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Bio-Nano Interactions www.small-journal.com

Formation of a Monolayer Protein Corona around

Polystyrene Nanoparticles and Implications
for Nanoparticle Agglomeration
Haixia Wang, Rui Ma, Karin Nienhaus, and Gerd Ulrich Nienhaus*

interacting with the molecular machinery

Nanoparticle (NP) interactions with cells and organisms are mediated of life. However, to ensure both efficacy
by a biomolecular adsorption layer, the so-called “protein corona.” An and safety of NPs in biomedical applica-
in-depth understanding of the corona is a prerequisite to successful and tions, their interactions with the biolog-
safe application of NPs in biology and medicine. In this work, earlier in situ ical environment have to be thoroughly
examined.[3–8]
investigations on small NPs are extended to large polystyrene (PS) NPs of
Pristine NP surfaces are typically highly
up to 100 nm diameter, using human transferrin (Tf ) and human serum reactive toward biomolecules, especially
albumin (HSA) as model proteins. Direct NP sizing experiments reveal a proteins.[9] As a consequence, whenever
reversibly bound monolayer protein shell (under saturating conditions) on a NP is immersed in a biological fluid, an
hydrophilic, carboxyl-functionalized (PS-COOH) NPs, as was earlier observed adsorption layer of proteins, known as the
“protein corona,”[10] quickly enshrouds the
for much smaller NPs. In contrast, protein binding on hydrophobic, sulfated
NP. As a consequence, the NP interacts
(PS-OSO3H) NPs in solvent of low ionic strength is completely irreversible; with its biological environment via this
nevertheless, the thickness of the observed protein corona again corresponds adsorption layer rather than its original
to a protein monolayer. Under conditions of reduced charge repulsion (higher surface.[11–13] As NP-based biomedical
ionic strength), the NPs are colloidally unstable and form large clusters compounds are often designed to expose
below a certain protein–NP stoichiometric ratio, indicating that the adsorbed functional groups for specific interaction
with biological targets, protein adsorp-
proteins induce NP agglomeration. This comprehensive characterization of
tion introduces an additional layer of
the persistent protein corona on PS-OSO3H NPs by nanoparticle sizing and complexity to their development.[6,14,15] In
quantitative fluorescence microscopy/nanoscopy reveals mechanistic aspects recent years, however, researchers have
of molecular interactions occurring during exposure of NPs to biofluids. devised strategies to control and exploit
the protein corona to enable specific capa-
bilities in biomedical applications.[16]
Owing to the enormous complexity of
1. Introduction the processes involved in protein corona formation, its detailed
exploration poses formidable challenges. Key properties of the
A vast variety of nanomaterials can nowadays be fabricated, protein corona have been revealed in recent years, but even fun-
featuring precisely defined molecular-scale structures and phys- damental issues are presently still controversially debated.[13,15]
icochemical properties that are often entirely new and unantici- NP–protein interactions can vary considerably in strength,
pated from the bulk materials.[1] Among these, nanoparticles depending on the properties of the protein and NP surfaces.
(NPs) promise to revolutionize biomedicine in many areas Weak binding results in a “soft corona,”[11] characterized by
including diagnostics and therapy.[2] NPs are of similar size fast dynamic exchange between adsorbed proteins and those in
as biomolecular complexes and, therefore, perfectly capable of solution, whereas a tightly bound, “hard corona” is essentially

Dr. H. Wang, R. Ma, Dr. K. Nienhaus, Prof. G. U. Nienhaus Prof. G. U. Nienhaus

Institute of Applied Physics Institute of Toxicology and Genetics
Karlsruhe Institute of Technology (KIT) Karlsruhe Institute of Technology (KIT)
76131 Karlsruhe, Germany 76344 Eggenstein-Leopoldshafen, Germany
E-mail: uli@uiuc.edu Prof. G. U. Nienhaus
Dr. H. Wang, Prof. G. U. Nienhaus Department of Physics
Institute of Nanotechnology University of Illinois at Urbana-Champaign
Karlsruhe Institute of Technology (KIT) Urbana, IL 61801, USA
76344 Eggenstein-Leopoldshafen, Germany
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smll.201900974.

