含浅层氮-空位色心金刚石垂直纳米针阵列的制备及其生物传感特性研究

Deciphering the neuronal code-the rule by which neuronal circuits store and process information-is a major scientific challenge. However, studies in this area are impeded by a lack of experimental tools that are scalable and sensitive enough to simultaneously analyze large number of neurons with single cell level. As proposed in this project, vertical aligned single crystal diamond nanoneedle arrays containing shallow Nitrogen-vacancy (NV) centers were prepared by reactive ion etching (RIE) and delta-doping methods. The excellent fluorescence and spin coherence properties of NV centers in diamond make it an attractive platform for sensitive and high-precision measurements of magnetic field, electric field, temperature in neurons. Moreover, vertical aligned diamond nanoneedle arrays with remarkable mechanical property and biocompatibility are perfect for intracellular delivery and sensing by pierce into the cells. In this proposal, the profile and crystal structure of the diamond nanoneedles will be tuned by RIE; binding form and concentration of the nitrogen defect in the delta-doping layer, the formation conditions and distribution of NV centers will be investigated systematically; finally effectively control the density and distribution of NV center in the diamond nanoneedles will be realized. The diamond nanoneedle arrays with excellent fluorescence and electric field sensing properties will be utilized to probing and analyzing the in situ response of neurons to external stimulations. The implementation of the proposed project is of great significance in the study of neuronal heterogeneity, and will promote the pathogenesis and mechanism studies of neurodegenerative diseases.

如何破解神经元密码，即神经元回路存储和处理信息的规则是科学上的重要难题。然而，这一领域的研究受限于缺乏能够在单细胞层面上同时分析大量神经细胞的不同反应，并进行精确量化测量的实验工具。在本项目的研究中，我们利用反应离子刻蚀和delta-掺杂技术制备含有浅层氮-空位（NV）色心的单晶金刚石纳米针阵列薄膜。NV色心具有优异的荧光特性和自旋相干性，可以实现对外界磁场、电场等物理量的高精度敏感测量。同时，金刚石纳米针具有优异的生物相容性和稳定性，能够直接深入到细胞内部确定位置进行原位检测。实验中我们调控金刚石纳米针的结构，系统研究delta-掺杂层中氮缺陷的结合形式和结合量，NV色心的产生条件和分布规律，实现有效控制NV色心的密度和分布。基于金刚石纳米针的荧光和电场传感性质，检测神经细胞对外界刺激的应激反应。本项目的实施对研究神经细胞异质性有重要的意义，并促进对神经退行性疾病的发病原因和机理的研究。

基于金刚石薄膜的垂直纳米结构阵列材料和缺陷-空位色心在细胞内分子检测和生理变化检测方面具有巨大的发展潜力和实用价值。在本项目的支持下，我们采用ECR-MPECVD反应离子刻蚀技术刻蚀择优取向金刚石薄膜，通过优化实验条件得到了单晶金刚石纳米针阵列，结合纳米针表面生长delta-掺杂金刚石层制备了浅层缺陷-空位色心，确立了CVD生长和掺杂，及热处理工艺对掺杂层中缺陷色心发光性能的影响规律，得到了分布和数量可控的缺陷-空位色心。本项目首创了基于金刚石纳米针阵列薄膜的细胞内活检技术，实现了对细胞内小分子核酸进行快速的、高灵敏度、高通量的检测。通过对小鼠胚胎干细胞分化成运动神经元的过程的研究，证明了该技术可用于捕捉同一批细胞内miRNA表达于时序上的动态变化，揭示了混合细胞群中细胞异质性的演变。利用结合了“鱼钩”的金刚石纳米针“鱼竿”直接从大鼠急性脑切片的大量细胞中对多种特定的miRNA标识物进行提取和检测，对诱导的精神分裂症中星形胶质细胞的失调进行了研究，证明了该技术可实现捕捉空间动力学信息的准单细胞miRNA原位分析。通过金刚石纳米针向细胞内递送传统的重编程基因，制造诱导干细胞（iPSCs）的效率比常规方法提高了两个数量级以上。本项目基本上达到了预期的研究目标，所开展的研究工作有助于推动高深宽比金刚石纳米针阵列在细胞内尺度的药物传输、生物传感、生物成像等生物医学领域更广泛的应用。