Real-Time Sensing of Neurotransmitters From Stem Cell-derived Neural Interface Using Hybrid Graphene-Nanostructures

神经递质是大脑中最重要的生物分子之一。它们在维持认知、记忆和行为等神经生理过程中起着至关重要的作用。例如，神经递质水平异常可能导致严重的神经系统疾病，如帕金森病、精神分裂症和亨廷顿氏病。在帕金森氏症的病例中，大多数患者已经失去了80%以上的多巴胺神经递质。以干细胞为基础的治疗创造多巴胺产生（多巴胺能）神经元，并将其植入患者体内，已成为治疗帕金森病的一种有希望的方法。干细胞来源的多巴胺能神经元的功能可以通过检测多巴胺来证实。该生物传感系统具有通过检测神经递质实时监测干细胞分化的能力。它将通过消除当前干细胞治疗中的一些瓶颈，加速干细胞治疗的发展。此外，所提出的生物传感器将成为推动再生医学和神经科学研究和发展的优秀传感平台。本提案的科学目标是研究干细胞如何通过选择性和有效地检测神经递质来产生与功能神经元的神经界面。传统的神经递质检测方法存在非特异性感知和缺乏原位分析的缺陷。为了解决当前神经递质检测方面的挑战，一种新型的原位纳米生物传感器将以氧化石墨烯覆盖的纳米结构阵列为平台，通过无创方式检测分泌的神经递质，实时监测干细胞成熟神经元的分化。为此，该提案侧重于合成化学上定义良好的氧化石墨烯纳米结构，并开发基于石墨烯的混合纳米电极阵列，以产生稳定且可重复的信号。基于石墨烯的杂化纳米电极阵列利用表面增强拉曼散射技术对多巴胺分子进行了选择性和灵敏度检测。拉曼染料（氧化石墨烯）标记的适体靶向多巴胺分子通过pi-pi相互作用附着在氧化石墨烯覆盖的纳米结构阵列表面，导致氧化石墨烯纳米结构的拉曼增强。在染料标记的适配体与多巴胺反应后，核苷酸与拉曼染料（孔雀石绿分子）标记的适配体之间的结合亲和力丧失，从而降低了定量分析的拉曼信号。整个过程能够检测到神经界面低浓度的多巴胺。一旦成功完成，所提出的纳米生物传感系统将通过监测复杂生物基质的细胞分泌物来促进生物现象的研究。该方案利用石墨烯功能化表面和高选择性生物识别元件，制造了一种新型的基于石墨烯-纳米混合结构的实时传感系统，作为多种分子检测的多用途传感器平台。考虑到单细胞水平神经递质原位检测的挑战，这种基于表面增强拉曼散射的检测方法可以代表一种独特的工具，用于研究与多巴胺或其他神经递质相关的单细胞机制，以及它们在神经过程中的作用。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Neurotransmitters are one of the most critical biomolecules in the brain. They play a vital role in preserving neurophysiological processes such as cognition, memory, and behavior. For example, the abnormal levels of neurotransmitters may result in severe neurological disorders such as Parkinson's disease, schizophrenia, and Huntington's disease. In the case of Parkinson's disease, most patients have lost more than 80% of their production of dopamine neurotransmitter. Stem cell-based therapy to create dopamine-producing (dopaminergic) neurons that can be implanted into patients has emerged as a promising approach for treating Parkinson's disease. The functionality of stem cell-derived dopaminergic neurons can be confirmed by the detection of dopamine. The proposed biosensing system has the capability of real-time monitoring of stem cell differentiation by detecting neurotransmitters. It will accelerate the development of stem cell therapies by removing some bottlenecks in current stem cell therapies. Moreover, the proposed biosensor will be an excellent sensing platform to advance research and development in regenerative medicine and neuroscience.The scientific goal of this proposal is to investigate how stem cells can generate a neural interface with functional neurons by detecting neurotransmitters selectively and effectively. Conventional neurotransmitter detection methods suffer from non-specific sensing and lack of in situ analysis. Addressing current challenges in the detection of neurotransmitters, a novel in situ nano-biosensor using graphene oxide-covered nanostructure arrays as a platform to real-time monitor mature neuronal differentiation of stem cells will be developed by detecting the secreted neurotransmitters in a noninvasive manner. For this purpose, the proposal focuses on the synthesis of chemically well-defined graphene oxide-nanostructures and the development of graphene-based hybrid nanoelectrode arrays to generate stable and reproducible signals. The developed novel graphene-based hybrid nanoelectrode arrays detect dopamine molecules selectively and sensitively using surface-enhanced Raman scattering technique. Raman dye (graphene oxide)-labeled aptamers targeting dopamine molecules are attached to the surface of graphene oxide-covered nanostructure arrays through the pi-pi interactions, resulting in a Raman enhancement from the graphene oxide nanostructures. The loss of binding affinity between nucleotides and the Raman dye (malachite green molecules)-labeled aptamers after the dye-labeled aptamers reacted with dopamine will consequently decrease the Raman signal for quantitative analysis. The whole processes enable the detection of low concentration of dopamine in the neural interface. Upon successful completion, the proposed nano-biosensing system will facilitate the study of a biological phenomenon by monitoring cell secretions from complex biological matrices. The proposal takes advantage of the graphene functionalized surface and the highly selective bio-recognition elements to fabricate a novel hybrid graphene-nanostructure-based real-time sensing system to act as a multi-purpose sensor platform for the detection of a variety of molecules. Given the challenges of in situ detection of neurotransmitters at the single cell level, this surface-enhanced Raman scattering-based detection method can represent a unique tool for investigating single-cell mechanisms associated with dopamine, or other neurotransmitters, and their roles in neurological processes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.