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, concentrations, and particle sizes of gold nanoparticles (AuNPs) is a
challenging issue due to the nanoparticle morphological
irregularity, surface roughness, and size distributions. A
ligand adsorption-based technique for determining AuNP
surface areas in solution is reported. Using a watersoluble, stable, and highly UV–vis active organothiol, 2mercaptobenzimidazole (MBI), as the probe ligand, we demonstrated that the amount of ligand adsorbed is proportional
to the AuNP surface area. The equivalent spherical AuNP
sizes and concentrations were determined by combining the
MBI adsorption measurement with Au3+ quantification of
Electronic supplementary material The online version of this article
(doi:10.1007/s00216-012-6489-2) contains supplementary material,
which is available to authorized users.
M. Gadogbe : S. M. Ansar : D. Zhang (*)
Department of Chemistry, Mississippi State University,
Mississippi State,
Starkville, MS 39762, USA
e-mail: [email protected]
W. E. Collier
Tuskegee University,
Tuskegee, AL 36088, USA
J. Rodriguez
Mississippi State Chemical Laboratory, Mississippi State,
Starkville, MS 39762, USA
G. He : D. Liu
Department of Mechanical Engineering, University of Houston,
N207 Engineering Building 1,
Houston, TX 77204-4006, USA
I.-W. Chu
Department of Chemical Engineering,
Mississippi State University, Mississippi State,
Starkville, MS 39762, USA
aqua regia-digested AuNPs. The experimental results from
the MBI adsorption method for a series of commercial colloidal AuNPs with nominal diameters of 10, 30, 50, and
90 nm were compared with those determined using dynamic
light scattering, transmission electron microscopy, and localized surface plasmonic resonance methods. The ligand
adsorption-based technique is highly reproducible and simple to implement. It only requires a UV–vis spectrophotometer for characterization of in-house-prepared AuNPs.
Keywords Gold nanoparticle (AuNP) . Ligand adsorption .
Surface area . Particle size
Introduction
Colloidal gold nanoparticles (AuNPs) have found applications in many areas including catalysis [1–4], drug delivery
[5, 6], imaging [7], and bio-sensing [8, 9] because they
exhibit unique chemical, catalytic, and electromagnetic
properties. These properties depend on the size, shape, and
composition of the AuNPs. Knowledge about AuNP size,
surface area, and concentration is vital to determine and
understand the correlation between AuNP structure and
function. For instance, Wang et al. observed that endocytosis of AuNPs, modified with single-stranded DNA for targeting of cancer cells, depends on the size of the
nanoparticles. It was observed that endocytosis decreases
with increasing size of AuNPs [10]. Alkilany and Murphy
showed that nanoparticle size plays an important role in the
rate and extent of cellular uptake [11]. Also, the catalytic
activity of AuNPs is critically dependent on particle size
[12–14].
Current methods for determining AuNP size can be divided into several subcategories. The first is light scattering
414
methods that include dynamic light scattering (DLS), static
light scattering (SLS) [15–17], and small-angle X-ray scattering [18]. The second category is the microscopy method
including atomic force microscopy (AFM), transmission
electron microscopy (TEM) [19], and scanning electron
microscopy (SEM). The third is the localized surface plasmonic resonance (LSPR) method, which takes advantage of
the fact that the LSPR features of AuNPs depend on the size,
shape, and concentration of the AuNPs [20–22]. A more
recent method is electrospray-differential mobility analysis
(ES-DMA) that involves the conversion of AuNP suspension into the gas phase using electrospray ionization. In ESDMA, the charged particles are sorted based on their electrical mobility and the number average diameter is measured
after counting [23–25]. While TEM, SEM, and AFM
images allow direct AuNP visualization [19, 26, 27], these
measurements require tedious sample preparation, sophisticated equipment, and lengthy and often subjective data
analysis to determine particle size and size distribution.
Since only a small population of the nanoparticles is probed,
the results are statistically less representative [27].
One key advantage of light scattering (e.g., DLS) and
LSPR-based methods is their ability to probe the AuNP in
situ, that is, the AuNP in solution, eliminating the tedious
sample preparation required for imaging-based methods.
