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Gold Nanoparticles Affect Thermoresponse and Aggregation Properties of Mesoscopic Immunoglobulin G Clusters Sondre Volden,† Loan T. T. Trinh,‡ Anna-Lena Kjøniksen,‡,§ Masahiro Yasuda,|| Bo Nystr€om,‡ and Wilhelm R. Glomm*,† †
)
Ugelstad Laboratory, Department of Chemical Engineering, Norwegian University of Science and Technology (NTNU), N-7491 Trondheim, Norway ‡ Department of Chemistry, University of Oslo, P.O.Box 1033, N-0315 Oslo, Norway § Department of Pharmacy, School of Pharmacy, University of Oslo, P.O. Box 1068, N-0316 Oslo, Norway Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, 5998531 Osaka, Japan ABSTRACT: Conjugation of proteins to nanomaterials such as gold nanoparticles (Aunp) is known to modulate the flocculation state, conformation, and functionality of the adsorbed protein. However, these changes in the adsorbed protein are also expected to manifest themselves in the temperature response relative to the native protein. Here, we have studied the thermoresponse of AunpImmunoglobulin G (Aunp IgG) constructs in the temperature range between 25 and 60 °C using UVvis and fluorescence spectroscopies, quartz crystal microbalance with dissipation monitoring (QCM-D), and dynamic light scattering (DLS). Under the conditions studied here, IgG forms multilayers on planar gold surfaces, whereas Aunps are embedded in mesoscopic protein clusters. We demonstrate that AunpIgG systems display irreversible temperatureinduced blue shifts and narrowing of the localized surface plasmon resonance (LSPR), which corresponds to a gradual breakup of AunpIgG clusters, as corroborated by DLS, steady-state (using both intrinsic and extrinsic probes), and time-resolved fluorescence. The modulation of thermal response depends on size of the Aunp core (30 or 80 nm), with the observed LSPR behavior being discussed within the framework of MieDrude theory. The findings presented here reveal that temperature response of AunpIgG constructs cannot be assumed to mirror that of the native protein.
’ INTRODUCTION It is well-known that when nanomaterials such as gold nanoparticles (Aunps) enter a biological fluid, they become coated with proteins, which in turn may elicit an immune response due to surface-induced protein conformation, altered protein function, or exposure to novel epitopes.15 Because of facile synthesis and functionalization, their unique optical properties, and biocompatibility, Aunpprotein constructs have been used extensively in cell-targeting studies (see e.g. Everts6 or works published by the Franzen research group711). While a remarkable number of studies have been published on interaction of human blood proteins with Aunps,5,1214 very little has been reported on the thermoresponsive behavior of Aunpprotein constructs. Teichroeb and co-workers15,16 have used denaturing of bovine serum albumin (BSA) adsorbed onto Aunps of different sizes, showing that denaturing of surface-bound proteins proceeds differently from free protein and that curvature strongly affects denaturing characteristics. Volden et al.17 recently reported reversible temperaturedependent LSPR shifts of 30 nm Aunps coated with anionic diblock and uncharged triblock copolymers based on poly (N-isopropylacrylamide) (PNIPAAM) in the temperature range r 2011 American Chemical Society
between 25 and 60 °C. PNIPAAM is a thermoresponsive polymer which undergoes a coil-to-globule transition and subsequent phase separation above its lower critical solution temperature (LCST). When adsorbed to Aunps, the resulting polymerAunp constructs displayed completely reversible red shifts with increasing temperature, which was ascribed to an increase in the refractive index of the shell layer caused by temperature-induced contraction of the PNIPAAM blocks, thus forming a denser structure. In the case of Aunps coated with an uncharged triblock PNIPAAM copolymer, much larger red shifts were observed, which was explained via an increase in the volume fraction of the shell layer, attributed to multilayer formation of the uncharged copolymer. The aim of this work is to examine the adsorption and thermal behavior of Aunpantibody constructs. Specifically, we have studied the adsorption and thermal behavior of immunoglobulin G onto Aunps. Immunoglobulin G (IgG) is the most abundant antibody found in plasma and is composed of four polypeptide Received: February 9, 2011 Revised: May 9, 2011 Published: May 19, 2011 11390
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The Journal of Physical Chemistry C chains connected by disulfide bonds and noncovalent forces. The four polypeptide chains are grouped in two identical Fab segments and one Fc segment, yielding a Y-shaped tertiary protein structure with antigen binding sites located on the far end of the Fab segments. Polyclonal IgG can form mesoscopic clusters in solution due to low solubility emanating from chemical variations between proteins of different physical properties.18 We demonstrate in this paper that Aunps covered with IgG display irreversible temperature-dependent LSPR shifts and that surface-bound proteins undergo thermal denaturing well below Tm of IgG. The irreversible LSPR shifts are caused by temperatureinduced breakup of protein aggregates, with the onset and nature of the breakup being strongly dependent on Aunp size. Our results show that changes in the optical signature of Aunps can be used to monitor temperature-induced changes in flocculation state as well as transitions in the adsorbed layer, expanding the range of plasmonic nanomaterials for tracking of environmentally induced changes in adsorbed macromolecules.