DOI: 10.1002/smll.201900974

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persistent.[11,17,18] Technically, hard coronae are easier to char-
acterize because the NPs can be separated from the biofluid
together with the adsorbed protein layer.[19] Major structural
changes of the adsorbed proteins up to complete denaturation
may occur during hard corona formation.[20–22] Even though the
polypeptide chains are tightly bound to the NP surface, they
can nevertheless be replaced by other ones adsorbing from the
biofluid (Vroman effect).[23] Thus, the hard protein corona is
dynamic and capable of slowly changing its physical composi-
tion over time.[7,17,24]
Biological fluids are complex mixtures of biomolecules in
aqueous solvent containing high concentrations of small ions,
and formation of a protein corona can markedly affect the
colloidal stability of NPs.[20,25–27] A dense adsorption layer of
globular proteins is highly hydrophilic due to its polyzwitteri-
onic nature and can enhance colloidal stability of NPs. How-
ever, charge screening by ions and adsorbed proteins may also
compromise the colloidal stability of charge-stabilized NPs
and induce agglomeration and, furthermore, the crosslinking
capability of strongly adsorbed polypeptide chains can also
give rise to NP clustering.[20] NP agglomerate formation can
be detrimental in many biomedical applications. Although
researchers are well aware of this problem, detailed studies are
still scarce.[10,28,29]
Investigations of simple model systems with well-
characterized NPs and relevant proteins (e.g., from blood
serum) allow a quantitative characterization of protein
adsorption and offer mechanistic insight into the forma-
tion of the protein corona. In earlier work, we have studied
the protein corona through the NP size increase due to
protein adsorption. Using in situ fluorescence correlation
spectroscopy (FCS) with various small, hydrophilic model
NPs (hydrodynamic radius, RH  < 10 nm) and buffer solu-
tions containing important serum proteins such human
serum albumin (HSA),[30] (apo-)transferrin (Tf),[31] and
apolipoproteins,[32] we have ubiquitously observed forma-
tion of a reversibly bound protein monolayer. Here, we have
extended these studies to much larger polystyrene NPs[33]
(for their physicochemical characterization, see Table  1 and
Figure S1, Supporting Information), using Tf and HSA as
model proteins. Specifically, we compare carboxylate-modified
Table 1. Schematic depictions and physicochemical properties of PS
NPs stabilized with different functional groups.
PS-NP PS-COOH NP PS-OSO3H NP PS-OSO3H NP
Structure
Excitationmax [nm]a) 545 474 504
Emissionmax [nm]a) 562 556 514
RH [nm] (DLS)a) 35.1 ± 1.3 50.0 ± 0.2 18.1 ± 0.2
RH [nm] (FCS)a) 36.3 ± 0.4 54.5 ± 0.1 18.0 ± 0.4
ζ-potential [mV]b) –47 ± 3 –58 ± 2 –45 ± 2
a)NPs suspended in PBS; b)NPs suspended in 0.1 × PBS.
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fluorescent polystyrene beads (PS-COOH NPs), which have a
fuzzy hydrophilic surface and are electrosterically stabilized,
and sulfate-functionalized NPs (PS-OSO3H NPs), which have
a smooth hydrophobic surface and are electrostatically stabi-
lized. By using dynamic light scattering (DLS) and quantitative
fluorescence microscopy including FCS and single molecule-
based localization microcopy (SMLM), we have characterized
the protein coronae formed around these NPs and investigated
the connection between NP–protein binding and protein-
induced NP agglomerate formation, an issue of fundamental
importance in the development of NP-based biomedicals.
2. Results and Discussion
2.1. Tf Protein Adsorption onto PS-COOH NPs
We incubated PS-COOH NPs (RH  ≈ 35 nm) with aqueous
solutions containing defined concentrations of Tf for 15 min to
measure the Tf concentration dependence of RH using FCS (or
DLS). FCS autocorrelation curves of fluorescent PS-COOH NPs
(0.7 × 10−9 m) in PBS solvent shift to the right with increasing Tf
concentration (Figure  1a), indicating an effective size increase
of the NPs due to Tf binding. A fit of the correlation data yields
the diffusion coefficient, D, from which RH is calculated with
the Stokes–Einstein relation
kBT
RH = (1)
6π ηD
with Boltzmann constant, kB, absolute temperature, T, and
solvent viscosity, η. The stepwise change of the hydrodynamic
radius, RH, with protein concentration (Figure 1b) can be
fitted perfectly with an expression that models protein binding
according to the Hill relation[34]
1
 3
 
 cN max  (2)
R H = R 0 1 + n 
 1 +  K D′  
  [P ]  

