SPR Imaging Studies of Nucleic Acid Microarrays for Biomarker Detection

描述（由申请人提供）：本研究的总体目标是开发增强型表面等离子体共振成像（SPRI）方法，该方法使用RNA适体微阵列对生物流体中低至飞摩尔浓度的高分子量和低分子量蛋白质生物标志物进行多路、超灵敏的检测。建议的研究包括两个主要领域：(i)开发新的RNA适体微阵列和（ii）创建新的增强的SPRI传感方法。新的RNA适体微阵列的创建既需要开发新的微阵列制造策略，也需要创建专门为增强的SPRI测量设计的新的RNA适体。阵列制造方法将采用表面酶反应策略，使用T4 RNA连接酶和T7 RNA聚合酶，结合微流体将合成的寡核苷酸和体外转录的RNA附着在金表面上。微阵列将使用先前发现的适配体，如血管内皮生长因子（VEGF）和茶碱的适配体，以及通过体外SELEX工艺鉴定的新RNA适配体。在SELEX筛选过程中，SPR和SPRI将用于筛选靶-适体相互作用。其中一些RNA序列将被整合到组合适体中，其中包括分子开关序列，其3D结构在目标结合时发生变化。新的增强的SPRI传感方法将利用核酸微阵列的各种表面酶反应，如T7 RNA聚合酶、RNase H和聚(A)聚合酶，结合吸附功能化纳米颗粒，以提高表面生物亲和力测量的特异性或灵敏度。最初的目标将是两种潜在的癌症生物标志物生长因子，VEGF和血小板衍生生长因子。最初的分子开关适体将采用已知的结合氨基葡萄糖-6-磷酸或茶碱的核糖开关序列；这些将与其他可以结合功能化金纳米颗粒的序列结合。一旦提出的增强SPRI生物传感方法实现，它们将应用于微阵列检测血清和其他生物体液中的癌症生物标志物VEGF、三种心脏生物标志物（肌钙蛋白T、NT-proBNP、b型利钠肽）和两种肾功能生物标志物（胱抑素C和β 2微球蛋白）。

DESCRIPTION (provided by applicant): The overall aim of this research is to develop enhanced surface plasmon resonance imaging (SPRI) methodologies that employ RNA aptamer microarrays for the multiplexed, ultrasensitive detection of both high and low molecular weight protein biomarkers in biological fluids down to femtomolar concentrations. The proposed research consists of two major areas: (i) the development of new RNA aptamer microarrays and (ii) the creation of new enhanced SPRI sensing methodologies. The creation of new RNA aptamer microarrays requires both the development of novel microarray fabrication strategies as well as the creation of new RNA aptamers specifically designed for the enhanced SPRI measurements. The array fabrication methods will employ surface enzyme reaction strategies using T4 RNA ligase and T7 RNA polymerase in conjunction with microfluidics to attach both synthetic oligonucleotides and in vitro transcribed RNA onto gold surfaces. The microarrays will use both previously discovered aptamers such as those for vascular endothelial growth factor (VEGF) and theophylline, as well as new RNA aptamers identified by the in vitro SELEX process. SPR and SPRI will be used in the screening of target-aptamer interactions during the SELEX selection. Some of these RNA sequences will be incorporated into combined aptamers which will include molecular switch sequences whose 3D structure changes upon target binding. The new enhanced SPRI sensing methodologies will employ various surface enzyme reactions of the nucleic acid microarrays such as T7 RNA polymerase, RNase H and poly(A) polymerase in conjunction with the adsorption of functionalized nanoparticles to either improve the specificity or the sensitivity of the surface bioaffinity measurements. The initial targets will be two growth factors that are potential cancer biomarkers, VEGF and platelet-derived growth factor. The initial molecular switch aptamers will employ known riboswitch sequences that bind Glucosamine-6-phosphate or theophyilline; these will be combined with other sequences that can bind to functionalized gold nanoparticles. Once the proposed enhanced SPRI biosensing methodologies have been realized, they will be applied to the microarray detection of the cancer biomarker VEGF, three cardiac biomarkers (troponin T, NT-proBNP, B-type natriuretic peptide) and two renal function biomarkers (cystatin C and beta 2-microglobulin) in serum and other biological fluids.