Anal. Chem.
2009, 81, 9425–9432
Dynamic Light Scattering as a Powerful Tool for
Gold Nanoparticle Bioconjugation and
Biomolecular Binding Studies
Hilde Jans,†,‡ Xiong Liu,§ Lauren Austin,§ Guido Maes,‡ and Qun Huo*,§
Interuniversity Microelectronics Center (IMEC), NEXT- Functional Nanosystems, Kapeldreef 75,
B-3001 Leuven, Belgium, Physical and Quantum Chemistry, Catholic University Leuven, Celestijnenlaan 200F,
B-3001 Leuven, Belgium, and Nanoscience Technology Center, University of Central Florida,
12424 Research Parkway Suite 400, Orlando, Florida 32826
Dynamic light scattering (DLS) is an analytical tool used
routinely for measuring the hydrodynamic size of nano-
particles and colloids in a liquid environment. Gold
nanoparticles (GNPs) are extraordinary light scatterers
at or near their surface plasmon resonance wavelength.
In this study, we demonstrate that DLS can be used as a
very convenient and powerful tool for gold nanoparticle
bioconjugation and biomolecular binding studies. The
conjugation process between protein A and gold nano-
particles under different experimental conditions and the
quality as well as the stability of the prepared conjugates
were monitored and characterized systematically by DLS.
Furthermore, the specific interactions between protein
A-conjugated gold nanoparticles and a target protein,
human IgG, can be detected and monitored in situ by
measuring the average particle size change of the assay
solution. For the first time, we demonstrate that DLS is
able to directly and quantitatively measure the binding
stoichiometry between a protein-conjugated GNP probe
and a target analyte protein in solution.
Protein-protein interaction plays an essential role in almost
all cellular processes and biological functions. Convenient, highly
sensitive, and low cost bioanalytical tools allowing fast and high
throughput screening and investigation of protein-protein interac-
tions are extremely important for biomolecular research.1,2
However, only a very limited number of techniques are available
for protein-protein interaction studies, especially for in situ, time-
dependent kinetic studies. Most traditional techniques, such as
affinity chromatography, affinity blotting, coimmunoprecipitation,
pull-down assay, yeast two-hybrid, and ligand-binding assay, are
mostly used for identifying protein-protein binding partners but
are not capable of monitoring the protein-protein binding process.
Kinetic studies are particularly important for understanding
transient protein-protein interactions. Such interactions control
a large number of cellular processes. Surface plasmon resonance
* To whom correspondence should be addressed. E-mail: qhuo@mail.ucf.edu.
†
NEXT- Functional Nanosystems.
‡
Catholic University Leuven.
§
University of Central Florida.
(1) Phizicky, E. M.; Fields, S. Microbiol. Rev. 1995, 59, 94–123.
(2) Russell, R. B.; Alber, F.; Aloy, P.; Davis, F. P.; Korkin, D.; Pichaud, M.;
Topf, M.; Sali, A. Curr. Opin. Struct. Biol. 2004, 14, 313–324.
10.1021/ac901822w CCC: $40.75  2009 American Chemical Society
Published on Web 10/05/2009
(SPR) is among one of the limited number of techniques available
for protein-protein interaction analysis and kinetics.3-9 SPR
measures complex formation by monitoring changes in the
resonance angle of the total internal reflected light from a gold
substrate. To conduct a typical protein-protein binding study, one
of the protein partners is immobilized on a thin gold film. When
the target protein from solution binds to the immobilized protein
partner, the binding will cause a refractive index change at the
surface layer, which is detected by SPR through resonance angle
change of the reflected light.8
Since its inception and commercialization, SPR has made a
revolutionary impact on biomolecular research.9 However, a
significant limit of the SPR technique is the high cost of the
instrument. This price of a typical SPR unit, $200 000-500 000,
is beyond the reach of many research laboratories and biotech
companies. Other than SPR, fluorescence-based techniques, such
as resonance energy transfer (FRET), fluorescence polarization,
or anisotropy with tagged molecules,1 have also been used for in
situ and kinetic monitoring of protein complex formation. How-
ever, because of some general problems of fluorophores and other
limitations, the application of these techniques has been far less
widespread than that of the label-free SPR technique.
Gold nanoparticles (GNPs) have attracted enormous attention
in recent years for both biosensor and bioassay development.
