Nanotrap technology for one step preservation and amplification of cancer biomark

描述（由申请人提供）：我们提出先进的开发和验证新技术，将最大限度地提高生物液体标本的质量和效用，并允许 测量以前不可见的低丰度生物标志物。该技术具有变革性和范式转变性，因为它立即经济地解决了使体液癌症生物标志物领域瘫痪的根本障碍。在一个步骤中，在几分钟内，我们的技术克服了生物标志物保存，低丰度和不需要的蛋白质掩蔽的严重问题。该技术是一种新型的多孔、浮力、核壳水凝胶纳米颗粒，含有高亲和力的反应性化学诱饵，可在体液中收获生物标志物。我们的纳米颗粒可以预先装入血液收集管或其他体液收集容器中。在与样品接触时，悬浮的纳米颗粒立即亲和螯合颗粒内的靶生物标志物，排除白蛋白，完全保护生物标志物免于降解（即使在升高的温度下），并将螯合的生物标志物大量浓缩到小体积中。该技术可以显著地（证明高达10，000倍）提高以下检测的下限和精度：a）质谱（MS）生物标志物发现，B）通过多反应监测（MRM）进行定量，或c）通过任何临床级免疫测定进行定量。已超出同品种器械IMAT NCI R21 CA 137706授权的所有目标和目标。我们已经发现了十几种对特定低丰度蛋白质分析物具有优先高亲和力（KD <10-11 M）的新型化学诱饵。我们发现了一种新的壳化学，它选择性地防止所有大小的白蛋白衍生肽的不必要的进入，而不阻碍非白蛋白小蛋白和肽的渗透。不稳定的体液生物标志物，否则会迅速降解，在收集时被完全保存，从而避免了昂贵的冷冻或蛋白酶抑制剂的需要。以前无法通过MS发现的低丰度蛋白质，以及以前无法通过MRM或临床免疫测定法测量的低丰度蛋白质，现在可以在测定的线性范围内以高精度进行定量。我们的技术是完全创新的，有3项已获得商业许可的许可（2项已颁发）专利和14项出版物。没有现有技术可以解决所有上述障碍，并获得接近100%的捕获/洗脱效率。该技术最近允许MS鉴定国际HUPO数据库中未列出的新血清和血浆蛋白。根据拟定的目标，我们将扩大该技术的规模，在两个大型（n = 400份血清和n = 74份血浆）对照良好的临床血清/血浆样本集（64种分析物）中对其性能精密度、准确度、提高的检测灵敏度和保存能力进行盲态验证。我们将把该技术扩展到尿液和汗液，将这些生物流体开辟为生物标志物研究的新类别，使用创新方法来解决这些生物流体的体积、易腐性和低蛋白浓度等基本问题。 公共卫生相关性：我们提出先进的开发和验证新的纳米技术，将最大限度地提高生物流体标本的质量和效用，并允许测量以前看不见的低丰度生物标志物。纳米技术是变革性的和范式转变的，因为它立即和经济地解决了使体液癌症生物标志物领域瘫痪的基本障碍，允许以高精度和准确度测量来自小（< 2 mm）早期癌症的生物标志物。

DESCRIPTION (provided by applicant): We propose advanced development and validation of novel technology that will maximize the quality and utility of biologic fluid specimens, and permit the measurement of previously invisible low abundance biomarkers. The technology is transformative and paradigm-shifting because it immediately and economically solves fundamental roadblocks paralyzing the body fluid cancer biomarker field. In a single step, in minutes, our technology overcomes the severe problems of biomarker preservation, low abundance, and masking by unwanted proteins. The technology is novel porous, buoyant, core-shell hydrogel nanoparticles containing high affinity reactive chemical baits that harvests biomarkers in body fluids. Our nanoparticles can be pre loaded into Vacutainer(R) blood collection tubes, or other body fluid collection vessels. Upon contact with the sample, the suspended nanoparticles immediately affinity-sequester target biomarkers inside the particles, exclude albumin, fully protect the biomarkers from degradation (even at elevated temperatures), and massively concentrate the sequestered biomarkers into a small volume. The technology can dramatically (demonstrated up to 10,000 fold) improve the lower limits of detection and the precision of: a) mass spectrometry (MS) biomarker discovery, b) quantitation by multiple reaction monitoring (MRM), or c) quantification by any clinical grade immunoassay. All of the Aims and Milestones of the predicate IMAT NCI R21 CA137706 grant have been exceeded. We have discovered more than a dozen novel chemical baits with preferential high affinities (KD <10-11 M) for specific low abundance protein analytes. We discovered a novel shell chemistry that selectively prevented unwanted entry of all size albumin-derived peptides without hindering the penetration of non-albumin small proteins and peptides. Labile body fluid biomarkers, that would otherwise rapidly degrade, are completely preserved at the time of collection, thus obviating the need for costly freezing or proteinase inhibitors. Low abundant proteins previously invisible to discovery by MS, and previously not measureable by MRM, or clinical immunoassays, can now be quantified with high precision within the linear range of the assay. Our technology is completely innovative, as documented by 3 allowed (2 issued) patents that have been commercially licensed, and 14 publications. No existing technology can solve all of the aforementioned roadblocks, and attain a capture/elution efficiency near 100%. The technology has recently permitted the MS identification of new serum and plasma proteins not listed in the international HUPO database. Under the proposed Aims we will scale up the technology to conduct blind validation of its performance precision, accuracy, improved detection sensitivity, and preservation capacity, in two large (n = 400 sera and n = 74 plasma) well-controlled clinical sera/plasma sample sets (64 analytes). We will extend the technology to urine and sweat to open up these biofluids as a new category for biomarker research, using innovative approaches to solve fundamental problems of volume, perishability, and low protein concentration for these biofluids. PUBLIC HEALTH RELEVANCE: We propose advanced development and validation of novel nanotechnology that will maximize the quality and utility of biologic fluid specimens, and permit the measurement of previously invisible low abundance biomarkers. The nanotechnology is transformative and paradigm-shifting because it immediately and economically solves fundamental roadblocks paralyzing the body fluid cancer biomarker field, permitting the measurement of biomarkers emanating from small (< 2 mm) early stage cancers with high precision and accuracy.