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Effect of Self-Association of Bovine Serum Albumin on the Stability of Surfactant-Induced Aggregates of Allylamine-Capped Silicon Quantum Dots Surajit Chatterjee and Tushar Kanti Mukherjee* Discipline of Chemistry, Indian Institute of Technology Indore, M-Block, IET-DAVV Campus, Khandwa Road, Indore 452017, M.P., India S Supporting Information *

ABSTRACT: The concentration-dependent self-association of bovine serum albumin (BSA) and subsequent altered interaction with sodium dodecyl sulfate (SDS) has been explored by means of photoluminescence (PL) spectroscopy, dynamic light scattering (DLS), circular dichroism (CD), PL imaging, and atomic force microscopy (AFM). By using an extrinsic luminescent probe, allylamine-capped silicon quantum dots (Si-QDs), we have demonstrated the unusual concentration-dependent altered BSA−SDS interaction. Allylamine-capped Si-QDs forms ordered aggregates in the presence of 1 mM SDS due to hydrogen bonding with the surfactants head groups at pH 7.4. Although these aggregates remain stable in the presence of monomeric BSA in the concentration range 1−8 μM, they form typical ring-shaped doughnut-like structures due to “necklace and bead”-like complex formation. However, beyond 10 μM BSA, these aggregates of Si-QDs slowly dissociate and complete dissociation occurs at 150 μM BSA. These anomalous results have been explained by considering the altered hydrophilicity of self-associated BSA.

1. INTRODUCTION The dynamics and mechanism of surfactant-induced denaturation of globular proteins have been thoroughly studied over the past few decades.1−7 It has been proposed that the initial interaction between very low concentrations of surfactants and proteins is predominantly ionic in nature with the surfactant head groups bound to oppositely charged groups on the protein. These initial electrostatic interactions lead to partially unfolded proteins, which subsequently bind with more surfactants in a cooperative fashion. The most well studied example of protein−surfactant interaction is between bovine serum albumin (BSA) and sodium dodecyl sulfate (SDS). Both intrinsic and extrinsic fluorescent probes have been used to illustrate the underlying mechanism and dynamics of BSA− SDS interactions.2,7−11 Earlier, Oakes has studied the binding isotherm between BSA and SDS by means of nuclear magnetic resonance signal of surfactant molecules and proposed that both the surfactant head groups and alkyl chains interact with the proteins at low surfactant concentrations.12 At higher surfactant concentrations, micelle-like complexes (“necklace and bead” model) are formed on proteins in which the surfactant alkyl chains are associated with protein apolar groups. Although, these earlier studies are very informative and describe the BSA−SDS interaction in detail, little is known about the effect of high protein concentration on the initial binding interaction between BSA and SDS. Serum albumins are the most abundant proteins in the bloodstream.13 The major physiological role of albumins is to carry various ligands, such as fatty acids, amino acids, steroids, © 2013 American Chemical Society

and metal ions, in the bloodstream to their respective target organs. BSA has a single polypeptide chain with a molecular weight of 66 kDa and consists of 583 amino acid residues.13 Unfolding and subsequent denaturation of native BSA under a variety of external conditions such as changes in pH, temperature, and the binding of chemical denaturants are well-known.14−16 Another important but less explored source of protein unfolding is self-association or aggregation at a high protein concentration. Proteins often exit in their physiological environment at high concentrations. Serum albumin, for example, is present in the blood at a high concentration of ∼50 mg/mL.13 Protein aggregation is a well-known phenomenon related to serious medical implications like Alzheimer’s disease, Parkinson’s disease, and amyloidosis.17−19 Conformation change is the primary requirement for initiation of association. The properties of unfolded and aggregated BSA at high concentration are poorly understood. Earlier, Brahma et al. have reported dimerization of BSA at mildly acidic conditions.14 They have identified a state of BSA at pH 4.2, which shows increased light scattering at 350 nm and assigned it to a partially unfolded dimeric intermediate with alteration of surface hydrophobicity. It has also been shown that thermal stress on BSA promotes protein aggregation through the formation of intermolecular β-sheets. However, there are very few reports of self-association of BSA in the absence of any external stress. Received: September 12, 2013 Revised: December 3, 2013 Published: December 4, 2013 16110

