Nanoparticle Conjugation Increases Protein Partitioning in Aqueous

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Anal. Chem. 2006, 78, 379-386

Nanoparticle Conjugation Increases Protein Partitioning in Aqueous Two-Phase Systems M. Scott Long and Christine D. Keating*

Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802

We describe the effect of bioconjugation to colloidal Au nanoparticles on protein partitioning in poly(ethylene glycol) (PEG)/dextran aqueous two-phase systems (ATPS). Horseradish peroxidase (HRP) was conjugated to colloidal Au nanoparticles by direct adsorption. Although HRP alone had very little phase preference, HRP/Au nanoparticle conjugates typically partitioned to the PEG-rich phase, up to a factor of 150:1 for conjugates of 15-nm colloidal Au. Other protein/Au nanoparticle conjugates exhibited partitioning of greater than 2000:1 to the dextran-rich phase, as compared with ∼5:1 for the free protein. The degree of partitioning was dependent on polymer concentration and molecular weight, nanoparticle diameter, and in some instances, nanoparticle concentration in the ATPS. The substantial improvements in protein partitioning achievable by conjugation to Au nanoparticles appear to result largely from increased surface area of the conjugates and require neither chemical modification of the proteins or polymers with affinity ligands, increased polymer concentrations, nor addition of high concentrations of salts. Adsorption to colloidal particles thus provides an attractive route for increased partitioning of enzymes and other proteins in ATPS. Furthermore, these results point to ATPS partitioning as a powerful means of purification for biomolecule/nanoparticle conjugates, which are increasingly used in diagnostics and materials applications.

Aqueous solutions of polymers such as poly(ethylene glycol) (PEG) and dextran can separate into two distinct aqueous phases, each enriched in one of the polymers. The resulting aqueous twophase systems (ATPS) are biocompatible and have been used for the purification of many types of biological materials, from proteins and nucleic acids to organelles or intact cells.1-4 Biomacromolecules partition between the two phases on the basis of their size and surface chemistry as well as the composition of the ATPS (chemistry; concentration; and molecular weight of the phase* E-mail: [email protected]. (1) Zaslavski, B. Y. Aqueous Two-Phase Partitioning: Physical Chemistry and Bioanalytical Applications; Marcel Dekker: New York, 1995. (2) Walter, H., Johansson, G., Eds. Aqueous Two-Phase Systems; Methods in Enzymology Series; Academic Press: San Diego, 1994; 228. (3) Albertsson, P-Å. Partition of Cell Particles and Macromolecules, 3rd ed.; John Wiley and Sons: New York, 1986. (4) Walter, H., Brooks, D. E., Fisher, D., Eds. Partitioning in Aqueous TwoPhase Systems; Academic Press: Orlando, FL, 1985. 10.1021/ac051882t CCC: $33.50 Published on Web 12/10/2005

© 2006 American Chemical Society

forming polymers; as well as any additives, such as salts or other small molecules).1-4 Partitioning is quantified as the partition coefficient, K, which is defined as the concentration ratio of solute in the top (PEG-rich) phase to that in the bottom (dextran-rich) phase. Not all biomolecules partition well in all ATPS. Indeed, much effort has focused on identifying conditions under which separations in ATPS can be improved.1-4 In general, this entails some combination of the following: (A) increasing the concentration of phase-forming polymers, (B) maximizing the difference in molecular weights of the two polymers, (C) changing pH, (D) increasing the concentration of salts, or (E) incorporation of an affinity ligand on one of the polymers. Perhaps the conceptually simplest and most dramatic improvements in K can be achieved using affinity ligands.5 Although in the absence of affinity ligands, ATPS optimization can be a process of trial and error, extensive experimental results have appeared, and in some cases, it was possible to isolate the effects of protein size, hydrophobicity, or charge.6-13 In addition, some groups have developed theory for understanding and predicting partitioning in ATPS.1,14-16 For example, Johansson et al. have used a modified Flory-Huggins approach to provide a set of simple equations to guide design of ATPS-based separations.14 More recent modifications of FloryHuggins theory to account for solvation of the polymers have appeared.15 A drawback of the experimental methods described above is that they all require changing the ATPS composition. The wide range of variables that impact partitioning in an ATPS may necessitate extensive trial and error to find an ATPS optimized (5) Flanagan, S. D.; Barondes, S. H. J. Biol. Chem. 1975, 250, 1484-1489. (6) Berggren, K.; Wolf, A.; Asenjo, J. A.; Andrews, B. A.; Tjerneld, F. Biochim. Biophys. Acta 2002, 1596, 253-268. (7) Andrews, B. A.; Schmidt, A. S.; Asenjo, J. A. Biotechnol. Bioeng. 2005, 90, 380-390. (8) Forciniti, D.; Hall, C. K.; Kula, M. R. Biotechnol. Bioeng. 1991, 38, 986994. (9) Franco, T. T.; Andrews, A. T.; Asenjo, J. A. Biotechnol. Bioeng. 1996, 49, 300-308. (10) Franco, T. T.; Andrews, A. T.; Asenjo, J. A. Biotechnol. Bioeng. 1996, 49, 309-315. (11) Walter, H.; Fisher, D.; Tilcock, C. FEBS 1990, 270, 1-3. (12) Abbott, N. L.; Blankschtein, D.; Hatton, T. A. Macromolecules 1993, 26, 825-828. (13) Sasakawa, S.; Walter, H. Biochemistry 1972, 11, 2760-2765. (14) Johansson, H.-O.; Karlstrom, G.; Tjerneld, F.; Haynes, C. A. J. Chromatogr., B 1998, 711, 3-17. (15) Pessoa Filho, P. A.; Mohamed, R. S. Process Biochem. 2004, 39, 20752083. (16) Hartounian, H.; Kaler, E. W.; Sandler, S. I. Ind. Eng. Chem. Res. 1994, 33, 2294-2300.