These sensors and assays are mostly based on the optical or
catalytic properties of GNPs. For example, using GNP probes, a
simple homogeneous colorimetric immunoassay was originally
developed by Leuvering et al.10 Antibody-conjugated GNPs will
form aggregates, upon mixing with antigen, causing a color
change of the gold colloid solution from a typical burgundy color
to blue or purple. Such a color change can be seen by the naked
(3) Malmqvist, M. Nature 1993, 361, 186–187.
(4) Jonsson, U. I.; Fagerstam, B.; Ivarsson, B.; Johnsson, R.; Kalsson, K.
BioTechniques 1991, 11, 620–627.
(5) Kalsson, R.; Michaelsson, A.; Mattsson, L. J. Immunol. Methods 1991, 145,
229–240.
(6) Teramura, Y.; Iwata, H. Anal. Biochem. 2007, 365, 201–207.
(7) Mason, S.; La, S.; Mytych, D.; Swanson, S. J.; Ferbas, J. Curr. Med. Res.
Opin. 2003, 19, 651–659.
(8) Englebienne, P.; Van Hoonacker, A.; Verhas, M. Spectroscopy 2003, 17,
255–273.
(9) Morgan, C. L.; Newman, D. J.; Price, C. P. Clin. Chem. 1996, 42, 193–
209.
(10) Leuvering, J. H. W.; Thal, R. J. H. M.; Van der Waart, M.; Shuurs,
A. H. W. M. J. Immunoassay 1980, 1, 77–91.
Analytical Chemistry, Vol. 81, No. 22, November 15, 2009 9425
eye or can be measured by a UV-vis spectrometer. Later this
phenomenon was adopted by Mirkin et al. for DNA detection.11
In addition to their surface plasmon resonance absorption, light
scattering is another optical property of GNPs that is of great
interest for biomolecular detection.12-20 The light-scattering cross-
section of a GNP with a diameter of 60 nm is 200-300 times
stronger than that of a polystyrene bead of the same size, and
4-5 orders of magnitude stronger than that of a strong fluores-
cence dye, e.g., fluorescein.12,13 Several groups have demonstrated
the use of GNPs to develop biomolecular assays based on either
the static, linear, or nonlinear scattering properties of GNPs.14-20
However, all these previous reports were focused on detecting
biomolecules through monitoring the scattered light intensity
change rather than the nanoparticle size change of the assay
solution.
Our group recently reported for the first time a unique
biooassay technology, named NanoDLSay, for biomolecular
detection.21-23 This technique couples the use of GNP probes as
a light-scattering enhancer and dynamic light scattering (DLS)
as a read-out system. The binding of an antigen or target DNA in
solution to the antibody or DNA-conjugated GNPs causes nano-
particle aggregation. The subsequent average particle size increase
was then measured by DLS and correlated to the analyte
concentration. By switching from detecting the light intensity
change to the particle size change using DLS, NanoDLSay opens
many more possibilities in biomolecular analysis.
We now further demonstrate that NanoDLSay can be used for
direct monitoring of biomolecular conjugation to GNPs and
biomolecular interaction studies between these conjugated GNPs
and their target analyte proteins, a capability which is very similar
to the SPR technique. In this work, we select the binding between
protein A and human IgG as a model system for the investigation.
Protein A is a cell wall constituent of Staphylococcus aureus.24 This
protein has specific binding sites to the Fc region of immunoglo-
bulin (IgG) from most mammalian species.24 Protein A-conjugated
GNPs can serve as a versatile nanoparticle agent for preparing
mammalian IgG-based immunoprobes. Because protein A binds
specifically to the Fc region of mammalian IgG, this nanoparticle
probe provides an excellent way of controlling the orientation of
IgG molecules so that the Fab and Fab′ fragment of the antibody
(11) Nam, J.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884–1886.
(12) Yguerabide, J.; Yguerabide, E. E. Anal. Biochem. 1998, 262, 137–156.
(13) Jain, P. K.; Lee, K. S.; El-Sayed, I. H.; El-Sayed, M. A. J. Phys. Chem. B
2006, 110, 7238–7248.
(14) Du, B.-A.; Li, Z.-P.; Liu, C.-H. Angew. Chem., Int. Ed. 2006, 45, 8022–8025.
(15) Russier-Antoine, I.; Huang, J.; Benichou, E.; Bachelier, G.; Jonin, C.; Brevet,
P.-F. Chem. Phys. Lett. 2008, 450, 345–349.
(16) Ray, P. C. Angew. Chem., Int. Ed. 2006, 45, 1151–1154.