Here, R0 is the radius of the bare PS NPs, K′D is the concen-
tration of half-saturation of binding, and [P] is the concentra-
tion of (free) protein in solution, Nmax denotes the maximum
number of protein molecules that can bind to a NP, assuming
that the volume of the adsorption layer is completely filled with
protein molecules. The coefficient c is the volume ratio between
a Tf molecule and a bare NP.[30,34] The fit yields a change of
RH from 36.2 ± 0.2 to 45.2 ± 0.8 nm, indicating formation of
a protein layer of thickness ΔRH  = 9.0 ± 0.8 nm under satu-
rating conditions. The molecular structure of Tf consists of
two homologous globular domains with overall dimensions of
4.2 × 7 × 10 nm3 (Figure S2, Supporting Information). Thus,
ΔRH ≈ 10 nm suggests formation of a Tf monolayer on the NPs
by preferentially adsorbing with a footprint of 4.2 × 7 nm2, so
that protein molecules are mainly oriented with their 10 nm
extension perpendicular to the NP surface. Assuming an
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Figure 1.  FCS study of Tf adsorption onto fluorescent PS-COOH NPs.
a) Selected autocorrelation curves (normalized to one for comparison),
measured with 0.7 × 10−9 m NPs immersed in PBS with 0 (black), 15.6
(orange), and 125 × 10−6 m (purple) Tf; symbols: data, lines: fits with the
model function.[34] b) Dependence of the hydrodynamic radius on the Tf
concentration of the PBS solution into which the NPs were immersed;
the solid line represents a fit based on Equation (2). Diamonds: Hydrody-
namic radius at 15.6 (orange) and 125 × 10−6 m (purple) Tf before (closed)
and after (open) an 8-fold decrease of the Tf concentration, as indicated
by the arrows. Insets: Depictions of a bare (green sphere) and a fully Tf-
coated PS-COOH NP.
equilibrium process, the midpoint concentration of the adsorp-
tion isotherm, K′D  = 13.0 ± 2.5 × 10−6  m, is a measure of the
binding affinity between Tf molecules and NPs. The Hill coef-
ficient, n  = 0.7 ± 0.1, controls the steepness of the transition;
its magnitude below one may indicate anti-cooperative pro-
tein binding due to competing protein–protein interactions.
The fit with Equation (2) also yields an estimate of the max-
imum number of proteins that can adsorb onto a single NP,
Nmax = 649 ± 46. We note that this number is an upper bound
because we assume that the space associated with the adsorption
layer can be densely packed with proteins. In reality, however,
this is not possible, and there will be gaps filled with solvent
molecules. Alternatively, we can estimate Nmax by dividing the
surface area of the bare NP by the size of the protein footprint,
as given by the protein structure. We note that this number,
Nmax = 560, is somewhat smaller than the previous one for geo-
metrical reasons rooted in the NP surface curvature.
As we will show further below, the excellent agreement of
such data with a binding isotherm (Figure 1b) does not per
se prove that a dynamic equilibrium rules the process.[9,35,36]
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Thus, we checked for reversibility by measuring RH of the
NPs immersed in PBS with specific Tf concentrations before
and after eightfold dilution of the protein.[6,37] RH decreased
after dilution as predicted by the binding curve (Figure 1b),
confirming reversibility of protein binding. For weak binding
with micromolar affinity, we would generally not expect major
structural changes of the proteins upon adsorption. To explore
this issue, we used circular dichroism (CD) spectroscopy to
measure the optical activity of the peptide bond electronic tran-
sitions, which reports on changes of the secondary structure.
The CD spectra of micromolar Tf solutions were identical with
and without NPs, suggesting that significant conformational
changes do not occur (Figure S3a, Supporting Information).
We note that the results obtained with PS-COOH NPs
(RH ≈ 35 nm) are in close agreement with those obtained with
much smaller carboxyl-functionalized NPs (RH ≈ 5 nm).[31,32,38,39]
Thus, we confirmed the presence of a soft monolayer corona
also for larger NPs, in contrast to other studies.[40–43]
2.2. Tf Protein Adsorption onto PS-OSO3H NPs
Next, we embarked on studying Tf adsorption onto large
PS-OSO3H NPs (RH = 50 nm, 0.7 × 10−9 m) in PBS solvent using
FCS (Figure 2a). At some Tf concentrations, e.g., 0.49 × 10−6  m
and 62.5 × 10−6  m, protein adsorption led to slightly increased
correlation times, corresponding to formation of a protein
corona of about 10 nm thickness. At other Tf concentrations,
e.g., 61 × 10−9  m, however, the correlation time τ was more than
30-fold greater than that of bare NPs, implying diffusion of very
large particles with RH  > 1.5 µm. As we will show below, this
enormous size increase is not caused by (multilayer) protein
adsorption onto 50 nm NPs, but rather by formation of large NP
clusters within a certain range of protein concentrations. Under
conditions of agglomeration, the fitted FCS curves do not track
the data well and exhibit undulations at long times (Figure 2a)
due to incomplete sampling of a small number of diffusing
agglomerates in the femtoliter-sized observation volume.
Thus, we turned to DLS for further investigations. The DLS
size distributions in Figure 2b, measured at the same Tf con-
centrations as the FCS data in Figure 2a, confirm that large
particles diffuse in the solution at 61 × 10−9  m Tf. Interestingly,
although the sizes of the NPs at 0.49 and 62.5 × 10−6  m Tf are
identical, the associated ζ-potential distributions are clearly dis-
placed from one another (Figure 2c), which may indicate more
bound Tf molecules at 62.5 × 10−6  m without any change of RH
and, thus, a densification of the protein corona at higher Tf con-
centrations.[35,44,45] In Figure 2d, we have plotted the average RH
values of the NPs (0.7 × 10−9  m) after immersion in PBS with
Tf at concentrations between 60 × 10−12 m and 125 × 10−6  m.
With increasing Tf concentration, there is first a gradual and
then a steep size increase to >1.5  µm. At 0.3 × 10−6  m Tf, the
size drops again steeply and remains close to 60 nm up to the
highest concentrations. Apparently, there is a threshold protein
concentration above which the NP surfaces are completely pas-
sivated against agglomeration.
To test if protein-mediated agglomeration also occurs for NPs
with different surface curvature, we repeated the experiment
with smaller PS-OSO3H NPs (RH  ≈ 18 nm). As for the larger
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Figure 2. Apparent size variation of large PS-OSO3H NPs on Tf
concentration as measured by FCS and DLS. NP concentration:
0.7 × 10−9 m. a) FCS autocorrelation curves (normalized to 1) measured
on NPs immersed in PBS with Tf at four selected concentrations (black,
0; red, 0.061 × 10−6 m; orange, 0.49 × 10−6 m; purple, 62.5 × 10−6 m). Sym-
bols: data, lines: fits according to ref. [34]. The fit at 0.061 × 10−6 m Tf was
restricted to τ < 50 ms. b) Size distributions in PBS (DLS) and c) zeta
potential in 0.1 × PBS; colors as in (a). d) Tf concentration dependence
of the apparent hydrodynamic radius of the NPs in PBS, as determined
by DLS; colors as in (a).
ones, we found agglomeration at intermediate Tf concentrations
(Figure S4a, Supporting Information); however, the average RH
only went up to 70 nm, which is much less than for the larger
particles. At Tf concentrations above the agglomeration region,
the protein layer had a thickness of about 7 nm, suggesting that
Tf binds to the smaller PS-OSO3H NPs in a different orienta-
tion, possibly as a result of the changed NP surface curvature.
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To examine if these effects are only a peculiarity of Tf or
perhaps of general relevance, we also studied HSA adsorption
onto 50 nm PS-OSO3H NPs and found a qualitatively similar
behavior as for Tf, with maximum agglomeration occurring
at about 0.2 × 10−6  m (Figure S4b, Supporting Information),
suggesting that this behavior also occurs with other proteins.
Above the concentration range of agglomeration (4 × 10−6  m),
the HSA corona thickness was 3.9 ± 0.9 nm, compatible with
a monolayer of HSA adsorbing with one of its triangular
faces (Figure S2, Supporting Information).[30] In summary,
at high HSA and Tf concentrations, where the proteins are
expected to be densely grafted to the NP surfaces, we observed
protein corona thicknesses on PS-OSO3H NPs as expected for
monolayers, with the protein molecules arranged in certain
orientations.
2.3. Microscopic Characterization of NP Agglomerates
The FCS and DLS data in Figure 2 gave clear evidence of large
diffusing particles forming upon immersion of PS-OSO3H NPs
in solutions containing Tf in a certain range of concentrations.
Because the NPs are fluorescent, we can unambiguously prove
formation of NP agglomerates by showing an enhanced bright-
ness of these clusters. To this end, we employed confocal optics
microscopy with single-fluorophore sensitivity (for technical
details, see ref. [46]). This method enables us to record time
traces of the fluorescence intensity emanating from the obser-
vation volume (≈1 fL) of the microscope due to fluorescent
particles diffusing through the volume (Figure 3a). These data
were compiled in histograms that depict the number of events
plotted against the number of photon counts within 1 ms time
bins (Figure 3b). The intensity time trace of bare PS-OSO3H
NPs shows photon bursts of up to 150 counts (Figure 3a,b),
representative of individual NPs diffusing through the con-
focal volume. In contrast, the time trace recorded at 61 × 10−9 m
contains only a few very intense bursts, indicating that a small
number of large NP clusters diffuse through the volume, as
is also clearly evident from the corresponding histogram. At
0.49  × 10−6  m Tf, the histogram exhibits a small tail extending
up to 300 counts, revealing that there is a tiny fraction of dimers
in addition to single NPs. The histograms at 0 and 62.5 × 10−6 m
Tf are essentially identical, so we can safely conclude that indi-
vidual NPs are present at the highest Tf concentrations.
To directly visualize the NP clusters, we immobilized PS-
OSO3H NPs sparsely on the surface of a cover glass after
15 min incubation in PBS solutions containing Tf at the speci-
fied concentrations. Subsequently, we acquired images of the
clusters using total internal reflection fluorescence (TIRF)
microscopy (Figure 3c and Figure S5, Supporting Informa-
tion). For quantitative analysis, we identified individual clusters
and integrated the photon counts of all pixels belonging to the
cluster to obtain its overall brightness. Then, we compiled histo-
grams of the number of fluorescent spots versus their bright-
ness (Figure 3d). Bare 50 nm PS-OSO3H NPs had an average
integrated intensity of 47 600 ± 1200 counts (mean ± SD),
as determined from a Gaussian fit. At 61 × 10−9  m Tf, large
clusters were visible, featuring extremely high integrated
intensities. At 0.49 × 10−6 and 62.5 × 10−6  m Tf, the spots again
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Figure 3.  Tf-induced 50 nm PS-OSO3H NP agglomeration, monitored by confocal and TIRF imaging. NPs (0.7 × 10−9 m) were immersed in PBS with
0 (black), 61 × 10−9 m (red), 0.49 × 10−6 m (orange), and 62.5 × 10−6 m (purple) Tf. a) Intensity time traces and b) the corresponding histograms (bin
size 10 counts) of the number of occurrences of counts in the associated interval within 1 ms, calculated from the intensity time traces in panel (a).
c) Exemplary regions of TIRF images of PS-OSO3H NPs, recorded after incubation with Tf for 15 min; scale bar, 10 µm. d) Histograms (bin size
10 000 counts) of the number of occurrences of individual spots with particular brightness (integrated emission), calculated from ten images each
by using the ImageJ software and plotted against the number of camera counts divided by 104. Lines: Gaussian fits. For 61 × 10−9 m Tf, the number of
events has been multiplied by 10.