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for a given material of interest. Changes to the concentration of polymers or salts or to the polymer chemistry (i.e., by addition of affinity ligands) will result in different ATPS properties, including interfacial tension, viscosity, osmolarity, or phase behavior. In some cases, such changes are detrimental. For example, in cell separations, it is important to avoid osmotic shock to the cells, limiting the range of usable salt concentrations.4Affinity-labeled polymers can be costly and limit the biomolecules that can be separated to those that bind to available affinity tags. Our laboratory is interested in using ATPS as primitive analogues of the cell cytoplasm.17-19 A key property of ATPS for this work is that temperature or osmotic pressure can be used to induce reversible aqueous phase transitions within giant vesicles (GVs),20 leading to dynamic control over protein microcompartmentation within the GV interior.17 This reversible phase behavior requires certain ATPS compositions, generally at low polymer weight percents.17 There is, therefore, a need to increase partitioning in ATPS GVs without increasing polymer weight percent. We have previously used affinity partitioning (i.e., lectins, which have native affinity for the dextran polymer, and streptavidin, which bound to biotinylated dextrans or PEGs). Here, we introduce a more general approach to improved partitioning in ATPS that does not limit protein selection to those having affinity for the polymers and requires no modification of the ATPS: attaching the biomolecule of interest to colloidal Au nanospheres. Protein/Au bioconjugates have been used for decades as electron-dense probes in transmission electron microscopy of cells and tissue samples.21,22 More recently, biomolecule/Au nanoparticle conjugates have found application as amplification tags in a wide range of bioanalytical applications23-26 and have generated enthusiasm as building blocks for bottom-up assembly.27-29 In the case of protein/Au nanoparticle conjugates, it is common to attach the protein to the nanoparticle through adsorption, typically resulting in some loss of activity.21,30,31 Although more elaborate methods of conjugation have been developed, providing for excellent retention of activity32 or for tunable enzyme inhibition,33 an advantage of the adsorption method is its ease of application to many different proteins.21,22 Biomolecule/nanoparticle conjugates are typically purified after synthesis by centrifugation and resuspension. This process readily separates the conjugates from free proteins or other materials of (17) Long, M. S.; Jones, C. D.; Helfrich, M. R.; Mangeney-Slavin, L. K.; Keating, C. D. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5920-5925. (18) Helfrich, M. R.; Mangeney-Slavin, L. K.; Long, M. S.; Djoko, K. Y.; Keating, C. D. J. Am. Chem. Soc. 2002, 124, 13374-13375. (19) Walter, H.; Brooks, D. E. FEBS Lett. 1995, 361, 135-139. (20) Luisi, P. L., Walde, P., Eds. Giant Vesicles. Perspectives in Supramolecular Chemistry; John Wiley and Sons: West Sussex, 2000; Vol. 6. (21) Colloidal Gold: Principles, Methods, and Applications; Hayat, M. A., Ed.; Academic Press: San Diego, CA, 1989; Vols. 1-3. (22) Geoghegan, W. D.; Ackerman, G. A. J. Histochem. Cytochem. 1977, 25, 1187-1200. (23) (a) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 227, 1078-1080. (b) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757-1760. (c) Park, S.-J.; Taton, A. T.; Mirkin, C. A. Science 2002, 295, 1503-1506. (d) Cao, C. Y. W.; Jin, R.; Mirkin, C. A. Science 2002, 297, 1536-1540. (e) Nam, J.-M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884-1886. (24) (a) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547-1562. (b) Fritzsche, W.; Taton, T. A. Nanotechnology 2003, 14, R63-R73. (c) Penn, S. G.; He, L.; Natan, M. J. Curr. Opin. Chem. Biol. 2003, 7, 609-615. (25) He, L.; Musick, M. D.; Nicewarner, S. R.; Salinas, F. G.; Benkovic, S. J.; Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000, 122, 9071-9077.

