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SAMPLE PREPARATION; The Achilles Heel of Mass Spectrometry Based Diagnostics

样品制备;

基本信息

批准号：
8979165
负责人：
Fred E Regnier
金额：
$ 14.72万
依托单位：
NOVILYTIC, LLC
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-01 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8979165
关键词：
Achievement Address Affinity Affinity Chromatography Air Aliquot Amino Acids Antibodies Automation Ballistics Biological Assay Blood Blood Plasma Volume Blood Tests Blood Volume Blood capillaries Blood specimen Caliber Centrifugation Chromatography Clinical Collection Column Chromatography Complex Coupled Development Diagnostic Disease Drops Drug Monitoring Energy-Generating Resources Enzyme-Linked Immunosorbent Assay Factor XIII FarGo Fingers Genetic Polymorphism Goals Hematocrit procedure Hemorrhagic Disorders Immune Immunosorbents Individual Label Laboratories Link Love Mass Spectrum Analysis Membrane Methods Microfluidics Nurses Peptides Pharmaceutical Preparations Pharmacologic Substance Phase Physicians Plasma Population Postage Stamps Preparation Process Protein Isoforms Proteins Proteomics Reagent Recovery Research Research Contracts Sampling Serum Site Spottings Staging Structure Suggestion System Technology Testing Therapeutic Time Training Venipunctures Water Work base capillary instrument mass spectrometer metabolomics migration miniaturize nanoparticulate nanoscale neonate particle protein metabolite public health relevance sample collection small molecule wasting

项目摘要

DESCRIPTION (provided by applicant): There is great excitement today about the use of mass spectrometry (MS) in clinical diagnostics, as we know from therapeutic drug monitoring. With μL volumes of plasma, modern MS can identify and quantify a hundred different drugs, metabolites, and proteins in seconds at sensitivities approaching ELISA. But MS goes far beyond ELISA assays. With coupled immunosorbent capture, MS can differentiate between proteoforms of a protein in seconds; identifying disease associated isoforms from among perhaps 50 very similar structures. ELISA can't do that. Single amino acid polymorphism is similar; in Factor XIII a Trp187Arg substitution leads to a hemorrhagic disorder in neonates that is easily recognized by MS. Clearly, MS analysis of μL blood volumes by immune selection (or some other type of structure specific affinity selection) will become the norm in diagnostics. Unfortunately blood is so complex it overwhelms MS instruments. Samples must be prepared in a laboratory by centrifugation and multiple preparation steps before MS analysis. More than 500 million blood samples are analyzed annually in the US using old sample preparation methods. The goal of this proposal is to miniaturize, simplify, and accelerate this process by developing a new sample preparation system that 1) extracts plasma from a finger-stick derived drop of blood, 2) collects 3.5 μL plasma aliquots, 3) adds labeled internal standards, 4) affinity capture analytes with nanosorbents in a collection membrane, 5) chemically modifies analytes in some cases, 6) purifies analytes in a 60 sec time frame, and 7) introduces samples into an MS. Technological leaps such as this will change clinical diagnostics; giving subjects at remote sites access to sophisticated MS analyses of their blood through transport of a dried sample collection disc the size of a postage stamp to analytical laboratory. Last year Novilytic introduced the Noviplex microfluidic sample preparation card consisting of a membrane stack that extracts 2.5 μL of plasma by capillary action from a finger-stick derived drop of blood. Preliminary studies with new, more advanced versions of Noviplex have shown that steps 1-5 can be executed automatically within a single small card without an energy source; enabling sampling at remote sites via a finger-stick, a substantial amount of sample preparation within each card, and dry transport to the analytical laboratory in a disc smaller than a postage stamp. With a large portion of the world's population far from advanced diagnostic laboratories, ease of sample transport is a major issue. We are proposing to develop technology that allows the first stages of sample preparation within Noviplex cards in-transit and final preparation in the LC-MS/MS platform; in-transit meaning post-plasma extraction An enabling feature is a new form of chromatography we will develop referred to here as "mobile-sorbent affinity chromatography" (MSAC) which involves the use of nanoscale affinity chromatography sorbents (NACS) in the mobile phase of chromatography columns. These nanosorbents will be synthesized in sizes too large to enter 50 nm pores in chromatography sorbents. NACS will be dispersed in water and added to the collection membrane on Noviplex cards where they will strongly sequester analytes within minutes. When the card arrives in the analytical lab, NACS will be eluted from the card and separated from other sample components in the LC-MS/MS by a C-18 restricted access media (RAM) column packed with particles of <50 nm pore diameter. NACS will be transported through the RAM column unretained at the velocity of the mobile phase while unbound substances are stripped from samples by the C-18 phase. Analytes will be recovered from NACS by denaturation of the affinity agent within the LC flow-train and eluted through a second RAM column where they are adsorbed and enriched while sorbents are transported to waste. Identification and quantification will be achieved by ballistic gradient elution of the secod RAM column into an MS in <60 sec. This allows the equivalent of many ELISA tests every min; an achievement of great significance in diagnostics.