had average intensities of 48 000 ± 1400 and 48 500 ± 1000
counts, respectively, identical to bare NPs. Thus, the results
from intensity time trace and image analysis are in complete
agreement, proving the existence of large NP agglomerates in a
certain range of Tf concentrations.
2.4. Effect of Electrostatic Repulsion on NP Agglomeration
PS-OSO3H NPs are strongly charged due to their surface decora-
tion with sulfate ions ensuring their colloidal stability (Table 1).
The strength of electrostatic repulsion can be modulated via the
concentration of mobile ions in the solvent.[47] Indeed, in 20-fold
diluted PBS (0.05 × PBS, pH 7.4, ionic strength: 8.1 × 10−3  m),
NP–NP repulsion is so high that agglomeration is absent for all
Tf concentrations (Figure  4a). Instead, after 15 min incubation,
we found a Tf concentration dependence of RH in the form of a
binding isotherm (Equation (2)), with ΔRH = 9.2 ± 0.8 nm, K′D =
2.9 ± 0.7 × 10−6 m and n  = 0.50 ± 0.07. The maximum number
of Tf molecules, Nmax  = 1178 ± 51, is in good agreement with
the ratio of NP surface area to Tf footprint, Nmax = 1070. We did
not find any further size change for incubation times up to 50 h
(Figure S6, Supporting Information). Even if the incubation
chamber was coupled to a large protein reservoir via a dialysis
membrane, thereby maintaining the Tf concentration at 15.3 ×
10−9 m, RH remained constant over the next 50 h (Figure S7, Sup-
porting Information).
In PBS (pH 7.4, ionic strength: 162.9 × 10−3  m), the electro-
static repulsion between bare NPs is still sufficient to keep them
colloidally stable. However, agglomeration becomes noticeable
as soon as there are similar numbers of NPs (0.7 × 10−9  m) and
Tf proteins in the solution (Figure 4a). By using fluorescently
labeled proteins, we verified that the majority of Tf proteins
associated with the NPs at nanomolar concentrations within
15 min (Figure S7, Supporting Information). The pronounced
size increase that is already visible around 1 × 10−9  m Tf
(Figure 4a) cannot be caused by binding of roughly one Tf pro-
tein per NP but clearly proves that even a single protein mole­
cule can act as “glue” for crosslinking two NPs. After mixing
protein and NP solutions, agglomeration will generally be
limited by the decreasing number of free Tf molecules due to
adsorption. However, if we couple the incubation chamber to a
large reservoir via a dialysis membrane to keep the Tf concen-
tration at 1 × 10−9  m, NP clustering continues (Figure S7, Sup-
porting Information).
We also investigated a sample with about half the ionic strength
(71.1  × 10−3  m) of PBS (Figure 4a). Comparison of the three
curves in Figure 4a suggests that the agglomeration-induced