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much lower mass, but it does not as readily remove unconjugated nanoparticles or nanoparticles with different biomolecules or different forms of a single biomolecule (e.g., native vs denatured). ATPS have not been explored as a platform for nanoparticle bioconjugate separations, but may offer several advantages. Excellent separations should be possible due to their high sensitivity to differences in bioconjugate surface chemistry, which may reflect different adsorbed biomolecules or different conformations of the same biomolecule. The ATPS compositions optimized for retention of bioactivity (pH, ionic strength, temperature) or other purposes (e.g., reversible phase separations) can still provide excellent purification. In addition, this approach avoids both centrifugation-induced pelleting and exposure to solid chromatographic supports, which may lead to irreversible aggregation or adsorption of marginally stable bioconjugates, respectively. We report the conjugation of proteins, focusing on the enzyme horseradish peroxidase (HRP), to colloidal Au for increased partitioning in PEG/dextran ATPS. HRP/Au nanoparticle bioconjugates typically partitioned to the PEG phase. The magnitude of this increase in partitioning was dependent on the ATPS polymer molecular weight; nanoparticle diameter; polymer concentrations; and in some instances, on nanoparticle concentration in the ATPS. Retention of activity was estimated as ∼25% for HRP conjugated to 15-nm colloidal Au. Gold nanoparticle conjugates of bovine serum albumin (BSA) and protein A exhibited an even larger degree of partitioning than those of HRP (greater than 450:1 and 2200:1 preference for the dextran phase, respectively). EXPERIMENTAL SECTION Chemicals. All reagents, including proteins, were obtained commercially and used as received except where noted. Deionized (26) (a) Lyon, L. A.; Musick, M. D.; Natan, M. J. Anal. Chem. 1998, 70, 51775183. (b) Ni, J.; Lipert, R. J.; Dawson, G. B.; Porter, M. D. Anal. Chem. 1999, 71, 4903-4908. (c) Dequaire, M.; Degrand, C.; Limoges, B. Anal. Chem. 2000, 72, 5521-5528. (d) Yguerabide, J.; Yguerabide, E. E. Anal. Biochem. 1998, 262, 157-176. (e) Schultz, S.; Smith, D. R.; Mock, J. J.; Schultz, D. A. Proc. Natl. Acad. Sci., U.S.A. 2000, 97, 996-1001. (f) Pavlov, V.; Xiao, Y.; Shlyahovsky, B.; Willner, I. J. Am. Chem. Soc. 2004, 126, 11768-11769. (g) Storhoff, J. J.; Lucas, A. D.; Garimella, V.; Bao, Y. P.; Muller, U. R. Nat. Biotechnol. 2004, 22, 883-887. (h) Glynou, K.; Ioannou, P. C.; Christopoulos, T. K.; Syriopoulou, V. Anal. Chem. 2003, 75, 41554160. (i) Hutter, E.; Pileni, M.-P. J. Phys. Chem. B 2003, 107, 6497-6499. (j) Hirsch, L. R.; Jackson, J. B.; Lee, A.; Halas, N. J.; West, J. Anal. Chem. 2003, 75, 2377-2381. (k) Su, M.; Li, S.; Dravid, V. P. Appl. Phys. Lett. 2003, 82, 3562-3564. (l) Levit-Binnun, N.; Lindner, A. B.; Zik, O.; Eshhar, Z.; Moses, E. Anal. Chem. 2003, 75, 1436-1441. (m) Cognet, L.; Tardin, C.; Boyer, D.; Choquet, D.; Tamarat, P.; Lounis, B. Proc. Natl. Acad. Sci., U.S.A. 2003, 100, 11350-11355. (n) Wang, Z.; Lee, J.; Cossins, A. R.; Brust, M. Anal. Chem. 2005, 77, 5770-5774. (27) (a) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (b) Alivisatos, A. P.; Johnsson, K. P.; Peng, X.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P., Jr.; Schultz, P. G. Nature 1996, 382, 609-611. (28) (a) Storhoff, J. J.; Mirkin, C. A. Chem. Rev. 1999, 99, 1849-1862. (b) Niemeyer, C. M.; Simon, U. Eur. J. Inorg. Chem. 2005, 3641-3655. (29) Goodrich, G. P.; Helfrich, M. R.; Overberg, J. J.; Keating, C. D. Langmuir 2004, 20, 10246-10251. (30) Jiang, X.; Jiang, J.; Yin, Y.; Wang, E.; Dong, S. Biomacromolecules 2005, 6, 46-53. (31) Keating, C. D.; Kovaleski, K. M.; Natan, M. J. J. Phys. Chem. B 1998, 102, 9404-9413. (32) Abad, J. M.; Mertens, S. F. L.; Pita, M.; Ferna´ndez, V. M.; Schiffrin, D. J. J. Am. Chem. Soc. 2005, 127, 5689-5694. (33) (a) Verma, A.; Simard, J. M.; Worrall, J. W. E.; Rotello, V. M. J. Am. Chem. Soc. 2004, 126, 13987-13991. (b) Hong, R.; Fischer, N. O.; Verma, A.; Goodman, C. M.; Emrick, T.; Rotello, V. M. J. Am. Chem. Soc. 2004, 126, 739-743. (c) Fischer, N. O.; McIntosh, C. M.; Simard, J. M.; Rotello, V. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5018-5023.

