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Article Cite This: Anal. Chem. 2018, 90, 785−793

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Quantification of Gold Nanoparticle Ultraviolet−Visible Extinction, Absorption, and Scattering Cross-Section Spectra and Scattering Depolarization Spectra: The Effects of Nanoparticle Geometry, Solvent Composition, Ligand Functionalization, and Nanoparticle Aggregation Joanna Xiuzhu Xu,†,∥ Kumudu Siriwardana,†,∥ Yadong Zhou,‡ Shengli Zou,*,‡ and Dongmao Zhang*,†,§ †

Department of Chemistry, Mississippi State University, Mississippi State Mississippi 39762, United States Department of Chemistry, University of Central Florida, Orlando, Florida 32816, United States § Department of Chemistry, Xihua University, Chengdu 610039, China ‡

S Supporting Information *

ABSTRACT: Using the recent polarized resonance synchronous spectroscopic (PRS2) technique, we reported the quantification of photon extinction, absorption, scattering cross-section spectra, and scattering depolarization spectra for AuNPs of different sizes and shapes. The effects of the solvent composition, ligand functionalization, and nanoparticle aggregation on the AuNP photon absorption and scattering have also been experimentally quantified. The light scattering depolarization is close to 0 for gold nanospheres (AuNSs) crossing the entire UV−vis region but is strongly wavelength-dependent for gold nanorods (AuNRs). Increasing the dielectric constant of the medium surrounding AuNPs either by solvents or ligand adsorption increases photon absorption and scattering but has no significant impact on the AuNP scattering depolarization. Nanoparticle aggregation increases AuNP photon scattering. However, even the extensively aggregated AuNPs remain predominantly photon absorbers with photon scattering-to-extinction ratios all less than 0.03 for the investigated AuNP aggregates at the AuNP peak extinction wavelength. The AuNP scattering depolarization initially increases with the AuNP aggregation but decreases when aggregation further progresses. The insights from this study are important for a wide range of AuNP applications that involve photon/matter interactions, while the provided methodology is directly applicable for experimental quantification of optical properties for nanomaterials that are commonly simultaneously photon absorbers and scatterers.

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Determination of the NP photon extinction spectrum and thereby its extinction cross-section spectrum is straightforward with conventional UV−vis spectrophotometric measurements. However, experimental decomposition of the NP extinction spectra into their absorption and scattering component spectra is difficult with existing methods. Current NP light scattering detections can be divided into three categories. The first is the integration-sphere-based methods in which the photons collected with the integration sphere are taken as the total photons scattered by the NPs.20 The second technique is the home-built UV−vis spectrophotometer in which the light collected 90° relative to the excitation direction is assumed to be the photons scattered by NPs.21 The third method uses the spectrofluorometer by assuming the resonance synchronous spectra (RS2) are the analyte scattering intensity spectra.22

ne key attraction of nanoscale materials is their sizedependent optical properties.1−4 While photon scattering is a universal property, metal and metal oxide nanoparticles (NPs) are often also active in photon absorption and emission because of the quantum confinement.5,6 Quantitatively understanding NP optical activities is critical for establishing the correlation among NP chemical structure, geometry, and optical properties. Such information is vital for providing guidelines for nanomaterial design for applications in display, solar energy harvesting, optical sensing, and optoelectronics.7−14 However, experimental determination of the NP optical properties including their photon absorption, scattering, and emission cross-sections is challenging because of the complex interplay of the photon absorption, scattering, and emission. As an example, existing fluorescence studies have focused exclusively on the Stokes-shifted fluorescence (SSF).15−17 The on-resonance fluorescence (ORF), photon emission occurring at the same wavelength as that for the excitation photons, has totally been ignored or mistakenly attributed to the material photon scattering.18,19 © 2017 American Chemical Society

Received: August 10, 2017 Accepted: November 24, 2017 Published: November 24, 2017 785

DOI: 10.1021/acs.analchem.7b03227 Anal. Chem. 2018, 90, 785−793

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and interpreting the experimental data. The combined experimental and computational results enable us to explore the possible correlation between AuNP structure and optical properties including the scattering-to-extinction ratio (SER), and scattering depolarizations. Besides, the effect of surface modification, AuNP aggregation, and the solvent composition on AuNP photon absorption and scattering was also quantified.

