Determination of Colloidal Gold Nanoparticle Surface Areas, Concentrations, and Sizes Through Quantitative Ligand Adsorption

Anal Bioanal Chem (2013) 405:413–422 DOI 10.1007/s00216-012-6489-2

ORIGINAL PAPER

Determination of colloidal gold nanoparticle surface areas, concentrations, and sizes through quantitative ligand adsorption

Manuel Gadogbe & Siyam M. Ansar & Guoliang He & Willard E. Collier & Jose Rodriguez & Dong Liu & I-Wei Chu & Dongmao Zhang

Received: 1 August 2012 /Revised: 1 October 2012 /Accepted: 8 October 2012 /Published online: 24 October 2012 # Springer-Verlag Berlin Heidelberg 2012

Abstract Determination of the true surface areas, concen- aqua regia-digested AuNPs. The experimental results from trations, and particle sizes of gold nanoparticles (AuNPs) is a the MBI adsorption method for a series of commercial col- challenging issue due to the nanoparticle morphological loidal AuNPs with nominal diameters of 10, 30, 50, and irregularity, surface roughness, and size distributions. A 90 nm were compared with those determined using dynamic ligand adsorption-based technique for determining AuNP light scattering, transmission electron microscopy, and local- surface areas in solution is reported. Using a water- ized surface plasmonic resonance methods. The ligand soluble, stable, and highly UV–vis active organothiol, 2- adsorption-based technique is highly reproducible and sim- mercaptobenzimidazole (MBI), as the probe ligand, we dem- ple to implement. It only requires a UV–vis spectrophotom- onstrated that the amount of ligand adsorbed is proportional eter for characterization of in-house-prepared AuNPs. to the AuNP surface area. The equivalent spherical AuNP sizes and concentrations were determined by combining the Keywords Gold nanoparticle (AuNP) . Ligand adsorption . MBI adsorption measurement with Au3+ quantification of Surface area . Particle size

