SUPPORTING INFORMATION
Lysozyme-Induced Spectral Changes in the Au nanoparticle solution Extinction Spectrum. Protein solutions of concentrations ranging from 32 µM to 3.2 nM (by factors of 10) were mixed with equal volumes of Au nanoparticle solutions (~10 pM). The control sample was prepared by the mixing of Au nanoparticle solution with an equal volume of the buffer used to prepare the lysozyme solutions. The photograph and extinction spectra shown in Figure S1 were taken after shaking the solution for 3 hours.
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Figure S1:
Extinction spectra and photograph of Au-NPs mixed with lysozyme of different
concentrations (a: 16 µM; b: 1.6 µM; c: 160 nM; d: 16 nM; e: 1.6 nM; f: 0 nM).
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Au nanoparticle Interaction with BSA. BSA/Au nanoparticle solution was prepared by mixing 20 µM of BSA solution, prepared in 10 mM phosphate buffer solution (pH=7.5) with an equal volume of ~10 pM Au nanoparticle solution.
Figure S2 shows the surface plasmon resonance (SPR) spectrum of
the BSA/Au nanoparticle solution measured at different times subsequent to sample preparation. The spectrum labeled as “0 minutes” was obtained with an equal volume mixture of Au nanoparticle solution and 10 mM phosphate buffer.
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Figure S2: SPR spectra of ~5 pM Au nanoparticle in 10 µM BSA solution measured at different times following sample preparation. Spectra obtained at 10 mins, 35 mins and 130 mins were scaled with a scaling factor of 1.000±0.003, to compensate for small intensity variations between spectral acquisitions. The inset shows the spectra in the wavelength region between 530 nm and 620 nm. Unlike the time-varying extinction spectrum of the Au nanoparticles mixed with lysozyme solution 2
(Fig. S1), there is essentially no temporal variation of the extinction spectra of the BSA/Au nanoparticle solution. Compared to the spectrum obtained for the BSA-free Au nanoparticle solution, however, the extinction spectra of Au nanoparticles in BSA is red-shifted 4 nm from 572 nm to 576 nm, indicating BSA adsorption onto the Au nanoparticles.36, 37 The lack of time-dependent spectral variation in the SPR spectra of the BSA/Au nanoparticle solution indicates that no Au nanoparticle aggregation occurs after BSA adsorption: aggregation would necessarily give rise to a new SPR peak at longer wavelength region (as in Fig. 1 in article).
This result is in contrast with the aggregate formation for the
lysozyme/Au nanoparticle solution, where the formation of aggregates is essentially completed in the first 30 minutes after the mixing of Au nanoparticle solution with lysozyme.
Quantification of Lysozyme Adsorbed onto Au nanoparticles with Tryptophan Fluorescence: Quantification of lysozyme adsorbed onto Au nanoparticles was performed with highly concentrated nanoparticles synthesized with the same protocol specified in the experimental section, but with a doubled amount of formaldehyde. The blue curve in Figure S3 is the extinction spectra of the stock Au nanoparticle solution after a 60 X dilution, while the red curve is the extinction spectrum calculated using Mie theory, assuming a particle size of 56 nm diameters. The overlapping of the peak maxima in the experimental and the calculated spectra indicates an average size of the synthetic Au nanoparticle is nominally 56 nm. The larger peak width at half maximum of the sample spectrum is due to some polydispersity of the synthetic Au nanoparticles. Stock concentration of the synthetic Au nanoparticle was estimated to be 2.8 nM according to its UvVis spectrum and the extinction coefficient calculated for 56 nm Au nanoparticles. The red oily stock solution of Au nanoparticles turned into black directly after it was mixed with an equal volume of 5 µM, 10 µM or 20 µM lysozyme solution respectively. Corresponding controls for each experiment solution were prepared by mixing the 5 µM, 10 µM or 20 µM lysozyme solutions with
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equal volumes of distilled water. After shaking both control and experimental solutions for two days under ambient conditions, all solutions were centrifuged for 30 minutes (400g) with an IEC LC21 multispeed centrifuge machine (Thermo Electron). Fluorescence intensities of tryptophan for each solution shown in Figure S5 were measured with a fluorolog-3 fluorimeter (Jobin Yvon Horiba Inc.). The calibration curve shown in Figure S4 (D) was obtained with three control solutions as well as two lysoyzme calibration solutions with concentrations of 3.75 µM and 6.5 µM, respectively.
Figure S3: Surface plasmon resonance spectra of Au nanoparticle solution. The blue trace was obtained with theAu nanoparticles after 60X dilution from a stock Au nanoparticle solution, and the red trace is the calculated SPR spectrum, assuming a particle size diameter of 56 nm. The intensity of the calculated spectrum is scaled to match the peak maximum of the Au nanoparticles.
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Figure S4: Fluorescence intensities of tryptophan in the supernatant of lysozyme solutions (red) with and (blue) without Au nanoparticles. Final lysozyme concentrations are 2.5 µM, 5 µM and 10 µM respectively. Concentration of Au nanoparticle is 1.4 nM in all the Au nanoparticle containing solutions. Plot (D) is the calibration curve. The peaks around 310 nm correspond to the Raman modes of H2O. With the results shown in Figure S4 (C)-(D), it is estimated that ~4000 lysozyme molecules were centrifuged per Au nanoparticle. Assuming all the precipitated proteins were accumulated onto the Au nanoparticle surfaces and the adsorbed proteins retain their solution hydrodynamic radius at pH~7,38 there would be over 6 layers of protein on each Au nanoparticle, consistent with our observation of multilayer protein deposition onto the nanoparticles.
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Cryo-TEM images taken with the Lysozyme/Au nanoparticle solution and control solution
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Figure S5: Cryo-TEM Images of structures obtained from (A)-(C) lysozyme/Au nanoparticle solution and (D) control solution. While most aggregates in images (A)-(C) appear to be amorphous, some of them have slightly more defined features, such as relatively straight edges. No aggregates were found in the control samples. Scale bar: 200 nm.
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SERS Spectra and Peak Assignment for Protein Adsorbed onto the Au nanoparticle Surfaces
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Figure S6: Representative SERS spectra acquired with three different protein/NP assemblies in solution. All spectra were acquired using a 63X water immersion objective with a laser power of 0.57 mW measured before sample (0.1% of the power used for normal Raman acquisition) and an integration time of 10 s. Spectra were offset for clarity.
References: 36. Shang, L.; Wang, Y.; Jiang, J.; Dong, S. Langmuir 2007, 23, (5), 2714-2721. 37. Tam, F.; Moran, C.; Halas, N. J. Phys. Chem. B 2004, 108, (45), 17290-17294. 38. Bonincontro, A.; De Francesco, A.; Onori, G. Colloids and Surfaces B: Biointerfaces 1998, 12, (1), 1-5.
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