Unfortunately, none of those techniques has simultaneously addressed the following four technical obstacles for accurate light scattering cross-section determination. The first is the separation of the NP photon scattering from its ORF for fluorescence-active materials. The second is the correction of the sample inner filter effect (IFE) induced by the material photon absorption of the excited and scattered/emitted photons. The third is the removal of the solvent Rayleigh scattering and cuvette background scattering in the determination of the NP photon scattering and ORF. The fourth is the undersampling problem. This refers to the fact that no existing approaches including the integration-sphere method are capable of collecting all scattered photons generated by the samples. The collected fraction of the scattered/emitted photons depends not only on the specific instrument configuration, such as the detector orientation, sample size, and acceptance angle, but also on the NP light scattering depolarization.23 Such a depolarization dependence has, to our knowledge, not been considered before in the determination of the light scattering cross-sections. We have recently developed a polarized resonance synchronous spectroscopic (PRS2) method that has enabled, for the first time, quantitative decoupling of the interplay of the photon absorption, scattering, and ORF emission.23 This method involves combined UV−vis extinction and PRS2 measurements conducted with a conventional UV−vis spectrophotometer and spectrofluorometer. The UV−vis extinction spectral measurements are important not only for determination of NP extinction cross-section but also for correcting the sample IFE in the PRS2 acquisition. The spectrofluorometer equipped with excitation and detection polarizers provides a simple way for direct PRS2 detection. The only difference between PRS2 and the conventional RS2 lies in the fact that both excitation and detected lights are polarized in PRS2, but neither one is polarized in the conventional RS2 technique. PRS2 enables one to quantitatively separate the light scattering and ORF signal by taking advantage of their significant difference in photon depolarization or anisotropy.23 AuNPs are among the most studied plasmonic nanoparticles that have found broad applications in optical spectroscopy, optoelectronics, and solar energy harvesting.12−14,24 Earlier works showed that the AuNP UV−vis extinction spectrum varies as a function of solvent composition,25,26 AuNP surface modifications,27,28 and AuNP aggregations.29 However, how much such variations are due to the AuNP photon absorption and scattering is unclear. Filling these knowledge gaps is important for a wide range of existing AuNP applications and for exploring new AuNP applications. This is because photon absorption and scattering are different optical processes that can be responsive differently to different experimental parameters and their impacts also differ on different targeted applications. The study of the effects of solvents, ligand functionalization, and the NP aggregation on the AuNP photon absorption and scattering activities should open doors for one to fine-tune the AuNP optical properties for its target applications. Using the combination of the UV−vis spectrophotometric and PRS2 measurements, reported herein is the experimental quantification of the UV−vis extinction, absorption, scattering cross-section spectra, and the scattering−depolarization spectrum for a series of AuNPs with different sizes and shapes. Meanwhile, computational simulations have been used for exploring the effect of AuNP geometries on its optical activities

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EXPERIMENTAL SECTION Materials and Equipment. All chemicals were obtained from Sigma-Aldrich unless indicated otherwise. Nanopure water (18.2 MΩ cm, Thermo Scientific) was used in all sample preparations. Mutated third IgG-binding domain of protein G (GB31) was provided by Prof. Nicholas Fitzkee at Mississippi State University. Wild-type GB3 protein does not contain cysteine residue. The lysine residue at the 19th position in wildtype GB3 protein is replaced by a cysteine residue in GB31 so that GB31 can bind specifically to AuNP through formation of a S−Au bond. The GB3 and GB31 sequences, preparation, and their binding to AuNPs were described in previous publications.28,31 Both GB31 and bovine serum albumin (BSA) proteins were purified through membrane dialysis before use. The gold nanorods (AuNRs) were obtained from Nanopartz. The catalog numbers of AuNR550, AuNR600, and AuNR650 are A12-25-550 (Lot No. E2973D), A12-25-600 (Lot No. RPB045D), and A12-25-650 (Lot No. RPD236AD), respectively. UV−vis measurements were taken with an Evolution 300 spectrophotometer (Thermo Scientific, Waltham, MA, USA). PRS2 spectra were acquired with a Fluoromax-4 spectrophotometer (Horiba Jobin Yvon) equipped with both excitation and detection polarizers. The instrumental G-factor of the spectrofluorometer needed for correcting the instrument polarization bias in the signal response in the calculation of the light scattering depolarization is determined and validated with a G-factor sample set (Raminescent LLC). This G-factor sample set enables determination of the G-factor crossing the entire spectral wavelength region with one sample. This is in contrast to the earlier G-factor determination that requires multiple fluorescent samples.23 Transmission electron microscopy (TEM) images were obtained by a JEOL 2100 instrument with an accelerating voltage of 200 kV. The AuNSs and AuNRs were deposited on a Cu grid covered by a Formvar carbon film. AuNS Preparation. AuNS13 was synthesized in house with the citrate reduction method,32,33 while AuNS50 and AuNS70 were obtained from nanoComposix. The brief procedure for the AuNS13 preparation is as follows. A 0.0415 g amount of gold(III) chloride trihydrate was dissolved in 100 mL of nanopure water. The solution was refluxed while stirring. Upon solution boiling, 10 mL of 38.8 mM sodium citrate dihydrate solution was added and the solution mixture was allowed to further reflux for 20 min. The peak localized surface plasmonic resonance (LSPR) extinction wavelength is 520 nm, indicating the average diameter is 12.3 ± 1.5 nm for the spherical AuNP. The concentration of the as-synthesized AuNS13 is 12 nM estimated according to the LSPR peak intensity. Effect of Solvent. Solvent effect on AuNS13 plasmonic properties was studied by water/glycerol mixture solvents containing 8.3%, 25%, and 33% glycerol. AuNSs (12 nM, 1 mL) were mixed with equal volumes of glycerol solutions prior to UV−vis and PRS2 spectral acquisitions. Solvent PRS2 spectra were acquired and subtracted from their respective IFE786