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the safety precautions before using it.] Each of these cleaning steps was followed by repeated washing with Milli-Q water. Finally, these washed slides were rinsed with methanol (SigmaAldrich) and dried in vacuum oven. The QDs sample was spincasted on washed cover slides at 2000 rpm for 3 min. 2.2. Instrumentation. Absorption spectra were recorded in a quartz cuvette (10 × 10 mm) using a Varian UV−vis spectrophotometer (Carry 100 Bio). PL spectra were recorded using Fluoromax-4 spectrofluorometer (HORIBA Jobin Yvon, model FM-100) with excitation and emission slit width at 4 nm. All measurements were performed at room temperature. CD spectra of BSA were acquired by a JASCO J-815 CD spectropolarimeter using a quartz cell of 1 mm path length. Scans were made with a slit width of 1 mm and speed of 20 nm/min. AFM images were recorded on a cleaned glass coverslip using AIST-NT microscope (model SmartSPM1000). Transmission electron microscope images were recorded on a JEOL electron microscope (JEM-2100). DLS experiments were performed on a Brookhaven particle size analyzer (model 90 Plus). All the samples for DLS measurements were prepared in pH 7.4 milli-Q water and filtered through a 0.22 μm syringe filter (Whatman). PL imaging of individual Si-QDs was performed on a custom-made fluorescence microscope setup based on an inverted fluorescence microscope (Nikon, Eclipse Ti−U) coupled to a back-illuminated EM-CCD camera (Andor, iXon X3 897). The schematic representation of the instrumental arrangement is shown in Figure S5 (Supporting Information). A collimated laser beam from an air-cooled argon ion laser (Melles Griot, 400-A03) was passed through a beam expander (Holmarc, India) and a neutral density filter (Sigma Koki, Japan). The beam was then focused on the back focal plane of an oil immersion objective (Apo TIRF, Nikon, 100X, NA = 1.49). With the help of a mirror (Sigma Koki, Japan) the beam was directed toward the center of the back aperture of the objective parallel to the objective axis. The samples at the interface were excited by the laser beam with an excitation wavelength of 457 nm. The PL signal was filtered by a 505 nm dichroic mirror and a 520 nm long-pass filter. Finally, the images were captured by the back illuminated EM-CCD camera at frame rate of 200 ms. The images were analyzed with ImageJ (Version 1.47v) NIH.

Earlier, Levi et al. have demonstrated reversible fast dimerization of BSA at room temperature in pH 5.8 buffer through fluorescence resonance energy transfer.20 They have shown that BSA is in monomer−dimer equilibrium with a dissociation constant of 10 ± 2 μM at 25 °C in pH 5.8 buffer. Although the dimeric form of BSA has been known for some time, its physiochemical properties have not been explored in details. In the present work, we have explored the dynamics of BSA−SDS interactions with an external luminescent marker allylamine-capped Si-QDs as a function of BSA concentration. Quantum dots are semiconductor nanocrystals that have emerged as far better candidates for light-emitting devices than conventional organic dyes due to their unique photoluminescence (PL) properties.21−23 In the past few decades Cd-based core−shell QDs have been studied thoroughly. However, these core−shell QDs do have a significant drawback in biomedical application due to their bigger size and cytotoxicity.24 Recently, silicon quantum dots (Si-QDs) have emerged as a most promising candidate for optoelectronics and biomedical application.25−27 Earlier, we have shown that in the presence of negatively charge SDS surfactant, allylamine-capped Si-QDs forms ordered aggregates that exhibit large Stokes shifted PL at 610 nm.28 These aggregates are highly dynamic in nature, as they subsequently dissociate into monomeric forms at higher surfactant concentrations due to formation of spherical micelles. The aim of the present study is 2-fold: first, whether the specific protein−surfactant interaction leads to deaggregation of surfactant-induced aggregates of Si-QDs and, second, the effectiveness of Si-QDs as an extrinsic luminescence marker for BSA−SDS interaction.

