Sensitive biomarker detection using organized nanoparticles in multiwell plates

描述(由申请人提供)：我们建议探索一种新的材料，该材料有望将多孔板中的ELISA分析的灵敏度提高至少一个数量级，并独特地使凝集素能够用于探测生物标记物的糖基化。这项技术将使癌症能够更早地被发现。该技术是基于多井板井底的胶体结晶膜。假设这些膜将提供较高的表面积来捕捉生物标志物，并控制扩散以防止凝集素在漂洗过程中解吸。结晶度是高透明度与高比表面积相结合的关键。使用更高的表面积意味着灵敏度的增强独立于其他增强检测的方法，例如量子点标记，这两种独立的方法可以组合在一起以获得更高的灵敏度。与多孔板的兼容性将允许多路复用，从而可以使用生物标志物面板。这种兼容性还将促进这项技术的广泛采用，因为多孔板在今天得到了广泛的使用。设计了三个特定的目标来测试建议的多孔板：选择二氧化硅涂层以最大限度地提高捕获和结合活性，同时减少非特异性结合；选择纳米颗粒直径以平衡高表面积与足够的孔径和凝集素的受控扩散；以及评估两种类型生物标记物分析板的灵敏度。第一个检测将是标准的夹心免疫检测，以前列腺特异性抗原为模型系统。第二种将是一种新型的检测方法，它使用凝集素来代替二级抗体来探测蛋白质的糖基化。该模型系统将再次使用前列腺特异性抗原，有样本证据表明差异糖基化是癌症的指示，以及来自Aleuria aurania的商业岩藻糖结合凝集素。这项拟议的工作将在两年内确定这些多孔板是否将广泛用作早期癌症诊断的新工具。 与公共健康相关：将研究一种基于纳米技术的薄膜，作为癌症早期和更具特异性的体外诊断的新工具。

DESCRIPTION (provided by applicant): We propose to explore a novel material that promises to enhance the sensitivity of ELISA assays in multi-well plates by at least an order of magnitude, and uniquely enable lectins to be used to probe glycosylation of biomarkers. This technology would enable earlier detection of cancer. The technology is based on colloidal crystalline film on the bottoms of the wells of multiwell plates. The hypothesis is that these films will impart a high surface area for capture of biomarkers, and also control diffusion to prevent lectins from desorbing during rinsing. The crystallinity is the key to combining high transparency with high surface area. The use of higher surface area means that the sensitivity enhancement is independent of other approaches that enhance detection, such as quantum dot labels, and the two independent approaches can be combined for even higher sensitivity. The compatibility with multiwell plates will allow multiplexing so that panels of biomarkers can be used. The compatibility will also facilitate the widespread adoption of this technology because multiwell plates are widely used today. Three specific aims are designed to test the proposed multiwell plates: choice of coating for the silica to maximize capture and binding activity while reducing nonspecific binding, choice of nanoparticle diameter to balance high surface area with sufficient pore diameter and controlled diffusion of lectins, and evaluation of the sensitivity of the plates for two types of biomarker assays. The first assay will be the standard sandwich immunoassay, with prostate specific antigen as the model system. The second will be a novel type of assay that uses lectins in place of the secondary antibody to probe the glycosylation of the protein. The model system will again be prostate specific antigen used, for which there is sample evidence that differential glycosylation is indicative of cancer, and a commercial fucose-binding lectin from Aleuria aurentia. The proposed work will determine in two years whether these multiwell plates will be broadly useful as a new tool for early stage cancer diagnostics. PUBLIC HEALTH RELEVANCE: A thin film based on nanotechnology will be studied as a new tool for early and more specific in vitro diagnostics of cancer.