Unfortunately, DLS and SLS suffer from poor robustness
and accuracy. For example, large uncertainties in particle
size analysis can occur in both DLS and SLS induced by
variations in the viscosity and refractive index of the solvent, particulate contaminants in the sample, and fluctuations in the solution temperature. Although DLS proves to
be a useful tool for monitoring slow aggregation processes,
Khlebtsov et al. reported that false peaks appear in the size
range of 5–10 nm in DLS measurements of colloidal AuNPs
[17]. This was attributed to rotational diffusion of nonspherical particles with sizes greater than 30–40 nm [17]. A
critical limitation of the LSPR method is its insensitivity to
small changes in particle size, which is particularly problematic for AuNPs smaller than 35 nm in diameter [28].
Theoretical calculations have shown that the change in the
peak LSPR wavelength is less than 5 nm when the particle
size changes from 5 to 35 nm in diameter [28]. Because of
their limited reliability, light scattering and LSPR methods
are better suited to study gross changes in AuNP sizes
induced by AuNP aggregation and protein/AuNP binding
than to determine the exact particle size. Although ES-DMA
can measure very small particle sizes (d<5 nm), the method
requires expensive instrumentation [23].
Accurate surface area values are crucial when studying
the molecular-level ligand interaction with AuNPs. However, compared to determining particle size, quantifying AuNP
surface area is much more challenging. Colloidal AuNPs are
rarely perfectly spherical or monodispersed, as evident from
M. Gadogbe et al.
the published TEM images [29–31]. They are polycrystalline containing different crystal facets with junctions,
vertices, and defects [32–36]. These low to subnanometer surface features are difficult to account for with
imaging methods such as TEM and SEM, or other
existing particle sizing methods, but they can have
significant impact on the nanoparticle surface areas.
Our hypothesis is that more realistic values for the true
AuNP surface areas can be estimated with molecular
probes that have small cross sections (<1 nm2/molecule,
for example) on the AuNPs.
Reported herein is a ligand adsorption method for determining the surface areas, concentrations, and sizes of colloidal AuNPs, using 2-mercaptobenzimidazole (MBI) as the
probe ligand. While ligand adsorption methods have been
used for determining the exposed surface area of AuNPs
(e.g., AuNP catalysts supported on solid substrates)
[37–39], these methods have, to our knowledge, not been
applied to AuNPs in solution. A recent report by Elzey et al.
showed that the ligand packing density of the organothiol 3mercaptopropionic acid (MPA) on AuNPs is size independent for AuNPs of 5, 10, 30, 60, and 100 nm in diameter
[40]. This suggests that ligand adsorption can be a reliable
method for quantification of the AuNP surface areas and
determination of the AuNP sizes.
Several factors led to choosing MBI as the probe ligand.
The first is the high binding affinity of MBI with AuNPs.
Previous research from our group revealed that MBI binds
bidentately with AuNPs at neutral pH with a binding constant of 4.44±(1.29)×106 M−1 [41]. The high binding affinity of MBI is important to ensure that saturation ligand
binding on the AuNPs can be achieved without using a large
excess of MBI, thereby reducing the cost of chemical
reagents and minimizing the potential environmental impact
of this experimental scheme. Using this Langmuir binding
constant, it can be shown that a 95 % monolayer MBI
packing can be achieved when the excess MBI is as low
as 5 μM in solution [41]. Secondly, MBI has excellent
solubility and stability in water. MBI solutions of up to
≈1 mM can be readily prepared in water, and there is no
detectable change in UV–vis spectra after 8 months of
sample storage (data not shown). Thirdly, the amount of
MBI adsorbed can be readily quantified by UV–vis spectroscopy, one of the most reliable and commonly available
analytical techniques. MBI has a relatively strong UV–vis
absorbance at 300 nm with a molar absorptivity of
27,400 M−1 cm−1. Finally, MBI has a small footprint on
the AuNP. On the basis of the saturation packing density of
0.574±0.006 nmol/cm2, we recently reported for MBI on
AuNPs of nominal size of 13 nm by assuming a spherical
shape of the AuNPs [41]; the footprint of MBI on AuNP is
0.29 nm2 per molecule. This small footprint is critical for
detecting subnanometer AuNP surface roughness.
