INL - International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330 Braga, Portugal.
Escola de Enxeñaría de Minas e Enerxía, University of Vigo, 36310 Vigo, Pontevedra, Spain.
ACS Appl Mater Interfaces. 2024 Nov 6;16(44):60828-60841. doi: 10.1021/acsami.4c12462. Epub 2024 Oct 24.
Intracellular sensing technologies necessitate a delicate balance of spatial resolution, sensitivity, biocompatibility, and stability. While existing methods partially fulfill these criteria, none offer a comprehensive solution. Nanodiamonds (NDs) harboring nitrogen-vacancy (NV) centers have emerged as promising candidates due to their sensing capabilities under biological conditions and their ability to meet all aforementioned requirements. This study focuses on expanding the application of NDs and NV center-based sensing to neuronal contexts by investigating their functionalization and subsequent effects on three distinct cell lines relevant to neurodegenerative disease research. Our study concentrates on positioning fluorescent NDs (FNDs) with NV center point defects onto neuronal cell surfaces. Achieving this through specific antibody attachment enhances the proximity of FND to neurites, facilitating the detection of local action potentials. Targeting voltage-dependent calcium channels (Cav2.2) with biotin-streptavidin-bound antibodies enables the precise positioning of FNDs. The functionalized FNDs (f-FNDs) show increased size and zeta potential, confirming the antibody presence without compromising cell viability. Two-color confocal imaging and co-localization algorithms are employed to further attest to the success of the functionalization. The f-FNDs are applied to cell cultures of three cell lines: SH-SY5Y, differentiated dopaminergic neurons, and hippocampal rat neurons; their biocompatibility and effects on synaptic activity are explored. Moreover, preliminary total internal reflection fluorescence - optically detected magnetic resonance (TIRF-ODMR) experiments across cellular sites demonstrate the magnetic field sensitivity of our sensor network. The successful establishment of this sensor network provides a platform for characterizing neuronal signaling in healthy models and conditions mimicking Parkinson's disease.
细胞内传感技术需要在空间分辨率、灵敏度、生物相容性和稳定性之间取得微妙的平衡。虽然现有的方法在一定程度上满足了这些标准，但没有一种方法提供了全面的解决方案。氮空位（NV）中心的纳米金刚石（NDs）由于其在生物条件下的传感能力以及满足上述所有要求的能力，已成为有前途的候选者。本研究通过研究其在与神经退行性疾病研究相关的三种不同细胞系中的功能化及其后续效应，将 NDs 和基于 NV 中心的传感的应用扩展到神经元环境中。我们的研究集中在通过特定抗体附着将具有 NV 中心点缺陷的荧光 ND（FND）定位到神经元细胞表面上。通过这种方式，可以增强 FND 与神经突的接近度，从而促进局部动作电位的检测。通过生物素-链霉亲和素结合的抗体靶向电压依赖性钙通道（Cav2.2），可以精确地定位 FND。功能化的 FND（f-FND）显示出增加的大小和 zeta 电位，证实了抗体的存在而不会损害细胞活力。使用双色共焦成像和共定位算法进一步证明了功能化的成功。将 f-FND 应用于三种细胞系的细胞培养物：SH-SY5Y、分化的多巴胺能神经元和海马大鼠神经元；探索它们的生物相容性和对突触活动的影响。此外，跨细胞位点的初步全内反射荧光 - 光学检测磁共振（TIRF-ODMR）实验证明了我们传感器网络的磁场敏感性。该传感器网络的成功建立为在健康模型和模拟帕金森病的条件下表征神经元信号提供了一个平台。