2. EXPERIMENTAL SECTION 2.1. Materials and Sample Preparation. Silicon tetrachloride (99%) and tetrahydrofuran (THF, 99.5%) were purchased from Merck. Allylamine (99%) was purchased from Spectrochem. Bovine serum albumin (BSA, ≥99%, essentially fatty acid free and globulin free), tetraoctylammonium bromide (TOAB, 98%), chloroplatinic acid hexahydrate, sodium dodecyl sulfate (SDS, 98.5%), and isopropyl alcohol (99%) were purchased from Sigma-Aldrich. Lithium aluminum hydride (LAH, 97%) and toluene (99%) were purchased from SD Fine Chemicals. Milli-Q water was obtained from a Millipore water purifier system (Milli-Q integral). Allylamine-capped Si-QDs were synthesized in reverse micelles according to literature.29 All reactions were carried out in an argon atmosphere. Reactants were removed by successive filtration through a 0.22 μm membrane filter. For all the experiments the final concentration of Si-QDs was kept constant at 300 nM so that the optical density (O.D.) should not exceed the value of 0.5 to avoid unnecessary selfaggregation and the inner filter effect.30 All solutions were prepared in pH 7.4 phosphate buffer. Different concentrations of BSA samples were prepared by dissolving the required amount (MBSA= 66 000 g mol−1) of BSA in pH 7.4 phosphate buffer. The final concentrations were determined spectrophotometrically using the extinction coefficient at 280 nm (ε280) of 44 720 M−1 cm−1.2,13 For microscopy experiments the samples were spin-coated with a spin-coater (Apex Instruments, Spin NXG-P1) on a clean cover slide. The cover slides were first cleaned with 2% Hellmanex III (Sigma-Aldrich) followed by piranha solution (3:1 concentrated sulfuric acid and 30% hydrogen peroxide). [Caution: Piranha solution is aggressive and explosive. Never mix piranha waste with solvents. Check

3. RESULTS AND DISCUSSION 3.1. PL Characteristics of Si-QDs Aggregates in the Presence of BSA. The synthesized allylamine-capped Si-QDs have a mean diameter of 3.65 ± 0.04 nm (Figure 1A). These SiQDs show continuous absorption between 200 and 600 nm with size-dependent PL (Figure S1 of the Supporting Information).28 Addition of 1 mM SDS into the QDs solution results in PL quenching at 455 nm with the appearance of a new large Stokes shifted PL at 610 nm (Figure 1). Earlier, we have assigned this Stokes shifted PL band to an ordered surfactant-induced aggregates of Si-QDs.28 Maximum aggregation has been observed at 1 mM SDS concentration and beyond that they slowly dissociates due to formation of spherical micelles. Here, it is important to note that even at 1 mM SDS concentration both monomeric and aggregated SiQDs remains in an equilibrium which indicate that there are no free surfactants present in the solution. The absorption and PL spectrum of allylamine-capped Si-QDs remains unchanged in the presence of BSA (Figure S1 of the Supporting Information). Figure 1B shows the changes in PL spectra of allylamine-capped Si-QDs in the presence of 1 mM SDS with 16111

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resulting in unfolding of the protein. The greater extent of quenching of tryptophan fluorescence in the presence of SiQDs aggregates signifies additional unfolding of the proteins. However, significant spectral changes for the aggregated SiQDs have been observed beyond the 10 μM BSA concentration. Figure 1C shows the effect of high concentration of BSA on the PL spectra of surfactant-induced aggregates of allylamine-capped Si-QDs. Here, it is important to note that these PL spectra were subtracted from the intrinsic fluorescence of BSA at 455 nm, which has been discussed in more detail in the next paragraph. With gradual addition of BSA, the PL intensity at 455 nm increases whereas that at 610 nm decreases with an isoemissive point at 542 nm. The aggregated emission at 610 nm totally disappears at 150 μM BSA. The changes in the ratio of the two PL band intensity with BSA concentration are shown in Figure 1D. The complete disappearance of the aggregated PL at 610 nm with increased PL from normal emission at 150 μM BSA suggests that SDS molecules specifically bind with the protein rather than allylamine-capped Si-QDs. Similar kinds of spectral changes have been observed for 460 nm excitation (Figure S3 of the Supporting Information). These results strongly signify the concentration-dependent conformational change of BSA with altered hydrophobicity and hydrophilicity. To know the origin of this concentration-dependent conformational dynamics of BSA, we performed intrinsic fluorescence measurement of BSA and DLS measurement. 3.2. Intrinsic Fluorescence and CD Spectroscopy of BSA. Figure 2A shows the normalized intrinsic BSA emission as a function of excitation wavelength at 150 μM concentration. Excitation at 295 nm results in normal tryptophan emission centered at 340 nm. However, excitation at and above 350 nm results in large Stokes shifted broad emission. A broad emission