DOI: 10.1021/acs.analchem.7b03227 Anal. Chem. 2018, 90, 785−793

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Figure 1. As-acquired PRS2 spectra obtained with the AuNS-containing solutions with excitation and detection polarization combinations of (A) “VV” and (B) “VH”, respectively. The AuNS PRS2 spectra of excitation and detection polarization combinations of (C) VV and (D) VH. Panels E−I are the AuNS extinction, absorption, scattering cross-section spectra, scattering depolarization spectra, and SER spectra, respectively. The solid lines are experimental data, and the dashed lines are computational results. Panels J−L are representative TEM images obtained with AuNS13, AuNS50, and AuNS70, respectively. TEM images for AuNS50 and AuNS70 are reprinted with permission from nanoComposix.

Theoretical Simulations. Discrete dipole approximation (DDA) method is used in the simulations. The detailed description of the DDA method can be found in the existing literature for calculation of scattering, absorption, and extinction cross-sections of metal NPs of different shapes. In calculating the depolarization properties of metal NPs with different aspect ratios, we arranged nanoparticles with over 200 different orientations in the space and then integrated the scattered light along the detector direction. The incident light is kept polarized in the horizontal direction, but both vertical and horizontal detection polarizations are used to mimic the experimental measurement.

corrected AuNS/glycerol PRS2 spectra before further data analysis. Effect of Ligand Binding. The effect of ligand functionalization on AuNS13 optical properties was studied using glutathione (GSH), BSA, and GB31 as model ligands. GSH, GB31, and BSA concentrations were changed from 2 to 100 μM, from 0.5 to 10 μM, and from 0.25 to 12.5 μM, respectively. Equal volumes of AuNS13 and model ligands were mixed and incubated overnight prior to UV−vis and PRS2 spectral acquisition. PRS2 spectra of the ligand and solvent mixtures were taken and subtracted from the IFE-corrected PRS2 spectra of the AuNS13/ligand mixture spectra before further data analysis. Effect of AuNP Aggregation. The effect of AuNP aggregation on the AuNP photon extinction, absorption, and scattering cross-sections and scattering depolarization was studied using AuNS13 as the model AuNPs. The aggregation was induced by using KNO3 as the aggregate initiator and 20 μM BSA as the aggregation quencher. Earlier research has shown that the earlier stage of AuNP aggregation can be readily quenched by BSA addition.34 The degree of the aggregation was controlled by treating AuNPs with different concentrations of KNO3 before quenching by BSA 10 min after the KNO3 and AuNP mixing. After vortex mixing the AuNPs with KNO3, BSA was added into the AuNP/KNO3 solution. The final solution is vortex mixed briefly before UV−vis and PRS2 measurements. The solvent background PRS2 spectra obtained with the BSA and KNO3 mixtures were subtracted from the IFE-corrected PRS2 spectra obtained with the (AuNP/KNO3)/BSA mixtures before further data analysis.

