Journal of the American Chemical Society ARTICLE Figure 5. Nanoparticles functionalized with 17 diﬀerent molecular baits (poly(NIPAm/ABB), poly(NIPAm/DB3), poly(NIPAm/RBB), poly- (NIPAm/PR1), poly(NIPAm/AB4), poly(NIPAm-co-VSA), poly(NIPAm/ DY3), poly(NIPAm/AB1), poly(NIPAm/DO3), poly(NIPAm/DY9), poly(NIPAm/R12), poly(NIPAm/TBO), poly(NIPAm-co-AAc), poly- (NIPAm/PR), poly(NIPAm/BB), poly(NIPAm/AB2), poly(NIPAm/ CB)) show preferential high aﬃnities for 13 speciﬁc low-abundance protein analytes as indicated by the color of the cell. The expected concentrations of the indicated proteins in serum/plasma were derived from the literature.36 48. ’ RESULTS AND DISCUSSION We identiﬁed a series of small novel organic dye molecules possessing extremely high protein-binding aﬃnity (KD < 10 11 M, Figure 2). These dyes act as molecular baits by binding proteins and peptides, likely through a combination of hydro- phobic and electrostatic forces, by inserting their aromatic rings into hydrophobic pockets present on the protein surfaces.30 We immobilized the baits by binding amino groups in the dyes to carboxylic groups in the particles (Figure 4 shows the changes in the hydrodynamic volume after dye functionalization). Zero- length cross-linking amidation strategies were used and opti- mized on the basis of hydrophilic/hydrophobic dye properties.33 As shown in Figure 5 when hydrogel nanoparticles containing one of 17 diﬀerent classes of organic chemistries were screened against a panel of 13 known low-abundance diagnostically relevant biomarker proteins, strong bait selectivity for speciﬁc proteins or classes of proteins was noted. For example, hepato- cyte growth factor (HGF) was captured by poly(NIPAm/DY3) particles and excluded by poly(NIPAm/DB3) particles (see also Figures 6 and 7). We determined that the aﬃnity of dye protein binding reactions was dependent on the type of reactive group substitu- tion (Figure 8A). Troponin-I, a biological marker for cardiac muscle tissue injury, is present in the blood at low concentrations (5 pg/mL),47 below the detection limit of routine mass spectro- metry. Remazol brilliant blue R (RBB)-functionalized particles sequestered more than 99.9% of troponin-I present in solution (estimated dissociation constant KD < 1.1 10 11 M) so that the troponin-I concentration in the supernatant outside the particle at equilibrium (<10 min) was reduced below the detec- tion limit (50 pg/mL) of the Immulite clinical immunoassay. Modiﬁcation in the chemical group substitution (keeping the bait molecules equimolar) reduced the capture eﬃciency due to a 10- fold reduction in the KD (2.4 10 10 M, Figure 8A). The advantage of such high-aﬃnity binding dye chemistries is several fold: (a) high ON/OFF rate ratio (estimated 9.2 1010 M 1 Figure 6. Heat map representation of serum proteins captured by nanoparticles functionalized with diﬀerent molecular baits (poly(NIPAm/- RBB), poly(NIPAm/CB), poly(NIPAm-co-VSA), poly(NIPAm/DY9), poly(NIPAm/DO3), poly(NIPAm/DY3), poly(NIPAm/PR1), poly- (NIPAm/AB4), poly(NIPAm/BB), poly(NIPAm-co-AAc), poly(NIPAm/ AB1), poly(NIPAm/ABB). Dyes incorporated in the particles are shown on the X axis, proteins identiﬁed with MS analysis of serum preprocessed with the particles are shown in the Y axis. Each dye bait is represented by a diﬀerent color. Example low-abundance proteins uniquely captured by speciﬁc dye baits are highlighted. Figure 7. Poly(NIPAm/DY9) and poly(NIPAm/DO3) particles cap- ture unique groups of proteins from serum. Addition of two types of hydrogel particles complement each other by combining their respective protein repertoire. Example low-abundance proteins are highlighted in the boxes. for poly(NIPAm/RBB) nanoparticles and 4.2 109 M 1 for poly(NIPAm/DB3)) nanoparticles achieves rapid complete 19183 dx.doi.org/10.1021/ja207515j |J. Am. Chem. Soc. 2011, 133, 19178–19188