Electronic Microflow Cytometry for Surface Expression Analysis of Tumor Cells

目的：细胞异质性是癌症的标志，单个癌细胞在其肿瘤启动潜力和治疗抗性方面存在差异。在系统生物学和医学中，肿瘤细胞的异质性常常被群体水平的测量所掩盖，但它在疾病进展中具有重要意义。该提案是关于生物分析技术的，这些技术能够分析单个癌细胞，并为早期检测和监测癌症提供令人兴奋的机会，同时它仍然是可管理的，并获得生物学洞察力以开发有效的疗法。非技术摘要：虽然能够详细分析单细胞，但由于其高成本，操作复杂性和笨重的仪器，目前的技术仅限于大型设备齐全的实验室。该提案将通过开发一种根本不同的单细胞分析平台来解决这些缺点，该平台快速，低成本，便携，因此比传统技术更适合现场使用。为了实现这一目标，我们将开发一个芯片实验室平台，通过利用包括微流体，电子和电信在内的多个技术学科的工程方法，将微流体细胞分选与电子传感相结合。为了进一步提高该计划的更广泛的影响，我们将参与外展活动，以（1）通过与当地和小学合作，吸引K-12和高中学生接受STEM教育，（2）让本科生参与研究活动，（3）通过利用格鲁吉亚理工学院的多元化学生群体，吸引工程专业代表性不足的学生，（4）通过对教师教育计划的贡献促进教学和培训，（5）通过媒体发布和实验室网站向社会传播拟议研究计划的成果。技术摘要：流式细胞术是一种用于单细胞综合生物物理和生物化学分析的宝贵技术。在流式细胞术中，流体流中的单细胞被逐个询问，随后基于测量的特性进行分选。该提案的目的是将固有的并行微流体细胞分选与并行电子传感器阵列集成，以创建根本不同的流式细胞仪。作为该项目的一部分，将承担的研究任务是（1）开发一种可扩展的电子传感器，该传感器利用电阻脉冲感测从单个电输出同时检测多个微流体通道中的颗粒，（2）设计一种基于磁泳的微流体细胞分选仪，并将其与开发的电子传感器阵列集成以量化分选的细胞，（3）通过磁性预标记和针对已建立的实验室技术的基准来分析癌细胞的表面表达。在这个建议中，我们联合收割机两个传统上不同的技术学科，微流控和电信设计一个可扩展的电子读出机制接口的微流控芯片。我们的技术依赖于多路复用微机械库尔特计数器阵列，每个计数器设计用于在检测到颗粒时产生不同的数字代码。这些数字代码是使用码分多址（CDMA）电信网络的相同原理开发的，并且可以通过简单的数学计算唯一地恢复。这种方法将用于增加传感器的数量，而不增加硬件的复杂性。相反，硬件的复杂性将转向计算算法来解码信号。然后，我们将把这种传感器与肿瘤细胞的磁泳分选相结合，以量化一种具有临床实用性的蛋白质的表达，用于管理和指导癌症治疗。

Electronic Microflow Cytometry for Surface Expression Analysis of Cancer CellsProposal Objectives:Cellular heterogeneity is the hallmark of cancer with individual cancer cells differing in their tumor initiating potential and therapeutic resistance. Tumor cell heterogeneity is often obscured by population level measurements in systems biology and medicine; yet it has significant implications in disease progression. This proposal is about bio-analytical technologies that are capable of analyzing individual cancer cells and offer exciting opportunities for early-detection and monitoring of cancer while it is still manageable, and for gaining biological insight to develop effective therapies. Non-Technical Abstract:While capable of analyzing single cells in detail, current technologies are exclusively limited to large well-equipped laboratories due to their high cost, operational complexity and bulky instrumentation. This proposal will address these shortcomings by developing a fundamentally different single cell analysis platform that is fast, low-cost, portable and hence more amenable to point-of-care use than conventional technologies. To achieve this, we will develop a lab-on-a-chip platform that integrates microfluidic cell sorting with electronic sensing by utilizing engineering approaches from multiple technical disciplines including microfluidics, electronics and telecommunications. To further enhance the broader impact of this program, we will be involved with outreach activities to (1) attract K-12 and high school students to STEM education by engaging with local and elementary schools, (2) engage undergraduate students in research activities, (3) attract underrepresented students in engineering by utilizing Georgia Tech's diverse student body, (4) promote teaching and training by contributing to teacher education programs, (5) disseminate the results of the proposed research program to the society through media releases and lab website.Technical Abstract:Flow cytometry is an invaluable technique for comprehensive biophysical and biochemical analysis of single cells. In flow cytometry, single cells in a fluid stream are interrogated one by one and subsequently sorted based on the measured properties. The objective of this proposal is to integrate inherently parallel microfluidic cell sorting with a parallel electronic sensor array to create a fundamentally different flow cytometer. The research tasks to be undertaken as part of this project are (1) to develop a scalable electronic sensor that utilizes resistive pulse sensing to simultaneously detect particles in multiple microfluidic channels from a single electrical output, (2) to design a microfluidic cell sorter based on magnetophoresis and integrate it with the developed electronic sensor array to quantify sorted cells, (3) to analyze surface expression of cancer cells by magnetic pre-labeling and benchmark against established laboratory techniques. In this proposal, we combine two traditionally distinct technical disciplines, microfluidics and telecommunications to design a scalable electronic readout mechanism for interfacing microfluidic chips. Our technology relies on multiplexing an array of micromachined Coulter counters each designed to produce a distinct digital code when a particle is detected. These digital codes are developed using the same principles of Code Division Multiple Access (CDMA) telecommunication networks and can be uniquely recovered through simple mathematical calculations. This approach will be used to increase number of sensors without increasing the hardware complexity. Instead, complexity in the hardware will be shifted towards computational algorithms to decode the signal. We will then integrate this sensor with magnetophoretic sorting of tumor cells to quantify the expression of a protein with clinical utility for managing and guiding cancer therapy.