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RESULTS AND DISCUSSION Quantification of Optical Cross-Section and Depolarization Spectra. Since AuNPs are simultaneous photon scatterers and absorbers with no significant fluorescence emission, the IFE-corrected AuNP PRS2 spectra contains only light scattering features from the solvent, cuvette, and the NP themselves, but not ORF. Therefore, AuNP scattering depolarization Psca AuNP(λ) can be readily determined by AuNP PRS2 spectra as described in eq 1, and the AuNP light scattering cross-section spectrum is quantified by using monodispersed polystyrene nanoparticles (PSNPs) as the sca external reference (eq 2).23 Psca AuNP(λ) and PPSNP(λ) in the equations are the AuNP and PSNP light scattering depolarizasca tion spectra, and Isca AuNP,VV(λ) and IPSNP,VV(λ) are the AuNP and PSNP PRS2 spectra determined using the excitation and detection polarization, both perpendicular to the measurement plane defined by the source, sample cuvette, and the detector. 787

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Figure 2. (A−D) Schematic representation of the shape of the simulated AuNRs. The computed (E) photon extinction, (F) absorption, and (G) scattering cross-section spectrum and the (H) depolarization spectrum for AuNRs with a constant volume of 8200 nm3 but different aspect ratio. Peak (I) extinction, (J) absorption, (K) scattering, and (L) depolarization as a function of the AuNR aspect ratio. The arrows in the second-row plots refer to the data with increasing aspect ratios.

refer to the combination of the excitation and detection polarizations. The effective path length of the spectrofluorometer, which is needed for correcting the sample IFE in the PRS2 measurement, is 1.14, and it was quantified on the basis of the IFE on solvent Raman signal.30,35 The procedures for the path length determination, sample IFE correction, and solvent background subtraction are available from the earlier publication.23 The fact that the AuNS PRS2 spectra differ significantly from the solution PRS2 spectra highlights the importance of removing the sample IFE and solvent background interference. The AuNS extinction cross-section spectra (Figure 1E) were calculated straightforwardly using the extinction spectra obtained with the AuNS extinction spectra (Figure S1, Supporting Information). The AuNS scattering depolarization spectra (Figure 1H) and scattering cross-section spectra were determined with eq 1 and eq 2, respectively. AuNS peak extinction (Figure 1E), absorption (Figure 1F), and scattering (Figure 1G) cross-sections, as well as the SER (Figure 1I) spectra increase with increasing particle sizes in diameters ranging from 13 to 70 nm. SER is a measure of the fractional contribution of sample light scattering to its UV−vis extinction. The fact that both the SER value and absorption cross-section increase with increasing particle size indicates that while AuNS photon absorption and scattering increases with increasing AuNS sizes, photon scattering increases faster than absorption crossing the entire spectral region. It also shows that photon scattering is more sensitive than AuNP photon extinction and absorption in detecting AuNS size variations. Overall, the computational AuNS extinction, absorption, scattering, and SER spectra are in good agreement with their experimental counterparts for all investigated AuNSs (Figures 1E−G,I). This provides a cross-validation of both the computational method and experimental strategy. However, the experimental AuNS light scattering depolarization is significantly higher than that of the computational counterparts

PSNPs are predominantly photon scatterers in the UV−vis wavelength region from 300 and 800 nm. Its scattering crosssections can be reliably quantified using UV−vis measurements.30 Detailed mathematic derivation of eq 1 and eq 2 and the experimental procedures for obtaining the parameters in the right-hand side of these equations are available from the recent literature.23 Since AuNPs are pure absorbers and scatterers as mentioned above, the scattering PRS2 spectra of AuNP sca IAuNP,VV (λ) in eq 2 should be equal to IPRS2 AuNP,VV(λ) in eq 1. sca PAuNP (λ ) = G (λ )

sca σAuNP (λ ) =

PRS 2 IAuNP , VH(λ) PRS 2 IAuNP , VV (λ)

(1)

sca sca (1 + 2PAuNP (λ)) CPSNPIAuNP , VV (λ) sca σPSNP(λ) sca sca (1 + 2PPSNP (λ)) CAuNPIPSNP , VV (λ)

(2)

The G(λ) in eq 1 is the G-factor spectrum. Detailed discussion of the G-factor spectrum and its applications in photon scattering, SSF, and ORF depolarization calculation is available from an earlier publication.23 After obtaining the AuNP extinction cross-section spectrum with its UV−vis spectra and its scattering spectrum with the PRS2 measurements (eq 2), the AuNP absorption cross-section spectrum is then obtained by subtracting its scattering cross-section spectrum from its extinction cross-section spectrum. AuNS and AuNR Optical Activities. Gold nanospheres (AuNSs) of different sizes are employed to investigate the effect of particle volume on optical properties. Figure 1 shows the data obtained for AuNSs with the diameters of 13 nm (AuNS13), 50 nm (AuNS50), and 70 nm (AuNS70). Panels A and B of Figure 1 are the as-acquired PRS2 spectra obtained with the AuNS-containing solutions, while the spectra in Figure 1C,D are AuNS PRS2 spectra obtained by subtracting the solvent background PRS2 spectra from the IFE-corrected AuNS solution PRS2 spectra. The superscripts “VV” and “VH” 788

