Journal of the American Chemical Society ARTICLE Figure 1. (A) Schematic representation of core shell hydrogel nano- particles. (B) Porous nature of the hydrogel particle shell has an eﬀective pore size that acts as a molecular sieve with a cutoﬀ determined by the cross-link density. Proteins small enough to enter the particle are captured by the high-aﬃnity bait. The high aﬃnity permits very rapid sequestration of target analytes and dissociation from carrier proteins. The shell contains a chemical species (vinylsulfonic acid, VSA) that selectively prevents entry of contaminating albumin-derived peptides of all sizes without hindering the capture of non-albumin proteins and peptides small enough to enter the particle. (C) Nanoparticles can be produced in large quantities with uniform size distribution (atomic force microscopy image). lability and perishability of candidate biomarkers ex vivo follow- ing clinical sample collection. Diagnostic proteins and peptides in body ﬂuids are subject to rapid enzymatic degradation following collection.8 In the present study we show that modiﬁed hydrogel nanoparticles can provide a means to overcome all of these technical and physiological barriers and signiﬁcantly improve the sensitivity of detection, and the preservation, of candidate bio- markers in a single step. Hydrogel nanoparticles have been extensively described in the literature as good candidate materials for drug delivery9 12 and in vivo imaging purposes.13 In contrast to their application as drug delivery vehicles, we have proposed the use of hydrogel nanoparticles for sequestering rather than releasing target mole- cules.14 19 In this study, open-meshwork core shell hydrogel nanopar- ticles were functionalized with combinations of novel organic dye baits. Speciﬁc dye chemistries immobilized in the particle core possess extremely high aﬃnity for select classes of proteins and peptides (Figure 1). Unwanted high-molecular weight proteins such as albumin and immunoglobulins are excluded from enter- ing the particles because of the restrictive, eﬀective pore size of the hydrogel that acts as a molecular sieve. Albumin fragments are also an unwanted contaminant present in the blood.20 To further exclude unwanted contaminating albumin fragments small enough to penetrate the shell, the shell contains a chemical species (vinylsulfonic acid, VSA) that selectively prevents entry of albumin-derived peptides of all sizes (Figure 1), without hindering the capture of non-albumin proteins and peptides small enough to enter the particle. Figure 2. High-aﬃnity chemical baits covalently incorporated into hydrogel nanoparticle cores or shells. ABB, DB3, PR1, AB48, DY3, AB1, DO3, DY9, R12, TBO, PR, and AB2 have not been previously used for protein aﬃnity chromatography. Some classes of organic dyes (indocyanine green,21 Cibacron Blue F3GA,22 Coomassie Blue,23 triarylmethane dyes,24 eosin dyes25) have been used in the past as protein binding agents and inhibitors of protein protein interactions.26 Cibacron Blue F3GA and related dyes are most extensively studied as ligands in aﬃnity chromatography. A widely accepted mechanism that has been proposed to explain the high-aﬃnity binding of Cibacron Blue F3GA and related dyes to proteins is based on hydrophobic and electrostatic forces.27 The speciﬁcity of the dye protein interaction depends mainly on hydrophobic inter- actions, whereas electrostatic forces contribute to the stability of interaction.27 The dye binding site on the surface of the target protein is a nonpolar pocket surrounded by hydrophilic amino acid residues.28 Moreover, the protein binding sites are often located in areas overlapping with the binding sites of biological ligands with respect to other parts of the protein surface.29 Organic reactive dyes can insert aromatic rings into nonpolar hydrophobic pockets of the protein surface, while the ﬂanking portions of the dye and protein molecules can rearrange, depending on energy constraints. Since both active protein sites and dye molecules have limited conformational freedom, some speciﬁcity is achieved.30 Increasing the size of the dye molecule ring system can enhance the speciﬁcity.28 For example, Cibacron Blue F3GA forms a strong bond with speciﬁc classes of enzymes (dehydro- genases and kinases) because the 1-amino-4-(40-aminopheny- lamino)-anthraquinone-2,30-disulfonic acid part of the dye ﬁts well to a structural element of the enzyme called the dinucleotide 19179 dx.doi.org/10.1021/ja207515j |J. Am. Chem. Soc. 2011, 133, 19178–19188