Determination of colloidal gold nanoparticle surface areas
415
Principles of the methodology
The general principles for the determination of the surface
area and particle size of AuNPs using quantitative ligand
adsorption are outlined below. After mixing a known volume of AuNP solution with a known amount of MBI, the
MBI/AuNP mixture sat at room temperature to allow the
MBI-adsorbing AuNP to aggregate and settle. The excess
MBI that remained free in the supernatant was quantified by
UV–vis spectroscopy. The amount of MBI adsorbed per
milliliter of AuNP solution, ΓMBI (nanomoles per milliliter
of AuNP solution), was determined as the difference between the amount of MBI added and the excess MBI in the
supernatant. The surface area afforded per milliliter of
AuNP solution (in square centimeters per milliliter of AuNP
solution) is then calculated using Eq. 1
S¼
Γ MBI
PMBI
ð1Þ
where PMBI (0.574 nmol/cm2) is the MBI packing density
on AuNPs.
The equivalent spherical particle diameter DAuNP (in nanometers), that is the diameter of the AuNPs that are assumed
perfectly spherical and monodispersed, can be calculated with
Eq. 2. Derivation of this equation is shown in the supporting
information. dAu (19.3 g/cm3) is the density of gold and a060
was obtained from unit conversions. The total gold ion content
(CAu3+ in parts per million) is experimentally quantified by
inductively coupled plasma optical emission spectroscopy
(ICP-OES) after digestion of a known volume of AuNP
solution with aqua regia.
DAuNP ¼
aPMBI C Au3þ
Γ MBI d Au
ð2Þ
The AuNP concentrations can also be easily calculated
using Eq. 3. Derivation of this equation is shown in supporting
information. The results of this ligand adsorption method are
compared with those obtained using the DLS, LSPR, and TEM
methods for a series of commercial citrate-capped AuNPs with
nominal particle sizes of 10, 30, 50, and 90 nm in diameter.
CAuNP ¼ 3:17 103 CAu3þ
3
DAuNP dAu
ð3Þ
Experimental section
Chemicals and equipment All chemicals, including MBI (M
3205), were obtained from Sigma-Aldrich. UV–vis measurements were made with a Fisher Scientific Evolution 300 UV–
visible spectrophotometer. Centrifugation was performed using a bench top Fisher Scientific centrifuge (Fisher 21000R).
An ICP-OES (Varian 715-ES) was used for Au3+ quantification. Sonication was performed using a Branson sonifier 250.
Nanopure water was used throughout the experiment. While
the solution of citrate-capped AuNPs with a nominal diameter
of 13 nm was synthesized in-house, the citrate-capped AuNPs
with nominal sizes of 10, 30, 50, and 90 nm in diameter were
obtained from Nanocomposix, Inc.
AuNP synthesis The in-house AuNP solution was prepared
using the citrate reduction method [42]. Briefly, 0.0788 g of
gold (III) chloride trihydrate (HAuCl4·3H2O) was added to
200 mL of Nanopure water, and then the solution was
brought to a boil. Twenty milliliters of 38.8 mM trisodium
citrate dihydrate was added, and the resulting solution was
boiled under constant rapid stirring for 20 min before cooling to room temperature.
AuNP titration of MBI solution The in-house-synthesized
AuNP solution was concentrated five times by centrifugation using the bench top centrifuge at 8,000 rpm and 20 °C
for 1 h. Four MBI/AuNP samples were prepared by titrating
1 mL of concentrated AuNP solution into 1 mL of MBI
(159.3 μM) using a burette at four different rates (≈1 drop/s,
≈1 drop/6 s, ≈1 drop/24 s, and ≈1 drop/600 s, respectively)
at room temperature. The MBI solutions were stirred during
the entire titration process. The solutions were covered with
paraffin to ensure no significant solvent evaporation during
the titration process. A fifth sample was prepared by adding
the entire 1 mL AuNP solution directly into the 1 mL MBI
solution. After preparation of the MBI/AuNP mixtures, the
samples were incubated overnight (≈15 h) to allow the MBIadsorbing AuNP aggregates to settle. The concentration of
free MBI (unbound) in each sample was quantified by
taking the UV–vis spectrum of its supernatant. The amount
of MBI adsorbed was calculated as the difference between
the amount of MBI added into each sample and that of the
free MBI in the supernatant.