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Figure 4.  Dependence of Tf adsorption to 50 nm PS-OSO3H NPs and Tf-
mediated NP aggregation on ionic strength and NP concentration. The
hydrodynamic radius of the PS-OSO3H NPs is plotted as a function of
the Tf concentration in the solution into which the NPs were immersed.
a) Comparison of three different solvents; black: PBS (ionic strength I =
162.9 × 10−3 m); red: 70 × 10−3 m NaCl, 0.5 × 10−3 m KH2PO4/Na2HPO4
(I = 71.1 × 10−3 m); blue: 0.05 × PBS (I = 8.1 × 10−3 m). Lines represent
binding isotherms, with estimated K′D values of 0.7 × 10−9 m (black),
11  × 10−9 m (red), and 2.9 × 10−6 m (blue). (b) Comparison of two NP
concentrations; open symbols, 0.7 × 10−9 m (same as panel a); filled sym-
bols, 0.088 × 10−9 m. Error bars indicate standard deviations from 3 inde-
pendent measurements.
contributions to RH ride on a sigmoidal binding curve like the
one observed for 0.05 × PBS. Naturally, the estimated midpoint
of the binding curve shifts to higher Tf concentration with
decreasing ionic strength because of the enhanced electrostatic
repulsion between the negatively charged NPs and Tf proteins
(isoelectric point, pI = 5.9). In comparison to the PBS sample,
the onset of aggregation is shifted by a factor of 40 to higher Tf
concentrations at an ionic strength of 71.1 × 10−3  m (Figure 4a).
Two effects likely contribute to this shift, the reduced ten-
dency of Tf-NP binding, as inferred from the shifted sigmoidal
binding curve, and the enhanced NP–NP repulsion. Notably,
the Tf concentration above which agglomeration disappears
remained unchanged with respect to PBS solvent, suggesting
that the NPs are completely passivated against agglomeration
above a certain protein-to-NP stoichiometric ratio.
In Figure 4b, we compare protein concentration-dependent
agglomeration in PBS for two different NP concentrations,
0.7 × 10−9  m and 88 × 10−12 m (8-fold diluted 0.7 × 10−9  m stock
solution). At 88 × 10−12  m, the average RH of the NP clusters
is reduced to about 1.2 µm. Overall, however, both curves look
very similar but are displaced by a factor of eight against each
other, as expected if stoichiometry controls agglomeration. RH
rises already at lower Tf concentration for 88 × 10−12  m NP
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concentration because fewer proteins are needed to crosslink
the smaller number of NPs, and the critical Tf concentration
to completely prevent agglomeration is also found to scale
with the NP concentration (Figure 4b and Figures S8–S11,
Supporting Information).
2.5. Probing Reversibility of Protein Adsorption
onto PS-OSO3H NPs
Reversibility of NP–protein binding can be tested with concen-
tration jump experiments, as shown above for PS-COOH NPs
(Figure 1b). Thus, we immersed PS-OSO3H NPs in PBS con-
taining Tf at concentrations for which we expect huge changes
of RH after a Tf dilution step if protein binding were in dynamic
equilibrium, i.e., 7.6 × 10−9  m, 61 × 10−9  m, and 0.49 × 10−6  m
(Figure 5a). RH, however, did not change at all after diluting the
sample with PBS to achieve an 8-fold lower Tf concentration,
indicating that the NP-bound proteins were attached irrevers-
ibly on the time scale of our experiments.
We further immersed the NPs in PBS with 7.6 × 10−9  m Tf
and then jumped the concentration up to 61 × 10−9 m Tf (at early
times, before further adsorption occurred). The large increase
of RH (Figure 5b, blue points) shows that the added proteins
acted as “glue” for further NP agglomeration. An eightfold con-
centration jump from 61 × 10−9  m to 0.49 × 10−6  m, i.e., to a
concentration at which NPs do not cluster upon immersion,
did not cause any change of RH (Figure 5b, red points). This
finding shows that the NPs are irreversibly agglomerated and
cannot disassemble to form individual NPs with an adsorbed
Tf layer. By using fluorescently labeled Tf molecules, we found
that NP clusters preformed at 61 × 10−9  m Tf bind additional
proteins from 0.49 × 10−6 m Tf solution (Figure S12, Supporting
Information). Immersion into 0.49 × 10−6  m Tf in PBS fully
passivates the NP surface against agglomeration; a subsequent
increase of the Tf concentration to 4 × 10−6 m had no any meas-
urable effect on the NP size (Figure 5b, orange points). Nev-
ertheless, further proteins adsorbed onto the NPs, as we will
show further below.
Next, we performed Tf concentration jump experiments to
examine the nature of Tf binding in 0.05 × PBS (Figure 5c–f).
The corresponding data in Figure 4a have been replotted in
Figure 5c,d with a greatly expanded vertical scale, showing
the excellent agreement with a binding curve according to
Equation (2). A 64-fold decrease of the Tf concentration did not
cause any change of RH (Figure 5c), proving that the proteins
cannot dissociate from the NPs (see Figure 5e for the time evo-
lution). In contrast, a 64-fold increase of the Tf concentration
led to a larger RH, whereas a subsequent concentration jump
down again had no effect on RH (Figure 5d). These experiments
show unambiguously that further proteins can adsorb irrevers-
ibly onto the NPs if the Tf concentration is increased. The RH
change is complete within the 15 min incubation time after
the Tf concentration jump, no further changes are visible up to
50 h (Figure 5f). We also note that, for the jump from 0.98 to
62.5 × 10−6  m, the RH increase was smaller than expected based
on the binding curve (Figure 5d). This result unambiguously
shows that the physical nature of the protein corona depends
on the prior history.
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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neutral particles, depends only on the linear

