Multifunctional Core–Shell Nanoparticles: Discovery of Previously 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 Sanità, Rome 00161, Italy

Department of Urology, S. Giovanni Bosco Hospital, Turin 10154, Italy

Department of Medicine and Experimental Oncology, University of Turin, 10125 Turin, Italy

J. Am. Chem. Soc., Article ASAP

DOI: 10.1021/ja207515j

Publication Date (Web): October 14, 2011

Copyright © 2011 American Chemical Society

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 immunoglobulins, 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 (K_D < 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.