pH dependence of the MBI adsorption The pH dependence
of MBI adsorption onto the AuNPs was investigated over
a pH range of 5 to 8. After adjusting the pH of the
AuNP solutions to the desired pH levels, 1 mL of MBI
solution (57 μM) was mixed with an equal volume of
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
for the AuNP solutions at each pH. The amount of MBI
adsorbed was calculated as the difference between the
amount of MBI added and the amount of free MBI in
the supernatant.
AuNP concentration dependence of MBI adsorption The
linearity of the MBI adsorption with the AuNP concentration
416
or surface area was demonstrated with the citrate-capped
commercial 30 nm AuNP solution. Briefly, the AuNP solution
was concentrated four times by centrifugation using the bench
top centrifuge at 5,500 rpm and 20 °C for 45 min. The
concentrated AuNP solution was then sonicated for ≈15 min
to redisperse any aggregated nanoparticles that might form
during centrifugation. Five different concentrations of AuNP
solutions were prepared at room temperature by mixing the
concentrated AuNP solution with water in volume ratios
(AuNP/water) of 1:4, 1:1.5, 1:0.67, 1:0.25, and 1:0, respectively. The UV–vis spectra for the resulting solutions, shown
in Fig. S1 in the Electronic supplementary material, confirmed
that no AuNP aggregation was induced by AuNP concentration or dilution. One milliliter of each of the five concentrations of AuNP solutions was mixed with 1 mL of MBI
(49.5 μM). The AuNP/MBI mixtures were incubated overnight (≈15 h), and the free MBI in the supernatant was
quantified by UV–vis spectroscopy at a wavelength of
300 nm. Three replicates were prepared for each concentration
of AuNP solution.
AuNP digestion and Au3+ quantification Briefly, a known
volume of AuNP solution was mixed with an equal volume
of aqua regia solution (Warning: Aqua regia solution is
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
Au3+ concentration in the resulting solution was analyzed
using ICP-OES at emission wavelengths of 267.594 and
242.794 nm. A calibration curve for Au3+ content from 0
to 100 mg/L was established for ICP-OES analysis using
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 undisclosed 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
[44]. The general procedures of the CSPI method are outlined as follows. By comparing the gray value of each
individual image pixel with a threshold value, the TEM
M. Gadogbe et al.
images were first converted into binary images (black and
white) where a black pixel represents a portion of AuNP and
a white pixel represents the background. The threshold
value was determined automatically from the method suggested by Otsu which evaluates the gray value histogram of
the original image [44]. Then the surface area of the individual AuNP was calculated by integrating the differential
surface elements at all cross-section locations along the
outer profile of the image (shown in Fig. 1), assuming that
the cross sections are circular.
The advantage of this CSPI method is that the AuNP
does not have to be perfectly spherical, as long as the cross
section of the AuNP can be approximated to be circular
(Fig. 1). After determining the surface areas of individual
AuNPs, the average equivalent spherical AuNP diameter D
is determined using Eq. 4
sffiffiffiffi
S
DCSPI ¼
ð4Þ
p
where S is the average surface area of the AuNPs analyzed
using the CSPI method. The concentration of the AuNP
solution is determined with Eq. 3 shown in the “Principles
of the methodology” section.