(17) Xie, H.; Gill-Sharp, K. L.; Oneal, D. P. Nanomed.: Nanotechnol., Biol. Med.
2007, 3, 89–94.
(18) Hirsch, L. R.; Jackson, J. B.; Halas, N. J.; West, J. L. Anal. Chem. 2003,
75, 2377–2381.
(19) Aslan, K.; Holley, P.; Davies, L.; Lakowicz, J. R.; Geddes, C. D. J. Am. Chem.
Soc. 2005, 127, 12115–12121.
(20) Xie, C.; Xu, F.; Huang, X.; Dong, C.; Ren, J. J. Am. Chem. Soc. 2009, 131,
12763–12770.
(21) Liu, X.; Dai, Q.; Austin, L.; Coutts, J.; Knowles, G.; Zou, J.; Chen, H.; Huo,
Q. J. Am. Chem. Soc. 2008, 130, 2780–2782.
(22) Dai, Q.; Liu, X.; Coutts, J.; Austin, L.; Huo, Q. J. Am. Chem. Soc. 2008,
130, 8138–8139.
(23) Liu, X.; Huo, Q. J. Immunol. Method 2009, 349, 38–44.
(24) Akerström, B.; Björk, L. J. Biol. Chem. 1986, 261, 10240–10247.
9426 Analytical Chemistry, Vol. 81, No. 22, November 15, 2009
are exposed on the nanoparticle surface for antigen binding.25-27
Although this work focuses on the study of protein A-human
IgG interactions, it is easy to see that the principle of NanoDLSay
may be extended to almost any other protein-protein binding
and interaction study.
EXPERIMENTAL SECTION
Reagents. Gold nanoparticles (GNPs) used throughout the
experiments were purchased from Ted Pella Inc. (Redding, CA).
Human IgG, protein A (prA), and bovine serum albumin (BSA)
were purchased from Sigma-Aldrich (Milwaukee, WI) and used
without further purification. Gluteraldehyde, buffer ingredients,
and all other chemicals were obtained from Sigma-Aldrich.
DLS Measurements. The hydrodynamic diameters of the
GNPs under investigation were measured using a Zetasizer Nano
ZS90 DLS system equipped with a red (633 nm) laser and an
Avalanche photodiode detector (APD) (quantum efficiency > 50%
at 633 nm) (Malvern Instruments Ltd., England). A Hellma cuvette
QS 3 mm was used as sample container. DTS applications 5.10
software was used to analyze the data. All sizes reported here
were based on intensity average. The intensity average particle
size was obtained using a non-negative least squares (NNLS)
analysis method. For each sample, two DLS measurements were
conducted with a fixed 10 runs and each run lasts 10 s. A detection
angle of 90° was chosen for the size measurement unless stated
otherwise.
Dark Field Optical Imaging Study. The dark field measure-
ments were performed using an Olympus BX51 microscope
equipped with a dark field condenser and an oily dispersed
objective (100×/1.3, UPlan FLN). The microscope was fitted with
Soft Imaging System’s Colorview III camera and AnalySIS Life
Sciences software. Prior to the measurement, the GNP solution
samples were dropped and then dried on a glass microscope slide.
The dried samples were covered with a glass cover slide and
mounted on the stage for imaging.
Preparation of Gold Nanoparticle-Protein A Conjugates
(adapted and modified according to the literature).26-28 To
900 µL of a GNP solution (concentration 10 pM) was added 100
µL of a Protein A solution at a concentration of 100 µg/mL. The
solution was previously adjusted to a pH value of 7-7.5 using 20
µL of 0.02 M NaOH. The solution was set at room temperature
for 1 h on a shaking device. Then 150 µL of bovine serum albumin
(BSA) (concentration 4 mg/mL) was added. The reaction mixture
was placed on a shaking device for an additional 1 h. The solution
was then centrifuged at 5 krpm for 3 min. After decantation of
the solution, the nanoparticle residue was redispersed in 1 mL of
10 mM phosphate buffer (PB) solution. The probes were ready
for use in the assay.
Salt-Induced Gold Nanoparticle (GNP) Aggregation Study.
Prior to all measurements, the nanoparticles were diluted in a
1:1 volume-ratio with different concentrations of NaCl solutions
(10, 50, 100, 500 mM). Different samples were incubated at room
temperature for 5 min, and then the size of each nanoparticle
(25) Wang, H.; Liu, Y.; Yang, Y.; Deng, T.; Shen, G.; Yu, R. Anal. Biochem. 2004,
324, 219–226.