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Figure 3. (Top row) Representative TEM images for the (A) AuNR550, (B) AuNR600, and (C) AuNR650. (Bottom row) AuNR (D) extinction, (E) absorption, and (F) scattering cross-section spectra of (black) AuNR550, (red) AuNR600, and (green) AuNR650. The experimental extinction spectra, the solution PRS2 spectra, the PSNP UV−vis and PRS2 spectra, the solvent background spectra required for calculation of the AuNS extinction, absorption, and scattering cross-section, and the scattering depolarization were shown in Supporting Information Figure S2.

notations of AuNRaspect ratio (Figure 2) and AuNRpeak wavelength (Figure 3) to represent the theoretical and experimental AuNRs is that it is currently impossible to prepare AuNRs with the same volume but different aspect ratios. It is also difficult for reliabe experimental determination of the sizes and shapes of the experimental AuNPs. This is clearly evident from the TEM images of a series of AuNRs from the same vendor (Figure 3). As an example, the average volume of the AuNR600 is most likely the highest among these three commercial AuNRs, but its aspect ratio is larger than that for AuNR550 but smaller than that for AuNR650 (Figure 3A−C). It is therefore impossible to perform a head-to-head comparison of the computational data with experimental values shown in Figure 2. Qualitatively, however, the experimental data obtained with AuNR550, AuNR600, and AuNR650 (Figure 3) are in reasonable agreement with the computational results. First, they both show that AuNRs remain predominantly photon absorbers with no significant scattering. The largest SER values as the peak extinction wavelengths are 0.09, 0.04, and 0.13 for AuNR550, AuNR600, and AuNR650, respectively. Second, experimental depolarization spectra (Figure S2, Supporting Information) all exhibit a wavelength dependence similar to that of the computed data (Figure 2H). The depolarization of the AuNRs is very close to zero in the wavelength region below 520 nm, then rapidly increasing to its maximum between 550 and 650 nm, followed by slowly decreasing. The reason that the depolarization maximum appears between 550 and 650 nm can be understood as the following. The peak light scattering wavelength of the transverse mode is around 520 nm followed by a gradual activity decrease with increasing wavelength, while the longitudinal mode activity increases with increasing wavelength before reaching its peak wavelength from 650 to 950 nm. The peak AuNR light scattering depolarization should appear at the wavelength when the light scattering cross sections of the transverse and longitudinal modes are most similar to each other. Solvent Effect on the AuNP Plasmonic Properties. Experimental data described in this and subsequent sections were all acquired with AuNS13 as the model AuNP. It is known that the AuNP UV−vis extinction increases with the increasing solvent dielectric constant.2,36 However, how much such increment is due to photon absorption and scattering is unclear. Moreover, the solvent effect on the AuNP light scattering depolarization has not been investigated. Figure 4

(Figure 1H). This discrepancy is due most likely to the fact that the AuNSs used in the computational simulations were assumed to be perfectly spherical, while essentially all actual AuNSs are nonspherical. This is evident from the AuNS TEM images (Figures 1J−L). Indeed, some particles can be oval shaped even like a rod. The deviation of the experimental and modeled light scattering depolarization also suggests that scattering depolarization might be very sensitive to shape variations, which is examined in the following section. The effects of AuNP shape on its optical properties are subsequently studied by computational simulations. The modeled AuNRs are constructed such that two hemispheres at each end are linked by a cylindrical rod in the middle (Figure 2). The aspect ratio is defined by the distance between the ends of the hemisphere divided by the diameter of the cylinder diameter. Aiming to reveal the trend of how shape variation could modify optical properties of AuNP, the volumes of the AuNPs were kept the same but the shapes varied from a perfect AuNS, that is, viewed as AuNR with an aspect ratio of 1.00, to AuNRs with an aspect ratio of 5.10. The computational modeling reveals that light scattering depolarization is extraordinarily sensitive for detection of shape transitions from nanosphere to nanorod (Figure 2). The data shown in Figure 2 indicate that AuNR peak extinction (Figure 2E), absorption (Figure 2F), scattering (Figure 2G), and scattering depolarization (Figure 2H) wavelengths all increase with increasing AuNR aspect ratios. Another key new learning is that the peak light scattering depolarization value is far more sensitive in detecting the AuNP shape changes from a perfect nanosphere to nanorod with an aspect ratio below 2 (Figure 2L), while the peak AuNP extinction, scattering, and absorption cross-sections are most sensitive to detect the AuNP shape change when the aspect ratio falls in the region of 2−4 (Figure 2I−K). This indicates the peak light scattering depolarization and peak scattering intensity are highly complementary in the detection of the AuNP sizes and shapes. Since both light scattering depolarization and cross-section spectra can be conveniently obtained with these PRS2 measurements, the data indicate that PRS2 can be used for in situ studying of the AuNP geometry in solutions. To experimentally verify the above assumptions, PRS2 measurements were made with AuNR with a nominal peak extinction wavelength of 550 nm (AuNR550), 600 nm (AuNR600), and 650 nm (AuNR650). The reason we use 789