Figure 1. (A) TEM image of allylamine-capped Si-QDs. The inset shows the size distribution histogram. PL spectra of allylamine-capped Si-QDs at λex = 375 nm in the presence of (B) (i) 0 mM SDS and 0 μM BSA, (ii) 1 mM SDS, (iii) 1 mM SDS and 2 μM BSA, (iv) 1 mM SDS and 4 μM BSA, (v) 1 mM SDS and 6 μM BSA, and (vi) 1 mM SDS and 8 μM BSA and of (C) (i) 0 mM SDS and 0 μM BSA, (ii) 1 mM SDS, (iii) 1 mM SDS and 20 μM BSA, (iv) 1 mM SDS and 50 μM BSA, (v) 1 mM SDS and 100 μM BSA, and (vi) 1 mM SDS and 150 μM BSA. (D) Changes in the PL intensity ratio of the normal band (IN) and aggregated band (IAgg) of Si-QDs with varying BSA concentrations.

varying concentration of BSA from 2 to 8 μM range. A marginal increase in the PL intensity at 455 nm with no noticeable change in the 610 nm band indicates that the surfactantinduced aggregates remain unperturbed in the presence of BSA. Earlier, it has been shown that BSA contains a high affinity binding site for very low concentrations of SDS.10 Addition of very low concentrations of SDS (0.0−0.2 mM) results in specific binding that follows a noncooperative binding region. The cooperative binding of SDS to BSA starts at or near 0.6 mM SDS concentration. Hence, the unchanged PL behaviors of these surfactantinduced aggregates of Si-QDs signify that SDS has more affinity to bind at the QDs surface than with the proteins. Even though the surfactant-induced aggregates of Si-QDs remain stable in the presence of BSA, we cannot rule out interaction of BSA with these aggregates as a whole due to the presence of free hydrocarbon chains on SDS. To verify this, we monitored the intrinsic fluorescence of BSA in the absence and presence of these Si-QDs aggregates. Earlier, it has been shown that the intrinsic tryptophan fluorescence of BSA is highly sensitive to any conformational change. 10 BSA shows an intense fluorescence band centered at 340 nm in pH 7.4 phosphate buffer at λex = 295 nm (Figure S2 of the Supporting Information). In the presence of 1 mM SDS this band shifts to 314 nm with 41% quenching in fluorescence. This quenching of fluorescence with 24 nm blue shift strongly signifies unfolding of native BSA where the tryptophan residues are surrounded by more hydrophobic hydrocarbon chains of the SDS molecules. Interestingly, addition of surfactant-induced aggregates of Si-QDs into BSA solution results in 18 nm blue shift with 54% quenching in fluorescence intensity of the intrinsic tryptophan emission (Figure S2 of the Supporting Information). These spectral changes indicate that these surfactant-induced aggregates of Si-QDs bind with BSA,

Figure 2. (A) Changes in intrinsic fluoresecnce of 150 μM BSA with different excitation wavelengths. (B) Intrinsic fluorescence spectrum of BSA as a function of concentration at 375 nm excitation. The inset shows the changes in PL intensity with varying BSA concentration. (C) Excitation spectrum of 150 μM BSA for 340 nm (blue) and for 455 nm (red) emission. 16112