dimensions, diffusion coefficients, and con-
centrations of the particles (Smoluchowski
equation). For charged particles, Coulomb
forces come into play as well. In our case,
there are repulsive interactions between the
negatively charged Tf and PS-OSO3H NPs;
they reduce the association and increase the
dissociation rate coefficient (see Figure 6 for
a schematic). The structural changes which
render the weakly bound NP–protein com-
plex irreversible mainly concern the proteins
because PS-OSO3H NPs, like many other
NPs, have rather rigid surfaces. By contrast,
proteins are flexible macromolecules that
can thermally fluctuate between many dif-
ferent conformations[50] and readily change
their structures to optimize interactions
with a rigid NP surface, as shown by mole­
cular dynamics simulation approaches.[51] In
contact with the PS-OSO3H NP surface, a
globular protein has the tendency to expose
its hydrophobic moieties, normally hidden
in the protein interior, to the interface for
optimal binding (Figure 6). Indeed, the CD
spectra of Tf adsorbed onto PS-OSO3H NPs
are markedly changed from Tf solution
spectra (Figure S3b,c, Supporting Informa-
tion), proving that major protein structural
changes occur during formation of the hard
corona. In order for these structural changes
Figure 5.  Probing the reversibility of protein corona formation on PS-OSO3H NPs (50 nm, to happen, the weakly associated, initial
0.7 × 10−9 m) by DLS. Hydrodynamic radii measured before (closed symbols) and after 8-fold NP–protein complex has to exist for a time
(open symbols) a) decrease and b) increase of the Tf concentration in PBS (see arrows). Initial long enough that the protein can (partially)
concentrations: 7.6 × 10−9 m (blue), 61 × 10−9 m (red), and 0.49 × 10−6 m (orange). Data are unfold and establish an irreversible interac-
plotted together with those in Figure 4a (black) for comparison. Hydrodynamic radii measured tion. At low protein concentration, only one
before (closed symbols) and after (open symbols) 64-fold c) decrease and d) increase of the or a few proteins weakly adhere to the NP on
protein concentration in 0.05 × PBS, as indicated by the solid arrows. Initial concentrations:
−9 −9 −6
15.3 × 10 m (magenta), 980 × 10 m (green), 62.5 × 10 m (purple). Dashed arrows in (d)
average, and protein–protein interactions are
indicate a 64-fold decrease after the initial 64-fold increase. Data are plotted together with less likely to occur. Thus, each weakly bound
those in Figure 4a (blue) for comparison. e,f) Hydrodynamic radii in 0.05 × PBS, measured as protein molecule unfolds individually, and its
a function of time before (t = 0) and after e) decreasing and f) increasing the Tf concentration linear polypeptide chain spreads across the
64-fold. Error bars indicate standard deviations from at least three independent measurements. surface and covers an area of several times its
Lines mark the average hydrodynamic radii. footprint in the folded state (Figure 6).[20,52–54]
The actual dimension of the footprint
2.6. Mechanistic Aspects of the Formation depends on the relative strength of the protein–surface interac-
of a Hard Protein Corona tion and the internal stability of the protein. In regard to the
binding curve, protein spreading leads to a smaller increase of
The data in Figure 5c,d raise the question as to why the pro- RH than expected from the dimensions of the folded protein
tein concentration dependence of RH in 0.05 × PBS solution and an effectively smaller number of binding sites on the NP
perfectly follows a relationship based on equilibrium binding surface. There is, however, another important issue raised by
(Equation (2)) even though Tf binding is clearly irreversible. the following observations. Even at low (nanomolar) protein
To solve this conundrum, we discuss the processes involved in concentrations, the RH value associated with a particular protein
protein adlayer formation in terms of a molecular-level, mecha- concentration is established within 15 min; no further change
nistic model (Figure  6). In general, protein adsorption can be of RH occurs at a constant (Figure S6, Supporting Information)
modeled as a two-step process, in which an adsorbing protein or decreased (Figure 5e) Tf concentration over the following
and the NP form an initial, weakly bound complex that sub- days, even though there is still plenty of space left on the NP
sequently evolves into a strongly bound complex by means of surface for more proteins to bind. A further size increase due to
structural changes at the interface.[9,48,49] In solution, proteins protein adsorption only takes place after an upward Tf concen-
and NPs diffuse and incessantly collide at a frequency that, for tration jump (Figure 5f). These observations indicate that the