’ EXPERIMENTAL SECTION Materials. Gold nanoparticles (Aunps; 30 and 80 nm) were purchased from British BioCell/Ted Pella Inc., while human serum Immunoglobulin G was acquired from Sigma-Aldrich (Prod. No. 56834). A typical procedure for adsorbing IgG onto Aunps involved mixing of 2.7 mL of the gold colloid suspension with 0.3 mL of an IgG solution (10 μM in citrate (10 mM)). All samples were measured within 2448 h after mixing. QCM-D Measurements. Quartz crystal microbalance measurements were performed on a D300 apparatus from Q-sense, Sweden, with gold-coated quartz crystals supplied by Biolin Scientific AB. Adsorption measurements were performed after a stable baseline was achieved with a 10 mM citrate solution, with subsequent addition of IgG solution (1 μM in citrate (10 mM)) to the measurement chamber. UVvis. UVvis measurements were conducted using a Shimadzu UV-2401PC instrument, equipped with a TCC-240 temperature control unit, over a wavelength range of 400 800 nm. Measurements were done in 5 °C intervals between 25 and 60 °C, with an equilibration time of 15 min at each temperature. Bradford assays were performed to determine the amount of adsorbed protein on Aunps,19 wherein a stock solution of IgG was mixed with gold colloid suspension to give final concentrations of IgG of 1 μM. Following equilibration, the samples were centrifuged to precipitate the AunpIgG constructs, and the remaining protein concentration in the supernatant was measured using the Bradford method, at the absorption wavelength of 595 nm. Dynamic Light Scattering Measurements. The dynamic light scattering (DLS) experiments were conducted using an ALV/CGS-8F multidetector version compact goniometer system with eight fiber-optical detection units, supplied by ALVGmbH, Langen, Germany. The beam from a Uniphase cylindrical 22 mW HeNe laser, operating at a wavelength of 632.8 nm with vertically polarized light, was focused on the sample cell (NMR tubes (10 mm) of highest quality from Wilmad Glass Co.) through a temperature-controlled cylindrical quartz container (containing two plane-parallel windows). The temperature accuracy was controlled within (0.01 °C through a heating/ cooling circulator filled with a refractive index matching liquid (cis-decalin). The protein/proteinAunp solutions were filtered
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Figure 1. Normalized average frequency shift and adsorbed mass acquired at the third harmonic as a function of time (A) and Df plot (B) for 1 μM IgG adsorbed onto planar citrate-coated Au surfaces. In panel A, the symbols refer to normalized frequency shifts, whereas the line represents adsorbed mass (ng/cm2, calculated using the Sauerbrey equation). The arrow in panel B indicates a breakpoint in the Df plot.
in an atmosphere of filtered air through a 5.0 μm filter (Millipore) directly into precleaned NMR tubes. Fluorescence Measurements. Steady-state fluorescence measurements were performed on a Fluorolog-3 apparatus equipped with a Peltier element thermostat cell holder from HORIBA Jobin Yvon. Excitation wavelength applied was 295 nm for the intrinsic probes (Tryptophan (Trp)) and 370 nm for the extrinsic probe (1-anilino-8-naphthalenesulfonic acid (ANS)). Time-correlated single photon counting experiments were done on the same instrument using a NanoLed with excitation wavelength 370 nm. Steady-state measurements were done in 5 °C intervals between 25 and 55 °C, with an equilibration time of 15 min at each temperature.
’ RESULTS AND DISCUSSION In order to gain a more complete understanding and correlation between associated amounts and temperature-dependent properties, the experimental matrix was divided into adsorptive properties on planar and curved gold surfaces, followed by studies of optical behavior of the formed Aunpprotein assemblies related to physical changes in the adlayer as a function of temperature. IgG Forms Multilayers on Planar Citrate-Coated Au Surfaces. In order to study the adsorption kinetics and concomitant
layer organization, the adsorption of IgG onto citrate-coated Au surfaces was studied using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique. Normalized frequency shifts (of the third harmonic of the fundamental frequency, n = 3) Δf/n as a time-dependent function at 25 °C for the adsorption of 1 μM IgG onto citrate-coated planar Au are shown in Figure 1A (left axis). Corresponding adsorbed mass, calculated using the Sauerbrey equation,20 are shown on the right axis of Figure 1A. Figure 1A reveals that IgG has a high affinity for the Au surface, approaching saturation after ∼1 h following introduction to the measurement chamber. Following 46 h of exposure, a saturation plateau of 76 Hz (∼1350 ng/cm2) is reached. To relate the changes in dissipation ΔD (i.e., how quickly the oscillation dissipates relative to the citrate-coated Au surface) to Δf as they occurred during the adsorption of IgG, a 11391
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Figure 2. UVvis absorption spectra of 1 106 M IgG adsorbed onto (A) 30 nm citrate-stabilized Aunps (0.3 nM) and (B) 80 nm citrate-stabilized Aunps (0.02 nM) obtained during heatingcooling cycles between 25 and 60 °C. Difference spectra for 30 and 80 nm Aunp as shown in (C) and (D), respectively, were obtained by subtracting the spectrum of Aunp only from the spectrum of the mixture at each temperature. The notation 1, 2, and 3 in each panel refers to citrate-coated Aunps only, IgGAunp mixtures prior to heating, and IgGAunp mixtures following completion of one heatingcooling cycle, respectively. Arrows indicate direction of shift upon heating and subsequent cooling.
