SAMPLE PREPARATION; The Achilles Heel of Mass Spectrometry Based Diagnostics

 描述(由申请人提供)：正如我们从治疗药物监测中了解到的那样，今天人们对质谱仪(MS)在临床诊断中的使用感到非常兴奋。随着μL血浆体积的增加，现代MS可以在几秒钟内以接近ELISA的灵敏度识别和定量上百种不同的药物、代谢物和蛋白质。但多发性硬化症远远超出了酶联免疫分析的范畴。通过结合免疫吸附捕获，MS可以在几秒钟内区分蛋白质的蛋白质形式；从大约50个非常相似的结构中识别与疾病相关的亚型。伊莉莎不能这么做。单一氨基酸多态性相似；在凝血因子XIII中，Trp187Arg替换会导致新生儿出现MS容易识别的出血性疾病。显然，通过免疫选择(或其他类型的结构特异性亲和选择)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，以至于MS仪器不堪重负。在MS分析之前，样品必须在实验室中通过离心法和多个制备步骤进行制备。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。这项建议的目标是通过开发一种新的样品制备系统来小型化、简化和加速这一过程，该样品制备系统1)从指棒提取的血滴中提取血浆，2)收集3，5μL血浆等分，3)增加标记的内标，4)在收集膜中使用纳米吸附剂亲和捕获分析物，5)在某些情况下对分析物进行化学修饰，6)在60秒的时间范围内对分析物进行纯化，以及7)将样品引入MS，如这将改变临床诊断；通过将一个邮票大小的干燥样本收集盘运送到分析实验室，偏远地点的受试者可以获得对他们血液的复杂的MS分析。去年，诺维莱特公司推出了Noviplex微流控样品制备卡，它由一个膜堆组成，通过毛细管作用从手指提取的一滴血中提取2.5μL的血浆。对新的、更先进的Noviplex版本的初步研究表明，步骤1-5可以在一张小卡片内自动执行，而不需要能源；能够通过手指杆在远程地点进行采样，在每张卡片内进行大量的样品准备，并在比邮资邮票更小的圆盘中干式运输到分析实验室。由于世界上有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。我们建议开发一种技术，允许在运输途中在Noviplex卡中进行样品制备的第一阶段，并在LC-MS/MS平台中进行最终制备；在运输中意味着血浆提取后的一个使能功能是我们将开发的一种新形式的层析，在此称为“移动-吸着剂亲和层析”(MSAC)，它涉及在层析柱的流动相中使用纳米级亲和层析吸附剂(NAC)。这些纳米吸附剂的合成尺寸太大，无法进入层析吸附剂中的50纳米孔。NAC将分散在水中，并添加到Noviplex卡片上的收集膜上，在那里它们将在几分钟内强烈隔离分析物。当卡片到达分析实验室时，NaCS将从卡片上洗脱，并通过C-18受限访问介质(RAM)柱与LC-MS/MS中的其他样品组分分离，该柱填充了&lt；50 nm孔径的颗粒。NAC将以流动相的速度不保留地通过RAM柱，而未结合的物质被C-18相从样品中剥离。分析物将通过LC流水线中亲和剂的变性从NAC中回收，并通过第二个RAM柱洗脱，在那里它们被吸附和浓缩，同时吸附剂被运输到废物中。鉴定和定量将通过在60秒内将Secod RAM柱弹道梯度洗脱到MS中来实现。这使得每分钟可以进行相当于许多次的酶联免疫吸附试验；这在诊断学上是一项非常重要的成就。

 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.