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protein binding sites on the NPs are heterogeneous in strength,
as has been beautifully shown for flat surfaces in single-mole-
cule fluorescence studies by Schwartz and co-workers.[55,56] At
the lowest protein concentration, only a single site on the NP
may bind proteins strongly enough (reversibly!) that it can be
appreciably populated for subsequent conformational changes
and the ensuing irreversible attachment to occur. As the protein
concentration is increased, successively more, but less strongly
binding sites become populated and permanently adsorb pro-
teins, so the binding curve rises continuously (Figure 5c).
Importantly, as more proteins bind reversibly, there is less area
per protein available to spread on the NP surface due to the
presence of neighboring chains (Figure 6). Thus, protein–pro-
tein interactions come into play that can modify the strength
of the binding sites. At the highest concentrations, proteins
become densely grafted onto the NP surface as a monolayer.
In general, the conformational state of the adsorbed proteins
depends on the balance between their internal stabilization
energy on the one hand and the strengths of protein–NP and
protein–protein interactions on the other hand.
Thus, in a nutshell, the apparent “equilibrium binding curve”
results from the fact that the irreversibly adsorbed, hard protein
corona evolves from a weakly bound adsorption layer formed
under equilibrium conditions. The complicating effects dis-
cussed above, heterogeneity of NP–protein interaction strengths,
and protein–protein interactions, can change the width of the sig-
moidal binding curve (Figure 5c,d) from that of a simple Lang-
muir isotherm, as reflected by a Hill coefficient deviating from
one. Single molecule detection-based experiments may yield fur-
ther insight into the dynamics of protein corona formation.[20,57]
Figure 6. Mechanistic scheme of protein adsorption onto charge-
stabilized, hydrophobic NPs. I) Initially, the protein, depicted as an oval
with hydrophobic core (blue) and hydrophilic (red) shell, and the NP,
with hydrophobic surface (blue) and charged functionalized groups (red)
form a weakly and reversibly bound complex with fast protein adsorp-
tion and desorption. II) The protein residing on the NP may change its
conformation and III) partially unfold to expose its hydrophobic core. IV)
Strong hydrophobic interactions establish an irreversible complex. Bound
water (hydration shells), which has to be removed when the NP–protein
interface forms, has not been included in this scheme.
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2.7. Quantitative Analysis of the Number of NP-bound Proteins
In the previous subsection, we showed that 0.7 × 10−9  m
PS-OSO3H NPs (50 nm) were fully protected against agglom-
eration upon immersion in a PBS solution containing Tf above
the critical concentration of 0.49 × 10−6  m. Moreover, RH stayed
essentially constant with increasing Tf concentration above
0.49  × 10−6  m, whereas the ζ-potential decreased (Figure 2c,d),
which suggests further protein binding.[44] This raises the
question if further proteins can indeed adsorb without
changing RH, i.e., by densification of the hard corona.
To quantify the absolute number of NP-bound Tf proteins,
we used dual-color SMLM, a super-resolution imaging method
with nanoscale resolution.[58] It exploits the fact that the position
of an individual fluorophore can be determined much more
precisely that the diffraction-limited width of its optical image.
For imaging, 50 nm PS-OSO3H NPs were immobilized on a
cover glass after 15 min incubation in 0.49 and 62.5 × 10−6 m Tf
solutions containing 1% and 0.2% Tf fluorescently labeled with
Atto647N (Atto647N-Tf), respectively, and subsequent washing
with plain buffer. Notably, the low labeling ratio was chosen
to ensure that only a small number of Atto647N-Tf molecules
resided on the NPs, which is a technical requirement for SMLM.
Green/red overlay images of these surfaces show spots associ-
ated with the green-emitting NPs and red-emitting Atto647N-Tf
molecules; yellow indicates colocalization (Figure  7a,b). From
the images in the green channel, we can extract the positions
of the NPs with nanometric precision (Figure 7c,d). From the
images in the red channel, we can unravel the positions and
positional uncertainties of the Atto647N-Tf molecules within
the protein corona (Figure 7c,d) by analyzing the intensity
time traces of Atto647N-Tf emission (Figure 7e,f, see also the
Experimental Section and Figures S13–S15, Supporting Infor-
mation). The intensity time traces were calculated by collecting
all photons in the red channel from circular regions of 150 nm
radius centered on the NP loci. They show a step-wise decrease
of the emission intensity due to sequential photobleaching of
individual Atto647N dyes (Figure 7e,f); further examples are
given in Figures S16 and S17 (Supporting Information). Most
traces displayed four or less steps, as shown by the histograms
in Figure 7g,h. These histograms agree very well with Poisson
distributions, with 1.75 ± 0.09 and 1.97 ± 0.10 photobleaching
steps on average for NPs exposed to PBS solutions with 0.49
and 62.5 × 10−6  m Tf, respectively. Taking the labeling ratios of
the Tf solutions into account, we conclude that 175 ± 9 (0.49 ×
10−6 m) and 985 ± 50 (62.5 × 10−6 m) Tf molecules were attached
to the NPs on average. The uncertainties are quoted as given by
the non-linear least-squares fitting routine.
Although there was no change in NP size between 0.49 ×
10−6 m (RH  = 57.8 ± 0.9 nm) and 62.5 × 10−6 m (RH  = 57.4 ±
0.1 nm, see Figure S18a, Supporting Information), our SMLM
data clearly show about 5 × more protein molecules adsorbed
onto the NPs in PBS at 62.5 × 10−6 m. This finding is further
supported by the different ζ-potentials at 0.49 and 62.5 × 10−6 m
(Figure 2c, Figure S18b, Supporting Information), indicating
better charge screening at higher Tf concentration due to the
denser protein corona.[44] Interestingly, the Tf concentration
dependence of the ζ-potential of the NPs in 0.05 × PBS is sim-
ilar to the one of RH but shifted by about an order of magnitude
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Figure 7.  Determination of the number of Tf molecules adsorbed onto surface-immobilized PS-OSO3H NPs (50 nm) by single molecule localization
microscopy and step-wise photobleaching of Atto647N-labeled Tf. Data were taken after 15 min immersion of the NPs in PBS with 0.49 (left column)
and 62.5 × 10−6 m (right column) Tf with labeling ratios of 1% and 0.2%, respectively. a,b) Exemplary regions of dual-color images of PS-OSO3H NPs
(green) and Atto647N-Tf (red). Scale bar, 5 µm. c,d) Examples of NP and Tf localizations. Green star: position of the PS-OSO3H NP; red stars: posi-
tions of Atto647N-Tf, determined from the individual localizations marked as gray dots; green spheres indicate the size of the NP. e,f) Exemplary
emission intensity time traces of PS-OSO3H NP showing e) two-step and f) three-step photobleaching. g,h) Histograms of the number of NPs, NNP,
detected with specific numbers of steps, Nstep (error bars: NNP ). Solid squares show Poissonian distributions with the same average Nstep value as
the experimental data.