ΔD vs Δf plot (hereafter referred to as a D-f plot) was created (Figure 1B). Plotting ΔD vs Δf offers the advantage of eliminating time as an explicit parameter. Moreover, information about kinetic regimes, multilayer formation and possible conformational changes can be deduced from changes in the slope of Df plots.2124 For adsorption of IgG onto citrate-coated Au, the Df plot reveals two linear regimes of significantly different slopes, with the breakpoint occurring at about 71 Hz (indicated by the arrow in Figure 1B), which corresponds to ∼94% of the saturation plateau. The higher slope of the second regime corresponds to a more viscoelastic layer, which is consistent with surface-induced conformational changes or multilayer formation.21,22 In this study the high adsorbed amounts and Df behavior determined from QCM experiments correspond to multilayer formation of IgG. Here, it should be noted that we cannot conclusively distinguish between (i) multilayers of monomeric IgG, (ii) mesoscopic protein clusters adsorbing onto monomeric IgG or vice versa, and (iii) multilayers of mesoscopic clusters. From the transition from a relatively rigid layer to a viscoelastic film revealed from Figure 1B, multilayer formation likely occurs by deposition of mesoscopic clusters onto a preadsorbed layer of monomeric IgG. Aunps Are Embedded in Mesoscopic IgG Clusters. In order to quantify IgG adsorption onto citrate-coated Aunps, Bradford assays were performed (see Experimental Section for details).19 Briefly, IgGAunp constructs were precipitated via centrifugation, and the concentration of the remaining IgG (i.e., not associated with Aunps) was determined via a colorimetric assay. At pH 8.55 and a protein concentration of 1 μM, IgG adsorption
resulted in IgG:Aunp ratios of 186 and 3153 for 30 and 80 nm Aunps at ambient conditions, respectively. Estimating an IgG diameter of 10 nm and by assuming that the protein stacks as closely packed spheres in a face-centered tetragonal lattice, monolayer coverage corresponds to IgG:Aunp ratios of ∼10 (30 nm Aunps) and ∼60 (80 nm Aunps). This would yield IgG: Aunp ratios of ∼3 monolayer coverage and ∼67 monolayer coverage for 30 and 80 nm Aunps, respectively, based on the Bradford assays. Thus, as IgG forms multilayer structures on both planar and curved gold surfaces, we conclude that Aunps are embedded in mesoscopic IgG clusters. Mesoscopic AunpIgG Clusters Display Irreversible Temperature-Dependent Shifts in the Localized Surface Plasmon Resonance (LSPR) Profile. Figure 2 displays UVvis absorbance spectra obtained during heating and cooling cycles of 30 nm (A) and 80 nm (B) Aunp with 1 μM IgG, with the respective difference spectra in panels C and D for 30 and 80 nm Aunps, respectively. The spectral profile of unmodified (citratecoated) Aunps remained constant throughout this temperature range, as reported earlier.17 Introduction of IgG to the Aunp suspension at 25 °C resulted in large red shifts, intensity reduction, and broadening of the LSPR peak for both particle sizes, as shown in Figure 3A,B, indicating strong interaction between IgG and Aunps. The severe broadening of the LSPR line shape upon introduction of IgG can be attributed to the Aunps residing as flocs or embedded in IgG mesoscopic clusters.11,25,26 Upon heating, the LSPR peak is gradually blue-shifted with a concomitant intensity increase and narrowing of the line shape, approaching that of single colloids. Interestingly, no changes are 11392
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Figure 3. Temperature-dependent LSPR peak positions and shifts for the systems shown in Figure 2. Panels A and B depict LSPR position as a function of temperature for 30 nm IgGAunp and 80 nm IgGAunp, respectively, compared to unmodified Aunps (gray triangles). The error bars represent width of the LSPR peak at the temperatures indicated. Panels C and D show the shift in LSPR relative to unmodified Aunps. Closed symbols correspond to spectra acquired from the heating part of the temperature loop, whereas open symbols correspond to the cooling down cycle of the experiment. Lines are only meant as a guide for the eyes.
