SPR Imaging Studies of Nucleic Acid Microarrays for Biomarker Detection

用于生物标志物检测的核酸微阵列 SPR 成像研究

• 批准号: 7318714
• 负责人: ROBERT M CORN
• 金额: $ 29.25万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7318714
• 关键词: Adsorption; Antibodies; Area; Binding; Biological; Biological Markers; Biopolymers; Biosensing Techniques; Brain natriuretic peptide; Cancer Detection; Cardiac; Classification; Complex; Cystatins; DNA; Detection; Development; Diagnosis; Endoribonucleases; Enzymes; Evolution; Exhibits; Fluorescence; Goals; Gold; Growth Factor; Heart; Heart Diseases; Image; In Vitro; Label; Ligands; Liquid substance; Liver diseases; Maize; Malignant Neoplasms; Measurement; Methodology; Methods; Microfluidics; Molecular; Molecular Weight; N-terminal; NT-proBNP; Nucleic Acids; Oligonucleotides; Pancreatic ribonuclease; Platelet-Derived Growth Factor; Polynucleotide Adenylyltransferase; Process; Proteins; RNA; RNA Ligase (ATP); RNA Sequences; Range; Reaction; Renal function; Research; Research Personnel; Ribonuclease H; Ribonucleases; Sampling; Screening procedure; Serum; Specificity; Surface; Surface Plasmon Resonance; T7 RNA polymerase; Testing; Theophylline; Troponin T; Vascular Endothelial Growth Factors; Zea mays; aptamer; base; beta-2 Microglobulin; design; glucosamine 6-phosphate; improved; nanoparticle; novel; post gamma-globulins; programs; research study; small molecule; three dimensional structure; toe corn

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.

描述（由申请人提供）：这项研究的总体目的是开发增强的表面等离子体共振成像（SPRI）方法学，这些方法使用RNA适体微阵列，用于对女性气流浓度的生物流体中高分子和低分子量蛋白质生物标记物的多重超敏感性检测。拟议的研究由两个主要领域组成：（i）新的RNA适体微阵列的发展，以及（ii）创建新的增强的Spri Sensing方法。新的RNA适体微阵列的创建既需要开发新型的微阵列制造策略，又需要创建专门为增强的SPRI测量设计的新RNA Aptamer。阵列制造方法将使用T4 RNA连接酶和T7 RNA聚合酶与微流体一起使用表面酶反应策略，以将合成寡核苷酸和体外转录RNA连接到金表面上。微阵列将使用先前发现的适体，例如血管内皮生长因子（VEGF）和茶碱，以及由体外SELEX过程鉴定的新RNA适体。 SPR和SPRI将在SELEX选择期间用于筛选目标APTAMER相互作用。这些RNA序列中的某些将被掺入合并的适体中，其中包括分子开关序列，其3D结构在目标结合时会发生变化。新的增强的SPRI传感方法将采用核酸微阵列的各种表面酶反应，例如T7 RNA聚合酶，RNase H和Poly（a）聚合酶以及官能化纳米颗粒的吸附，以提高表面生物具有测量的特异性或敏感性。最初的目标将是两个潜在的癌症生物标志物，VEGF和血小板衍生的生长因子的生长因子。初始分子开关适合学剂将采用已知的核糖开关序列，结合葡萄糖胺-6-磷酸盐或茶氨基氨酸；这些将与其他可以与功能化的金纳米颗粒结合的序列结合使用。一旦实现了提出的增强的SPRI生物传感方法，它们将应用于癌症生物标志物VEGF的微阵列检测，三种心脏生物标志物（Troponin T，NT-ProboBNP，B-Type Natriaretic Natriaretic peptide）和两个肾功能生物标志物（cystatin c和beta cystaTin c和beta 2-Micr）流体。