Electronic supplementary material The online version of this article (doi:10.1007/s00216-012-6489-2) contains supplementary material, Introduction which is available to authorized users. : : * Colloidal gold nanoparticles (AuNPs) have found applica- M. Gadogbe S. M. Ansar D. Zhang ( ) – Department of Chemistry, Mississippi State University, tions in many areas including catalysis [1 4], drug delivery Mississippi State, [5, 6], imaging [7], and bio-sensing [8, 9] because they Starkville, MS 39762, USA exhibit unique chemical, catalytic, and electromagnetic e-mail: [email protected] properties. These properties depend on the size, shape, and W. E. Collier composition of the AuNPs. Knowledge about AuNP size, Tuskegee University, surface area, and concentration is vital to determine and Tuskegee, AL 36088, USA understand the correlation between AuNP structure and function. For instance, Wang et al. observed that endocyto- J. Rodriguez Mississippi State Chemical Laboratory, Mississippi State, sis of AuNPs, modified with single-stranded DNA for tar- Starkville, MS 39762, USA geting of cancer cells, depends on the size of the : nanoparticles. It was observed that endocytosis decreases G. He D. Liu with increasing size of AuNPs [10]. Alkilany and Murphy Department of Mechanical Engineering, University of Houston, N207 Engineering Building 1, showed that nanoparticle size plays an important role in the Houston, TX 77204-4006, USA rate and extent of cellular uptake [11]. Also, the catalytic activity of AuNPs is critically dependent on particle size I.-W. Chu [12–14]. Department of Chemical Engineering, Mississippi State University, Mississippi State, Current methods for determining AuNP size can be di- Starkville, MS 39762, USA vided into several subcategories. The first is light scattering 414 M. Gadogbe et al. methods that include dynamic light scattering (DLS), static the published TEM images [29–31]. They are polycrys- light scattering (SLS) [15–17], and small-angle X-ray scat- talline containing different crystal facets with junctions, tering [18]. The second category is the microscopy method vertices, and defects [32–36]. These low to subnanom- including atomic force microscopy (AFM), transmission eter surface features are difficult to account for with electron microscopy (TEM) [19], and scanning electron imaging methods such as TEM and SEM, or other microscopy (SEM). The third is the localized surface plas- existing particle sizing methods, but they can have monic resonance (LSPR) method, which takes advantage of significant impact on the nanoparticle surface areas. the fact that the LSPR features of AuNPs depend on the size, Our hypothesis is that more realistic values for the true shape, and concentration of the AuNPs [20–22]. A more AuNP surface areas can be estimated with molecular recent method is electrospray-differential mobility analysis probes that have small cross sections (<1 nm2/molecule, (ES-DMA) that involves the conversion of AuNP suspen- forexample)ontheAuNPs. sion into the gas phase using electrospray ionization. In ES- Reported herein is a ligand adsorption method for deter- DMA, the charged particles are sorted based on their elec- mining the surface areas, concentrations, and sizes of col- trical mobility and the number average diameter is measured loidal AuNPs, using 2-mercaptobenzimidazole (MBI) as the after counting [23–25]. While TEM, SEM, and AFM probe ligand. While ligand adsorption methods have been images allow direct AuNP visualization [19, 26, 27], these used for determining the exposed surface area of AuNPs measurements require tedious sample preparation, sophisti- (e.g., AuNP catalysts supported on solid substrates) cated equipment, and lengthy and often subjective data [37–39], these methods have, to our knowledge, not been analysis to determine particle size and size distribution. applied to AuNPs in solution. A recent report by Elzey et al. Since only a small population of the nanoparticles is probed, showed that the ligand packing density of the organothiol 3- the results are statistically less representative [27]. mercaptopropionic acid (MPA) on AuNPs is size indepen- One key advantage of light scattering (e.g., DLS) and dent for AuNPs of 5, 10, 30, 60, and 100 nm in diameter LSPR-based methods is their ability to probe the AuNP in [40]. This suggests that ligand adsorption can be a reliable situ, that is, the AuNP in solution, eliminating the tedious method for quantification of the AuNP surface areas and sample preparation required for imaging-based methods. determination of the AuNP sizes. Unfortunately, DLS and SLS suffer from poor robustness Several factors led to choosing MBI as the probe ligand. and accuracy. For example, large uncertainties in particle The first is the high binding affinity of MBI with AuNPs. size analysis can occur in both DLS and SLS induced by Previous research from our group revealed that MBI binds variations in the viscosity and refractive index of the sol- bidentately with AuNPs at neutral pH with a binding con- vent, particulate contaminants in the sample, and fluctua- stant of 4.44±(1.29)×106 M−1 [41]. The high binding affin- tions in the solution temperature. Although DLS proves to ity of MBI is important to ensure that saturation ligand be a useful tool for monitoring slow aggregation processes, binding on the AuNPs can be achieved without using a large Khlebtsov et al. reported that false peaks appear in the size excess of MBI, thereby reducing the cost of chemical range of 5–10 nm in DLS measurements of colloidal AuNPs reagents and minimizing the potential environmental impact [17]. This was attributed to rotational diffusion of nonspher- of this experimental scheme. Using this Langmuir binding ical particles with sizes greater than 30–40 nm [17]. A constant, it can be shown that a 95 % monolayer MBI critical limitation of the LSPR method is its insensitivity to packing can be achieved when the excess MBI is as low small changes in particle size, which is particularly prob- as 5 μM in solution [41]. Secondly, MBI has excellent lematic for AuNPs smaller than 35 nm in diameter [28]. solubility and stability in water. MBI solutions of up to Theoretical calculations have shown that the change in the ≈1 mM can be readily prepared in water, and there is no peak LSPR wavelength is less than 5 nm when the particle detectable change in UV–vis spectra after 8 months of size changes from 5 to 35 nm in diameter [28]. Because of sample storage (data not shown). Thirdly, the amount of their limited reliability, light scattering and LSPR methods MBI adsorbed can be readily quantified by UV–vis spec- are better suited to study gross changes in AuNP sizes troscopy, one of the most reliable and commonly available induced by AuNP aggregation and protein/AuNP binding analytical techniques. MBI has a relatively strong UV–vis than to determine the exact particle size. Although ES-DMA absorbance at 300 nm with a molar absorptivity of can measure very small particle sizes (d<5 nm), the method 27,400 M−1 cm−1. Finally, MBI has a small footprint on requires expensive instrumentation [23]. the AuNP. On the basis of the saturation packing density of Accurate surface area values are crucial when studying 0.574±0.006 nmol/cm2, we recently reported for MBI on the molecular-level ligand interaction with AuNPs. Howev- AuNPs of nominal size of 13 nm by assuming a spherical er, compared to determining particle size, quantifying AuNP shape of the AuNPs [41]; the footprint of MBI on AuNP is surface area is much more challenging. Colloidal AuNPs are 0.29 nm2 per molecule. This small footprint is critical for rarely perfectly spherical or monodispersed, as evident from detecting subnanometer AuNP surface roughness. Determination of colloidal gold nanoparticle surface areas 415

