Disease-associated blood biomarkers exist in exceedingly low concentrations within complex mixtures of high-abundance proteins such as albumin. We have introduced an affinity bait molecule into N-isopropylacrylamide to produce a particle that will perform three independent functions within minutes, in one step, in solution: a) molecular size sieving b) affinity capture of all solution phase target molecules, and c) complete protection of harvested proteins from enzymatic degradation. The captured analytes can be readily electroeluted for analysis.There is an urgent need to discover novel biomarkers that provide sensitive and specific disease detection1 , 2. Cancer is rapidly becoming the leading cause of death for many population groups in the United States, largely due to the fact that the disease is usually diagnosed after the cancer has metastasized and treatment is ineffective. It is widely believed that early detection of cancer prior to metastasis will lead to a dramatic improvement in treatment outcome. Biomarkers are nucleic acids, proteins, protein fragments or metabolites indicative of a specific biological state, that are associated with the risk of contraction or presence of disease3. Biomarker research has revealed that low-abundance circulating proteins and peptides present a rich source of information regarding the state of the organism as a whole 4 . Two major hurdles have prevented these discoveries from reaching clinical benefit: 1) disease-relevant biomarkers in blood or body fluids may exist in exceedingly low concentrations within a complex mixture of biomolecules and could be masked by high-abundance species such as albumin, and 2) degradation of protein biomarkers can occur immediately following the collection of blood or body fluid as a result of endogenous or exogenous proteinases. The goal of this study was to create "smart" nano-particles that allow enrichment and encapsulation of selected classes of proteins and peptides from complex mixtures of biomolecules such as plasma, and protect them from degradation during subsequent sample handling. The captured analytes can be readily extracted from the particles by electrophoresis allowing for subsequent quantitative analysis. This nanotechnology provides a powerful tool that is uniquely suited for the discovery of novel biomarkers for early stage diseases such as cancer.SUPPORTING INFORMATION AVAILABLE: Available in the Supplementary Information are details on particles synthesis protocol, SDS PAGE analysis on molecular sieving properties and enzymatic degradation, and tables (Table S1 and S2) listing proteins (with peptide coverage lists) identified via LC-MS/MS (ESI) on material electroeluted from NIPAm and NIPAm/AAc particles. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public AccessAuthor Manuscript Nano Lett. Author manuscript; available in PMC 2010 May 28. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe concentration of proteins and peptides comprising t...
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