(26) Ghitescu, L.; Bendayan, M. J. Histochem. Cytochem. 1990, 38, 1523–1530.
(27) Horisberger, M.; Clerc, M.-F. Histochem. 1985, 82, 219–223.
(28) Hermanson, G. T. Bioconjugate Techniques, 2nd ed.; Pierce Biotechnology,
Thermo Fisher Scientific: Rockford, IL, 2008; Chapter 24.
solution was measured by DLS. UV-vis absorption spectra were
measured using a Cary 300 Bio UV-visible spectrophotometer
from Varian Inc. (Palo Alto, CA).
Binding Study between Protein A-Conjugated GNPs and
Human IgG. The binding of human IgG onto the protein
A-conjugated GNPs was evaluated by DLS. Hereto, equal volumes
of protein A conjugates and human IgG solution (PB 10 mM) were
mixed together for exactly 5 min. The concentration of human
IgG was varied (0, 1.6, 6.2, 12.5, 25 µg/mL). The samples were
measured by DLS without purification of the solution from the
unbound fraction of human IgG.
Direct Binding Study of Protein A-Conjugated GNP (100
nm) with Human IgG. To monitor the direct binding of human
IgG by DLS, GNP-protein A conjugates were mixed in a quartz
cuvette with a human IgG solution to a concentration of 1 µg/
mL. The size increase was monitored at different incubation time
intervals.
Cross-Linked Protein A-Conjugated Gold Nanoparticles.
Hereto, 225 µL amounts of protein A-conjugated GNPs were
incubated for exactly 1 h with 25 µL gluteraldehyde solutions of
0.1%, 1%, 2%, 5% (V/V) in H2O. The cross-linking reaction was
stopped by removing the supernatants after centrifugation of
the reaction mixture at a speed of 5 krpm for 3 min. The GNP
residue was resuspended in PB (10 mM).
RESULTS AND DISCUSSION
Detection Limit Study of Gold Nanoparticles (GNPs) by
DLS. To demonstrate the high sensitivity of DLS toward GNPs
detection, we examined the detection limit of DLS on a series of
GNPs with an average diameter ranging from 40 to 250 nm.
Different from UV-vis and fluorescence spectrophotometry, the
detection limit of DLS varies from instrument to instrument
significantly, because the detected scattering light intensity is
related to the wavelength and power of the incident light, the type
of detector, and the detection angle.29 The scattered light intensity
of each GNP with a different diameter measured by an Avalanche
photodiode detector of the Malvern ZS90 system at different
concentrations is plotted in Figure 1. From the plot, it can be seen
that the light-scattering intensity decreases linearly with decreas-
ing GNP concentration. Such linear relationships were observed
for GNPs at all sizes investigated in this study. It is apparent that
larger nanoparticles scatter light much more strongly than smaller
nanoparticles, as predicted by theoretical calculations.12,13 The
lower limit of detection (LLOD) for each size GNP is estimated
according to the standard σ + 3s rule, where σ and s are the base
values of the scattered light intensity and the standard deviation
of a blank sample (which is pure water in this case), respectively.
As seen from the inserted table in Figure 1, the LLOD for
nanoparticles larger than 100 nm can easily reach femtomolar (fM)
or lower concentrations, without using any amplification strategy.
The high sensitivity of DLS toward GNP detection establishes the
base for highly sensitive bioanalytical tool development using GNP
probes and DLS as a read-out system.
Stability Study of GNPs in High Salt Environment by DLS
and Dark Field Imaging. Citrate-protected GNPs are the most
commonly used gold nanoparticles for conjugation with proteins
(29) Berne, B. J.; Pecora, R. Dynamic light scattering: with applications to
chemistry, biology, and physics; Wiley: New York, 1976.
