SBIR-STTR Award

?There is great excitement today about the use of mass spectrometry (MS) in clinical diagnostics, as we know from therapeutic drug monitoring. With ?L 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 ?L 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 ?L 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 ?L 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

Public Health Relevance Statement:


Public Health Relevance:
Even though the advance of mass spectrometry allows us to analyze small molecules or peptides in seconds, sample preparation becomes a rate-limiting step for the Metabolomics and Proteomics. The nanoparticulate affinity chromatography sorbents (NPACS) will be developed to purify target analytes from the plasma, which is combined with a restricted access media (RAM) column to isolate the selected analytes while they are transported in a mobile phase. The goal of the research is to develop the new system for mass linked immune-selective analysis (MALISA), where the eluted analyte can be identified and quantified by a mass spectrometer (MS) in minutes. This technology implements a new paradigm where sample preparations are directly connected to high throughput mass spectrometry.

Project Terms:
Achievement; Address; Affinity; Affinity Chromatography; Air; Aliquot; Amino Acids; Antibodies; Automation; Ballistics; base; Biological Assay; Blood; Blood capillaries; Blood Plasma Volume; Blood specimen; Blood Tests; Blood Volume; Caliber; capillary; 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; instrument; Label; Laboratories; Link; Love; mass spectrometer; Mass Spectrum Analysis; Membrane; metabolomics; Methods; Microfluidics; migration; miniaturize; nanoparticulate; nanoscale; neonate; Nurses; particle; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Physicians; Plasma; Population; Postage Stamps; Preparation; Process; Protein Isoforms; protein metabolite; Proteins; Proteomics; public health relevance; Reagent; Recovery; Research; Research Contracts; sample collection; Sampling; Serum; Site; small molecule; Solutions; Spottings; Staging; Structure; Suggestion; System; Technology; Testing; Therapeutic; Time; Training; Venipunctures; wasting; Water; Work

LC-MS provides superior data in clinical diagnostics relative to immunoassays, but it is slow and costly. As the old saying goes, time is money. The technique must become simpler, faster, and less expensive to displace older, less accurate immunological assay (IA) methods. The focus of this proposal is to make LC-MS analytics more competitive in clinical diagnostics by increasing throughput 10 fold with existing instrumentation. The objective in Phase I was to use analyte sequestering transport particles (ASTPs) to circumvent the above limitations by i) structure specifically sequestering analytes of interest, ii) separating them from non-analytes within a min, ii) rapidly transferring them to an MS, and iv) enabling the MS to identify all analytes of interest in less than 60 sec. This was achieved by preparing 2 megadalton (mDa) ASTPs that sequester analytes at their surface; elevating their effective Mw to 2 mDa and allowing them to be purified by mobile affinity sorbent chromatography (MASC) in 60 sec. Thermal or solvent dissociation was used to recover analytes for ESI-MS or LDTD-MS wells. This proposal has four specific aims. One is to complete manufacturing protocols for antibody and protein A/G based ASTP products that capture, resolve, and transport analytes to MS or fluorescence instruments in research, big pharma, and CRO laboratories. The second is to integrate the above ASTP products into miniature membrane laboratories (MemLabs); producing point-of-collection products that extract plasma from a drop of blood and prepare samples in situ for biomarker detection. The third aim was to evaluate and optimize the release and detection of analytes from ASTPs via LDTD-MS, MALDI-MS, and ESI-MS. The fourth specific aim is to adapt ASTP technology to multiplexed drug testing and the analysis of protein i) primary structure, ii) post-translational modifications, and iii) conformation.

Public Health Relevance Statement:
PROJECT NARRATIVE Delivery of high quality medicine without bankrupting the economy is of major importance today; mass spectrometry (MS) based assays being a good example. Although MS is inherently high throughput, sample preparation is slow and costly. The significance of this proposal is in making high quality MS based immunoassays widely available to the life science world by eliminating conventional sample preparation laboratories and reducing analysis costs 10X. This will be achieved by advances in sample collection, preparation, and throughput that circumvent conventional mode of preparing samples for LC-MS; among the more innovative being 1) a miniature membrane laboratory bearing 2) nanoparticulate analyte sequestering transport particles that select clusters of targeted biomarkers and rapidly transport them through 3) mobile sorbent affinity chromatography (MASC) to an MS. The net effect of implementing these technologies is that superior MS assay methods will be costs competitive with conventional immunoassays.

Project Terms:
Affinity; Affinity Chromatography; Aliquot; Antibodies; base; Biological Assay; Biological Markers; Biological Sciences; Blood; C-reactive protein; Cells; Chromatography; clinical diagnostics; Clinical Research; Collection; Complex; cost; Cost Analysis; Coupled; Data; Detection; detector; Diagnostic; Dissociation; Drops; drug testing; Excision; Fingers; Fluorescence; Funding; Goals; GTP-Binding Protein alpha Subunits, Gs; Immunoassay; Immunology procedure; In Situ; innovation; instrument; instrumentation; interest; ionization; Laboratories; Mass Spectrum Analysis; Medicine; Membrane; Methods; Molecular Conformation; nanoparticulate; particle; Performance; Phase; Plasma; Post-Translational Protein Processing; Preparation; Protein Analysis; Proteins; Protocols documentation; Recovery; Research; Resolution; reversed phase chromatography; Route; sample collection; Sampling; Series; Solvents; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; Surface; System; targeted biomarker; Techniques; Technology; Time; tool; Variant