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centered at 455 nm has been observed at 375 nm excitation. The quantum yield of this band increases with an increase in BSA concentration (Figure 2B). The inset of Figure 2B shows the variation of 455 nm band intensity with BSA concentration (2−150 μM). It is evident that the band at 455 nm appears above 10 μM BSA concentration. To know the origin of this large Stokes shifted emission band, we measured its excitation spectrum. Figure 2C shows the excitation spectrum of 150 μM BSA at 340 and 455 nm. The excitation spectrum at 340 nm shows a single peak at 293 nm, which exactly overlaps with the absorption spectrum, whereas that at 455 nm shows two peaks: one at 293 nm and a very broad peak at 360 nm. The presence of a second broad peak at 360 nm in the excitation spectrum clearly highlights ground state association or aggregation.31 It is evident from these results that the self-association of BSA starts above 10 μM BSA concentration, which is in good agreement with the earlier reported dissociation constant of 10 ± 2 μM.20 To know whether this self-association of BSA alters the secondary structure of BSA, we recorded the far-UV CD spectra of BSA at 2 and 150 μM concentration (Figure S4 of the Supporting Information). It has been shown earlier that native BSA is mainly α-helix (∼66%) and exhibits two minima at 208 and 222 nm in the CD spectrum.32 It is evident from Figure S4 of the Supporting Information that BSA at a concentration of 2 μM remains in native form, which exhibits two characteristic negative bands at 208 and 222 nm with 64% α-helix. However, at 150 μM BSA concentration, the spectrum loses its α-helical character and resembles more a β-sheet structure with only 10% α-helix content. Similar kinds of spectral changes have been observed earlier for BSA.33 These spectral changes clearly demonstrate the alteration in secondary structure of self-associated BSA at high concentration. To the best of our knowledge, this type of direct spectroscopic evidence of concentration-dependent self-association or aggregation has not been reported earlier for BSA. 3.3. Dynamic Light Scattering (DLS) Measurement. Direct evidence of self-association of BSA at high concentration comes from DLS measurement. Figure 3 shows the changes in the intensity histogram as a function of BSA concentration. BSA at a concentration of 2 μM shows a narrow monodispersed peak with a mean diameter of 4.260 ± 0.001 nm. However, BSA at a concentration of 50 μM shows a bimodal distribution with one peak at 4.04 ± 0.03 nm and the second one at 59.40 ± 0.47 nm (Figure 3B). The second peak shifts to higher size with increasing BSA concentration and at 150 μM BSA the second peak shows a value of 104.66 ± 1.26 nm (Figure 3C,D). The first peak with a mean diameter of 4.260 ± 0.001 nm observed for 2 μM BSA is in good agreement with the hydrodynamic diameter (RH) reported in the literature for monomeric BSA at pH 7.4.34−36 The second larger diameter peak that appears at higher BSA concentration can be assigned due to the self-associated BSA as the value of RH increases with an increase in BSA concentration. This kind of concentrationdependent self-association without any external perturbation is very rare in literature. Earlier, Zhau et al. have studied the intermolecular interactions between a polysaccharide, sodium alginate, and BSA by means of ζ potential analyzer, DLS, and turbidimetric analysis.36 The reported ζ potential of diluted (2.0 mg/mL) BSA aqueous solution is −33.9 ± 0.6 mV whereas that for a concentrated (20.0 mg/mL) BSA solution is −27.8 ± 0.8 mV. A similar ascending trend of the ζ potential has been shown for the heat-denatured BSA. On the basis of these observations, they proposed that at high concentration

Figure 3. Size distribution histogram from DLS measurement for (A) 2 μM, (B) 50 μM, (C) 100 μM, and (D) 150 μM BSA.