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Figure 4. Comparison of (first and third columns) experimental (labeled as “Exp.”) and (second and fourth columns) computational (labeled as “Comp.”) optical properties of AuNS13 in glycerol/water mixtures. (A) Experimental and (B) computational UV−vis extinction cross-section spectra, (E) experimental and (F) computational absorption cross-section spectra, (I) experimental and (J) computational scattering cross-section spectra of AuNS13 in glycerol/water mixtures. (C) Peak experimental and (D) computational UV−vis extinction cross-section, (G) peak experimental and (H) computational absorption cross-sections, (K) peak experimental and (L) computational scattering cross-sections as a function of (black) glycerol percentage and (red) refractive index (n) of glycerol/water mixtures. (Fourth row) (M) Experimental and (N) computational depolarization and (O) experimental and (P) computational SER values at the AuNS13 peak extinction wavelength as a function of glycerol percentage.

compared with experimental and computational data obtained with the AuNS13 dissolved in the water/glycerol mixture solvents. The as-acquired UV−vis extinction and PRS2 spectra used for the cross-section calculation were shown in Figure S2 in the Supporting Information. The dielectric constants of the glycerol/water mixture were calculated according to previous literature.37 Evidently, the experimental AuNS extinction (Figure 4A,B), absorption (Figure 4E,F), and scattering crosssection spectra (Figure 4I,J) are all in excellent agreement with their computational counterparts, all increasing linearly with increasing glycerol concentrations and the solvent refractive index. However, the light scattering depolarization is totally independent of the glycerol concentrations for the studied nanoparticles (Figure 4M,N). This is, to our knowledge, the first experimental and computational investigation of the solvent effects on photon absorption and scattering contribution to the AuNP UV−vis extinction spectra and on the AuNP scattering depolarization. The fact that solvent dielectric has no detectable effect on the AuNS light scattering depolarization is consistent with the theoretical consideration that increasing solvent dielectric constant is equivalent to increasing the size of the AuNS but has no effect on the shape of the AuNS. Since the AuNS light scattering depolarizations are negligibly small in the size range from 13 and 70 nm in diameter, it is not surprising that the

small change in the AuNP size induced by increasing the solvent dielectric constant is inadequate to cause significant change on the AuNS depolarization. Effect of AuNP Surface Functionalization. Ligand binding has significant impact on the AuNP localized surface plasmonic resonance (LSPR) properties. Figure 5 compares the AuNP UV−vis extinction, scattering, and absorption crosssection spectra as functions of the nominal concentration of model ligands including GSH, GB31, and BSA. The as-acquired UV−vis and PRS2 spectra used for the cross-section and light scattering depolarization spectrum calculation were shown in Figures S4 and S5 in the Supporting Information. The molecular weights of GSH, GB31, and BSA are 307, 6208, and 67000 g/mol, respectively. This set of the ligands were chosen because, first, they all contain thiols that can bind to AuNPs through the Au−S bond formation; second, they are highly hydrophilic and their binding has no significant effect on the AuNP dispersion stability;28,38,39 and third, the large differences in their molecular weights/sizes also provide an opportunity for us to study the possible ligand size effects on the AuNP photonic activity. When the ligand concentration is low, the bindings of GSH, GB31, and BSA all increase the AuNP UV−vis extinction, scattering, and absorption cross-sections as they increase with increasing ligand concentrations in the AuNS/ligand mixture 790

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Figure 5. Effect of the ligand functionalization on the AuNS13 optical properties. The model ligands are (top row) GSH, (middle row) GB31, and (bottom row) BSA. AuNS (A) UV−vis extinction, (B) absorption, and (C) scattering cross-section spectra as a function of GSH concentration. AuNS (D) UV−vis extinction, (E) absorption, and (F) scattering cross-section spectra as a function of GB31 concentration. AuNS (G) UV−vis extinction, (H) absorption, and (I) scattering cross-section spectra as a function of BSA concentration. The insets are the peak photon extinction, absorption, and scattering cross-sections as a function of the nominal ligand concentrations. The light scattering depolarization spectra and SER spectra as a function of the ligand concentrations are shown in Supporting Information Figure S5.