Principles of the methodology An ICP-OES (Varian 715-ES) was used for Au3+ quantification. Sonication was performed using a Branson sonifier 250. The general principles for the determination of the surface Nanopure water was used throughout the experiment. While area and particle size of AuNPs using quantitative ligand the solution of citrate-capped AuNPs with a nominal diameter adsorption are outlined below. After mixing a known vol- of 13 nm was synthesized in-house, the citrate-capped AuNPs ume of AuNP solution with a known amount of MBI, the with nominal sizes of 10, 30, 50, and 90 nm in diameter were MBI/AuNP mixture sat at room temperature to allow the obtained from Nanocomposix, Inc. MBI-adsorbing AuNP to aggregate and settle. The excess MBI that remained free in the supernatant was quantified by AuNP synthesis The in-house AuNP solution was prepared UV–vis spectroscopy. The amount of MBI adsorbed per using the citrate reduction method [42]. Briefly, 0.0788 g of milliliter of AuNP solution, ΓMBI (nanomoles per milliliter gold (III) chloride trihydrate (HAuCl4·3H2O) was added to of AuNP solution), was determined as the difference be- 200 mL of Nanopure water, and then the solution was tween the amount of MBI added and the excess MBI in the brought to a boil. Twenty milliliters of 38.8 mM trisodium supernatant. The surface area afforded per milliliter of citrate dihydrate was added, and the resulting solution was AuNP solution (in square centimeters per milliliter of AuNP boiled under constant rapid stirring for 20 min before cool- solution) is then calculated using Eq. 1 ing to room temperature. Γ S ¼ MBI ð1Þ AuNP titration of MBI solution The in-house-synthesized PMBI AuNP solution was concentrated five times by centrifuga- 2 tion using the bench top centrifuge at 8,000 rpm and 20 °C where PMBI (0.574 nmol/cm ) is the MBI packing density on AuNPs. for 1 h. Four MBI/AuNP samples were prepared by titrating The equivalent spherical particle diameter D (in nano- 1 mL of concentrated AuNP solution into 1 mL of MBI AuNP μ ≈ meters), that is the diameter of the AuNPs that are assumed (159.3 M) using a burette at four different rates ( 1 drop/s, ≈ ≈ ≈ perfectly spherical and monodispersed, can be calculated with 1 drop/6 s, 1 drop/24 s, and 1 drop/600 s, respectively) Eq. 2. Derivation of this equation is shown in the supporting at room temperature. The MBI solutions were stirred during 3 the entire titration process. The solutions were covered with information. dAu (19.3 g/cm ) is the density of gold and a060 was obtained from unit conversions. The total gold ion content paraffin to ensure no significant solvent evaporation during 3+ the titration process. A fifth sample was prepared by adding (CAu in parts per million) is experimentally quantified by inductively coupled plasma optical emission spectroscopy the entire 1 mL AuNP solution directly into the 1 mL MBI (ICP-OES) after digestion of a known volume of AuNP solution. After preparation of the MBI/AuNP mixtures, the ≈ solution with aqua regia. samples were incubated overnight ( 15 h) to allow the MBI- adsorbing AuNP aggregates to settle. The concentration of ¼ aPMBI CAu3þ ð Þ free MBI (unbound) in each sample was quantified by DAuNP 2 – Γ MBI dAu taking the UV vis spectrum of its supernatant. The amount of MBI adsorbed was calculated as the difference between The AuNP concentrations can also be easily calculated the amount of MBI added into each sample and that of the using Eq. 3. Derivation of this equation is shown in supporting free MBI in the supernatant. information. The results of this ligand adsorption method are compared with those obtained using the DLS, LSPR, and TEM pH dependence of the MBI adsorption The pH dependence methods for a series of commercial citrate-capped AuNPs with of MBI adsorption onto the AuNPs was investigated over nominal particle sizes of 10, 30, 50, and 90 nm in diameter. a pH range of 5 to 8. After adjusting the pH of the AuNP solutions to the desired pH levels, 1 mL of MBI ¼ : 3 CAu3þ ð Þ CAuNP 3 17 10 3 3 solution (57 μM) was mixed with an equal volume of D dAu AuNP each AuNP solution, and the resulting solution was briefly vortexed and left to sit at room temperature overnight (≈15 h). Three replicate