and at high temperature, BSA undergoes conformational change where the positively charged amino acids are surfaceexposed and the system became unstable. In the present study the obtained RH of the self-associated BSA at a concentration of 150 μM is in good agreement with the reported RH of 91 ± 14 nm for the heat-denatured BSA. Hence, the present DLS results signify that BSA at high concentration undergoes self-association and both monomeric and self-associated BSA remains in equilibrium. 3.4. PL Microscopy. To investigate the luminescent behavior of individual Si-QDs aggregates in the absence and presence of BSA, we performed PL imaging. PL imaging of individual Si-QDs aggregates was performed by a 457 nm excitation source and the detailed schematic diagram of our home-built PL microscopic setup has been shown in Figure S5 of the Supporting Information. Figure 4 shows the PL images of surfactant-induced aggregates of Si-QDs in the absence and presence of BSA at pH 7.4. Surfactant-induced aggregates of SiQDs shows bright luminescent spots with broad fwhm ranging from 2 to 7 pixels (Figure 4A) similar to what has been reported earlier at pH 4.5.28 The intensity trajectory of three representative luminescent spots of Si-QDs aggregates are shown in figure S6 of the Supporting Information. They show gradual intensity decay with no clear photobleaching step (Figure S6 of the Supporting Information). However, when these QDs aggregates spin-cast in the presence of 150 μM BSA, we observed diffraction limited spots (fwfm ∼ 2−3 pixels) with characteristic fluorescence blinking and single step photobleaching (Figure S6 of the Supporting Information). Blinking (fluorescence on−off) is a well-known phenomenon for single molecule or single QDs that distinguishes between 16113

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Scheme 1. Proposed Model for Concentration-Dependent Differential Interaction of BSA with SDS-Induced Si-QDs Aggregates: (A) Low and (B) High Concentration of BSA

Figure 4. PL images of Si-QDs with (A) 1 mM SDS, (B) 1 mM SDS and 150 μM BSA, and (C) 1 mM SDS and 2 μM BSA. (D) Intensity line profile of the selected doughnut-shaped ring.

aggregated spots against single dot spots.28,37 This is in accordance with our steady state results where SDS specifically binds with the self-associated BSA, leaving monomeric QDs in solution. Interestingly, in the presence of 2 μM BSA these aggregates exhibit uniform ring-shaped doughnut emission patterns. Similar kinds of ring-shaped doughnut patterns have been observed earlier for z-oriented molecular structures in total internal reflection (TIR) excitation mode.38−40 However, ring-shaped emission patterns are very rare in epi-fluorescence excitation mode, as it does not have electric-field components perpendicular to the surface. Ring-type structural morphology has also been reported earlier by various groups.41−45 Earlier, Cavallini et al. have demonstrated the mechanism of nanometric ring formation by Mn12 clusters on polycarbonate film.44 They observed that drop-casting of Mn12 clusters in dichloromethane solvent on polycarbonate film results in formation of nanometric rings due to demixing of solute upon solvent treatment. Here, it is important to note that these earlier rings were observed from thin film prepared by drop casting and the technique was based on the transformation of droplets to rings due to evaporation of volatile organic solvent and subsequent solvent treatment. However, the present system is unique in the sense that samples were prepared by spin coating in phosphate buffer solution on the glass surface. Here, it is important to remember that SDS forms “necklace and bead”-like structure in the presence of BSA in the concentration range of 1−7 μM (Scheme 1). Hence, the observed ring-shaped emission patterns of SiQDs aggregates in the presence of BSA is due to the formation of “necklace and bead” structures where the micelle-like aggregates of SDS are associated with the unfolded protein through their free hydrocarbon chain. To the best of our knowledge, this is the first direct microscopic evidence of “necklace and bead”-like structures between BSA−SDS. 3.5. Atomic Force Microscopy (AFM). To determine the morphology and size of these fluorescent ring structures, we performed AFM (Figure 5). Clear ring-shaped doughnut structures have been observed in the presence of 2 μM BSA concentrations. Figure 5B shows a close up look of one of the selected ring with its line scan analysis. This type of characteristic line profile in AFM image with no appreciable amount of QDs in the center of the ring clearly

Figure 5. (A) AFM image of SDS-induced aggregates of allylaminecapped Si-QDs in the presence of 2 μM BSA. (B) AFM image of the selected ring with its line scan analysis. Size distribution of (C) ring diameter and (D) hole diameter.