water, resistivity g18.2 MΩ, from a Barnstead NANOpure Diamond unit (Van Nuys, CA) was used for all experiments. Approximately 31- and 50-nm colloidal Au (coefficient of variation 61 and e73 HRP molecules per Au nanoparticle were required for protection from NaCl-induced aggregation (Figure S1). This is in approximate agreement with a literature value of 61 HRP35 and somewhat higher than the amount needed for an estimated monolayer surface coverage (between 37 and 57 molecules of HRP, depending on which side of HRP is adsorbed into the nanoparticle).36 We added an excess (97 HRP/Au, 80 µg/ mL HRP in solution) for preparation of HRP/15-nm Au conjugates in all subsequent studies. More HRP was required to stabilize Au nanoparticles of larger diameter; this is expected on the basis of both the larger surface area of these particles and their reduced stability toward aggregation.37 Flocculation experiments such as those described above do not directly report the Au/protein stoichiometry, but rather give an upper limit, since they cannot distinguish between bound and unbound protein molecules. As such, they give an upper limit to the HRP/Au ratio (i.e., it may be necessary to add more protein molecules to the colloidal Au than a monolayer in order to drive the adsorption process). To determine what fraction of the HRP molecules necessary for stabilization were actually bound to the particles, we compared HRP activity measurements for the sample containing 80 µg/mL HRP (97 HRP/Au) to the supernatant from this sample after removal of HRP/Au bioconjugates by centrifugation. These experiments indicated that essentially all of the ∼73 molecules (97%, or ∼71 molecules) required for stabilization in the flocculation experiments were, in fact, bound to the particles. Protein/Au Bioconjugate Preparation. The citrate reduction method38-40 was used to prepare ∼15-nm (15.3 ( 1.33 nm) and (34) Keating, C. D.; Musick, M. D.; Keefe, M. H.; Natan, M. J. J. Chem. Ed. 1999, 76, 949-956. (35) de Roe, C.; Courtoy, P. J.; Baudhuin, P. J. Histochem. Cytochem. 1987, 35, 1191-1198. (36) This assumes HRP dimensions of 3.6 × 3.6 × 5.6 nm, obtained from the Protein Data Bank, PDB ID 1HCH. (37) Frens, G. Kolloid-Z. Z. Polym. 1972, 250, 736-741. (38) Handley, D. A. In Colloidal Gold: Principles, Methods, and Applications; Hayat, M. A., Ed.; Academic Press: San Diego, CA, 1989; Vol. 1, pp 1332. (39) Grabar, K. C.; Freeman, R. C.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735-743. (40) Frenz, G. Nat. Phys. Sci. 1973, 240, 20-22.

∼13-nm (12.5 ( 1.02 nm) Au nanospheres. One hundred microliters of 40 mM pH 7.4 sodium phosphate buffer was added to a centrifuge tube, followed by 800 µL of Au nanoparticle solution. This solution was vortexed for several seconds, after which 100 µL of protein was added. The concentration of the protein stock solution was 800 µg/mL for 15- and 13-nm colloidal Au, 4 mg/ mL for 31-nm colloidal Au, and 8 mg/mL for 50-nm colloidal Au. These are in excess of that required for complete coverage of the nanoparticles. This solution was vortexed for several seconds, and the protein was allowed to adsorb onto the nanoparticles for 20 min. Maximal surface coverage of HRP on colloidal Au can be estimated at this point through varying the initial concentration of added HRP and adding 1.5 M NaCl to the conjugate solution after adsorption, noting any peak broadening at ∼520 nm. After adsorption, the conjugate solution was centrifuged (20 min at 16000g for 15-nm nanoparticles and larger, 40 min at 16000g for 13-nm nanoparticles) to separate the unbound HRP from the conjugate. The supernatant was removed, and the conjugates were resuspended to 100 µL (8× Au concentration). Unless explicitly noted, 10 such solutions were combined into one tube, and these centrifugation and resuspension steps were repeated once. The final resuspension volume was 100 µL (80× Au concentration).41 The average retention of Au after the typical conjugation, centrifugation, and resuspension steps was ∼80% in the case of 15-nm Au nanoparticle conjugates. Partitioning. Partitioning of colloidal Au and protein/colloidal Au conjugates was determined through UV/vis spectroscopy, adding 30 µL of the conjugate solution to 2970 µL of ATPS at 25 °C. Partitioning of “bare” Au nanoparticles (i.e., capped with citrate reductant remaining from the synthesis rather than with a protein layer) was determined through substituting a Au nanoparticle stock solution in place of some of the water comprising the ATPS.42 Partitioning of fluorophore-tagged proteins was determined through fluorimetry, adding 30 µL of a 0.1-0.5 w/w% protein solution to 2970 µL of ATPS at 25 °C. When fluorophoretagged polymers were partitioned in addition to conjugates (to determine the effect of conjugate concentration on polymer partitioning), protein/Au nanoparticle conjugates were also added, and the Au was dissolved before fluorescence spectral acquisition through adding KCN and K3Fe(CN)6 (final concentrations in sample: 70 mM KCN, 0.72 mM K3Fe(CN)6, ∼1.6 nM nanoparticles) in a manner similar to a literature protocol.43 In such cases, the absorbance due to Au determined through UV/vis spectroscopy disappeared after at most several minutes, as previously reported.43 In all cases, the ATPS solutions were allowed to phase-separate at ∼3 °C overnight. The next day, 200-µL aliquots of both the top (41) These concentration steps were performed to increase absorbance from the conjugate in both phases after partitioning in the ATPS through UV/ vis spectroscopy. This does not aggregate the nanoparticles, as confirmed by UV/vis spectroscopy. Neither are conformational changes exhibited in the adsorbed protein as a consequence; conjugates prepared from one centrifugation step exhibit the same partitioning as conjugates prepared from 10 such solutions that are diluted by a factor of 10 after concentration. When the amount of conjugate in the PEG phase was still too low, an ∼1.5mL aliquot of a PEG-phase sample containing protein-Au nanoparticle conjugate was centrifuged, and most of the supernate was extracted for further concentration of the sample. (42) Concentration of unconjugated Au nanoparticles by centrifugation is not possible because this leads to irreversible aggregation. (43) Demers, L. M.; Mirkin, C. A.; Mucic, R. C.; Reynolds, R. A., III; Letsinger, R. L.; Elghanian, R.; Viswanadham, G. Anal. Chem. 2000, 72, 5535-5541.