observed upon cooling; i.e., temperature-induced changes to the LSPR profiles of the IgGAunp constructs are irreversible. From the difference spectra (obtained by subtracting the spectrum of Aunp only from the mixture at each temperature), the temperature-induced shifts and narrowing of the line shape are clearly evident, with both broadening and LSPR shifts being distinguishable. Upon completion of a heatingcooling cycle, the LSPR is still red-shifted relative to unmodified Aunps (11 and 15 nm shifts for 30 and 80 nm Aunps, respectively), indicating that IgG remains associated with Aunps.17 The UVvis profile of the IgGAunp mixtures remained unchanged for several weeks after completion of the heatingcooling cycle, revealing that spectral shifts emanate from changes in the IgGAunp interface rather than from flocculation of Aunps. To elucidate the temperature-induced effects for the IgGAunp systems, the LSPR peak positions (panels A and B for 30 and 80 nm Aunps, respectively) as well as the shift in LSPR relative to unmodified Aunps (panels C and D for 30 and 80 nm Aunps, respectively) are shown as a function of temperature in Figure 3. The error bars in panels A and B correspond to the apparent width of the LSPR peaks. Figure 3 reveals that both Aunp sizes used here result in the same overall trend: (1) The LSPR is increasingly blue-shifted with a progressively narrower line shape upon heating. (2) Spectral changes induced by heating are irreversible, as revealed by the absence of spectral changes upon cooling. However, there are two significant differences between the temperature-dependent LSPR profiles for the two different Aunp sizes studied here. First, the initial red shift and spectral broadening upon interaction with IgG are much larger
for 80 nm Aunps than for 30 nm Aunps (the initial LSPR shifts are 40 and 160 nm for 30 and 80 nm Aunps, respectively). Second, the temperature-dependent behavior is different for the different Aunp sizes. While the blue shift occurs gradually over a wide temperature range (∼3050 °C) for the 30 nm Aunps, there appears to be a more abrupt temperature response for the larger Aunp species; the entire shift of the LSPR occurs between 35 and 45 °C, with the onset of the blue shift occurring in the range between 35 and 40 °C. The optical properties of nanoparticles made from noble metals such as Au and Ag are usually described within the framework of MieDrude theory (for a theoretical treatment, see for example Franzen27 or Mulvaney2830). MieDrude theory is used to predict the effect of particle size, interparticle distance, dielectric function of the surrounding medium, and core charge state on the LSPR absorption band of noble metal nanoparticles. In the case of nanoparticles coated with a passivating shell layer of surfactants, polymers, or proteins, the LSPR band is also known to be influenced by the thickness and volume fraction (g) of the adsorbed shell layer. Specifically, an increase in the thickness (via an increase in the refractive index) or volume fraction of the shell layer results in the LSPR band being redshifted. In this study, the reverse trend is observed from what was reported by Volden et al. for Aunps coated with anionic and uncharged PNIPAAM block copolymers,17 in that increasing temperature results in blue-shifted LSPR bands and that the temperature-induced changes are irreversible. Assuming that the size of the Aunp core is kept constant, MieDrude theory 11393
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The Journal of Physical Chemistry C predicts a blue shift of the LSPR (i) upon an increase in the interparticle distance following breakup of flocs or increased separation of very tightly spaced particles, (ii) with a decrease of the refractive index of the solvent (εm = nm2), (iii) with decreasing the refractive index of the shell layer (εs = ns2), (iv) with decreasing the thickness of the shell layer, thus lowering the volume fraction g of the shell, and (v) with an electron excess with respect to the point of zero charge (EPZC). It has been documented31,32 that strong interparticle coupling and a concomitant red shift occurs as the interparticle distance D approaches a critical value of D/(2r) = 1.2 for two nanoparticles of radius r. For flocculated Aunp suspensions, this red shift is accompanied by a significant broadening of the LSPR.25,26 Aslan and co-workers25 have shown that streptavidin-induced flocculation of biotinylated Aunps can be reversed by addition of soluble biotin, dissociating streptavidin from the Aunp surfaces so that the LSPR returned to the position and width prior to protein addition. While interparticle coupling resulting from flocculation of the Aunps may serve as an explanation for the initial red-shift and line shape broadening following introduction of IgG, this does not fully account for the temperature-induced spectral changes observed in this study, as no additives were used—only temperature was varied to obtain the results, and as the LSPR remains red-shifted compared to unmodified Aunps following heating and cooling, indicating that IgG remains adsorbed onto the Aunps. Moreover, the upper temperature studied here (60 °C) is well below Tm of IgG (∼75 °C), and the UVvis spectrum of IgG only remained unchanged after one temperature cycle, ruling out protein denaturation and subsequent precipitation as the cause of the LSPR shifts. The refractive index of water decreases by ∼0.55% from 25 to 70 °C,33 which according to MieDrude theory should result in a slight blue shift of LSPR for Aunps.29 However, as the LSPR bands of unmodified Aunps were unaffected by temperature within the intervals studied here, and as the temperature-induced reduction of the refractive index of water is reversible upon cooling, we conclude that changes in the dielectric function of the solvent—mechanism ii above—are not responsible for the irreversible temperature-dependent spectral changes of the IgGAunps observed here. Also, we do not consider mechanism v—an excess of electrons compared to the EPZC—to be responsible for the effects reported here, as temperature changes within the interval studied here are unlikely to cause any changes in the core charge state of the Aunps. Mechanism iii—decreasing the refractive index of the shell layer—and mechanism iv—lowering the volume fraction g of the shell—are likely to contribute to the spectral changes observed here. It is well-known18,3436 that a fraction of IgG aggregates irreversibly in the temperature range 4060 °C, which corresponds well to the majority of changes in the LSPR profiles observed here. Moreover, protein conformation is known to be affected by interaction with surfaces, with the degree of native structure retention being proportional to curvature.5 The UVvis results presented in Figures 2 and 3 reveal that the changes in the LSPR profile vary between the two Aunp sizes studied here. Specifically, the higher magnitude of the LSPR shift and the more abrupt onset of the temperature-induced blue shift are larger for systems containing 80 nm Aunps than for 30 nm Aunps (Figure 3), which indicates differences in the state of the adsorbed protein for the Aunp sizes studied here. These differences can likely be attributed to differences in the degree of partial unfolding of the surface-bound IgG as a function of
