Biospecimen preparation technologies to enable high throughput and highly sensiti
生物样本制备技术可实现高通量和高灵敏度
基本信息
- 批准号:8471943
- 负责人:
- 金额:$ 21.08万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2013
- 资助国家:美国
- 起止时间:2013-06-01 至 2016-05-31
- 项目状态:已结题
- 来源:
- 关键词:AddressAnteriorAntibodiesAutomationBindingBiologicalBiological AssayCancer PatientCharacteristicsChromatographyClinicalDataDetectionDiffuseDigestionDiseaseDisease ProgressionEnzyme-Linked Immunosorbent AssayEvaluationExhibitsFutureHIV Core Protein p24HeatingHigh Pressure Liquid ChromatographyImmunoassayIndividualLiquid substanceMagnetismMalignant NeoplasmsMalignant neoplasm of prostateMass Spectrum AnalysisMedicalMicellesMolecularMolecular ProfilingMonitorMonoclonal AntibodiesOutcomePerformancePhasePolymersPreparationProcessProtein MicrochipsProteinsProteomicsReactionReagentSamplingSerumSolidSolubilitySolutionsSpecimenStimulusStromal CellsSurfaceSystemTechnologyTemperatureTimeTissuesUrineVariantanalytical toolantibody conjugatecancer diagnosiscancer proteomicsimprovedinnovationmagnetic fieldmultiple reaction monitoringnanoparticleneoplastic cellnovelparticlepre-clinicalprotein expressionpublic health relevanceresearch clinical testingscreeningsuccess
项目摘要
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.
描述(由申请人提供):许多疾病导致生物液体分子谱的特异性和特征性变化(例如,尿)和组织。 对于癌症,这些变化可以用于筛查,或作为疾病进展或治疗相关变化的指标,这可以显着改善医疗结果。 质谱(MS),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的水平,因此诸如尿液的样本需要大量的制备(即,HPLC)。 这些制备过程可能导致分析物的显著损失,并且是麻烦的、耗时的、不适合自动化的,并且更容易受到污染,这显著影响了总体测定性能,例如检测限(LOD)。 为了满足这一需求,该项目旨在开发一种新型的生物标本制备技术,该技术可以通过浓缩多个目标分析物(e 102倍)来快速处理整个可用尿液样本(d 30分钟),以实现高通量多重靶向癌症蛋白质组学,并显着降低LOD。 所提出的样品制备利用刺激响应二元试剂系统进行有效和快速的无固相色谱。 关键的创新在于刺激响应试剂的组合,包括磁性纳米颗粒(mNPs)和聚合物-抗体缀合物,以最大限度地提高分析物结合效率,同时保持有效的磁性分离。 这种二元试剂系统可以通过简单地施加更大量的抗体缀合物而不增加磁性纳米颗粒的质量来实现比现有磁性微粒显著更高的分析物结合,因为抗体不附着于颗粒表面。 当刺激(例如,加热)应用于溶液时,两种刺激响应性试剂(包括mNP和聚合物-抗体缀合物)形成大的聚集体,其可以使用适度的磁场分离以浓缩结合的分析物。 我们将开发一种温度响应的分离模块,用于同时分离尿液中的多种分析物。 分离模块将进行优化,以处理整个可用的尿液(约200 ml),并与MS连接以进行靶蛋白定量。 此外,我们将利用临床尿液样本评价拟定标本制备工艺的多重能力以及MS测定的改进。 该项目的成功将产生一种生物标本制备技术,该技术可以快速(d 30分钟)处理整个可用的尿液样本,以产生多种浓缩的目标分析物(e 102倍),从而通过MS实现高通量多重目标蛋白质组学,并显著降低检测LOD。 该技术适用于不同的标本(例如,血清)用于不同的分析物面板,这将大大加快癌症蛋白质组学的发展,并促进未来高质量的癌症诊断。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
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James Lai其他文献
James Lai的其他文献
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Biospecimen preparation technologies to enable high throughput and highly sensiti
生物样本制备技术可实现高通量和高灵敏度
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