mixed with BSA monotonically increase with increasing BSA concentration (insets in Figure 5G−I). Effect of AuNP Aggregation on Its Optical Properties. UV−vis spectroscopy is the most convenient, effective, and broadly applied method to monitor the AuNP aggregation.43 This is because AuNP aggregation induces a red shift and broadening of the AuNP LSPR peaks.44 However, quantitative understanding of the effect of AuNP aggregation on the photon absorption and scattering contribution to the AuNP extinction spectra is currently lacking. Furthermore, there is no analytical method capable of providing the in situ shape information on the AuNP aggregates in solutions. Figure 6 shows that with the increasing degree of AuNP aggregation, the UV−vis extinction peak becomes increasingly broader in which the spectral intensity at the long wavelength region becomes increasingly prominent. The photon absorption at the 520 nm region, which is assigned to the monomeric AuNPs, decreases with increasing AuNP aggregations. These results are expected because the number of monomeric AuNPs reduces as the AuNP aggregate proceeds. The increased UV− vis intensity at the long wavelength region is due to the LSPR feature of the aggregated AuNPs. There are two most noteworthy observations for the data in Figure 6. First, regardless of the interplay of the photon absorption and scattering at different wavelengths, the investigated AuNP aggregates remain predominantly a photon absorber across the entire investigated wavelength region. Indeed, the largest SER ratio for the aggregated AuNPs at the peak extinction wavelength is 0.027 (Figure 6), indicating that light absorption contributes at least 97% of the AuNP peak UV−vis extinction intensity. In contrast, the photon scattering only contributes less than 3% of the AuNP UV−vis extinction. The second important observation is that the light scattering depolarization provides a simple measurement of the shape evolution of the AuNP aggregates. The peak depolarization increases initially with the degree of the AuNP aggregation but

solutions. These results are expected because the ligand binding increases the dielectric constant of the medium immediately surrounding the AuNP surfaces. However, depending on the ligand identities, further increasing ligand concentration has different effects on the AuNP optical properties. In the case of GSH and GB31, the AuNP optical cross-section constants reach a plateau when the ligand concentration is higher than 15 μM for GSH and 3 μM for GB31. In the case of BSA, however, the AuNP extinction, scattering, and absorption cross-sections monotonically increase with increasing BSA concentrations. The difference between BSA and the two other ligands is due most likely to their sizes. Ligands can modify the optical properties of the AuNP-containing solutions through three pathways. The first is the direct ligand adsorption that increases the dielectric constant of the medium immediately surrounding the AuNP surfaces. Such effect is clearly in effect for all three ligands, and it should occur only before the ligand reaches saturation adsorption. The second is the ligand-induced AuNP aggregation. This commonly occurs when the ligands are hydrophobic molecules,39,40 or electrolytes with high concentrations,41 or electrolytes that can form ion pairs on the AuNP surfaces.42 However, this AuNP aggregation pathway can be excluded because the GSH-, GB31-, and BSA-functionalized AuNPs all have excellent dispersion stability in water. The third mechanism is the solvent dielectric changes induced by the excess ligands. BSA is a macromolecule with the 67000 g/mol molecular weight, which is 120 and 10 times higher than those of GSH and GB31, respectively. Presumably, excess BSA is much more effective to change the solution dielectric constant than excess GSH and GB31 of similar concentrations. This explains why, for GSH and GB31, AuNS photon extinction, absorption, and scattering increase with increasing ligand concentration until saturation adsorption of the ligand was reached in the investigated concentration range (insets in Figure 5A−F), but the optical cross-sections of the AuNP 791