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Scheme 1. Temperature-Induced Behavior of Mesoscopic AunpIgG Assemblies, Illustrating the Transition from Aunps Imbedded in a Protein Matrix to Smaller AunpIgG Flocs upon Elevating the Temperature
curvature, which in turn would affect the refractive index of the shell layer. The LSPR profile of the IgGAunps transitions irreversibly from a flocculated state or a loose network to approaching single colloids with increasing temperature, which corresponds to a breakup of mesoscopic IgG clusters with embedded Aunps, thus decreasing the volume fraction g of the shell layer, as illustrated in Scheme 1. To elucidate the role of possible temperature- and particle-induced IgG unfolding in breakup of mesoscopic clusters, steady-state and time-resolved fluorescence data were acquired, as discussed later. IgG forms Mesoscopic Clusters in Solution at Ambient Conditions. DLS measurements on pure IgG solutions (data not shown) reveal the presence of large clusters in the micrometer range, which is outside the size range of where the dynamic light scattering technique can accurately measure, revealing that IgG resides as mesoscopic clusters. It should be noted that for the DLS experiments shown here an IgG concentration of 0.1 μM was used, i.e., 1/10 of the concentration used in the other experiments included in this study (QCM-D, UVvis, steadystate and time-resolved fluorescence). This is due to multiple scattering from the large clusters, rendering any analysis difficult even at ambient conditions for higher IgG concentrations. The hydrodynamic radius of 30 nm AunpIgG as a function of temperature is shown in Figure 4A,B together with the data for the pure 30 nm Aunp. The 30 nm AunpIgG was found to exhibit two modes, from which the hydrodynamic radii Rh,f (fast diffusive mode) and Rh,s (slow nondiffusive mode) have been calculated. Rh,f is in the same size range as the Aunps used, and it is reasonable to assume that this mode corresponds to free (single) Aunps which may be associated with IgG. At high temperatures Rh,f for the 30 nm Aunp in the presence of IgG appears to be lower than the size of the pure Aunps. This suggests that the citrate layer that covers the pure Aunps is replaced with a thinner layer of IgG. The slow mode (Rh,s) of the 30 nm AunpIgG system is nondiffusive; i.e., calculated radii are dependent on the angle of observation. The apparent radii shown in Figure 4B are therefore only a rough estimate of the sizes. From the UVvis results above, the Aunps are likely embedded within mesoscopic IgG clusters, implying that Rh,s corresponds to mesoscopic IgG clusters with embedded Aunps. Figure 4B reveals that at temperatures above 35 °C the size of the mesoscopic clusters is reduced, which is consistent with the interpretation of the UVvis results (Figures 2 and 3) for the AunpIgG systems, with the LSPR bands being blue-shifted and narrower at elevated temperatures. Thus, the IgGAunp mesoscopic clusters are irreversibly broken up at higher temperatures. 11394
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Figure 4. Hydrodynamic radii of 0.1 μM IgG þ Aunp compared with the pure Aunp suspensions.
As the large clusters are broken up, the size of the Aunps decreases. This could be caused either by a partial desorption of IgG from the surface of the particles or by a contraction of the adsorbed layer. It is unlikely that IgG is fully desorbed from the Aunp surface, since sizes below that of pure Aunp indicate that the Aunps have lost some of their stabilizing citrate layer. Without any stabilizing layer, the Aunps will aggregate and we would not expect to observe any unaggregated Aunps at 60 °C. The Aunps are therefore probably stabilized by an adsorbed layer of IgG. Interestingly, the 80 nm AunpIgG system only displayed one size distribution corresponding to Rh,f (Figure 4C). Here, the temperature-dependent size corresponds roughly to the 80 nm Aunps coated with an IgG monolayer at ambient conditions, with a contraction or desorption of the adsorbed layer at elevated temperatures. As was observed for the 30 nm Aunps, at high temperatures the size of the 80 nm Aunps with adsorbed IgG is lower than what is observed for the pure Aunps, which might indicate desorption of the citrate layer which is replaced by an IgG layer instead. A contraction of the adsorbed layer at elevated temperatures is consistent with temperature-induced unfolding of the IgG adlayer. As the UVvis results for the 80 nm AunpIgG system (Figures 2 and 3) reveal the same trend as for 30 nm AunpIgG, the absence of a slow nondiffusive mode could be attributed to the collected intensity being dominated by the large scattering cross section of the 80 nm Aunps, since the
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pure 80 nm Aunp scatters at least 50 times more than the pure IgG solutions while the 30 nm Aunp only scatter about 8 times as much as the pure IgG solutions. At 25 °C the 80 nm AunpIgG is significantly larger than the pure Aunp, while for the 30 nm Aunps the size of the Aunp and the AunpIgG is much closer to each other. This is in agreement with the much larger shift in the LSPR observed at this temperature for the 80 nm AunpIgG compared with the 30 nm AunpIgG in Figures 2 and 3. Aunps Affect Temperature-Dependent Intrinsic IgG Emission. Steady-state intrinsic emission spectra collected during heating and cooling of IgG are shown in Figure 5A. Heating of IgG results in a gradual intensity reduction, with the highest temperature (55 °C) resulting in the lowest emission intensity. Upon cooling, partial intensity recovery is seen. The temperature-dependent emission intensity reduction is consistent with quenching by increased exposure to solvent, which could be caused by irreversible conformational changes in IgG well below Tm, by breakup of mesoscopic IgG clusters. As the temperatureinduced spectral changes were found to be irreversible, a combination of unfolding and cluster breakup is the most likely cause, with irreversible conformational changes on a singleprotein level affecting the energy barriers for reformation of mesoscopic clusters. The same overall trend with respect to temperature was observed for IgGAunp constructs. As none of the IgG-containing systems studied here displayed any spectral shifts either as a function of temperature or upon introduction of Aunps, only normalized (with respect to IgG) maximum