signify that surfactant-induced aggregates of Si-QDs warp around the protein to form “necklace and bead”-like structures (Scheme 1). These rings show a mean diameter of 1.30 ± 0.04 μm with a mean hole diameter of 0.47 ± 0.01 μm (Figure 5C,D). On the basis of our experimental results, we propose a model where BSA in its monomeric native state undergoes unfolding in the presence of SDS-induced Si-QDs aggregates and form “necklace and bead”-like structures mainly due to hydrophobic interaction with the free hydrocarbon chain of SDS (Scheme 1). Uniform ring-shaped emission patterns in PL imaging with ring-shaped morphology in AFM clearly support our proposed model. Interestingly, the extent of SDS-induced aggregation of Si-QDs remains unaltered in these “necklace and bead”-like structures. However, BSA at high concentration undergoes selfassociation and adopts a unique conformation where its positively charged residues expose to surrounding environment. We believe that these exposed positively charged residues on 16114

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(3) Mesa, C. L. Polymer-Surfactant and Protein-Surfactant Interactions. J. Colloid Interface Sci. 2005, 286, 148−157. (4) Chakraborty, T.; Chakraborty, I.; Moulik, S. P.; Ghosh, S. Physicochemical and Conformational Studies on BSA-Surfactant Interaction in Aqueous Medium. Langmuir 2009, 25, 3062−3074. (5) Naidu, K. T.; Prabhu, N. P. Protein-Surfactant Interaction: Sodium Dodecyl Sulfate-Induced Unfolding of Ribonuclease A. J. Phys. Chem. B 2011, 115, 14760−14767. (6) Otzen, D. Protein-Surfactant Interactions: A Tale of Many States. Biochim. Biophys. Acta 2011, 1814, 562−591. (7) Anand, U.; Jash, C.; Mukherjee, S. Spectroscopic Probing of the Microenvironment in a Protein-Surfactant Assembly. J. Phys. Chem. B 2010, 114, 15839−15845. (8) Chakraborty, A.; Seth, D.; Setua, P.; Sarkar, N. Photoinduced Electron Transfer in a Protein-Surfactant Complex: Probing the Interaction of SDS with BSA. J. Phys. Chem. B 2006, 110, 16607− 16617. (9) Sahu, K.; Roy, D.; Mondal, S. K.; Karmakar, R.; Bhattacharyya, K. Study of Protein-Surfactant Interaction using Excited State Proton Transfer. Chem. Phys. Lett. 2005, 404, 341−345. (10) Paul, B. K.; Samanta, A.; Guchhait, N. Exploring Hydrophobic Subdomain IIA of the Protein Bovine Serum Albumin in the Native, Intermediate, Unfolded, and Refolded States by a Small Fluorescence Molecular Reporter. J. Phys. Chem. B 2010, 114, 6183−6196. (11) De, D.; Santra, K.; Datta, A. Prototropism of [2,2′-Bipyridyl]3,3′-diol in Albumin-SDS Aggregates. J. Phys. Chem. B 2012, 116, 11466−11472. (12) Oakes, J. Protein-Surfactant Interactions. Nuclear Magnetic Resonance and Binding Isotherm Studies of Interactions between Bovine Serum Albumin and Sodium Dodecyl Sulfate. J. Chem. Soc., Faraday Trans. 1 1974, 70, 2200−2209. (13) Carter, D.; Ho, J. X. Advances in Protein Chemistry; Academic Press: New York, 1994; Vol. 45, pp 153−215. (14) Brahma, A.; Mandal, C.; Bhattacharyya, D. Characterization of a Dimeric Unfolding Intermediate of Bovine Serum Albumin under Mildly Acidic Condition. Biochim. Biophys. Acta 2005, 1751, 159−169. (15) Takeda, K.; Wada, A.; Yamamoto, K.; Moriyama, Y.; Aoki, K. Conformational Change of Bovine Serum Albumin by Heat Treatment. J. Protein Chem. 1989, 8, 653−659. (16) Jiménez, M. O.; Pozzo, D. C. Structural Analysis of Protein Denaturation with Alkyl Perfluorinated Sulfonates. Langmuir 2012, 28, 17749−17760. (17) Smith, M. A. Alzheimer Disease. Int. Rev. Neurobiol. 1998, 42, 1−54. (18) Olanow, C. W.; Tatton, W. G. Etiology and Pathogenesis of Parkinson’s Disease. Annu. Rev. Neurosci. 1999, 22, 123−144. (19) Wetzel, R. Immunoglobulin Deposition Disorder. Adv. Protein Chem. 1997, 50, 183−242. (20) Levi, V.; Flecha, F. L.G. Reversible Fast-Dimerization of Bovine Serum Albumin Detected by Fluorescence Resonance Energy Transfer. Biochim. Biophys. Acta 2002, 1599, 141−148. (21) Henglein, A. Small Particle Research: Physiochemical Properties of Extremely Small Colloidal Metal and Semiconductor Particles. Chem. Rev. 1989, 89, 1861−1873. (22) Weller, H. Quantized Semiconductor Particles: A Novel State of Matter for Materials Science. Adv. Mater. 1993, 5, 88−95. (23) Murray, C. B.; Norris, D. J.; Bawendi, M. G. Synthesis and Characterization of Nearly Monodisperse CdE (E = Sulfur, Selinium, Tellurium) Semiconductor Nanocrystallites. J. Am. Chem. Soc. 1993, 115, 8706−8715. (24) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Probing the Cytotoxicity of Semiconductor Quantum Dots. Nano Lett. 2004, 4, 11−18. (25) Bley, R. A.; Kauzlarich, S. M.; Davis, J. E.; Lee, H. W. H. Characterization of Silicon Nanoparticles Prepared from Porous Silicon. Chem. Mater. 1996, 8, 1881−1888. (26) Erogbogbo, F.; Yong, K. T.; Roy, I.; Xu, G.; Prasad, P. N.; Swihart, M. T. Biocompatible Luminescent Silicon Quantum Dots for Imaging of Cancer Cells. ACS Nano 2008, 2, 873−878.

