Nanotechnology for One Step Concentration and Preservation of Labile Biomarkers i

描述(申请人提供)：与癌症相关的血液生物标记物存在于白蛋白和免疫球蛋白等高丰度蛋白质的复杂混合物中，浓度极低。此外，血液中的生物标志物在运输和储存过程中可能会发生降解。这种降解是癌症生物标记物测量和发现的一个重要偏差来源。我们已经创建了包含内部亲和力诱饵的N-异丙基丙烯酰胺多孔筛选核壳“SMART”纳米颗粒，在几分钟内在溶液(血清或血浆)中一步完成三项独立功能：a)与白蛋白和免疫球蛋白等高丰度滞留蛋白完全分离的分子大小筛选，b)亲和捕获所有溶液相目标分子，以及c)完全保护收获的蛋白质免受酶降解。捕获的分析物可以很容易地电洗脱进行分析。在我们的初步研究中，我们制造了大量均匀的多孔纳米颗粒，其中含有特定的诱饵化学物质，可以结合高度不稳定的低丰度目标癌症生物标志物蛋白(例如，血小板衍生生长因子)。我们已经证明，这种颗粒可以捕获和浓缩血清中的目标蛋白，从而在5分钟内将目标分子从散装溶液中完全去除，并完全排除白蛋白和免疫球蛋白。即使在37摄氏度下，捕获的蛋白质即使变性，也可以通过高浓度的蛋白酶(胰蛋白酶或胰凝乳酶)保护其不被降解。这项拟议的可行性R21研究的可交付目标是将这项“智能”纳米颗粒技术与标准血液采集管相结合，无论是否有添加剂/抗凝剂，以快速包裹已知非常罕见和不稳定的癌症生物标记物。这项设想的技术是一组干燥的冷冻干燥亚微米大小的收获颗粒，这些颗粒携带着已知生物标志物的特定诱饵。在引入血液或体液后，相应的颗粒群体将在溶液中一步将其所有目标分子从整个样本体积中移除，并将隔离的分析物集中在颗粒内。然后分析物将从颗粒中洗脱出来，产生比起始样品高得多的浓度和纯度。根据血液的起始量，这项技术可以在几分钟内将生物标记物浓缩数百倍，并防止降解。 公共卫生相关性：迫切需要早期癌症的生物标记物，以便在转移之前开始治疗。不幸的是，癌症生物标记物的浓度很低，而且非常不稳定。该项目将提供一种新的纳米技术，可以在血液采集管中一步快速采集、浓缩全血或血清中不稳定的生物标志物，并防止其降解。

DESCRIPTION (provided by applicant): Cancer-associated blood biomarkers exist in exceedingly low concentrations within complex mixtures of high-abundance proteins such as albumin and immunoglobulins. Moreover, biomarkers in the blood may be subjected to degradation during transportation and storage. Such degradation is a significant source of bias for cancer biomarker measurement and discovery. We have created N-isopropylacrylamide porous sieving core shell "smart" nanoparticles containing an internal affinity bait to perform three independent functions within minutes, in one step, in solution (serum or plasma): a) molecular size sieving with complete separation from high abundance residence proteins such as albumin and immunoglobulin, b) affinity capture of all solution phase target molecules, and c) complete protection of harvested proteins from enzymatic degradation. The captured analytes can be readily electroeluted for analysis. In our preliminary studies we have manufactured large quantities of uniform porous nanoparticles containing specific bait chemistries that bind highly labile low abundance target cancer biomarker proteins (e.g., platelet derived growth factor). We have demonstrated that the particles can capture and concentrate target proteins from serum such that the target molecule is completely removed from the bulk solution within 5 minutes, with completed exclusion of albumin and immunoglobulins. The captured proteins, even if denatured, are protected from degradation by high concentrations of proteases (trypsin or chymotrypsin) even at 37 oC overnight. The deliverable goal of this proposed feasibility R21 study is to combine this "smart" nanoparticle technology with standard blood collection tubes, with or without additives/anticoagulants, to rapidly encapsulate cancer biomarkers known to be very rare and labile. The envisioned technology is a panel of dry lyophilized sub-micron sized harvesting particles that carry specific bait for known biomarkers. Following introduction of the blood or body fluid, the respective particle populations will remove all of their target molecules, in one step, in solution, from the entire volume of the sample and concentrate the sequestered analytes inside the particles. Analytes will then be eluted from the particles to yield a much higher concentration and purification compared to the starting sample. Depending on the starting volume of the blood, this technology can concentrate a biomarker many hundred fold, and prevent degradation, within minutes. PUBLIC HEALTH RELEVANCE: Biomarkers for early stage cancer are urgently needed so that treatment can be initiated prior to metastasis. Unfortunately, cancer biomarkers exist in very low concentrations and are highly labile. This project will deliver a novel nanotechnology that can rapidly harvest, concentrate, and protect from degradation, labile biomarkers in whole blood or serum, in one step, in the blood collection tube.