Figure 1. The linear relationship between the scattered light intensity
measured by DLS and the concentration of gold nanoparticles with
different average diameters. The lower limit of detection (LLOD) of
each gold nanoparticle is summarized in the inserted table. Each data
point was repeated for three times, solid lines are linear fitting curves,
and LLOD data were calculated according to the standard σ + 3s
method.
for both biomolecular detection and bioimaging. However, a
problem of citrate-protected GNPs is that the nanoparticles cluster
easily in high salt environment. Once these nanoparticles are
successfully conjugated with a layer of proteins such as antibodies,
the nanoparticles can be stabilized in high salt content buffer
solutions. This phenomenon has actually been used as one method
to confirm the successful conjugation of proteins to GNPs.28 So
far, UV-vis absorption spectroscopy has been used as a common
tool to monitor GNP aggregation, because the aggregation causes
a shift or broadening of the surface plasmon resonance of the
GNPs. However, such measurements are often only semiquanti-
tative with low sensitivity. The aggregation can be detected by
UV-vis spectroscopy only when the aggregation level is signifi-
cant enough to cause a color change.
As a particle size measurement tool, it is natural to hypothesize
that DLS could be a more sensitive tool for nanoparticle aggrega-
tion study. We examined the aggregation behavior of GNPs with
a diameter of 40, 60, 80, and 100 nm at different NaCl concentra-
tions using DLS. As shown in Figure 2A, the measured average
particle size starts to show obvious increase at a NaCl concentra-
tion around 25 mM after mixing the nanoparticles with the NaCl
solution for only 5 min. This particle size increase is due to the
formation of nanoparticle clusters in NaCl solution, as evidenced
from a broadening of the particle size distribution (DLS data not
shown) and also supported by the dark field imaging study as
discussed below. By comparing the size increase of GNPs with
different diameters, it is also noticed that nanoparticles with
smaller sizes such as 40 nm are less stable in high salt content
solution than larger particles. At 25 mM of NaCl, the size of the
40 nm GNP is more than doubled. In contrast, from the UV-vis
absorption spectra of a 40 and 100 nm GNP solution (Figure 2B),
almost no SPR peak shift or broadening is observed at a NaCl
concentration of 25 mM. Typically, one only starts to observe a
clear SPR peak shift or broadening from the citrate-protected
GNPs at a NaCl concentration around 30 mM.
Because large particles and particle aggregates scatter light
more intensely at forward angle than at 90° or back scattering
angle, and smaller particles scatter light very weakly at forward
angle,29 DLS measurement at forward angle is heavily biased
Analytical Chemistry, Vol. 81, No. 22, November 15, 2009 9427
Figure 2. A: Stability study of GNPs (40, 60, 80, 100 nm) at different salt concentrations. Each nanoparticle solution was incubated with a
NaCl solution for exact 5 min prior to the DLS measurement. B: UV-vis absorption spectra of 40 and 100 nm GNPs at different salt concentrations.
C: Detection sensitivity study of nanoparticle aggregates using different detection angles. Hereto, an equal volume of 40 or 100 nm GNPs
solution and NaCl solution were mixed together and incubated for exactly 5 min, prior to the measurement at different DLS detection angles
(12.8° and 90°). D: Dark field optical microscopic images of 40 nm gold nanoparticles after incubation with different concentrations of NaCl: (a)
0 mM, (b) 5 mM, (c) 25 mM, (d) 50 mM.

toward larger particles or particle aggregates and neglects the
smaller ones in a specific particle population. For this reason,
nanoparticle aggregation should be detected more sensitively
by DLS using forward angle detection. A comparison study on
the particle size change of a 40 and 100 nm GNP under different
salt concentrations was conducted at 12.8° and 90° detection
angle, respectively, and the results are summarized in Figure
2C. As predicted, a more substantial size increase of nanopar-
ticles induced by salt addition is detected from forward angle
measurement than from 90° angle measurement. Even at a
NaCl concentration as low as 5 mM, a significant nanoparticle
size increase of ∼10% is observed from both 40 and 100 nm
nanoparticles, while the UV-vis absorption spectra of these
nanoparticle solutions do not show any observable change
(peak shift or broadening) in their SPR absorption band. These
results suggest that DLS is indeed a more sensitive and suit-
9428 Analytical Chemistry, Vol. 81, No. 22, November 15, 2009
able tool than UV-vis absorption spectroscopy to monitor gold
nanoparticle aggregation.
To confirm that the measured GNP nanoparticle size increase
by DLS is indeed due to nanoparticle aggregation, we also
conducted a dark field optical imaging analysis of the nanoparticle
samples treated with different concentrations of NaCl solution.
As shown in Figure 2D, the gold nanoparticles exhibit a bright
orange color under a dark field optical microscope and can be
easily identified. Upon an increase in NaCl concentration, it is
clearly seen that the particles tend to cluster in groups. The
number of particles per group increases with increased salt
concentration. The nanoparticle clustering observed by dark field
microscopy confirmed that the average particle size increase
obtained from DLS measurement is indeed due to salt-induced
nanoparticle aggregate/cluster formation, not due to size increase
of individual nanoparticles.