measurements were carried out Experimental section for the AuNP solutions at each pH. The amount of MBI adsorbed was calculated as the difference between the Chemicals and equipment All chemicals, including MBI (M amount of MBI added and the amount of free MBI in 3205), were obtained from Sigma-Aldrich. UV–vis measure- the supernatant. ments were made with a Fisher Scientific Evolution 300 UV– visible spectrophotometer. Centrifugation was performed us- AuNP concentration dependence of MBI adsorption The ing a bench top Fisher Scientific centrifuge (Fisher 21000R). linearity of the MBI adsorption with the AuNP concentration 416 M. Gadogbe et al. or surface area was demonstrated with the citrate-capped images were first converted into binary images (black and commercial 30 nm AuNP solution. Briefly, the AuNP solution white) where a black pixel represents a portion of AuNP and was concentrated four times by centrifugation using the bench a white pixel represents the background. The threshold top centrifuge at 5,500 rpm and 20 °C for 45 min. The value was determined automatically from the method sug- concentrated AuNP solution was then sonicated for ≈15 min gested by Otsu which evaluates the gray value histogram of to redisperse any aggregated nanoparticles that might form the original image [44]. Then the surface area of the indi- during centrifugation. Five different concentrations of AuNP vidual AuNP was calculated by integrating the differential solutions were prepared at room temperature by mixing the surface elements at all cross-section locations along the concentrated AuNP solution with water in volume ratios outer profile of the image (shown in Fig. 1), assuming that (AuNP/water) of 1:4, 1:1.5, 1:0.67, 1:0.25, and 1:0, respec- the cross sections are circular. tively. The UV–vis spectra for the resulting solutions, shown The advantage of this CSPI method is that the AuNP in Fig. S1 in the Electronic supplementary material, confirmed does not have to be perfectly spherical, as long as the cross that no AuNP aggregation was induced by AuNP concentra- section of the AuNP can be approximated to be circular tion or dilution. One milliliter of each of the five concentra- (Fig. 1). After determining the surface areas of individual tions of AuNP solutions was mixed with 1 mL of MBI AuNPs, the average equivalent spherical AuNP diameter D (49.5 μM). The AuNP/MBI mixtures were incubated over- is determined using Eq. 4 night (≈15 h), and the free MBI in the supernatant was sffiffiffiffi quantified by UV–vis spectroscopy at a wavelength of S D ¼ ð4Þ 300 nm. Three replicates were prepared for each concentration CSPI p of AuNP solution. where S is the average surface area of the AuNPs analyzed AuNP digestion and Au3+ quantification Briefly, a known using the CSPI method. The concentration of the AuNP volume of AuNP solution was mixed with an equal volume solution is determined with Eq. 3 shown in the “Principles of aqua regia solution (Warning: Aqua regia solution is of the methodology” section. extremely corrosive and reactive. Care is needed in its preparation and usage). Complete dissolution of the AuNPs was observed within ≈30 min of the sample incubation. The Results and discussion Au3+ concentration in the resulting solution was analyzed using ICP-OES at emission wavelengths of 267.594 and AuNP titration of MBI Our previous research demonstrated 242.794 nm. A calibration curve for Au3+ content from 0 that MBI binding induces rapid AuNP aggregation resulting to 100 mg/L was established for ICP-OES analysis using in complete AuNP settlement within 3 to 4 h of sample Fisher single-element gold standard (ICP-MS/ICP-AES). The ICP-OES method was also applied to determine the percent conversion of Au3+ to AuNPs in the AuNP synthesis. Briefly, 6 mL of the in-house-synthesized AuNP solution was taken and aggregated using a sufficient amount of solid KCl. After AuNP settlement, 5 mL of the supernatant was taken and diluted to 10 mL with water. The resulting solution was analyzed for any detectable Au3+. To calculate the percent conversion, the concentration of Au3+ in this solution was compared with the concentration of Au3+ in a sample where the whole sample was digested and analyzed for Au3+ content.