Results and discussion
AuNP titration of MBI Our previous research demonstrated
that MBI binding induces rapid AuNP aggregation resulting
in complete AuNP settlement within 3 to 4 h of sample
Fig. 1 Scheme of CSPI determination of AuNP surface area using
AuNP TEM images. Circles (a–c) correspond to the cross section at
points i–k, respectively, along the horizontal axis shown in red. The
AuNP surface area is calculated by integrating the perimeters (shown
in red in circles a–c) of all the cross sections along the selected axis
Determination of colloidal gold nanoparticle surface areas
Fig. 2 Amount of MBI adsorbed onto AuNPs (nominal size of 13 nm)
in samples where 1 mL of in-house AuNP solution was titrated into
1 mL MBI solution at different titration rates. a Simultaneous AuNP/
MBI mixing, b ≈1 drop/s, c ≈1 drop/6 s, d ≈1 drop/24 s, and e ≈1 drop/
600 s. The concentration of MBI is 0.16 mM. The volume fraction of
AuNPs in the AuNP/MBI mixtures is ≈0.5. The as-synthesized AuNP
solution was concentrated five times before the experiment. The
dashed line shows the mean of the results
preparation in a typical 4-mL vial [45]. This can compromise 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 different pH values of AuNP/
MBI mixtures
pH of AuNP/
MBI mixture
5.3
6.2
7.2
8.2
417
Fig. 3 AuNP concentration dependence of MBI adsorbed onto
AuNPs. The AuNP solutions were prepared by mixing the concentrated 30-nm AuNPs with water to yield AuNP volume fractions from 0.2
to 1 (Detailed information is in the “Experimental section”). Inset:
UV–vis spectra of supernatants of the MBI binding solutions with
AuNP volume fractions of (a to f) 1, 0.8, 0.6, 0.4, 0.2, and a control
(MBI mixed with water)
impact on the MBI adsorption offers a significant advantage
for this ligand adsorption-based surface area measurement
ΓMBI (nmol/mL
AuNP solution)
16.2±0.5
15.7±0.4
16.3±0.4
16.8±0.5
Fig. 4 UV–vis spectra and TEM images of commercial AuNPs with
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.
Table 2 MBI adsorbed onto the AuNPs and concentration of Au3+
after aqua regia AuNP digestion
Nominal size (nm)
10
30
50
90
In-house AuNP
ΓMBI (nmol/mL AuNP solution)
CAu3+ (ppm)
7.9±0.5
3.0±0.1
2.0±0.1
1.2±0.1
15.7±0.4
43.1
45.3
45.9
49.0
84.0
volume fraction of AuNPs indicates that this ligand adsorption method can be used to predict the AuNP surface
area afforded by AuNPs in the sample volume or the
nanoparticle concentration if the equivalent AuNP size is
known.
AuNP surface areas, sizes, and concentrations Figure 4
shows the UV–vis spectra and the TEM images of the
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 difference as large as 2-fold in diameter can be readily
observed for AuNPs with nominal sizes of 30, 50, and
90 nm, respectively, whereas the size distribution of the
10-nm AuNPs is relatively narrow. The amount of MBI
adsorbed onto the AuNPs and Au3+ ion concentrations of
the digested AuNPs for both the commercial and inhouse-prepared AuNPs are shown in Table 2. These were
determined according to the procedures described in the
experimental sections.
Tables 3, 4, and 5 show the equivalent spherical
particle sizes, AuNP concentrations, and AuNP surface
areas afforded by 1 mL colloidal AuNP solutions of the
commercial AuNPs determined using the MBI adsorption, TEM, DLS, and LSPR methods. The MBI packing
density of 0.574 ± 0.006 nmol/cm2 determined in our
recent work [41] was used in the calculation of the
MBI adsorption-based AuNP sizes. The LSPR particle
sizes were determined from the peak UV–vis absorption
wavelength (λSPR) using the formula reported by Haiss
et al. [28], while the LSPR AuNP concentrations were
calculated according to Liu et al. [46]. It should be
noted that the equivalent spherical particle size and
AuNP concentration for this MBI adsorption method
are calculated on the assumption that all the nanoparticles are spherical and monodispersed, which is the
same assumption used by the current AuNP LSPR
method.
technique because it allows convenient separation of excess
MBI from the AuNP/MBI aggregates. Since the amount of
adsorbed MBI is independent of the rate of AuNP/MBI
mixing, AuNPs and MBI were mixed simultaneously in all
our subsequent ligand binding studies.