fluorescence intensities are shown as a function of temperature during heating (closed symbols) and cooling (open symbols) in Figure 5BD. From Figure 5B, heating to 55 °C gradually reduces the emission intensity to 58% of the system prior to heating. Upon cooling, a partial recovery up to 66% of the emission intensity prior to heating is observed. Addition of Aunps resulted in reduced emission intensity compared to IgG only (Figure 5BD), with the initial intensity reduction being larger for the 30 nm than for the 80 nm AunpIgG (70% and 88% of the IgG-only intensity, respectively). This can be attributed to static quenching when the protein is physi- or chemisorbed to Aunps,37 further confirming interaction between IgG and Aunps. The higher degree of quenching observed for the 30 nm AunpIgG system is likely due to the higher particle concentration and particle surface area in the case of the smaller Aunp diameter (see Experimental Section for particle concentrations). While both Aunp sizes display the same overall trend, i.e., an initial emission intensity reduction, with concomitant emission intensity reduction upon heating, followed by partial recovery during cooling, the temperature dependence varies between the two Aunp diameters. Specifically, while the emission intensity decrease occurs more or less gradually with increasing temperature for the 80 nm AunpIgG sample (Figure 5D), there appears to be a breakpoint between 35 and 40 °C for the 30 nm AunpIgG sample (Figure 5C), with the latter temperature region displaying a higher rate of intensity reduction. Moreover, the degree of emission intensity recovery varies with Aunp diameter. For the 30 nm AunpIgG system, the intensity recovery relative to the reference system (IgG only prior to heating) was found to be 49%, whereas the recovery in the 80 nm AunpIgG system was determined to be 76%. Thus, the recovery for the AunpIgG systems are significantly different than what was found for IgG only (66%), revealing that the interaction with Aunps affects the temperature-dependent intrinsic fluorescence of IgG. 11395
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Figure 5. Temperature dependence of the intrinsic steady-state IgG fluorescence (A) and normalized (with respect to 1 μM IgG) temperaturedependent fluorescence intensity at 346 nm for IgG (B), 30 nm AunpIgG (C), and 80 nm AunpIgG (D). Closed circles denote results acquired during heating, whereas open symbols denote results acquired from the cooling part of the temperature loop.
While the temperature-dependent fluorescence intensities of IgG only can be attributed to changes in solvent access due to irreversible unfolding below Tm or temperature-induced breakup of mesoscopic protein clusters, we consider the latter to be the primary mechanism behind the temperature-dependent fluorescence intensities of the AunpIgG systems, as this theory is corroborated by the UVvis results. However, we cannot rule out that irreversible unfolding occurs simultaneously or indeed that unfolding is the cause of the cluster breakup. Depending on the extent to which unfolding occurs, this could be a contributing cause to the difference between the two different sizes of Aunps in how they affect IgG fluorescence, as particle diameter is known to affect protein conformation.5 From the intrinsic fluorescence results, the effect of Aunps cannot be attributed solely to either varying degrees of surface-induced protein unfolding or to different AunpIgG construct sizes following initial cluster breakup. It should be noted that the temperature-dependent intrinsic fluorescence trends observed for the AunpIgG constructs are slightly different from the trends observed by UVvis (Figures 2 and 3). This is expected, as the UVvis results refer to changes in the immediate vicinity of the Aunps, whereas the intrinsic fluorescence results refer to changes in the protein. Aunps Induce Irreversible Breakup of Mesoscopic IgG Clusters upon Heating. In order to obtain more information about the intrinsic IgG fluorescence, the IgG and IgGAunp systems were characterized using time-correlated single photon counting (TCSPC) spectroscopy before and after going through a heating and cooling cycle. For more information about the technique, see, e.g., Glomm et al.38 and references therein. All the IgG-containing samples were best modeled using a triexponential decay, with the lifetimes (in nanoseconds) T1 (0.5 ( 0.1),
T2 (1.8 ( 0.1), and T3 (5.8 ( 0.2). No significant changes in the three calculated lifetimes were found for the IgGAunp constructs relative to IgG only. However, the relative population between the three lifetimes was found to vary both upon introduction of Aunps, upon Aunp size, and upon whether the systems had undergone a temperature loop. The fluorescence populations under the various conditions are given in Table 1. Since a 280 nm LED was used for lifetime measurements, contributions from all intrinsic fluorophores are included. However, the response is expected to be dominated by tryptophan emission. Because of the large number of intrinsic fluorophores in IgG, the calculated lifetimes do not represent specific residue locations. Rather, we interpret the three lifetimes as corresponding to fluorophores residing in three different chemical environments with respect to quencher proximity: a polar chemical environment close to the external protein surface and thus to solvent (short lifetime, T1), a nonpolar chemical environment with little or no contact with solvent, such as the hydrophobic interior of a protein (long lifetime, T3), and one chemical environment of intermediate polarity (medium lifetime, T2). Table 1 reveals irreversible changes in the distributions between lifetimes as a result of the temperature cycle as well as upon addition of Aunps. For IgG only, the temperature cycle results in an increase in the medium lifetime at the expense of the short lifetime, while the population associated with the long lifetime remains relatively unchanged. This decrease in the fraction of solvent-exposed fluorophores is likely due to irreversible unfolding of a fraction of IgG as described above. Introduction of Aunps to mesoscopic IgG clusters results in population changes (Table 1). Specifically, the presence of both 11396
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Table 1. Fluorescence Populations (B1, B2, B3) Determined by TCSPC of IgG Only, 30 nm AunpIgG, and 80 nm AunpIgG before and after a 25 °C55 °C25 °C Temperature Cycle (Excitation Was Done at 280 nm)a short lifetime B1 (%)
a
medium lifetime B2 (%)
long lifetime B3 (%)
before
after
before
after
before
after
IgG only
18
14
54
59
28
27
30 nm AunpIgG
13
20
57
57
30
23
80 nm AunpIgG
16
22
54
53
30
25
“Short”, “medium”, and “long” lifetimes refer to T1 (0.5 ( 0.2), T2 (1.8 ( 0.1), and T3 (5.8 ( 0.2), respectively. Here, lifetimes T are given in ns.
