Engineered Self-Assembling Fvs for Piezoimmunosensors

描述(申请人提供)：压电免疫传感器(PZ)是一种生物传感器，将抗体放置在石英晶体微天平(QCM)上，以检测抗体与抗原结合时质量的微小变化。然而，由于整个抗体的复杂性质，在生物传感器中使用PZ技术是有问题的，这会导致非特异性相互作用和分子捕获的问题。为了克服这些问题，提出了一种利用自组装单层(SAM)中的重组抗体可变片段(FV)作为生物识别剂的方法。基因工程的FV将形成覆盖在QCM表面的抗原结合位点的“草坪”，提供化学稳定的感应层，包含大量的结合位点，并形成薄而均匀的表面。最初的研究将集中在开发a-人/山羊抗体FV-SAM以测试其基本原理。将使用四种生物工程方法来创建具有必要属性的FV-SAM。电化学、网络阻抗分析和原子力显微镜(AFM)将同时用于表征固定化FV-SAM层的缺陷/针孔结构、刚性、取向、稳定性和表面覆盖率。与其他免疫分析和/或传统方法(GC/LC或GC/MS、LC/MS)相比，FV-SAM压电免疫传感器将在灵敏度、选择性、线性、稳定性、检测限和可靠性方面进行评估和验证。一系列针对各种抗原/毒素的传感器可以自动化或与流动注射系统相结合，以在线显示结果来分析复杂样品中的不同分析物。该项目可能导致生产一种廉价、易于使用的生物传感器，这种传感器可以快速、准确地识别生物制剂。

DESCRIPTION (provided by applicant): Piezoimmunosensors (PZs) are biosensors in which antibodies are placed on a quartz crystal microbalance (QCM) to detect minute changes in mass as the antibodies bind with antigens. However, use of PZ technology in biosensors is problematic due to the complex nature of whole antibodies, which cause problems with non-specific interactions and molecule trapping. To overcome these problems, an approach in which recombinant antibody variable fragments (Fv) in self assembling monolayers (SAM) serve as the biorecognition agent is proposed. Engineered Fvs will form a "lawn" of antigen-binding sites coating the surface of the QCM, providing a sensing layer which is chemically stable, contains high numbers of binding sites, and forms a thin uniform surface. The initial study will focus on developing an a-human/goat IgG Fv- SAM to test the underlying principle. Four bioengineering methods will be used to create an Fv-SAM that possesses the necessary attributes. Electrochemistry, network impedance analysis, and Atomic Force Microscope (AFM) will be used in parallel to characterize the immobilized Fv-SAM layers for defects/pin hole structure, rigidity, orientation, stability, and surface coverage. The Fv-SAM piezoimmunosensor will be evaluated and validated for sensitivity, selectivity, linearity, stability, detection limits, and reliability in comparison to other immunoassays and/or traditional methods (GC/LC or GC/MS, LC/MS). An array of sensors specific for various antigens/toxins could be automated or combined with flow injection systems to assay different analytes in complex samples with on-line display of results. The project could lead to production of an inexpensive, easy-to-use biosensor which provides fast, precise identification of biological agents