Figure 3. A: GNP-protein A conjugation study. The final sizes of the conjugates are plotted versus the final concentration of prA used in the
conjugation protocol. B: Conjugation study of GNP of different sizes (40, 60, 80, 100 nm) with protein A and the stability study of the conjugates
in electrolytic environment. C: An illustration of the elongated fibrous shape of protein A and its three possible orientations on the nanoparticle
surface after adsorption. D: pH effect on protein A conjugation to GNPs.

Direct Monitoring and Characterization of Biomolecule
Conjugation to Gold Nanoparticles. Proteins are biomacromol-
ecules, and their size is usually larger than a few nanometers.
Proteins can be detected and measured directly by DLS. For
example, bovine serum albumin (BSA) is a protein with a
molecular mass of 60 kDa and a diameter around 7-8 nm (data
from Protein Data Bank). BSA is used by many DLS manufactur-
ers as a standard to calibrate DLS instruments. However, because
of the intrinsic weak light-scattering intensity of biomacromol-
ecules, proteins can only be detected by DLS at very high
concentrations, usually in the mg/mL range. Because of the strong
light-scattering intensity of GNPs, proteins adsorbed to GNPs can
be detected by DLS much more sensitively. When a complete
layer of proteins is adsorbed on the GNP, the diameter of the
GNP will increase at least by two times the diameter of the protein
molecule. The Malvern ZS90 system has an average detection
error of 1-2 CV%. For a nanoparticle with a diameter of 100 nm,
a size change of 3-5 nm can be reliably measured by DLS
according to the standard σ + 3s rule (σ and s are the base values
and standard deviation of a negative control sample, respectively).
This means that, if a protein or any other biomacromolecule has
a diameter of at least 3 nm, the adsorption and formation of a
stable monolayer of such biomacromolecules onto a gold nano-
particle surface will cause an approximately 6 nm size increase,
which is readily detectable by DLS.
Our study reveals that indeed, the adsorption process of protein
A to GNPs can be conveniently monitored in situ by DLS. Figure
3A shows the observed size increase of the GNPs after incubating
them with protein A solution at different protein concentrations.
The initial size of the citrate-protected GNPs was about 106 nm.
When particles are incubated with protein A solution, the particle
size increases clearly, and at a protein A concentration of 5 µg/
Analytical Chemistry, Vol. 81, No. 22, November 15, 2009 9429
Figure 4. A: Direct binding study of protein A-conjugated GNP (100 nm) with human IgG. Human IgG was mixed with the GNP conjugate
solution to a final IgG concentration of 1 µg/mL in a quartz cuvette. The size increase of the mixed solution was monitored by DLS in situ at
different incubation time intervals. B: Binding study of protein A-conjugated GNPs of different sizes (40, 60, 80, 100 nm) with human IgG at
different concentrations. Each GNP conjugate solution was incubated with a human IgG solution for exactly 5 min, and the average particle size
was measured immediately after. The net particle size increase was plotted versus the human IgG concentration. In all the measurements, the
unbound fraction of human IgG was not separated from the assay solution.

mL, the particle size increase reaches a maximum of 22-25 nm.
We also prepared nanoparticle-protein A conjugates using nano-
particles with an initial size of approximately 40, 60, 80, 100 nm
and a protein A concentration of 10 µg/mL. After conjugation with
protein A, the hydrodynamic diameter of all the gold nanoparticles
increases roughly by about 25 nm (Figure 3B). The size increase
is independent of the original nanoparticle size, which means that
this size increase can only be due to the adsorption of protein A
on the gold surface.
The molecular mass of protein A as determined via several
methods resulted in values ranging from 40 kDa to 44 kDa with
an average of 42 kDa.30 Although protein A is a relatively small
protein, it has an elongated fibrous shape structure as illustrated
in Figure 3C, with a length of 25-30 nm and a diameter of 2.5
nm.31 From the maximum particle size increase observed from
the DLS study, we gained further understanding on the orientation
of protein A adsorbed on the gold nanoparticles. If protein A is
oriented vertically to the nanoparticle surface, a size increase of
50 nm would be expected from a nanoparticle fully covered by
protein A (Figure 3C, scenario A); on the other hand, if protein A
lies totally flat on the nanoparticle surface, the nanoparticle size
should increase by approximately 5 nm (Figure 3C, scenario C).
