Sensors: A Novel Protein Immobilization Technique for Protein Array Sensors with High Stability, Multiple Functionalities, and Excellent Sensitivity

摘要-0528605华盛顿大学随着传感器技术的快速发展，以处理复杂介质中的多种分析物，芯片级传感器系统变得越来越小，迫切需要开发一种适合于长期芯片存储而不失去其生物活性的相应蛋白质固定化技术，并在一个流动通道中方便地产生多个功能点。目前，蛋白质阵列通常使用点样方法生成。限制蛋白质阵列广泛应用的最大缺点可能是蛋白质芯片一旦“点样”就稳定性（寿命）差。最近的研究表明，虽然一些抗体比其他抗体更适合（或稳定）在抗体阵列上，但许多蛋白质阵列将在两周后开始逐渐失去其生物活性。此外，目前可用的用于蛋白质固定的表面化学方法，如物理吸附、共价固定、蛋白A或G以及生物素/链霉亲和素，都没有选择性，只能在一个流动通道内提供一个功能点。最近，在PI小组的概念验证实验中证明，DNA-直接蛋白质固定技术非常适合于实现芯片稳定性和功能性的目标。通过ssDNA-抗体缀合物实现DNA定向的抗体固定，每个缀合物由化学连接到ssDNA的抗体组成，并且被设计为使得ssDNA具有与连接到表面的ssDNA序列之一互补的序列。在检测之前，将不同ssDNA-抗体缀合物的混合物施加到用ssDNA探针分子预功能化的芯片上。每种抗体偶联物将通过DNA杂交自固定到指定斑点。这样，芯片将作为DNA芯片存储，并用作蛋白质芯片。这不仅解决了蛋白质芯片的长期存储问题，而且还提供了一种简单方便的方法来在一个流动通道中创建多个功能点。此外，已经表明这种新平台比常用的生物素/链霉亲和素平台灵敏50倍。这项工作将集中在工程方面，以实现各种芯片实验室生物传感器的这项技术。它由五个任务组成-（a）开发便于快速生成DNA阵列和合成ssDNA-抗体缀合物的新表面平台，（B）设计具有高特异性的多个ssDNA序列，（c）使用最先进的八通道SPR传感器测试多个ssDNA-抗体缀合物的特异性和交叉活性，（d）使用改进的喷墨打印机用不同的ssDNA序列图案化芯片，以及（e）在玻璃基底上实现该技术。这项技术不仅将消除传统蛋白质阵列面临的芯片长期稳定性的关键障碍，而且还提供了一种选择性蛋白质固定化技术以在一个流动通道中产生多个功能点。这种蛋白质固定化技术与生物传感器（所有类型）的集成将使生物传感器非常强大，包括它们对多个通道的鲁棒性，长期芯片储存的稳定性，以及除了一个通用芯片用于所有应用和方便的芯片再生之外的高灵敏度和特异性。PI的团队正在与各种研究人员合作，使用传感器解决现实世界的问题。这项工作的支持将使这些人能够获得传感器技术来解决他们的问题。PI实验室的许多本科生都参与了传感器相关的工作。支持这一建议将提供更多的多学科研究和教育机会，这些学生，并将提供宝贵的信息，一个新的生物传感器和生物材料的生物接口的PI正在开发的课程。

ABSTRACT - 0528605University of WashingtonAs sensor technology is developing rapidly to handle multiple analytes in complex media and a chip-scale sensor system is becoming smaller, there is an urgent need to develop a corresponding protein immobilization technique suitable for long-term chip storage without losing its bioactivity and for the convenient generation of multiple functional spots in one flow channel. Currently, protein arrays are often generated using spotting methods. The biggest drawback limiting widespread application of protein arrays is perhaps the poor stability (lifetime) of protein chips once "spotting". Recent studies show that while some antibodies are more suited (or stable) on antibody arrays than others, many protein arrays will start to lose their bioactivity gradually after two weeks. Furthermore, surface chemistries for protein immobilization currently available, such as physical adsorption, covalent immobilization, protein A or G, and biotin/streptavidin, are not selective and will provide only one functional spot within one flow channel.Intellectual Merit: Recently, it is demonstrated in proof-of the-concept experiments by the PI's group that the DNA-direct protein immobilization technique is well suited to achieving the goals for chip stability and functionality. The DNA-directed antibody immobilization is achieved via ssDNA-antibody conjugates, each of which consists of an antibody chemically linked to an ssDNA and is designed such that the ssDNA has a sequence complementary to one of the ssDNA sequences attached to the surface. Before detection, a cocktail of different ssDNA-antibody conjugates is applied to the chip pre-functionalized with ssDNA probe molecules. Each antibody conjugate will be self-immobilized to a designated spot via DNA hybridization. In this way, the chip will be stored as a DNA chip and used as a protein chip. This will not only resolve the long-term storage issue of a protein chip, but also provide a simple and convenient way to create multiple functional spots in one flow channel. Furthermore, it has been shown that this new platform is 50 times more sensitive than the commonly used biotin/streptavidin platform. This work will focus on engineering aspects to realize this technology for various lab-on-a-chip biosensors. It consists of five tasks - (a) developing a new surface platform convenient for the fast generation of DNA arrays and synthesizing ssDNA-antibody conjugates, (b) designing multiple ssDNA sequences with high specificity, (c) testing the specificity and cross-activity of multiple ssDNA-antibody conjugates using a state-of-the-art eight-channel SPR sensor, (d) patterning a chip with different ssDNA sequences using a modified inkjet printer, and (e) realizing this technology on a glass substrate.Broader Impact: This technology will not only remove the key obstacle of long-term chip stability faced in conventional protein arrays, but also provide a selective protein immobilization technique to create multiple functional spots in one flow channel. The integration of this proposed protein immobilization technique with biosensors (all types) will make biosensors very powerful, including their robustness for multiple channels, stability for long-term chip storage, and high sensitivity and specificity in addition to one universal chip for all applications and convenient chip regeneration. The PI's group is collaborating with a variety of researchers to solve real-world problems using sensors. Support of this work will allow these people to access sensor technology to solve their problems. Many of undergraduate students in the PI's laboratory have been involved in sensor-related work. Support of this proposal will provide more multiple-disciplinary research and educational opportunities to these students and will provide valuable information to a new course on biological interfaces in biosensors and biomaterials under development by the PI.