Journal of the American Chemical Society 68.84 mg of O-benzotriazole-N,N,N0,N0-tetramethyl-uronium-hexa- fluorophosphate (HBTU, Peptides International), 24.52 mg of N- hydroxybenzotriazole (HOBT, GL Biochem), and 40 μL of N-methyl- morpholine (Nmm; Fluka Biochemika) were added to the particle suspension and reacted at room temperature at medium stirring rate for 5 min under a nitrogen atmosphere. After 5 min, an appropriate amount of dye (molar ratio of dye/AAc = 10:1) dissolved in DMF was added to the three-neck round flask. The reaction was maintained under nitrogen atmosphere for 6 h. Poly(NIPAm/dye) particles were washed five times by centrifugation (19,000 rpm, 50 min, 25 C) with decreasing concentra- tions of DMF (90%, 75%, 50%, 25%, and 5%) in water in order to eliminate unreacted dye and to favor particle rehydration. Supernatants were discarded, and the particles were resuspended in 10 mL of water. Synthesis of Poly(NIPAm-co-VSA) Nanoparticles. NIPAm (0.475 g, 0.0042 mol), BIS (0.040 g, 0.026 mol), and vinyl sulfonic acid (VSA; Sigma-Aldrich, 0.80.4 μL, 0.00073 mol) were dissolved in 60 mL of H2O, filtered using a nitrocellulose membrane disk filter (0.45 μm pore size), and transferred into a three-neck round-bottom flask. The solution was purged with nitrogen for 1 h at room temperature, at medium stir rate, and then heated to 70 C. KPS (0.028 g, 0.0001 mol) was dissolved in 2.5 mL of H2O and was added to the solution to initiate the polymerization. The reaction was maintained at 70 C under nitrogen for 6 h. The particles were washed five times in water by centrifugation (19,000 rpm, 50 min, 25 C) and then resuspended in 60 mL of H2O. Poly(NIPAm/CB) Core Poly(NIPAm-co-VSA) Shell Nano- particle Preparation. Poly(NIPAm-co-Allylamine, AA) particles were prepared as follows. NIPAm (0.89 g, 7.83 mmol) and BIS (0.042 g, 0.27 mmol) were dissolved in 30 mL of water and then passed through a 0.2 μm nylon membrane filter. The solution was purged with nitrogen for 15 min at room temperature with a medium stirring rate. AA (Sigma, 0.051 g, 0.90 mmol) was added; the solution was purged with nitrogen for 15 min and then heated to 70 C. KPS (0.0070 g, 0.025 mmol) was dissolved in 1.0 mL of water and was added to the solution to initiate the polymerization. The reaction was maintained at 70 C under nitrogen for 3 h. The reaction was subsequently allowed to cool to room temperature and stirred overnight. Particles were washed five times by centrifugation (19,000 rpm, 50 min, 25 C) with water in order to eliminate the unreacted monomer and then resuspended in 30 mL of H2O. In order to incorporate Cibacron Blue F3GA (CB) dye, CB (0.76 g, 0.90 mmol) was dissolved in 10 mL of 0.1 M aqueous sodium car- bonate. The poly(NIPAm-co-AA) particle suspension (10 mL volume) was purged with nitrogen for 15 min with a medium stirring rate in a three-neck round-bottom flask. Solid sodium carbonate (0.106 g, 1.0 mmol) was added to the suspension. The suspension was then stirred at room temperature under nitrogen for about 1 min. The CB solution was then added to the poly(NIPAm-co-AA) particle suspen- sion, and the combined reaction mixture was then stirred at room temperature under nitrogen for 48 h. The resulting poly(NIPAm/CB) particles were harvested and washed using centrifugation (19,000 rpm, 50 min, 25 C). In order to build a VSA shell on the poly(NIPAm/CB) core, 20 mL of poly(NIPAm/CB) particle suspension was heated at 70 C and purged with nitrogen for 1 h. NIPAm (0.156 g, 1.38 mmol), BIS (0.013 g, 0.084 mmol), VSA (26 ul, 0.334 mmol), and KPS (0.092 g, 0.328 mmol) were dissolved in 20 mL of water and passed through a 0.2 μm nylon membrane filter. Three milliliters of this solution was added to the poly(NIPAm/CB) particle suspension, and the remaining 17 mL of solution was added in aliquots of 3 mL every 5 min. Poly(NIPAm/CB) core poly(NIPAm-co-VSA) shell particles were washed five times in water by centrifugation (19,000 rpm, 50 min, 25 C). Nanoparticle Characterization. The temperature dependence of particle size was determined via photon correlation spectroscopy (Submicrometer Particle Size Analyzer, Beckman Coulter). The measure- ment of particle diameter was performed using water as a diluent (refractive ARTICLE