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and bottom phases of the ATPS solutions were obtained by pipet for analysis. The partition coefficient, K, was determined by dividing the concentration of the material in the PEG (top) phase by that in the dextran (bottom) phase. All ATPS used were all one phase at 25 °C and two phases at 3 °C/5 °C. All ATPS were also between ∼10 and 30 volume percent dextran phase to keep relative positions along the tie lines1-4 approximately constant. Activity. The general method of HRP activity determination provided by Sigma-Aldrich was followed. A blank spectrum of OPD solution was obtained through UV/vis spectroscopy. Five hundred nanoliters of HRP (either a 500-fold dilution of a standard concentration of HRP or a 100-fold dilution of an 8× HRP/Au nanoparticle conjugate solution) was added to a 1.0-cm quartz cuvette, which was placed in the spectrometer sample holder. Two hundred microliters of OPD was then added, and the acquisition of spectra over 30-s intervals for 2.5 min at 25 °C started immediately. RESULTS AND DISCUSSION BSA/Au, protein A/Au, and HRP/Au nanoparticle bioconjugates were prepared by direct adsorption of the proteins to Au nanoparticles, followed by removal of excess protein by centrifugation and resuspension in protein-free buffer. Assuming ∼71 HRP per 15-nm Au nanoparticle (see Experimental Section for details), the percent activity retention after conjugation was ∼25%, with rather large variations from batch to batch ((15%). The reduction in activity observed for HRP bound to Au nanoparticles is due to a combination of steric inhibition via blocking of the active site by the Au surface or adjacent HRP molecules and loss of activity due to denaturation. Denaturation can result when various portions of the protein unfold to bind to the metal surface. This effect is to some extent dependent upon the curvature of the surface to which the proteins are bound, with higher-curvature surfaces generally thought to preserve greater function, as compared to flatter surfaces.44,45 We note that biomolecule/Au conjugates prepared with various antibodies or oligonucleotides have been shown to retain excellent bioactivity for selective binding.24,46 Enzyme/ nanoparticle conjugates prepared by direct adsorption often show some loss in bioactivity;44,45 however, there have also been reports of improved activity or robustness for some adsorbed enzymes.47 More elaborate bioconjugation protocols, such as attachment of His-tagged proteins to Co(II)-nitriloacetic acid groups on the surface of Au nanospheres,32 have potential as general methods for attaching enzymes to particles without loss of activity. Partitioning of Protein/Au Conjugates in ATPS. Table 1 compares partitioning of HRP, BSA, and protein A with that of their Au nanoparticle bioconjugates and with unconjugated Au nanoparticles. A 7.5 wt % PEG 8 kDa/8 wt % dextran 10 kDa ATPS and 13-nm-diameter Au nanoparticles were used for these experiments. The partition coefficient of HRP was 0.72 in this ATPS; i.e., its concentration in the dextran-rich phase was ∼1.4× its (44) Vertegel, A. A.; Siegel, R. W.; Dordick, J. S. Langmuir 2004, 20, 68006807. (45) Lundqvist, M.; Sethson, I.; Jonsson, B.-H. Langmuir 2004, 20, 1063910647. (46) El-Kouedi, M.; Keating, C. D. In Nanobiotechnology: Concepts, Methods, and Perspectives; Niemeyer, C., Mirkin, C. A., Eds.; Wiley-VCH Verlag Gmbh & Co.: Weinheim, 2004; pp 429-443. (47) Gole, A.; Dash, C.; Ramakrishnan, V.; Sainkar, S. R.; Mandale, A. B.; Rao, M.; Sastry, M. Langmuir 2004, 17, 1674-1679.

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Table 1. Partitioning of Proteins and Protein/Au Conjugates protein or conjugatea

Kb

HRPc HRP/Au 13-nm Au BSAd BSA/Au protein Ae protein A/Au

0.72 ( 0.020 60 ( 6.3 35.0 ( 3.4 0.190 ( 0.0027 (2.20 ( 0.011) × 10-3 0.230 ( 0.0058 (4.5 ( 0.37) × 10-4

a Concentrations of Au, HRP/Au, and BSA/Au conjugates were 1418 nM, and of protein A/Au, were ∼4 nM. All Au nanoparticles were 13 nm in diameter. b ATPS: 7.5 wt % PEG 8000 Da/8 wt % dextran 10 000 Da/5 mM pH 7.4 sodium phosphate buffer, T ) 3 °C. c FITCHRP. d Fluorescein/BSA. e Alexa488/protein A.