Figure 6. Steady-state ANS fluorescence from IgG (circles) and 30 nm AunpIgG (squares) at 25 °C before and after a heatingcooling cycle (closed symbols refer to the system prior to temperature cycle, and open symbols refer to the system after a temperature cycle) (A). Temperature dependence of ANS fluorescence, shown as integrated areas between 450 and 600 nm for IgG (B), 30 nm AunpIgG (C), and 80 nm AunpIgG (D). In parts BD, closed symbols denote results acquired during heating, whereas open symbols denote results acquired from the cooling part of the temperature loop.
sizes Aunps results in a decrease in the short lifetime population and an increase in the long lifetime population. The effects are more pronounced upon addition of 30 nm Aunps, which can be attributed to their higher concentration and total surface area (Aunp concentrations are 0.3 and 0.02 nM for 30 and 80 nm Aunps, respectively, yielding a 30 nm Aunp/80 nm Aunp surface area ratio of ∼2.6). As revealed in Table 1, the thermoresponse of the AunpIgG constructs is markedly different from that of IgG only. While the population of the short lifetime decreases for IgG only upon exposure to a heating/cooling cycle, the reverse is true for the AunpIgG systems; there is a marked increase in the population associated with the short lifetime at the expense of the long lifetime. We interpret this result as more of the intrinsic fluorophores being exposed to solvent following a temperature cycle, which is consistent with temperature-induced breakup of mesoscopic IgG clusters with embedded Aunps. As IgG has a large number of intrinsic fluorophores in various chemical environments, we also employed the common extrinsic
fluorescent probe ANS for the study of temperature-induced conformational changes. ANS is highly fluorescent when associated with the hydrophobic interior of the protein but is almost completely quenched in water due to solvent effects.39,40 Consequently, conformational changes due to temperature-induced unfolding below Tm or breakup of mesoscopic IgG clusters are expected to result in changes in ANS intensity. Steady-state fluorescence spectra of IgG and 30 nm AunpIgG at 25 °C before and after a heatingcooling cycle are shown in Figure 6A. Samples containing only ANS or Aunps with ANS in water gave negligible signal intensity compared to the protein-containing samples. Figure 6A reveals that upon completion of a heating cooling cycle for IgG only the emission intensity increases and the fluorescence peak is blue-shifted ∼10 nm. This is consistent with an increased access to hydrophobic domain via partial unfolding, which is in agreement with the steady-state and TCSPC results for intrinsic fluorophores. Conversely, a heating cooling cycle results in a marked reduction in ANS emission 11397
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The Journal of Physical Chemistry C intensity for the AunpIgG system (only the 30 nm AunpIgG is shown). Moreover, the AunpIgG systems also display a marked temperature-induced change in line shape—with increasing temperature, the emission peak goes from monomodal (peak maximum ∼515 nm) to bimodal, with peak maxima observed at ∼490 and ∼580 nm. This change in line shape can be attributed to energy transfer between ANS and Aunps, caused by the Aunps becoming more accessible to ANS molecules with increasing temperature. Because of this change in ANS emission line shape for the Aunp-containing systems, the temperature response is plotted as the normalized (with respect to the highest observed signal—IgG only after completion of a temperature cycle) areas in the region 450650 nm for the systems studied here (Figure 6, panels BD). From Figure 6BD, the temperature response is markedly different between IgG only and the AunpIgG systems, but also for the two different Aunp sizes. While heating results in an increased emission intensity for IgG only (Figure 6B), both AunpIgG systems display an irreversible reduction in signal intensity upon heating (Figure 6C). Between the Aunp sizes, the largest temperature-induced signal reduction is observed for the 30 nm AunpIgG (Figure 6C), whereas the 80 nm AunpIgG displays the largest initial reduction in emission intensity (Figure 6D). The temperature-induced reduction in emission for the AunpIgG systems can be attributed to increased contact with solvent from breakup of mesoscopic clusters, which is in agreement with UVvis, DLS, and intrinsic fluorescence data presented above. A breakup of clusters results in more surface area being exposed and thus to increased ANSsolvent contact. Thus, as the Aunps are embedded into large IgG clusters, the initial contact with the polar ANS molecule is quite limited. However, as the mesoscopic Aunp-containing IgG clusters are broken up as a result of heating, the Aunps are closer to the external cluster surface and thus more accessible to ANS, resulting in the observed line shape changes. Differences between Aunp sizes are likely attributed to a combination of (i) different particle concentrations and total surface areas leading to variations in cluster location and ANS access and (ii) IgG unfolding on Aunp surfaces being dependent on particle size.5 As there is a high degree of covariance between these two parameters, we cannot distinguish between their contributions within the experimental setup used here.