The observed size increase of 25 nm indicates that the elongated
protein A molecules are oriented at a tilted angle on the particle
surface as illustrated in the scenario B of Figure 3C.
After the protein A-nanoparticle conjugates were prepared,
the stability of the conjugates in a high salt content environment
was also studied by DLS. From the data shown in Figure 3B, three
conjugates, prepared using nanoparticles with an initial size of
approximately 40, 60, 80 nm, remained very stable in NaCl solution
up to a concentration of 850 mM. The stability of the 100 nm
nanoparticle conjugate is not as good as that of the smaller
particles. While citrate-protected GNPs are highly unstable in
electrolyte solution, the properly bioconjugated GNPs are very
(30) Björk, I.; Petersson, B.; Sjöquist, J. Eur. J. Biochem. 1972, 29, 579–584.
(31) Ohnishi, S.; Murata, M.; Hato, M. Biophys. J. 1998, 74, 455–465.
9430 Analytical Chemistry, Vol. 81, No. 22, November 15, 2009
stable in high salt content solution. This good stability is important
for using GNP probes in biological sample studies.
The DLS measurement also allows convenient investigation
of the optimum experimental conditions for the nanoparticle-protein
conjugation. We compared the different pH effects on protein
A-nanoparticle conjugation and found that the best condition for
conjugation is pH 7-9. In this pH range, a maximum size increase
of 25-30 nm corresponding to a full coverage of protein A on the
nanoparticle surface is observed (Figure 3D).
Direct Binding Study of Protein A-Conjugated Nanopar-
ticles with Human IgG. Protein A has a high affinity toward the
Fc fragment of mammalian immunoglobulins. The protein A layer
can serve as a universal surface for the attachment of a wide
variety of immunoglobulins to the GNPs. Here we demonstrate
that the binding kinetics of human IgG to protein A-conjugated
GNPs can be directly monitored by DLS. As shown in Figure 4A,
the nanoparticle probe size increases gradually over time after
the probe solution was mixed and incubated with the human IgG
solution at a concentration of 1 µg/mL. We also measured the
size increase of various conjugates upon incubation with human
IgG at different concentrations for 5 min. For all four nanoparticle
conjugates, the nanoparticle size increase reaches a maximum
value of approximately 60 nm at h-IgG concentrations around 6-12
µg/mL (Figure 4B). Further increase of the h-IgG concentration
does not lead to larger particle size increase.
The maximum size increase of 60 nm upon binding of protein
A-GNP conjugate with h-IgG is very interesting and worth further
discussion. When pure h-IgG is physically adsorbed to citrate-
protected GNPs directly, a size increase of 15-20 nm was
observed (data not shown here). As a matter of fact, we observe
similar particle size increases of 15-20 nm from all IgG-GNP
conjugates (more than 10 different kinds) that we have studied
so far. The molecular mass of an IgG molecule is around 150 KDa,
corresponding to a hydrodynamic diameter of approximately 7-10
nm (data from Protein Data Bank). The observed size increase
of 15-20 nm from h-IgG directly adsorbed to citrate-protected
Figure 5. A: Stability study of cross-linked protein A-conjugated GNPs. The protein A-conjugated GNPs were incubated for 1 h with different
gluteraldehyde (GA) concentrations (0.1, 1, 2, 5% (v/v) in H2O). The cross-linking was stopped by centrifugation, and the average particle size
of each treated GNP solution was measured. Additionally, the stability was evaluated by mixing equal volumes of cross-linked protein A-GNP
conjugates and NaCl solutions of different concentrations for exact 5 min. B: Binding affinity study of human IgG onto protein A-conjugated
GNPs before and after cross-linking by different concentrations of gluteraldehyde. The different cross-linked protein A-GNP conjugates were
mixed with an equal volume of human IgG, with an incubation time of exact 5 min prior to the DLS measurement.

GNPs confirms that the h-IgG product used in the binding study
is purely or mainly monomeric IgG. The substantial nanoparticle
size increase of 60 nm upon binding of h-IgG with protein
A-conjugated GNPs can only be attributed to the binding of
multiple h-IgG molecules per protein A molecule. We estimate
that each protein A binds with approximately two to three human
IgG molecules. This observation corresponds very well to a
previously known fact that each protein A molecule can bind with
at least two IgG molecules with high affinity (1010-1011 M-1).24
For the first time, we demonstrate that DLS is able to
quantitatively and directly measure the binding stoichiometry
between a protein-conjugated GNP probe and a target analyte
protein in solution.
