostics,ACS Nano,2018, 12, 12380
  • Qing Chen, Yang Du, Kai Zhang, Zeyu Liang, Jinquan Li, Hao Yu, Rong Ren, Jin Feng, Zhiming Jin, Fangyuan Li, Jihong Sun, Min Zhou, Qinggang He, Xiaolian Sun, Hong Zhang, Mei Tian*,Daishun Ling*, Tau-Targeted Multifunctional Nanocomposite for Combinational Therapy of Alzheimer's Disease,ACS Nano,2018, 12, 1321
  • Xi Hu, Jihong Sun, Fangyuan Li, Ruiqing Li, Jiahe Wu, Jie He, Nan Wang, Jianan Liu, Shuaifei Wang, Fei Zhou, Xiaolian Sun, Dokyoon Kim, Taeghwan Hyeon,Daishun Ling*, Renal-Clearable Hollow Bismuth Subcarbonate Nanotubes for Tumor Targeted Computed Tomography Imaging and Chemoradiotherapy,Nano Letters,2018, 18, 1196
  • Xi Hu, Fangyuan Li,* Shuying Wang, Fan Xia,Daishun Ling*, Biological Stimulus-Driven Assembly/Disassembly of Functional Nanoparticles for Targeted Delivery, Controlled Activation, and Bioelimination,Advanced Healthcare Materials,2018, 7, 1800359
  • Jingxiong Lu, Jin Wang,Daishun Ling*, Surface Engineering of Nanoparticles for Targeted Delivery to Hepatocellular Carcinoma,Small,2018, 14, 1702037
  • Haibin Wu, Fangyuan Li, Shuaifei Wang, Jingxiong Lu, Jinquan Li, Yang Du, Xiaolian Sun, Xiaoyuan Chen, Jianqing Gao*,Daishun Ling*, Ceria Nanocrystals Decorated Mesoporous Silica Nanoparticle based ROS-Scavenging Tissue Adhesive for Highly Efficient Regenerative Wound Healing,Biomaterials,2018, 151, 66
  • Shuying Wang, Fangyuan Li, Xi Hu, Min Lv, Chunhai Fan*,Daishun Ling*, Tuning the Intrinsic Nanotoxicity in Advanced Therapeutics,Advanced Therapeutics,2018, 1, 1800059
  • Meng Wang, Jiahe Wu, Ying Li, Fangyuan Li, Xi Hu, Gang Wang, Min Han,Daishun Ling*, Jianqing Gao*, A Tumor Targeted Near-Infrared Light-Controlled Nanocomposite to Combat with Multidrug Resistance of Cancer,Journal of Controlled Release,2018, 288, 34
  • Pengfei Cui, Wanru Zhuang, Xi Hu, Lei Xing, Ruyi Yu, Jianbin Qiao, Yujing He, Fangyuan Li,Daishun Ling*, Hulin Jiang*, A New Strategy for Hydrophobic Drugs Delivery Using Hydrophilic Polymer Equipped with Stacking Units,Chemical Communications,2018, 54, 8218
  • Xia Guo, Xiaotong Li, Shufang Kou, Xianfeng Yang, Xi Hu,Daishun Ling*, Jian Yang*, Plasmon-enhanced Electrocatalytic Hydrogen/oxygen Evolution by Pt/Fe–Au Nanorods,Journal of Materials Chemistry A,2018, 6, 7364
  • Taegyu Kang, Fangyuan Li, Seungmin Baik, Wei Shao,Daishun Ling*, Taeghwan Hyeon*, Surface design of magnetic nanoparticles for stimuli-responsive cancer imaging and therapy,Biomaterials,2017, 136, 98
  • Xi Hu, Fangyuan Li, Nabila Noor,Daishun Ling*, Platinum drugs: from Pt(II) compounds, Pt(IV) prodrugs, to Pt nanocrystals/nanoclusters,Science Bulletin,2017, 62, 589
  • Fangyuan Li, Jingxiong Lu, Xueqian Kong, Taeghwan Hyeon,Daishun Ling*, Dynamic Nanoparticle Assemblies for Biomedical Applications,Advanced Materials,2017, 29, 1605897
  • Hongping Xia, Fangyuan Li, Xi Hu, Wooram Park, Shuaifei Wang, Youngjin Jang, Yang Du, Seungmin Baik, Soojeong Cho, Taegyu Kang, DongHyun Kim,Daishun Ling*, Kam Man Hui*, Taeghwan Hyeon*, pH-Sensitive Pt Nanocluster Assembly Overcomes Cisplatin Resistance and Heterogeneous Stemness of Hepatocellular Carcinoma,ACS Central Science,2016, 2, 802
分享到