’ CONCLUSIONS Throughout this study it has been documented that a commercially available human immunoprotein (IgG) tends to form mesoscopic aggregates in citrate solution at pH 8.55. Although multiple studies exist reporting on optical and physical properties of proteins in bulk solution or even conjugated to nanoparticles, very little has been done regarding temperature-dependent behavior of these types of systems. By employing nanoparticles with inherent LSPR properties, it has been shown that both intrinsic and extrinsic optical properties can be utilized to follow protein flocculation behavior. More specifically, we have demonstrated that protein-embedded gold nanoparticles tend to irreversibly break up AunpIgG clusters as a function of increased temperature. Protein adsorption on gold surfaces and the resulting mesoscopic aggregates have been followed by means of QCM, UVvis, fluorescent steady state, and lifetime techniques and is corroborated by light scattering measurements, all revealing that
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the presence of Aunps alters the optical and physical properties of the concomitant constructs. The presented work is expected to expand the current knowledge of thermal response in protein and proteinnanomaterial constructs, and it also illustrates how correct choice of solid nanoparticle surface and size allows for tunable interactions and optical behavior. As is evident from the acquired results, the optical signature of Aunps can be utilized to monitor changes in the nanoparticles’ immediate environment, whether it is flocculation state and/or transitions in the adsorbed protein layer(s). Moreover, there is an inherent potential to affect the breakup and reversibility of protein plaque formation through use of nanoparticle constructs and subsequent thermal treatment.
’ AUTHOR INFORMATION Corresponding Author
*Fax: þ47 73 59 40 80. E-mail:
[email protected].
’ ACKNOWLEDGMENT This work has been made possible through funding from the Research Council of Norway within the FRINAT program, project no. 177556/V30. ’ REFERENCES (1) Cedervall, T.; Lynch, I.; Lindman, S.; Berggard, T.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2050–2055. (2) Lindman, S.; Lynch, I.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Nano Lett. 2007, 7, 914–920. (3) Linse, S.; Cabaleiro-Lago, C.; Xue, W. F.; Lynch, I.; Lindman, S.; Thulin, E.; Radford, S. E.; Dawson, K. A. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 8691–8696. (4) Lynch, I.; Cedervall, T.; Lundqvist, M.; Cabaleiro-Lago, C.; Linse, S.; Dawson, K. A. Adv. Colloid Interface Sci. 2007, 13435, 167–174. (5) Kane, R. S.; Stroock, A. D. Biotechnol. Prog. 2007, 23, 316–319. (6) Everts, M.; Saini, V.; Leddon, J. L.; Kok, R. J.; Stoff-Khalili, M.; Preuss, M. A.; Millican, C. L.; Perkins, G.; Brown, J. M.; Bagaria, H.; Nikles, D. E.; Johnson, D. T.; Zharov, V. P.; Curiel, D. T. Nano Lett. 2006, 6, 587–591. (7) Liu, Y. L.; Franzen, S. Bioconjugate Chem. 2008, 19, 1009–1016. (8) Ryan, J. A.; Overton, K. W.; Speight, M. E.; Oldenburg, C. M.; Loo, L.; Robarge, W.; Franzen, S.; Feldheim, D. L. Anal. Chem. 2007, 79, 9150–9159. (9) Tkachenko, A. G.; Xie, H.; Coleman, D.; Glomm, W.; Ryan, J.; Anderson, M. F.; Franzen, S.; Feldheim, D. L. J. Am. Chem. Soc. 2003, 125, 4700–4701. (10) Tkachenko, A. G.; Xie, H.; Liu, Y. L.; Coleman, D.; Ryan, J.; Glomm, W. R.; Shipton, M. K.; Franzen, S.; Feldheim, D. L. Bioconjugate Chem. 2004, 15, 482–490. (11) Xie, H.; Tkachenko, A. G.; Glomm, W. R.; Ryan, J. A.; Brennaman, M. K.; Papanikolas, J. M.; Franzen, S.; Feldheim, D. L. Anal. Chem. 2003, 75, 5797–5805. (12) Brewer, S. H.; Glomm, W. R.; Johnson, M. C.; Knag, M. K.; Franzen, S. Langmuir 2005, 21, 9303–9307. (13) Kaufman, E. D.; Belyea, J.; Johnson, M. C.; Nicholson, Z. M.; Ricks, J. L.; Shah, P. K.; Bayless, M.; Pettersson, T.; Feldoto, Z.; Blomberg, E.; Claesson, P.; Franzen, S. Langmuir 2007, 23, 6053–6062. (14) Lacerda, S. H. D.; Park, J. J.; Meuse, C.; Pristinski, D.; Becker, M. L.; Karim, A.; Douglas, J. F. ACS Nano 2010, 4, 365–379. (15) Teichroeb, J. H.; Forrest, J. A.; Jones, L. W. Eur. Phys. J. E 2008, 26, 411–415. 11398
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