TEM imaging analysis Two TEM image analysis methods were used to determine AuNP sizes. The first was an undis- closed technique that the vendor used to determine the AuNP sizes. The second method is a cross-section perimeter integration (CSPI) method which was based on the image processing algorithms by Ziou and Tabbone [43] and Otsu Fig. 1 Scheme of CSPI determination of AuNP surface area using – [44]. The general procedures of the CSPI method are out- AuNP TEM images. Circles (a c) correspond to the cross section at points i–k, respectively, along the horizontal axis shown in red. The lined as follows. By comparing the gray value of each AuNP surface area is calculated by integrating the perimeters (shown individual image pixel with a threshold value, the TEM in red in circles a–c) of all the cross sections along the selected axis Determination of colloidal gold nanoparticle surface areas 417

Fig. 3 AuNP concentration dependence of MBI adsorbed onto Fig. 2 Amount of MBI adsorbed onto AuNPs (nominal size of 13 nm) AuNPs. The AuNP solutions were prepared by mixing the concentrat- in samples where 1 mL of in-house AuNP solution was titrated into ed 30-nm AuNPs with water to yield AuNP volume fractions from 0.2 1 mL MBI solution at different titration rates. a Simultaneous AuNP/ to 1 (Detailed information is in the “Experimental section”). Inset: MBI mixing, b ≈1 drop/s, c ≈1 drop/6 s, d ≈1 drop/24 s, and e ≈1 drop/ UV–vis spectra of supernatants of the MBI binding solutions with 600 s. The concentration of MBI is 0.16 mM. The volume fraction of AuNP volume fractions of (a to f) 1, 0.8, 0.6, 0.4, 0.2, and a control AuNPs in the AuNP/MBI mixtures is ≈0.5. The as-synthesized AuNP (MBI mixed with water) solution was concentrated five times before the experiment. The dashed line shows the mean of the results impact on the MBI adsorption offers a significant advantage for this ligand adsorption-based surface area measurement preparation in a typical 4-mL vial [45]. This can compro- mise the accuracy of this ligand adsorption-based method if the AuNPs aggregate before MBI reaches saturation adsorption and the nanoparticle junctions between the aggregated AuNPs become inaccessible for further MBI adsorption. To investigate such a possibility, a series of AuNP titration experiments described in the Experimental section were conducted to investigate the interplay between AuNP aggregation and MBI adsorption. Slow titration of MBI with AuNPs is expected to slow the rate of AuNP aggregation and maximize MBI adsorption onto AuNPs if AuNP aggregation has a significant effect on MBI adsorption. Our experimental data showed that the amount of adsorbed MBI is essentially independent of the rate of mixing of the MBI/AuNP solutions (Fig. 2). The amount of adsorbed MBI is the same regardless of whether the 1 mL of AuNP solution is added into 1 mL of MBI solution at once, or added dropwise with a total titration time for more than 12 h (≈ 1 drop per 600 s). This result implies that either MBI has reached near saturation adsorption before AuNP aggregation or the fraction of the AuNP junction area that is inaccessible for further MBI adsorption is insignificant. The fact that the AuNP aggregation and settlement has no significant

Table 1 Amount of MBI adsorbed at differ- pH of AuNP/ ΓMBI (nmol/mL ent pH values of AuNP/ MBI mixture AuNP solution) MBI mixtures 5.3 16.2±0.5 6.2 15.7±0.4 7.2 16.3±0.4 Fig. 4 UV–vis spectra and TEM images of commercial AuNPs with 8.2 16.8±0.5 diameters; a 10, b 30, c 50, and d 90 nm. The scale bars for the top two images are 30 nm while the scale bars for the bottom two are 100 nm 418 M. Gadogbe et al.