concentration in the PEG-rich phase. Thus, free HRP does not have a strong preference for either phase, in agreement with literature reports.48 HRP/Au conjugates, in contrast, partitioned strongly to the PEG-rich phase, with K ) 60, a 60:1 PEG/dextran concentration ratio. To the best of our knowledge, the only prior reports on improving HRP partitioning in ATPS are from the laboratory of Kula and co-workers.48,49 These authors bound HRP to an antibody for HRP, increasing HRP partitioning due to dramatically increased size of the conjugate.48,50 The degree of partitioning was ∼5:1 or less in all cases.49 Unconjugated 13-nm-diameter Au nanospheres also partitioned to the PEG-rich phase, in agreement with our previous work in more concentrated ATPS.51 The large surface area and high surface charge (due to adsorbed citrate) of the Au nanospheres contributes to their excellent partitioning (K ) 35 in this ATPS). Both factors have previously been described as providing increased biomolecule partitioning, as compared with smaller, or less-charged biomolecules.1-4 The larger K observed for HRP/ Au as compared with unconjugated Au indicates changes in the surface chemistry as well as the increased size of the HRP-coated Au. Both BSA and protein A partition into the dextran-rich phase of this ATPS, with K ∼ 0.2 (a 5-fold concentration difference between the phases). Conjugation to 13-nm-diameter Au nanoparticles leads to substantial decreases in K (i.e. increased preference for the dextran-rich phase) for both proteins, 2 orders of magnitude for BSA (to 2.2 × 10-3, which corresponds to more than a 450:1 concentration ratio in the dextran-/PEG-rich phases), and nearly 3 orders of magnitude for protein A (to 4.5 × 10-5, a >2200:1 concentration ratio). Collectively, these data illustrate the generality of the nanosphere conjugation approach to increased partitioning of biomolecules in ATPS. Note that the ATPS evaluated is close to compositions that exist as a single phase; i.e., the PEG and dextran contents of the two phases are not very different. A phase diagram determined via cloud point titration is shown for this ATPS in Supporting Information Figure S2. In our previous investigations of single-stranded DNA/Au bioconjugates, in which the DNA strands are not expected to adopt (48) Elling, L.; Kula, M.-R.; Hadas, E.; Katchalski-Katzir, E. Anal. Biochem. 1991, 192, 74-77. (49) Elling, L.; Kula, M.-R. Biotechnol. Appl. Biochem. 1991, 13, 354-362. (50) Albertsson, P.-Å.; Cajarville, A.; Brooks, D. E.; Tjerneld, F. Biochim. Biophys. Acta 1987, 926, 87-93. (51) Helfrich, M. R.; El-Kouedi, M.; Etherton, M. R.; Keating, C. D. Langmuir 2005, 21, 8478-8486.

Table 2. Partitioning of HRP/Au, HRP, and Au as a Function of ATPS Composition wt % PEG

MW PEG, kDa

wt % dex

MW dex, kDa

9.0 7.5 4.875 4.75 4.125 4.125

4.6 8.0 8.0 8.0 8.0 8.0

9.0 8.0 6.5 5.0 5.0 3.0

10 10 40 70 150 500

K HRPa,b

K Aua,c

K HRP/Aua,c

0.76 ( 0.027 0.72 ( 0.02

73 ( 3.2 69 ( 2.1 77 ( 1.1 78 ( 0.65 1.6 ( 0.16 1.9 ( 0.31

150 ( 2.3 130 ( 7.9 140 ( 1.8 150 ( 7.8 3.6 ( 0.031 3.0 ( 0.061

1.10 ( 0.024

a All ATPS were prepared in 5 mM pH 7.4 sodium phosphate buffer. T ) 3 °C. b 1 µg/mL FITC-labeled HRP was used for determination of free HRP. c Concentrations of Au or HRP/Au conjugates were 14-18 nM, and Au diameter was 15 ( 1.3 nm.

substantially different conformations on the particles as compared to in solution, we found that the bioconjugate-like, free, singlestranded DNA partitioned into the dextran-rich phase.51 For HRP, conjugation to Au nanoparticles changed the phase preference of the enzyme, whereas for the other two proteins tested, conjugation amplified existing phase preferences of the proteins. This suggests that although the surface interactions between the protein A or BSA and the ATPS were not significantly altered by adsorption to the Au nanospheres, those between HRP and the ATPS were. These changes presumably arise from differences in the exposed protein chemistry due to adsorption orientation, changes in conformation (possibly including some denaturation) for the Aubound HRP, or both. In comparison to other methods of increased partitioning in ATPS, conjugation to colloidal Au nanospheres requires no alteration of the ATPS composition and can be used to generate excellent partitioning, even for ATPS compositions close to the single-phase region of the phase diagram. In addition, no covalent modification of the protein or polymer structure is required (e.g., with affinity ligands). Loss of biological activity, native structure, or both upon adsorption to the Au nanoparticles is a potential drawback of this attachment method. Nonetheless, given the long and successful history of protein/Au bioconjugates in immunostaining for electron and optical microscopies, we do not anticipate that these drawbacks will overwhelm the advantages of bioconjugation to colloidal Au. Indeed, ATPS separations may prove to be an excellent means of purification for immunohistochemistry probes and other nanoparticle bioconjugates. Effect of ATPS Composition on HRP/Au Partitioning. Since the degree of protein partitioning is known to depend on ATPS composition, we were interested in determining the effect of ATPS composition on protein/Au nanoparticle bioconjugates. Table 2 shows partitioning results for a range of PEG/dextran ATPS compositions. Note that the polymer weight percents were somewhat lower as the dextran MW increased, to keep the ATPS composition close to that separating the single- and two-phase regions of the phase diagram. Phase diagrams for two of the ATPS used here are shown in Supporting Information Figures S2 and S3). Substantial improvements in K were observed for HRP/Au conjugates over free HRP in all of the ATPS investigated. For the 9% PEG 4.6 kDa/9% dextran 10 kDa ATPS, in which HRP alone had a K of 0.76, K for the HRP/Au bioconjugates was 150. A K of 130 was observed in the 7.5% PEG 8 kDa/8.0% dextran 10 kDa ATPS and a somewhat higher K as the dextran molecular weight was increased to 40 and 70 kDa, with K ) 150 for the latter. For