Cross-Linked Nanoparticle-Protein A Conjugate: Stability
and Binding Affinity Study. From the stability study of the
protein A-conjugated GNPs as shown in Figure 3B, it was noticed
that the stability of the 100 nm conjugate is not as good as the
other three smaller particles. To increase the stability of this
conjugate, we tried to cross-link the protein A layer slightly using
gluteraldehyde (GA). Gluteraldehyde is often used to cross-link
enzymes and proteins immobilized on a solid substrate or
microparticles to prevent desorption of proteins from the
substrate.28,32 Gluteraldehyde is a bifunctional cross-linking
reagent which reacts with lysine residues on the exterior of
the proteins through amide bond formation. There are 14% lysine
residues available in the amino sequence of a protein A molecule
to react with gluteraldehyde.33 Protein A-conjugated GNPs were
treated with gluteraldehyde at different concentrations, and their
stability in high salt content solution was further investigated by
DLS. From Figure 5A, it can be seen that after cross-linking with
gluteraldehyde, the size of the nanoparticle conjugate decreases
slightly. This result confirms that the cross-linking occurs between
proteins within the same nanoparticle. Cross-linking of proteins
from different nanoparticles will substantially increase the average
(32) Bang, L. B.; Meza, M. B. Microspheres Part II-ligand attachment and test
formulation, IVD Technology Magazine, Canon Communications: Los
Angeles, March 1995.
(33) Sjöholm, I.; Sjödin, T. Eur. J. Biochem. 1974, 47, 491–498.
size of the nanoparticle probe solution. With increased degree of
cross-linking, the stability of the conjugate increases clearly, as
evidenced by the much smaller particle size increase of the probes
at NaCl concentration of 500 mM. However, when we examined
the binding affinity of the cross-linked nanoparticle conjugate with
human IgG, unfortunately, the conjugate lost its binding affinity
at the increased cross-linking level toward IgG almost completely
(Figure 5B). Although cross-linking can help to stabilize the
nanoparticles, it appears that such a treatment compromises the
binding affinity of the nanoparticle bioconjugates.
CONCLUSIONS
In summary, dynamic light scattering is a very powerful tool
for gold nanoparticle bioconjugation study and for in situ
monitoring of biomolecular binding and protein-protein inter-
actions. While gold nanoparticles are being investigated ex-
tensively for biomolecular detection and bioimaging because
of their many attractive optical properties, the characterization
of gold nanoparticle bioconjugates is often not performed
sufficiently and properly due to the lack of low cost and
convenient analytical techniques. Our work reported here
demonstrates that DLS can be used very conveniently for
comprehensive and quantitative studies of gold nanoparticle
bioconjugation, including monitoring the conjugation process,
identifying the best conjugation conditions, confirming their
specific binding affinity with target analytes, and investigating
the stability of the bioconjugates. DLS also has a much higher
sensitivity for monitoring gold nanoparticle aggregation forma-
tion/process. More importantly, the binding of target analyte
proteins to the bioconjugated nanoparticles can be monitored
in situ by DLS to obtain kinetic information of the protein-protein
interactions. Compared to the surface plasmon resonance
technique, DLS is a low cost and low maintenance instrument.
The preparation of gold nanoparticle probes is also much easier
than the immobilization of biomolecules on a gold thin film
substrate. Furthermore, because gold nanoparticle probes are
homogeneously dispersed in solution, the binding between
target proteins to gold nanoparticle-bound biomolecules is
Analytical Chemistry, Vol. 81, No. 22, November 15, 2009 9431
much faster than the binding of target analytes to molecules
immobilized on a two-dimensional solid substrate. Protein and
protein-protein interactions can be detected and monitored
by DLS in a more timely fashion compared to the surface
plasmon resonance technique.
ACKNOWLEDGMENT
This work is supported by the GROW program in the World
Gold Council and partially supported by the National Science
9432 Analytical Chemistry, Vol. 81, No. 22, November 15, 2009
Foundation award #0506531. Hilde Jans was supported by a grant
from the “Institute for the Promotion of Innovation through
Science and Technology in Flanders” (IWT-Vlaanderen).
Received for review August 12, 2009. Accepted September
18, 2009.
AC901822W