3+ Table 2 MBI adsorbed onto the AuNPs and concentration of Au volume fraction of AuNPs indicates that this ligand ad- after aqua regia AuNP digestion sorption method can be used to predict the AuNP surface 3+ Nominal size (nm) ΓMBI (nmol/mL AuNP solution) CAu (ppm) area afforded by AuNPs in the sample volume or the nanoparticle concentration if the equivalent AuNP size is 10 7.9±0.5 43.1 known. 30 3.0±0.1 45.3 50 2.0±0.1 45.9 AuNP surface areas, sizes, and concentrations Figure 4 90 1.2±0.1 49.0 shows the UV–vis spectra and the TEM images of the In-house AuNP 15.7±0.4 84.0 series of commercial citrate-capped AuNPs with nominal sizes of 10, 30, 50, and 90 nm in diameter. It is apparent from the TEM images that the AuNPs are neither strictly spherical nor monodispersed. Particles having a size dif- technique because it allows convenient separation of excess ference as large as 2-fold in diameter can be readily MBI from the AuNP/MBI aggregates. Since the amount of observed for AuNPs with nominal sizes of 30, 50, and adsorbed MBI is independent of the rate of AuNP/MBI 90 nm, respectively, whereas the size distribution of the mixing, AuNPs and MBI were mixed simultaneously in all 10-nm AuNPs is relatively narrow. The amount of MBI our subsequent ligand binding studies. adsorbed onto the AuNPs and Au3+ ion concentrations of the digested AuNPs for both the commercial and in- pH dependence of the MBI adsorption It was found that house-prepared AuNPs are shown in Table 2. These were citrate-reduced AuNPs are slightly acidic, and the pH varies determined according to the procedures described in the from 5.8 to 6.8 for AuNPs with particle sizes from 10 to experimental sections. 50 nm in diameter (data not shown). Previous research from Tables 3, 4,and5 show the equivalent spherical our lab indicates that MBI saturation packing density on particle sizes, AuNP concentrations, and AuNP surface AuNPs is statistically the same in the pH 7 and pH 12 areas afforded by 1 mL colloidal AuNP solutions of the solutions, but is different for a pH 2 solution in which commercial AuNPs determined using the MBI adsorp- MBI adopted a thione tautomeric form on the AuNP surface tion, TEM, DLS, and LSPR methods. The MBI packing [41]. In this study, we further determine the possible pH density of 0.574±0.006 nmol/cm2 determined in our dependence of MBI adsorption onto AuNPs in the pH range recent work [41] was used in the calculation of the that is most relevant in practical AuNP characterizations MBI adsorption-based AuNP sizes. The LSPR particle (Table 1). The results show that the amounts of MBI sizes were determined from the peak UV–vis absorption adsorbed onto AuNPs are essentially the same over the wavelength (λSPR) using the formula reported by Haiss entire investigated pH range (over three pH units), indicat- et al. [28], while the LSPR AuNP concentrations were ing the applicability of this MBI adsorption method for calculated according to Liu et al. [46]. It should be general AuNP characterization. noted that the equivalent spherical particle size and AuNP concentration for this MBI adsorption method Linearity of MBI adsorption To validate this ligand adsorp- are calculated on the assumption that all the nanopar- tion method, we studied the AuNP concentration depen- ticles are spherical and monodispersed, which is the dence of MBI adsorption (Fig. 3). The near perfect linear same assumption used by the current AuNP LSPR correlation between the amounts of MBI adsorbed and the method.