pH dependence of the MBI adsorption It was found that
citrate-reduced AuNPs are slightly acidic, and the pH varies
from 5.8 to 6.8 for AuNPs with particle sizes from 10 to
50 nm in diameter (data not shown). Previous research from
our lab indicates that MBI saturation packing density on
AuNPs is statistically the same in the pH 7 and pH 12
solutions, but is different for a pH 2 solution in which
MBI adopted a thione tautomeric form on the AuNP surface
[41]. In this study, we further determine the possible pH
dependence of MBI adsorption onto AuNPs in the pH range
that is most relevant in practical AuNP characterizations
(Table 1). The results show that the amounts of MBI
adsorbed onto AuNPs are essentially the same over the
entire investigated pH range (over three pH units), indicating the applicability of this MBI adsorption method for
general AuNP characterization.
Linearity of MBI adsorption To validate this ligand adsorption method, we studied the AuNP concentration dependence of MBI adsorption (Fig. 3). The near perfect linear
correlation between the amounts of MBI adsorbed and the
Table 3 Average equivalent spherical diameter of AuNPs (in nanometers)
Nominal size (nm)
TEMa
TEMb
MBI
DLSc
DLSd
10
30
50
10.1±0.7
32.0±3.3
49.6±6.4
8.8±0.6
25.5±3.0
38.4±6.2
9.7±0.6
26.7±0.9
40.2±2.0
11.6±2.9
31.5±3.1
46.7±3.6
19.3±4.2
39.9±2.5
54.1±3.1
10
30.9
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
LSPR
Determination of colloidal gold nanoparticle surface areas
419
Table 4 Concentration of equivalent spherical AuNPs (in nanomoles per liter)
Nominal
size
(nm)
TEMa
TEMb
MBI
DLSc
DLSd
LSPR
10
30
50
90
6.9±1.4
0.2±0.1
0.1±0.02
0.01±0.003
10.4±2.1
0.4±0.2
0.1±0.06
0.02±0.007
7.8±1.5
0.4±0.04
0.1±0.02
0.02±0.004
4.5±3.4
0.2±0.07
0.07±0.01
0.03±0.02
1.0±0.6
0.1±0.02
0.05±0.008
0.01±0.002
8.18
0.22
0.022
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
surface areas per unit volume obtained with different
techniques vary widely, illustrating the difficulties in
reliable AuNP characterization. The differences are particularly large for the 30, 50, and 90-nm citrate-capped
AuNPs where the polydispersities are relatively high.
These differences can be attributed to the inherent bias
associated with different techniques. For example, the
DLS particle sizes (Table 3) are consistently larger than
those obtained with other techniques. This may be related to the fact that DLS measures the hydrodynamic
radius of the AuNP and its surface adsorbates. Because
the AuNP surface is usually coated with citrate or other
stabilizing agent in the solution, DLS measurement is
expected to give larger particle sizes. The deviation of
the DLS results, in particular the ones obtained with
intensity histograms from that of TEM, might also have
resulted from the artifacts related to DLS measurement
procedure or data analysis. The relatively larger LSPR
particle sizes are not surprising either as the molar
extinction coefficients of larger nanoparticles are significantly higher than those of smaller nanoparticles.
Importantly, the results obtained from the TEM analysis using the CSPI method and the MBI adsorption
method are remarkably similar for all the AuNPs we
tested. The largest relative difference between the particle
sizes from these two methods is less than 10 %. In
addition, AuNP surface areas per unit volume of AuNP
solutions determined with these two methods are consistently larger than that obtained with other techniques
with the only exception being the DLS result for the
90-nm AuNP. This result provides critical cross validation of these two analytical methods. It should not be
surprising because by design the CSPI method and ligand
adsorption method can both accommodate some morphological irregularity of the AuNPs. However, compared to
the TEM CSPI method, the MBI adsorption technique is
much simpler to implement.