the largest dextran molecular weights, a much lesser degree of partitioning was observed, to be discussed in a separate section below. The data in Table 2 were acquired using HRP conjugated to 15-nm-diameter Au nanospheres, as compared to the 13-nmdiameter particles used in Table 1. This increase in bioconjugate size appears to have led to a substantial improvement in partitioning: from 60 to 130 in the 7.5% PEG 8 kDa/8.0% dextran 10 kDa ATPS. Effect of Protein/Au Bioconjugate Size on Partitioning. Solute size (i.e., protein molecular weight) has been suggested as a key factor in determining the extent of partitioning in an ATPS.1-4,19,52 However, it has proven difficult to elucidate the impact of protein molecular weight due to the lack of control over other variables, such as shape and the presence and distribution of charged, hydrophobic, and hydrophilic groups on the protein surface.1,6,19,50,53,54 The protein/Au nanoparticle bioconjugates used here provide a simple route for testing the effect of size on partitioning, in that their size can be changed by simply using a differently sized Au nanosphere. Au nanospheres can be synthesized with reasonable monodispersity in diameters from a few nanometers up to a few hundred nanometers.21 HRP/Au prepared with larger diameters of Au nanoparticles (concentration subnanomolar in ATPS) exhibited increased partitioning in the PEG 8 kDa/dextran 500 kDa ATPS (Figure 1). The effect is striking: an increase in Au nanoparticle diameter from 13 nm to 31 nm (an ∼6-fold increase in surface area) improved the partition coefficient by a factor of >40. This not only illustrates that Au nanoparticle diameter can dramatically increase the degree of partitioning, but also demonstrates that conjugates can exhibit significant partitioning, even in ATPS consisting of high dextran molecular weights. Note that the HRP/Au conjugates accumulated in the dextran-rich phase under the conditions used for this experiment. K < 1 is observed for HRP/Au conjugates in high-MW dextran ATPS when low concentrations of conjugate are used, as will be described in the following section. Similar experiments could not be performed in ATPS that provided good partitioning (i.e., K ) 150) for the HRP/Au conjugates prepared with 15-nm Au, since the conjugate concentrations in the dextranrich phase became so low as to be undetectable. A strong correlation between protein/Au partitioning and conjugate size was also observed for BSA/Au added to the 7.5 wt (52) Albertsson, P.-Å. Nature 1958, 182, 709-711. (53) Abbott, N. L.; Blankschtein, D.; Hatton, T. A. Bioseparation 1990, 1, 191225. (54) Huddleston, J.; Veide, A.; Ko¨hler, K.; Flanagan, J.; Enfors, S.-O.; Lyddiatt, A. TRENDS Biotechnol. 1991, 9, 381-388.

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Figure 1. Protein/Au conjugate partitioning as a function of conjugate surface area. For HRP/Au, the ATPS composition was 4.125 wt % PEG 8 kDa/3 wt % dextran 500 kDa, and for the BSA/ Au, the ATPS was 7.5 wt % PEG 8 kDa/8.0 wt % dextran 10 kDa. Both ATPS contained 5 mM, pH 7.4 sodium phosphate buffer. T ) 3 °C. Concentrations of protein/Au conjugates were in the subnanomolar range in the PEG 8 kDa/dextran 500 kDa ATPS and 14-18 nM in the PEG 8 kDa/dextran, 10 kDa ATPS.

% PEG 8 kDa/8.0 wt % dextran 10 kDa ATPS (Figure 1). The magnitude of the size effect was smaller for BSA/Au in this ATPS as compared to HRP/Au in the 4.125 wt % PEG 8 kDa/3 wt % dextran 500 kDa ATPS. Nonetheless, K for the BSA/Au was improved from 450:1 to the dextran phase when conjugated to 13-nm Au nanospheres to 1700:1 to the dextran phase when conjugated to 31-nm-diameter Au spheres. Together, these data on particle size effects support the importance of solute surface area in ATPS partitioning and point to the use of larger-diameter particles as a route to highly efficient separations. Anomalous Partitioning in High-Molecular-Weight Dextran ATPS. For ATPS containing dextran 150 kDa and 500 kDa, partitioning of HRP/Au (or Au alone) was substantially worse than for the other ATPS investigated (K ∼ 3, as compared with K > 100, Table 2). This somewhat puzzling result was further complicated by the dependence of K on the amount of HRP/Au added to these two ATPS. In the PEG 8000 Da/dextran 150 000 Da ATPS, this concentration difference only affected the partition coefficient by a factor of 2, with no change in phase preference. A more dramatic effect was observed in the PEG 8000 Da/dextran 500 000 Da ATPS. Figure 2 shows the concentration of HRP/Au conjugates in each phase of the PEG 8000 Da/dextran 500 000 Da ATPS as a function of the total concentration of conjugate added. At 1 nM HRP/Au, the conjugates prefer the dextran-rich phase (as in Figure 1). As the concentration of HRP/Au is increased, however, K becomes greater than 1, indicating partitioning to the PEGrich phase. The same trend is observed for Au particles alone (Figure 2): in both cases, the dependence upon conjugate concentration appears to be roughly linear in the 1-10 nM concentration range. Conjugate concentration did not appreciably impact K in the PEG 8000 Da/dextran 10 000 Da ATPS (Figure 3). Although K for macromolecules is generally assumed to be independent of the macromolecule concentration, this is not always true. Indeed, Zaslavsky has stressed the importance of measuring K for a given solute over a range of different concentrations.1 Differences in K for different protein concentrations can point to changes in structure (e.g., due to dimerization) or to 384 Analytical Chemistry, Vol. 78, No. 2, January 15, 2006

Figure 2. Effect of Au and HRP/Au nanoparticle conjugate concentration in the ATPS on partitioning. Au nanoparticle diameter ) 13 ( 1.0 nm. Solid symbols indicate HRP/Au, and open symbols, unconjugated Au nanoparticles. ATPS: 4.125 wt % PEG 8000 Da/3 wt % dextran 500 000 Da, in 5 mM, pH 7.4 sodium phosphate buffer for the HRP/Au, and in 3.5 mM citrate for the unconjugated Au. T ) 3 °C.