Table 3 Average equivalent spherical diameter of AuNPs (in nanometers)

Nominal size (nm) TEMa TEMb MBI DLSc DLSd LSPR

10 10.1±0.7 8.8±0.6 9.7±0.6 11.6±2.9 19.3±4.2 10 30 32.0±3.3 25.5±3.0 26.7±0.9 31.5±3.1 39.9±2.5 30.9 50 49.6±6.4 38.4±6.2 40.2±2.0 46.7±3.6 54.1±3.1 51 90 90.5±8.0 73.0±7.9 71.7±4.1 67.9±17.8 97.9±8.6 93 a TEM particle sizes provided by the vendors b TEM CSPI method c DLS particle sizes based on mass histogram d DLS particle sizes based on intensity histogram Determination of colloidal gold nanoparticle surface areas 419

Table 4 Concentration of equivalent spherical AuNPs (in nanomoles per liter)

Nominal TEMa TEMb MBI DLSc DLSd LSPR size (nm)

10 6.9±1.4 10.4±2.1 7.8±1.5 4.5±3.4 1.0±0.6 8.18 30 0.2±0.1 0.4±0.2 0.4±0.04 0.2±0.07 0.1±0.02 0.22 50 0.1±0.02 0.1±0.06 0.1±0.02 0.07±0.01 0.05±0.008 0.022 90 0.01±0.003 0.02±0.007 0.02±0.004 0.03±0.02 0.01±0.002 0.005

Calculated using the particle sizes determined with a TEM particle sizes provided by the vendors b TEM CSPI method c DLS based on mass histogram d DLS based on intensity histogram

The particle sizes, AuNP concentrations, and AuNP extinction coefficients of larger nanoparticles are signif- surface areas per unit volume obtained with different icantly higher than those of smaller nanoparticles. techniques vary widely, illustrating the difficulties in Importantly, the results obtained from the TEM analy- reliable AuNP characterization. The differences are par- sis using the CSPI method and the MBI adsorption ticularly large for the 30, 50, and 90-nm citrate-capped method are remarkably similar for all the AuNPs we AuNPs where the polydispersities are relatively high. tested. The largest relative difference between the particle These differences can be attributed to the inherent bias sizes from these two methods is less than 10 %. In associated with different techniques. For example, the addition, AuNP surface areas per unit volume of AuNP DLS particle sizes (Table 3) are consistently larger than solutions determined with these two methods are consis- those obtained with other techniques. This may be re- tently larger than that obtained with other techniques lated to the fact that DLS measures the hydrodynamic with the only exception being the DLS result for the radius of the AuNP and its surface adsorbates. Because 90-nm AuNP. This result provides critical cross valida- the AuNP surface is usually coated with citrate or other tion of these two analytical methods. It should not be stabilizing agent in the solution, DLS measurement is surprising because by design the CSPI method and ligand expected to give larger particle sizes. The deviation of adsorption method can both accommodate some morpho- the DLS results, in particular the ones obtained with logical irregularity of the AuNPs. However, compared to intensity histograms from that of TEM, might also have the TEM CSPI method, the MBI adsorption technique is resulted from the artifacts related to DLS measurement much simpler to implement. procedure or data analysis. The relatively larger LSPR The excellent agreement between the TEM CSPI particle sizes are not surprising either as the molar results and that obtained with MBI ligand adsorption

Table 5 Surface area of AuNPs in per unit volume (in square centimeters per milliliter)

Nominal size (nm) TEMa TEMb MBI DLSc DLSd LSPR

10 13.3±1.8 15.1±2 13.8±0.9 11.5±6 7.0±3.0 13.4 30 4.4±0.9 5.5±1.3 5.3±0.2 4.5±0.9 3.5±0.4 4.6 50 2.9±0.7 3.7±1.2 3.6±0.2 3.1±0.5 2.6±0.3 2.8 90 1.7±0.3 2.1±0.4 2.1±0.1 2.2±1.2 1.6±0.3 1.6