BSA specifically interact with negatively charged sulfate head groups of SDS and results in deaggregation of Si-QDs. However, we cannot rule out alteration of surface hydrophobicity of self-associated BSA that may also contribute to the overall deaggregation process.

4. CONCLUSIONS In summary, we have demonstrated the concentration-dependent self-association of BSA and its altered hydrophilicity. At high concentrations both monomeric and self-associated BSA remain in equilibrium. The altered hydrophilicity of selfassociated BSA has been explored through its differential interaction with SDS-induced aggregates of allylamine-capped Si-QDs. Even though the monomeric BSA undergoes unfolding in the presence of SDS-induced Si-QDs aggregates, it is not able to destabilize these aggregates. However, BSA at high concentrations destabilizes these aggregates due to selective binding of SDS to its altered conformation. Our microscopic results show that SDS-induced Si-QDs aggregates form doughnut-like ring structures due to the formation of “necklace and bead”-like complexes with monomeric BSA. These results are potentially useful to understand the interaction of these hydrophilic QDs with the cell membrane and associated proteins.

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ASSOCIATED CONTENT

* Supporting Information S

Changes in absorption and PL spectra of allylamine-capped SiQDs with BSA, changes in intrinsic tryptophan fluorescence in the absence and presence of Si-QDs aggregates, changes in PL spectra of Si-QDs aggregates as a function of BSA at 460 nm excitation, concentration-dependent changes in CD spectra of BSA, PL imaging setup, PL intensity profiles of Si-QDs aggregates in the absence and presence of BSA. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*T. K. Mukherjee: e-mail [email protected]; phone, +91-7312438-738. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank IIT Indore for providing the infrastructure, experimental facilities, and financial support. This work is supported by Council of Scientific and Industrial Research grant no. 01(2695)/12/EMR-II. The authors sincerely thank Professor Anindya Datta and Tuhin Khan from the Indian Institute of Technology Bombay for their help during dynamic light scattering experiment. The authors thank the sophisticated analytical instrument facility of North-Eastern Hill University, Shillong, for TEM measurements.

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REFERENCES

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dx.doi.org/10.1021/jp4091188 | J. Phys. Chem. B 2013, 117, 16110−16116