A high throughput platform for rapid single cell surface mapping

细胞表面电荷是细胞表面的净电荷，是一种很有前途的新的生物标志物，用于稀有细胞的检测、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，这对于膜调节的细胞功能如内吞作用、肌肉细胞收缩、营养转运、T细胞活化和胰岛素释放至关重要。最近的研究表明，通过调控细胞表面电荷模式，可以调控细胞的聚集、分裂和迁移等多种功能。 因此，单细胞表面电荷模式的快速绘图将提供一个独特的和重要的方法，在推进细胞的分析和调节其功能。然而，迄今为止，活细胞表面电荷的快速映射仍然是一个很大的挑战，因为缺乏强大的技术，能够在微米和纳米尺度的测量。 扫描离子电导显微镜已被用于定量细胞表面电荷映射。该方法采用单个纳米移液器逐点接近细胞表面，导致效率低、劳动密集和破坏性测量。拟议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，拟议的研究有望增强本科生，研究生和K-12学生的跨学科学习体验。该项目的目标是探索一个芯片实验室平台，用于快速，非破坏性的活细胞表面电荷映射。这种装置将1）促进发现用于细胞识别、分选和真实的时间监测的新生物标志物的突破，以及2）极大地推进理解细胞电特性对细胞功能的作用的知识。为实现这一目标，将开展以下工作：1）演示声学细胞操纵器，其将以连续流将单个活细胞快速聚焦到探针表面上/从探针表面释放，2）研究纳米孔阵列，其可以以高时间分辨率和准确度准确地映射细胞表面电荷，3）实现信号多路复用，其将使得能够经由纳米孔阵列同时测量表面电荷分布，和4）使用人皮肤成纤维细胞验证用于快速细胞表面电荷映射的平台。 非侵入性细胞操作的使用使得能够将细胞快速聚焦/释放到纳米孔阵列的表面上/从纳米孔阵列的表面释放，从而允许在连续流中快速、高通量的表面电荷映射。纳米孔阵列与信号多路复用相结合的使用能够在没有复杂的测量电子设备的情况下一次性快速绘制细胞表面电荷特性。该奖项反映了NSF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.