描述（由申请人提供）：正如我们从治疗药物监测中所了解的那样，目前质谱法（MS）在临床诊断中的应用令人非常兴奋。使用μL体积的血浆，现代MS可以在几秒钟内以接近ELISA的灵敏度识别和定量100种不同的药物，代谢物和蛋白质。但MS远远超出了ELISA检测。通过偶联免疫吸附捕获，MS可以在几秒钟内区分蛋白质的蛋白质型;从大约50个非常相似的结构中识别疾病相关的亚型。伊莉莎不能这么做。单个氨基酸多态性是相似的;在因子XIII中，Trp 187 Arg取代导致新生儿出血性疾病，这很容易被MS识别。显然，通过免疫选择（或其他类型的结构特异性亲和力选择）对μL血容量进行MS分析将成为诊断的标准。不幸的是，血液是如此复杂，它使MS仪器变得复杂。样品必须在实验室中通过离心和多个制备步骤制备，然后进行MS分析。在美国，每年有超过5亿份血液样本使用旧的样本制备方法进行分析。本提案的目标是通过开发一种新的样品制备系统来简化、简化和加速该过程，该系统1）从手指针刺衍生的血滴中提取血浆，2）收集3.5 μL血浆等分试样，3）添加标记的内标物，4）在收集膜中使用纳米吸附剂亲和捕获分析物，5）在某些情况下化学修饰分析物，6）在60秒的时间范围内纯化分析物，以及7）将样品引入MS。诸如此类的技术飞跃将改变临床诊断;通过将邮票大小的干燥样品收集盘运输到分析实验室，使远程站点的受试者能够对其血液进行复杂的MS分析。去年，Novilytic推出了Noviplex微流体样品制备卡，该卡由一个膜堆组成，可通过毛细管作用从手指针刺的血滴中提取2.5 μL血浆。对新的、更先进的Noviplex版本的初步研究表明，步骤1-5可以在单个小卡片中自动执行，而无需能源;通过手指针刺，在每个卡片中制备大量样品，并在小于邮票的圆盘中干燥运输到分析实验室，可以在远程站点进行采样。由于世界上很大一部分人口远离先进的诊断实验室，样本运输的便利性是一个主要问题。我们建议开发技术，使第一阶段的样品制备在Noviplex卡在运输和最后准备在LC-MS/MS平台;一个有利的特征是我们将开发的一种新形式的层析，在此称为“流动吸附剂亲和层析”（MSAC），其涉及使用纳米级亲和层析吸附剂（NACS）。在色谱柱的移动的相中。这些纳米吸附剂将以太大而不能进入色谱吸附剂中的50 nm孔的尺寸合成。NACS将分散在水中，并添加到Noviplex卡上的收集膜上，在那里它们将在几分钟内强烈螯合分析物。当卡到达分析实验室时，NACS将从卡上洗脱，并通过填充有<50 nm孔径颗粒的C-18限制性介质（RAM）柱与LC-MS/MS中的其他样品组分分离。NACS将以移动的速度通过RAM色谱柱运输，而未结合的物质将通过C-18相从样品中剥离。通过在LC流动链内使亲和剂变性，从NACS中回收分析物，并通过第二个RAM柱洗脱，在RAM柱中吸附和富集分析物，同时将吸附剂转运至废物。将通过在<60秒内将secod RAM柱弹道梯度洗脱到MS中来实现鉴定和定量。这使得每分钟可以进行相当于许多ELISA测试的测试;这在诊断学上具有重要意义。