A high throughput platform for rapid single cell surface mapping

细胞表面电荷，即细胞表面的净电荷，是一种有前景的新型生物标志物，可用于罕见细胞的检测、细胞分选和恶性细胞的病理分期监测。细胞表面电荷模式也反映了细胞膜的原位动态状态，对细胞内吞作用、肌肉细胞收缩、营养物质运输、T细胞活化和胰岛素释放等膜调控细胞功能至关重要。最近的证据表明，多种细胞功能，包括细胞聚集、细胞分裂和细胞迁移可以通过操纵细胞表面电荷模式来调节。因此，快速绘制单细胞表面电荷模式将为推进细胞分析及其功能调控提供一种独特而重要的方法。然而，到目前为止，由于缺乏能够在微纳米尺度上进行测量的可靠技术，活细胞表面电荷的快速测绘仍然是一个巨大的挑战。扫描离子电导显微镜已被用于定量绘制细胞表面电荷图。该方法采用单个纳米移液器逐点接近细胞表面，测量效率低，劳动强度大，破坏性大。提出的研究旨在通过探索一种快速，非破坏性表征和绘制细胞表面电荷的通用方法来解决这一长期存在的挑战。此外，通过教育和推广活动，拟议的研究将增强本科生，研究生和K-12学生的跨学科学习经验。这个项目的目标是探索一个芯片上的实验室平台，用于快速，非破坏性的活细胞表面电荷映射。这种装置将1)促进发现用于细胞鉴定、分选和实时监测的新生物标志物的突破，2)极大地推进对细胞电学性质在细胞功能中的作用的认识。为实现这一目标，将执行下列任务：1)声学细胞操纵器的演示，它将在连续流动中快速聚焦/释放单个活细胞到/离开探针表面；2)纳米孔阵列的研究，它可以以高时间分辨率和精度精确地绘制细胞表面电荷图；3)信号复用的实现，它将使通过纳米孔阵列同时测量表面电荷分布成为可能。4)利用人真皮成纤维细胞快速绘制细胞表面电荷图谱的平台验证。使用非侵入式细胞操作可以快速聚焦/释放细胞到/离开纳米孔阵列的表面，允许在连续流动中快速，高通量的表面电荷映射。纳米孔阵列与信号复用相结合的使用使得无需复杂的测量电子设备即可一次快速绘制细胞表面电荷特性。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。

Cell surface charge, net electricity on cell surface, is a promising new biomarker for rare cell detection, cell sorting, and pathology stage monitoring of malignant cell. Cell surface charge pattern also reflects the in situ dynamic status of cell membrane, which is critical for membrane-regulated cell functions such as endocytosis, muscle cell contraction, nutrient transport, T cell activation and insulin release. Recent evidences show that a variety of cell functions including cell aggregation, cell division and cell migration can be modulated by manipulating cell surface charge patterns. Therefore rapid mapping of single cell surface charge patterns will provide a unique and important approach in advancing analysis of cells and regulation of their functions. However, to date rapid mapping of living cell surface charge remains a large challenge owing to lack of robust techniques capable of measurements at the micro and nano scale. Scanning ion conductance microscopy has been used for quantitative cell surface charge mapping. This method employs a single nano pipette to approach the cell surface point by point, resulting in a low efficiency, labor intensive and destructive measurement. The proposed research aims to address this long standing challenge by exploring a universal method for rapid, non-destructive characterization and mapping of cell surface charge. In addition, with educational and outreach activities, the proposed research is poised to enhance the interdisciplinary learning experience for undergraduate, graduate and K-12 students. The objective of this project is to explore a lab-on-a- chip platform for rapid, non-destructive living cell surface charge mapping. Such a device will 1) foster the breakthrough of discovering new biomarkers for cell identification, sorting and real time monitoring, and 2) greatly advance the knowledge in understanding the roles cell electrical properties play on cell functions. To achieve the objective, the following tasks will be pursued: 1) demonstration of acoustic cell manipulator that will rapidly focus/release individual living cell onto/off the probe surface in a continuous flow, 2) study of an array of nanopores that can accurately map the cell surface charge with high temporal resolution and accuracy, 3) implementation of signal multiplexing that will enable simultaneous measurement of surface charge distributions via a nanopore array, and 4) validation of the platform for rapid cell surface charge mapping using human dermal fibroblasts cells. The use of non-intrusive cell manipulation enables quick focus/release cells onto/off the nanopore array's surface, allowing rapid, high-throughput surface charge mapping in continuous flow. The use of nanopore array in combination with signal multiplexing enables rapid mapping of the cell surface charge characteristics at one time without complex measurement electronics.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.