The excellent agreement between the TEM CSPI
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
10
30
50
90
13.3±1.8
4.4±0.9
2.9±0.7
1.7±0.3
15.1±2
5.5±1.3
3.7±1.2
2.1±0.4
13.8±0.9
5.3±0.2
3.6±0.2
2.1±0.1
11.5±6
4.5±0.9
3.1±0.5
2.2±1.2
7.0±3.0
3.5±0.4
2.6±0.3
1.6±0.3
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
LSPR
13.4
4.6
2.8
1.6
420
method indicates that the MBI packing density on the
AuNPs is independent of the particle size of AuNPs in
the size range from 10 to 100 nm in diameter. This
result is consistent with what was reported by Elzey et
al. who demonstrated that the packing densities of MPA
on AuNPs are the same for AuNPs in the size range
from 5 to 100 nm [40]. MBI and MPA adsorption
results suggest that the difference in the curvature of
AuNPs with diameter equal to or larger than 5 nm does
not have significant impact on the organothiol packing
density. This result is not surprising, given the subsquare-nanometer footprint of MBI and MPA on the
AuNP surfaces [40, 41].
Characterization of the in-house-synthesized AuNPs The
in-house-prepared AuNP solution (Fig. 5) was characterized with the MBI adsorption method and ICP-OES
determination of the Au3+ concentration, and the results
were compared with data obtained using the other techniques (Table 6). Again, TEM analysis by CSPI and the
MBI adsorption method give the smallest particle size
and highest AuNP surface area per unit volume of AuNP
solution.
The MBI adsorption method can be drastically simplified for characterization of in-house-prepared AuNPs by
eliminating the aqua regia AuNP digestion and the ICPOES Au3+ concentration determination. Indeed, the total
Au content of the AuNPs can be approximated as the
amount of Au3+ used for synthesis. Under our experimental conditions, about 97 % of the Au3+ added into
the AuNP synthesis solution was converted to AuNPs
(data not shown), which is consistent with previous
reports [47]. It is important to note that it can be erroneous to use the weight of HAuCl4·3H2O to calculate
the concentration of the Au3+ because of the hygroscopicity of HAuCl4·3H2O. Instead, one should use the UV–
vis absorbance of the HAuCl4·3H2O solution to determine the Au3+ concentration. A molar absorptivity of
2.56 × 103 M−1 cm−1 at 290 nm was determined for
HAuCl4·3H2O with solutions in the concentration range
Fig. 5 UV–vis spectrum and TEM image for in-house-synthesized
AuNP solution. The scale bar represents 25 nm
M. Gadogbe et al.
Table 6 Particle size, AuNP concentration, and AuNP surface area of
in-house-prepared AuNP solution
Methods
Particle size
(nm)
AuNP concentrations
(nM)
Surface area per
unit volume
AuNP (cm2/mL)
TEMa
MBI
DLSb
DLSc
LSPR
10.2±1.3
9.5±0.2
12.6±1.2
15.9±2.2
12.6
13.0±5.0
16.0±1.0
6.9±2.0
3.4±1.4
7.89
25.6±7.0
27.4±0.8
20.7±4.0
16.4±5.0
20.7
a
TEM CSPI
b
DLS particle size based on mass histogram
c
DLS particle size based on intensity histogram
from 0.25 to 0.5 mM (Electronic supplementary material, Fig. S2). This value is similar to that estimated from
the reported UV–vis spectrum of the HAuCl 4·3H 2O
solution at 290 nm [48].
Conclusion
Determination of the true surface area of colloidal
AuNPs is difficult with existing particle sizing techniques
due to the AuNP morphological irregularity, broad size
distribution, and surface roughness. Large discrepancies
exist in the particle sizes, concentrations, and surface
areas determined with existing particle sizing techniques
(DLS, TEM, and LSPR). The MBI adsorption method
presented in this work likely provides a more accurate
estimation of the true AuNP surface area because of its
capability to accommodate variations in AuNP morphology, shape, and size distributions. The main uncertainty
with this approach likely lies in the saturation MBI
packing density on AuNPs that is needed for estimation
of the AuNP surface area. Unfortunately, we do not have
a commonly agreed on AuNP standard that allows precise determination of ligand packing density. Nevertheless, 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
be particularly useful for quality control in nanoparticle
synthesis.
Determination of colloidal gold nanoparticle surface areas
Acknowledgments This work was supported in part by NSF funds
(EPS-0903787) provided to D.Z.
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