Magni Ruben, Luchini Alessandra
Center for Applied Proteomics and Molecular Medicine, George Mason University, 10920 George Mason Circle, MS1A9, Manassas, VA, 20110, USA.
Methods Mol Biol. 2017;1606:103-113. doi: 10.1007/978-1-4939-6990-6_7.
In the recent years, a lot of emphasis has been placed on the discovery and detection of clinically relevant biomarkers. Biomarkers are crucial for the early detection of several diseases, and they play an important role in the improvement of current treatments, thus reducing patient mortality rate. Because biofluids account to 60% of the body mass, they represent a goldmine of significant biomarkers. Unfortunately, because of their low concentration in body fluids, their lability, and the presence of high abundance proteins (i.e., albumin and immunoglobulins), low abundance biomarkers are difficult to detect with mass spectrometry or immunoassays. Nanoparticles made of poly(N-isopropylacrylamide) (NIPAm) and functionalized with affinity reactive baits allow researchers to overcome these physiological barriers and in one single step capture, concentrate, and preserve labile biomarkers in complex body fluids (i.e. urine, blood, sweat, CSF). Although hydrogel nanoparticles have been largely studied and used as a drug delivery tool, our application focuses on their capturing abilities instead of the releasing of specific drug molecules. Once the functionalized nanoparticles are incubated with a biological fluid, small biomarkers are captured by the affinity baits while unwanted high abundance analytes are excluded. The potentially relevant biomarkers are then concentrated into small volumes. The concentration factor (up to 10,000-fold) successfully enhances the detection sensitivity of mass spectrometry and immunoassays allowing the detection of previously invisible proteins.
近年来，人们非常重视临床相关生物标志物的发现和检测。生物标志物对于多种疾病的早期检测至关重要，并且在改善当前治疗方法中发挥着重要作用，从而降低患者死亡率。由于生物流体占体重的60%，它们是重要生物标志物的宝库。不幸的是，由于它们在体液中的浓度低、不稳定以及存在高丰度蛋白质（即白蛋白和免疫球蛋白），低丰度生物标志物难以用质谱法或免疫测定法检测。由聚（N-异丙基丙烯酰胺）（NIPAm）制成并用亲和反应诱饵功能化的纳米颗粒使研究人员能够克服这些生理障碍，并在一步中捕获、浓缩和保存复杂体液（即尿液、血液、汗液、脑脊液）中不稳定的生物标志物。尽管水凝胶纳米颗粒已被大量研究并用作药物递送工具，但我们的应用重点在于它们的捕获能力而非特定药物分子的释放。一旦将功能化纳米颗粒与生物流体孵育，小的生物标志物就会被亲和诱饵捕获，而不需要的高丰度分析物则被排除。然后将潜在相关的生物标志物浓缩到小体积中。浓缩因子（高达10000倍）成功提高了质谱法和免疫测定法的检测灵敏度，从而能够检测到以前不可见的蛋白质。
