Biospecimen preparation technologies to enable high throughput and highly sensiti

描述(由申请人提供)：许多疾病导致生物体液(例如尿液)和组织的分子图谱发生特殊和特有的变化。对于癌症，这些变化可以用于筛查，或者作为疾病进展或与治疗相关的变化的指标，这可以显著改善医疗结果。质谱仪(MS)是一种具有多重分析能力的高灵敏度分析技术，已被用于检测蛋白质表达的变化。MS的灵敏度本质上取决于可用的分析物浓度和背景干扰水平，因此尿样等样品在分析阶段之前需要广泛的准备(即高效液相色谱)。这些准备过程可能会导致分析物的大量损失，并且繁琐、耗时、不适合自动化，并且更容易受到污染的影响，这些污染会显著影响整体分析性能，如检测限(LOD)。为了满足这一需求，该项目旨在开发一种新的生物样品制备技术，该技术可以通过浓缩多目标分析物(e 102倍)来快速处理全部可用尿样(d 30分钟)，从而以显著较低的LOD实现高通量多重靶向癌症蛋白质组学。所提出的样品制备利用刺激响应的二元试剂系统进行有效和快速的无固相层析。关键的创新在于结合刺激反应试剂，包括磁性纳米颗粒(MNPs)和聚合物-抗体结合物，以最大限度地提高分析物结合效率，同时保持有效的磁分离。这种二元试剂系统通过简单地应用更大量的抗体结合物而不增加磁性纳米粒子的质量，可以实现比现有磁性微粒子更高的分析物结合，因为抗体不附着在粒子表面。当对溶液施加刺激(例如加热)时，两种刺激响应性试剂，包括mNPs和聚合物-抗体结合物，都形成大的聚集体，可以使用适度的磁场来分离这些聚集体，以浓缩结合的分析物。我们将开发一种温度响应型分离模块，用于同时分离尿液中的多种分析物。分离模块将进行优化，以处理全部可用尿液(约200毫升)，并与MS对接以进行目标蛋白质定量。此外，我们将利用临床尿样来评估拟议的标本制备过程与MS分析改进相结合的多重能力。该项目的成功将带来一种生物样品制备技术，该技术可以快速(d 30分钟)处理整个可用的尿样，以产生多个浓缩目标分析物(e 102倍)，从而实现通过MS高通量多重目标蛋白质组学，同时显著降低分析LOD。该技术适用于不同分析板的不同样本(例如血清)，这将显著加快癌症蛋白质组学，促进未来高质量的癌症诊断。

DESCRIPTION (provided by applicant): Many diseases result in specific and characteristic changes in the molecular profiles of biological fluids (e.g., urine) and tissues. For cancer, thes changes can be utilized for screening, or as indicators of disease progression or of treatment-associated changes, which can significantly improve the medical outcome. Mass spectrometry (MS), a highly sensitive analytical technology with multiplex analysis capability, has been utilized for detecting changes in protein expression. The sensitivity of MS is inherently dependent on the available analyte concentration and the level of background interference, so specimens such as urine require extensive preparation (i.e., HPLC) prior to the analytical phase. These preparatory processes can result in significant loss of the analyte and are cumbersome, time consuming, not amenable to automation, and more susceptible to contaminations, which significantly impact the overall assay performance such as limit of detection (LOD). To address the need, this project aims to develop a novel biospecimen preparation technology that can process entire available urine samples rapidly (d 30 minutes) by concentrating multiple target analytes (e 102- fold) to enable high throughput multiplex targeted cancer proteomics with significantly lower LOD. The proposed sample preparation utilizes the stimuli-responsive binary reagent system for effective and rapid chromatography without solid phase. The key innovation lies in the combination of stimuli-responsive reagents, including magnetic nanoparticles (mNPs) and polymer-antibody conjugates to maximize the analyte binding efficiency while maintaining effective magnetic separation. This binary reagent system can achieve significantly higher analyte binding then the existing magnetic microparticle by simply applying a larger amount of the antibody conjugates without increasing the mass of magnetic nanoparticles because the antibodies are not attached to the particle surfaces. When a stimulus (e.g., heating) is applied to the solution, both stimuli- responsive reagents, including mNPs and polymer-antibody conjugates, form large aggregates that can be separated using a modest magnetic field to concentrate the bound analytes. We will develop a temperature- responsive separation module for isolating multiple analytes simultaneously in urine. The separation module will be optimized for processing the entire available urine (ca. 200 ml) and interfacing with MS for target protein quantification. Additionally, we will utilize clinical urine samples to evaluate the multiplex capability of the proposed specimen preparation process in conjunction with the MS assay improvement. The success of the project will result in a biospecimen preparation technology that can rapidly (d 30 minutes) process the entire available urine sample to produce multiple concentrated target analytes (e 102-fold) to enable high throughput multiplex target proteomics via MS with significantly lower assay LOD. The technology is applicable to different specimens (e.g., serum) for different analyte panels, which will significantly accelerate cancer proteomics and facilitate high quality cancer diagnosis in the future.