index (RI) = 1.333, diluent viscosity = 0.890 cP). The test angle was 90 . Average values were calculated for three measurements using a 200 s integration time, and the solutions were allowed to thermally equilibrate for 10 min before each set of measurements. Measured values were then converted to particle sizes via the Stokes Einstein relationship.34 The particles were further characterized by atomic force microscopy (AFM) using an NSCRIPTOR DPN System (NanoInk). Particles in suspension (1 μg/mL) were deposited on freshly cleaved mica in a humid atmosphere for 15 min and dried under nitrogen before measurement. Images were acquired under AC mode using a silicon tip with a typical resonance frequency of 300 kHz and a radius smaller than 10 nm. Incorporation of dye in the particles was assessed by weighing the dry particles before and after dye coupling. Nanoparticle Incubation with Serum, Plasma, Blood, and Protein Solutions. Aliquots of 500 μL of serum and EDTA plasma were diluted 1:2 with 50 mM TrisHCl pH 7, mixed with 500 μL of hydrogel particles functionalized with the molecular baits described above, and incubated for 15 min at room temperature. Particles were separated by centrifugation (16.1 rcf, 25 C, 10 min) and washed with buffers specific for the molecular baits listed in Table 1. The particle pellet was incubated for 15 min at room temperature with 600 μL of elution buffer specific for the molecular baits listed in Table 1 and then centrifuged (16.1 rcf, 25 C, 10 min). The elution step was repeated twice, and the eluates were combined in the same vial and then dried in a vacuum concentrator. Blood was collected in EDTA-containing blood collection tubes (Vacutainer EDTA Tubes, Becton Dickinson). Aliquots of 1 mL of blood were diluted 1:2 with physiologic solution (0.9% NaCl). One milliliter of hydrogel particle suspension was added to the blood and incubated for 15 min. In order to separate the blood cells, the blood particle mixture was centrifuged (200 rcf, 25 C, 10 min), and the plasma supernatant was saved. The plasma was then centrifuged at higher speed (16.1 rcf, 25 C, 10 min), the supernatant was discarded, and the particle pellet was washed and eluted as previously described, using appropriate buﬀers for each type of particle (Table 1). Proteins (human growth hormone (Lilly), hepatocyte growth factor (Antigenix America Inc.), ﬁbroblast growth factor 22 (Cell Sciences), vascular endothelial growth factor (Cell Signaling Technologies), eotaxin 2 (AbD Serotec), IGF binding protein 7 (IGFBP7) (Preprotech), interleukin-6 (AbD Serotech), chemokine (C C motif) ligand 22 (CCL22) (R&D Systems), survivin (Novus Biologicals), B-cell lym- phoma 2 (bcl-2) (Fisher Scientiﬁc), osteopontin (Novus Biologicals), troponin-I (AbD Serotec), and kallikrein-6 (ARP American Research Products)) were each dissolved in 25 μL of 50 mM TrisHCl pH 7 at a concentration of 0.05 μg/μL, mixed with 25 μL of particle suspension, and incubated for 15 min at room temperature. Particles were separated by centrifugation (16.1 rcf, 25 C, 10 min), washed with water (16.1 rcf, 25 C, 10 min), and analyzed by SDS PAGE. Interleukin-8 (IL8) was dissolved in synthetic sweat (Corkig) at a concentration of 0.05 μg/μL, and the pH of the solution was adjusted to 8 with 1 M NaOH. Trypsin (Promega) was added to the IL8 solution at a 100:1 protein to protease w/w ratio. Poly(NIPAm/DY9) particles were added to the IL8, trypsin-containing solution and the mixture left to incubate for 3 h at 37 C. Aliquots of 100 μL of poly(NIPAm-co-VSA) particles were mix- ed with 2 μg of trypsin-digested human serum albumin (Michrom Bioresources) and allowed to incubate for 30 min. Particles were washed and eluted with the buﬀers indicated in Table 1. The eluate was dried in a SpeedVac and injected into an LTQ-Orbitrap mass spectrometer (ThermoFisher). Isoelectric points and molecular weights of trypsin- generated peptides derived from albumin were calculated online at http://isoelectric.ovh.org/. Immunoassay Measurement of Troponin-I. Poly(NIPAm/ DB3) and poly(NIPAm/RBB) particles were mixed with a solution of troponin-I (in 50 mM Tris HCl pH 7), incubated for 15 min at room 19181 dx.doi.org/10.1021/ja207515j |J. Am. Chem. Soc. 2011, 133, 19178–19188