Journal of the American Chemical Society Figure 3. One step, in solution aﬃnity capture and separation of low- abundance target biomarkers in whole blood (plasma). Core shell hydrogel particles are introduced into the Vacutainer collection tube and are allowed to incubate with blood for 15 min. Low-speed centrifugation is used to pellet the blood cells, while buoyant, open-meshwork hydrogel particles remain in suspension in the plasma. High-speed centrifugation is then used to separate the particles from the plasma. Proteins are eluted from the nanoparticle pellet and analyzed by mass spectrometry (MS), multiple reaction monitoring (MRM), or any other analytical platform (e.g., immunoassay). binding fold.31 In addition to high speciﬁc binding of some classes of proteins, Cibacron Blue F3GA also forms nonspeciﬁc complexes with additional proteins. This latter attribute is most likely due to the ability of the dye to act as weak cation exchanger through its sulfonic groups.28 Most of the bait dyes used in this work have never been used for aﬃnity chromatography or protein capture in the past and were found to have high aﬃnity for select classes of proteins (see Figure 2). In fact it can be hypothesized, on the basis of the proposed mechanism of binding, that all ionic aromatic com- pounds, separate from Cibacron Blue F3GA and related dyes, should be capable of binding all classes of proteins and peptides that have a suﬃciently large exposed hydrophobic region.28 Dye-functionalized nanoparticles can be introduced into whole blood at the time of collection (Figure 3). In one step, in solution, and in minutes, the nanoparticle (a) aﬃnities capture all the target analytes in the entire ﬂuid phase (plasma), (b) strip target analytes oﬀ of the carrier proteins, (c) protect the captured target protein from degradation, and (d) exclude all size classes of albumin-derived peptides. The buoyant, open-meshwork, sphe- rical nanoporous particles (0.5 0.7 μm diam) remain as a colloid in suspension in the ﬂuid phase and are in open exchange with the surrounding plasma. Target molecules not excluded by the shell freely enter the hydrogel inner meshwork to be captured by the bait(s). The separation process takes advantage of the enormous surface area aﬀorded by the nanoparticles. The open- meshwork hydrogel particles are 95% occupied by the sur- rounding solution phase and do not have the disadvantages of boundary layers, pressure gradients, and volume requirements of aﬃnity bead columns. Since the nanoparticles do not settle by gravity, the plasma containing the hydrogel particles can be separated from the cellular elements of the blood by low-speed centrifugation (Figure 3). ’ EXPERIMENTAL SECTION Nanoparticle Synthesis and Dye Coupling. Hydrogel nano- particles, poly(N-isopropylacrylamide-co-acrylic acid) (poly(NIPAm- co-AAc)), were synthesized by precipitation polymerization32 and covalently functionalized with amino-containing dyes using amida- tion chemistry.33 Zero-length cross-linking amidation strategies were ARTICLE performed in water or organic solvents on the basis of the hydrophilic/ hydrophobic properties of the dyes. Amine-containing hydrogel nano- particles, poly(NIPAm-co-AA), were created by precipitation polymer- ization and covalently functionalized with Cibacron Blue F3GA (CB) dye by nucleophilic substitution of the amine groups in the nanoparticles and a chloride atom in the CB dye. An outer shell containing vinylsulfonic acid (VSA) copolymer was created on the dye-functionalized particles by a second polymerization reaction as described below. Poly(NIPAm-co-AAc) Nanoparticle Synthesis. N-Isopropyla- crylamide (NIPAm, Sigma-Aldrich, 4.750 g, 0.042 mol), N,N0-methy- lene bisacrylamide (BIS Sigma-Aldrich, 0.400 g, 0.0026 mol), and acrylic acid (AAc, Sigma-Aldrich, 0.525 μL, 0.0073 mol) were dissolved in 500 mL of H2O, filtered using a nitrocellulose membrane disk filter (0.45 μm pore size, Millipore), and transferred in a three-neck round-bottom flask. The solution was purged with nitrogen for 1 h at room tempera- ture, at medium stirring rate, and then heated to 70 C. Potassium persulfate (KPS, Sigma-Aldrich, 0.276 g, 0.001 mol) was dissolved in 5 mL of H2O and was added to the solution to initiate the polymeri- zation. The reaction was maintained at 70 C under nitrogen for 6 h. Particles were washed five times by centrifugation (19,000 rpm, 50 min, 25 C) to eliminate the unreacted monomer and then resuspended in 600 mL of H2O. Functionalization of Poly(NIPAm-co-AAc) Nanoparticles with Amino Dyes in Water. Dye molecules containing an amine group were coupled by condensation to the carboxylic group of acrylic acid present in the poly(NIPAm-co-AAc) particles. The reaction was conducted in a water-based solvent for the following dyes: Acid Blue 22 (AB2, Sigma), Remazol Brilliant Blue R (RBB, Sigma), Acid Black 1 (AB1, Sigma), Brilliant Blue R250 (BB, Fisher), Toluidine Blue O (TBO, J.T. Baker), Rhodamine 123 (R12, Sigma), Disperse Yellow 3 (DY3, Fluka), and Disperse Blue 3 (DB3, Sigma). A preliminary activation of the carboxylic group present in the nanoparticles was performed. Briefly, 10 mL of the poly(NIPAm-co-AAc) particle suspen- sion was centrifuged (16.1 rcf, 25 C, 15 min), the supernatant was discarded, and the particle pellet was resuspended in 10 mL of 0.2 M NaH2PO4 pH 5. The particle suspension was transferred in a three-neck round flask, and to this were added 1 mL of 1% SDS (w/v), 824 mg of N-(3 dimethylaminopropyl)-N0-ethyl carbodiimide hydrochloride (EDC; Fluka Analitical), and 612 mg of solid N-hydroxy succinimide (NHS; Sigma-Aldrich). The reaction was held at room temperature and medium stirring rate for 15 min. Then, the suspension was centrifuged (19,000 rpm, 50 min, 25 C), the supernatant was discarded, and the particle pellet was resuspended in 20 mL of 0.2 M Na2HPO4 pH > 8. After this activation step, an appropriate amount of dye (molar ratio of dye/acrylic acid 10:1) was dissolved in 180 mL of 0.2 M Na2HPO4 buffer pH > 8, that was filtered by 0.22 μm CA filter (Corning) and added to the activated particles; the reaction was held at room temperature at medium stirring rate overnight. The number of acrylic acid moles in 10 mL of particle suspension was 0.0000121. In order to eliminate the unreacted dye, poly(NIPAm/dye) particles were washed five times with water by centrifugation (19,000 rpm, 50 min, 25 C). Supernatants were discarded, and particles were resuspended in 10 mL of water. Functionalization of Poly(NIPAm-co-AAc) Nanoparticles with Amino-Dyes in Dimethylformamide (DMF). The amida- tion reaction was conducted by phosponium/uranium activation in dimethylformamide (DMF) for the following dyes: Disperse Orange 3 (DO3, Sigma), Pararosaniline Base (PR, Sigma), Disperse Yellow 9 (DY9, Sigma), Pigment Red 177 (PR1, International Laboratory U.S.A.), and Acid Black 48 (AB4, Acros Organic). Briefly, 10 mL of poly(NIPAm-co-AAc) nanoparticles (acrylic acid moles = 0.0000121) was freeze-dried and resuspended in 10 mL of DMF (Sigma-Aldrich) in a three-neck round flask. The particle suspension was purged with nitrogen for 15 min at room temperature under medium stirring. Then, 19180 dx.doi.org/10.1021/ja207515j |J. Am. Chem. Soc. 2011, 133, 19178–19188