权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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.