Nanotechnology for One Step Concentration and Preservation of Labile Biomarkers i

描述（由申请人提供）：癌症相关血液生物标志物以极低浓度存在于高丰度蛋白质（如白蛋白和免疫球蛋白）的复杂混合物中。此外，血液中的生物标志物可能在运输和储存期间发生降解。这种降解是癌症生物标志物测量和发现的偏差的重要来源。我们已经创建了N-异丙基丙烯酰胺多孔筛分核壳“智能”纳米粒子含有内部亲和诱饵执行三个独立的功能在几分钟内，在一个步骤中，在溶液中（血清或血浆）：a）分子大小筛选，与高丰度的驻留蛋白如白蛋白和免疫球蛋白完全分离，B）所有溶液相靶分子的亲和捕获，和c）完全保护收获的蛋白质免于酶促降解。捕获的分析物可以容易地电洗脱用于分析。在我们的初步研究中，我们已经制造了大量含有特定诱饵化学物质的均匀多孔纳米颗粒，所述诱饵化学物质结合高度不稳定的低丰度靶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，颗粒可以从血清中捕获和浓缩靶蛋白，使得靶分子在5分钟内从本体溶液中完全去除，完全排除白蛋白和免疫球蛋白。捕获的蛋白质，即使变性，即使在37 ° C过夜，也不会被高浓度的蛋白酶（胰蛋白酶或糜蛋白酶）降解。这项拟议的可行性R21研究的可交付目标是将这种“智能”纳米颗粒技术与标准采血管结合起来，无论是否有添加剂/抗凝剂，以快速封装已知非常罕见和不稳定的癌症生物标志物。设想的技术是一组干燥冻干的亚微米尺寸的收获颗粒，其携带已知生物标志物的特异性诱饵。在引入血液或体液之后，相应的颗粒群将在一个步骤中在溶液中从样品的整个体积中去除它们的所有靶分子，并将螯合的分析物浓缩在颗粒内。然后，分析物将从颗粒中洗脱，以产生与起始样品相比高得多的浓度和纯化。根据血液的初始体积，这项技术可以在几分钟内将生物标志物浓缩数百倍，并防止降解。 公共卫生相关性：迫切需要早期癌症的生物标志物，以便在转移之前开始治疗。不幸的是，癌症生物标志物以非常低的浓度存在，并且非常不稳定。该项目将提供一种新的纳米技术，可以在采血管中一步快速采集、浓缩和保护全血或血清中的不稳定生物标志物免受降解。

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.