Calculated using the particle sizes determined with a TEM particle sizes provided by the vendors b TEM CSPI method c DLS based on mass histogram d DLS based on intensity histogram 420 M. Gadogbe et al. method indicates that the MBI packing density on the Table 6 Particle size, AuNP concentration, and AuNP surface area of AuNPs is independent of the particle size of AuNPs in in-house-prepared AuNP solution the size range from 10 to 100 nm in diameter. This Methods Particle size AuNP concentrations Surface area per result is consistent with what was reported by Elzey et (nm) (nM) unit volume 2 al. who demonstrated that the packing densities of MPA AuNP (cm /mL) on AuNPs are the same for AuNPs in the size range TEMa 10.2±1.3 13.0±5.0 25.6±7.0 from5to100nm[40]. MBI and MPA adsorption MBI 9.5±0.2 16.0±1.0 27.4±0.8 results suggest that the difference in the curvature of DLSb 12.6±1.2 6.9±2.0 20.7±4.0 AuNPs with diameter equal to or larger than 5 nm does DLSc 15.9±2.2 3.4±1.4 16.4±5.0 not have significant impact on the organothiol packing LSPR 12.6 7.89 20.7 density. This result is not surprising, given the sub- square-nanometer footprint of MBI and MPA on the a TEM CSPI AuNP surfaces [40, 41]. b DLS particle size based on mass histogram c DLS particle size based on intensity histogram Characterization of the in-house-synthesized AuNPs The in-house-prepared AuNP solution (Fig. 5) was charac- terized with the MBI adsorption method and ICP-OES from 0.25 to 0.5 mM (Electronic supplementary materi- determination of the Au3+ concentration, and the results al,Fig.S2). This value is similar to that estimated from were compared with data obtained using the other tech- the reported UV–vis spectrum of the HAuCl4·3H2O niques (Table 6). Again, TEM analysis by CSPI and the solution at 290 nm [48]. MBI adsorption method give the smallest particle size and highest AuNP surface area per unit volume of AuNP solution. Conclusion The MBI adsorption method can be drastically simpli- fied for characterization of in-house-prepared AuNPs by Determination of the true surface area of colloidal eliminating the aqua regia AuNP digestion and the ICP- AuNPs is difficult with existing particle sizing techniques OES Au3+ concentration determination. Indeed, the total due to the AuNP morphological irregularity, broad size Au content of the AuNPs can be approximated as the distribution, and surface roughness. Large discrepancies amount of Au3+ used for synthesis. Under our experi- exist in the particle sizes, concentrations, and surface mental conditions, about 97 % of the Au3+ added into areas determined with existing particle sizing techniques the AuNP synthesis solution was converted to AuNPs (DLS, TEM, and LSPR). The MBI adsorption method (data not shown), which is consistent with previous presented in this work likely provides a more accurate reports [47]. It is important to note that it can be erro- estimation of the true AuNP surface area because of its neous to use the weight of HAuCl4·3H2Otocalculate capability to accommodate variations in AuNP morphol- the concentration of the Au3+ because of the hygroscop- ogy, shape, and size distributions. The main uncertainty icity of HAuCl4·3H2O. Instead, one should use the UV– with this approach likely lies in the saturation MBI vis absorbance of the HAuCl4·3H2Osolutiontodeter- packing density on AuNPs that is needed for estimation mine the Au3+ concentration. A molar absorptivity of of the AuNP surface area. Unfortunately, we do not have 2.56×103 M−1 cm−1 at 290 nm was determined for a commonly agreed on AuNP standard that allows pre-

HAuCl4·3H2O with solutions in the concentration range cise determination of ligand packing density. Neverthe- less, the organothiol adsorption has been used extensively for determination of the exposed surface areas of AuNPs immobilized on solid substrates [37–39]. This work demonstrated for the first time that, by judiciously selecting a water-soluble and stable organothiol probe, this ligand adsorption method can be used for characterizing AuNPs in solution. In addition, this MBI adsorption method is very easy to implement as it does not require tedious sample preparation and sophisticated instruments. It only requires a UV–vis spectrophotometer to characterize of in-house-prepared AuNPs. Therefore, the technique can Fig. 5 UV–vis spectrum and TEM image for in-house-synthesized be particularly useful for quality control in nanoparticle AuNP solution. The scale bar represents 25 nm synthesis. Determination of colloidal gold nanoparticle surface areas 421

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