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Figure 6. Experimental data obtained with (first column) AuNS13/BSA, (second column) (AuNS13/25 mM_KNO3)/BSA, (third column) (AuNS13/30 mM_KNO3)/BSA, and (fourth column) (AuNS13/50 mM_KNO3)/BSA. The AuNP concentrations are identical in all four samples. (First row) TEM images of (A) AuNS13/BSA, (B) (AuNS13/25 mM_KNO3)/BSA, (C) (AuNS13/30 mM_KNO3)/BSA, and (D) (AuNS13/50 mM_KNO3)/BSA. (Second row) UV−vis extinction cross-section spectra of (E) AuNS13/BSA, (F) (AuNS13/25 mM_KNO3)/BSA, (G) (AuNS13/30 mM_KNO3)/BSA, and (H) (AuNS13/50 mM_KNO3)/BSA. (Third row) Absorption cross-section spectra of (I) AuNS13/BSA, (J) (AuNS13/25 mM_KNO3)/BSA, (K) (AuNS13/30 mM_KNO3)/BSA, and (L) (AuNS13/50 mM_KNO3)/BSA. (Fourth row) Scattering crosssection spectra of (M) AuNS13/BSA, (N) (AuNS13/25 mM_ KNO3)/BSA, (O) (AuNS13/30 mM_ KNO3)/BSA, and (P) (AuNS13/50 mM_ KNO3)/BSA. (Fifth row) (red) AuNS scattering depolarization and (black) SER ratio spectra of (Q) AuNS13/BSA, (R) (AuNS13/25 mM_ KNO3)/BSA, (S) (AuNS13/30 mM_ KNO3)/BSA, and (T) (AuNS13/50 mM_ KNO3)/BSA.

13 nm are aggregated linearly but ∼0.15 when they aggregate into an equilateral triangle. This further indicates the sensitivity of light scattering depolarization for monitoring the shape variations of the AuNP aggregates.

then decreases as the aggregation further progresses. The most likely reason is that the initial AuNP aggregation proceeds through the formation of dimer and trimer AuNP that is likely in a chain-/rod-like structure. These aggregates have a large depolarization as shown by the experimental data obtained with AuNS and AuNR. However, further aggregations most likely proceed by the agglomeration of the oligomerized AuNPs. Such AuNP agglomerate presumably adopts a globular structure with negligible photon scattering depolarization. This hypothesis is consistent with the experimental observation made with spherical polystyrene beads that have no detectable scattering depolarization.23 It is also supported by the TEM images acquired with the BSA stabilized as-synthesized AuNPs and the aggregated AuNPs. The as-synthesized AuNPs are mostly isolated (Figure 6A), but a few AuNP dimers and trimers are observed in the samples with a relatively small degree of AuNP aggregation and large depolarization. The AuNPs are apparently piled on the top of each other in the TEM images obtained with the heavily aggregated AuNP sample (Figure 6D). Computational simulation reveals that the peak depolarization is ∼0.5 when three AuNSs with a diameter of

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CONCLUSIONS Using a combination of UV−vis spectrophotometric measurements in combination with the recently developed PRS2 method, we have experimentally quantified the photon extinction, absorption, and scattering cross-sections and scattering depolarizations for AuNPs of different sizes and shapes. The effects of the solvent dielectric, ligand binding, and nanoparticle aggregation on the AuNP photon extinction, absorption, and scattering cross-section and scattering depolarization have also been quantified. While increasing the AuNS size simultaneously increases its photon absorption and scattering, the percentage of light scattering contribution to the AuNS photon extinction remains small for the AuNSs and AuNRs investigated in this work. Increasing the solvent dielectric increases the photon extinction, absorption, and scattering cross-sections at the peak extinction wavelengths. 792

DOI: 10.1021/acs.analchem.7b03227 Anal. Chem. 2018, 90, 785−793

Article

Analytical Chemistry

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Ligand binding also increases the AuNP photon extinction, absorption, and scattering cross-sections. The AuNP aggregation has profound effect on its surface plasmonic properties as it changes not only the AuNP extinction, absorption, and scattering cross-sections but also the scattering depolarizations. Besides providing a series of new insights on the effect of AuNP geometry, solvent composition, ligand functionalization, and nanoparticle aggregation on the AuNP optical properties, the technique presented in this work should lead to ways for in situ monitoring of AuNP structure modifications including crystal growth, aggregation, and digestion.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b03227. UV−vis extinction spectra of AuNS with different sizes, experimental data obtained with AuNRs, data for the effect of solvent (glycerol/water) on AuNP optical properties, data for the effect of ligand functionalization on the AuNP optical properties, and data for the effect of aggregation on the AuNP optical properties (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(S.Z.) E-mail: [email protected]. Fax: 407-823-2252. *(D.Z.) E-mail: [email protected]. Fax: 662325-1618. ORCID

Yadong Zhou: 0000-0003-3357-3553 Shengli Zou: 0000-0003-1302-133X Dongmao Zhang: 0000-0002-2303-7338 Author Contributions ∥

J.X.X. and K.S. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSF funds (Grants CHE 1151057 and EPS-0903787) provided to D.Z. REFERENCES

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DOI: 10.1021/acs.analchem.7b03227 Anal. Chem. 2018, 90, 785−793