ARTICLE pubs.acs.org/JACS Shell Nanoparticles: Discovery of Previously Multifunctional Core Invisible Biomarkers Davide Tamburro,†,‡,§ Claudia Fredolini,†,‡,|| Virginia Espina,† Temple A. Douglas,† Adarsh Ranganathan,† ‡ † † † Leopold Ilag, Weidong Zhou, Paul Russo, Benjamin H. Espina, Giovanni Muto, Emanuel F. Petricoin, III, Lance A. Liotta,† and Alessandra Luchini*,† † †Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia 20110, United States ‡Department of Analytical Chemistry, Stockholm University, Stockholm 106 91, Sweden §Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanita, Rome 00161, Italy ^Department of Urology, S. Giovanni Bosco Hospital, Turin 10154, Italy D)epartment of Medicine and Experimental Oncology, University of Turin, 10125 Turin, Italy bS Supporting Information ABSTRACT: Many low-abundance biomarkers for early detection of cancer and other diseases are invisible to mass spectrometry because they exist in body ﬂuids in very low concentrations, are masked by high-abundance proteins such as albumin and immunoglo- bulins, and are very labile. To overcome these barriers, we created porous, buoyant, core shell hydrogel nanoparticles containing novel high aﬃnity reactive chemical baits for protein and peptide harvesting, concentration, and preservation in body ﬂuids. Poly(N- isopropylacrylamide-co-acrylic acid) nanoparticles were functionalized with amino-containing dyes via zero-length cross-linking amidation reactions. Nanoparticles functionalized in the core with 17 diﬀerent (12 chemically novel) molecular baits showed preferential high aﬃnities (KD < 10 11 M) for speciﬁc low-abundance protein analytes. A poly(N-isopropylacrylamide-co-vinylsulfonic acid) shell was added to the core particles. This shell chemistry selectively prevented unwanted entry of all size peptides derived from albumin without hindering the penetration of non-albumin small proteins and peptides. Proteins and peptides entered the core to be captured with high aﬃnity by baits immobilized in the core. Nanoparticles eﬀectively protected interleukin-6 from enzymatic degradation in sweat and increased the eﬀective detection sensitivity of human growth hormone in human urine using multiple reaction monitoring analysis. Used in whole blood as a one-step, in-solution preprocessing step, the nanoparticles greatly enriched the concentration of low-molecular weight proteins and peptides while excluding albumin and other proteins above 30 kDa; this achieved a 10,000-fold eﬀective ampliﬁcation of the analyte concentration, enabling mass spectrometry (MS) discovery of candidate biomarkers that were previously undetectable. ’ INTRODUCTION A critical need within the biochemical and biomedical research sector is the identiﬁcation of low-abundance biomarkers that are predictive of early-stage disease or that correlate with therapeutic outcome or toxicity.1 While the interest in the potential and value of biomarkers has never been greater, the research investment in biomarker discovery and clinical validation has yielded a very poor return to date.1,2 This poor return is due in part to the low abundance of early disease biomarkers that exist at a concentra- tion below the detection limit of biomarker discovery platforms. Protein biomarker discovery and quantitation by mass spectro- metry (MS) and multiple reaction monitoring (MRM) are powerful approaches1,2 but are severely limited in their practical application for complex clinical samples because of their low eﬀective sensitivity.3 The analyte detection sensitivity for MS or MRM applied directly to a complex body ﬂuid is typically greater than 50 ng mL 1.4 In contrast, the vast majority of diagnostic analytes measured in the clinical laboratory by immunoassay platforms fall in the range between 50 pg mL 1 and 10 ng mL 1 5 . Thus, the most important protein biomarkers, particularly those derived from early-stage disease,6 are invisible to conven- tional MS or MRM.7 MS and MRM lack practical sensitivity because of technical and physiological constraints. The volume of sample introduced into the MS is very small (<1 μL) containing only a small number of target analyte molecules and thereby limiting the detection sensitivity. In addition, low-abundance, low- molecular weight proteins and peptides are masked by billion- fold excess quantities of resident proteins such as immunoglo- bulin and albumin. The MS input sample is strictly limited in the maximum total protein (<50 μg) content, a value lower than the serum or plasma protein content in 1 μL, the volume of the input sample for the mass spectrometer. Consequently, increasing the sensitivity is not simply a matter of concentrating the sample because this will overwhelm the total protein capacity introduced into the MS. A further barrier to biomarker discovery is the Received: August 9, 2011 Published: October 14, 2011 r 2011 American Chemical Society 19178 dx.doi.org/10.1021/ja207515j | J. Am. Chem. Soc. 2011, 133, 19178–19188