Figure 3. Effect of BSA/Au nanoparticle conjugate concentration in the ATPS on partitioning. ATPS: 7.5 wt % PEG 8000 Da/8 wt % dextran 10 000 Da/5 mM, pH 7.4 sodium phosphate buffer. T ) 3 °C.

protein precipitation. In our experiments, only the high-MW dextran-containing ATPS (PEG 8 kDa with either dextran 150 or 500 kDa) showed a dependence of K on conjugate concentration. In this case, aggregation of the HRP/Au conjugates is unlikely. The conjugates were stable at much higher concentrations than are used in these experiments (indeed, they were routinely pelleted and resuspended as part of our purification protocol) and were stable at these concentrations in the lower MW dextran ATPS. In the high-MW dextran ATPS where changes in K are observed, no sedimentation or color change was detectable. Addition of colloidal polystyrene and silica particles to PEG/ dextran ATPS has been shown to alter the phase behavior of these systems, causing phase separation in single-phase polymer solutions that were close to phase separation prior to addition of the particles.55 These observations were explained as the effects of surface modification of the particles due to polymer adsorption and capillary-induced phase separation (CIPS).55 CIPS refers to the ability of gaps between particles or larger surfaces to provide a more favorable site for phase separation, as compared to the (55) Olsson, M.; Joabsson, F.; Piculell, L. Langmuir 2005, 21, 1560-1567.

Table 3. Effect of HRP/Au Bioconjugates on Polymer Partitioning in ATPS polymer

Ka

PEG 5 kDa (no conjugate) PEG 5 kDa (with conjugate)b dextran 500 kDa (no conjugate) dextran 500 kDa (with conjugate)

2.0 ( 0.043 1.9 ( 0.11 0.12 ( 0.0014 0.074 ( 0.0015

a ATPS: 4.125 wt % PEG 8000/3 wt % dextran 500 000/5 mM, pH 7.4 sodium phosphate buffer, 3 °C. b HRP/Au bioconjugates prepared using 13-nm-diameter Au were added to a final concentration of several nanomolar in the ATPS.

Figure 4. Concentration of Au in the PEG and dextran phases of an ATPS with increasing concentrations of Au added to the ATPS. ATPS: 4.125 wt % PEG 8000 Da/3 wt % dextran 500 000 Da. Au nanoparticle diameter ) 13 ( 1.0 nm. T ) 3 °C.

bulk, and results in an attractive force between the particles.55-58 If either CIPS or polymer-adsorption-induced surface effects play an important role in the concentration dependence of HRP/Au and Au partitioning in our ATPS, we would expect a change in the position of the binodal, which would result in a different K for the polymers themselves and in our ATPS. Because the composition of our ATPS is close to (below) the binodal at 25 °C, these effects might also result in a change in phase state of the system. We do not observe a change in the phase state of the ATPS at 25 °C, and the partitioning of fluorescently tagged PEG and dextran polymers was not dramatically affected by the increased concentration of HRP/Au bioconjugates (Table 3). There was a slight increase in partitioning of the dextran (K ) 0.120 to 0.074) in the presence of several nM HRP/Au. Thus, while the nanoparticles do alter the composition of the ATPS phases, the effect does not appear to be very large. Figure 4 shows the concentration of (unconjugated) Au nanoparticles in each phase of the ATPS as a function of total Au nanoparticle concentration in the system. These data show that the increase in K with increasing conjugate concentration results from increased concentration in the PEG-rich phase, whereas the concentration in the dextran-rich phase (preferred by the conjugates at low conjugate concentrations) initially increases rapidly and then much more slowly with increased Au concentration in (56) Freyssingeas, E.; Thuresson, K.; Nylander, T.; Joabsson, F.; Lindman, B. Langmuir 1998, 14, 5877-5889. (57) Olsson, M.; Joabsson, F.; Piculell, L. Langmuir 2004, 20, 1605-1610. (58) Wennerstrom, H.; Thuresson, K.; Linse, P.; Freyssingeas, E. Langmuir 1998, 14, 5664-5666.

Figure 5. Partitioning (top panel) and concentration (bottom panel) of BSA/Au bioconjugates in the PEG and dextran phases of the 4.125 wt % PEG 8 kDa/3 wt % dextran 500 kDa/5 mM pH 7.4 sodium phosphate buffer ATPS as a function of bioconjugate concentration in the overall ATPS. Au nanoparticle diameter ) 13 ( 1.0 nm. T ) 3 °C.

the overall ATPS, never rising above ∼4 nM. Similar behavior is observed for the BSA/Au conjugates (Figure 5), which in lowerMW dextran ATPS partitioned strongly into the dextran-rich phase (Table 1). In this high dextran molecular weight ATPS, the BSA/ Au bioconjugates accumulate in the PEG-rich phase with K close to that for the HRP/Au. Asenjo and co-workers have reported this type of saturation behavior for several proteins in PEG/phosphate ATPS.59 These authors note that “true partitioning” is observed only at low protein concentrations (