to smaller Tf concentrations (Figure S18c, Supporting Infor-
mation); a fit with the Hill equation yields K′D = 0.20 ± 0.03 ×
10−6  m and n  = 0.60 ± 0.05. This shift may result from the
concentration-dependent degree of protein structural change
upon adsorption (Figure 6), as discussed in the previous sub-
section. At low Tf concentration, individual polypeptide chains
denature, flatten out and spread over a large area. They can
screen sulfate groups in a larger region on the NP surface com-
pared with folded proteins, so that the effect of Tf binding on
the ζ-potential is amplified with respect to RH, and the change
of the ζ-potential turns on at lower Tf concentrations. Toward
higher Tf concentrations, this amplification is continuously
reduced, as protein–protein interactions lead to confinement
of the Tf chains upon adsorption. A concentration jump from
0.98  × 10−6 to 62.5 × 10−6  m Tf decreased the ζ-potential, but
less so than expected from the binding curve (i.e., upon direct
exposure to 62.5 × 10−6  m Tf); the corresponding behavior was
seen for RH (Figure S18c,d, Supporting Information). Presum-
ably, Tf molecules initially adsorbed and (partially) denatured
at 0.98 × 10−6  m interfere with further binding after jumping
to 62.5 × 10−6  m. This dependence of the ζ-potential and RH on
the preparation history (Figure S18, Supporting Information)
clearly shows the nonequilibrium nature of hard protein corona
formation.
3. Conclusions
There are many studies in the literature reporting protein mul-
tilayer formation on the surface of nanomaterials.[14,40–43,59,60]

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However, in our own work with small NPs (RH  ≈ 5–10 nm),
in which we directly determined the NP size increase due to
protein adsorption as a function of the concentration of pro-
tein solutions[30–32,38,61,62] and blood serum,[35] we ubiquitously
observed only thin coronae compatible with protein mono­
layers. This apparent discrepancy led us to hypothesize that,
perhaps, the NP size and/or surface curvature have an effect on
the physical nature of the protein adlayer. To explore this issue,
we have here investigated the adsorption of two model serum
proteins, Tf and HSA, onto PS NPs (RH  ≈ 18–50 nm) with
two distinctly different types of surfaces. Tf molecules were
found to adsorb onto hydrophilic PS-COOH NPs (RH ≈ 35 nm)
in a reversible manner. The step-wise NP size increase with
increasing Tf concentration indicated formation of a monolayer
of proteins in a specific orientation at saturating concentra-
tions. The analogous experiments with strongly hydrophobic
PS-OSO3H NPs (RH ≈ 50 nm) in 0.05 × PBS showed a similar
binding curve and again monolayer formation at saturation,
although protein binding was completely irreversible. Thus, it
appears that NP size and/or surface curvature do not qualita-
tively change the properties of the protein corona. Taking the
results from all these direct NP sizing experiments together, we
believe that formation of monolayer coronae on NPs is the rule.
Note that globular proteins, especially blood proteins, are colloi-
dally very stable and thus do not tend to agglomerate or adhere
to a protein layer around an NP. For some exceptional proteins,
however, it is known that structural changes as those occurring
upon adsorption can trigger protein–protein association and
fibrillation. Moreover, in the living organism, proteins of the
immune system can further decorate the NPs (opsonization).[39]
So why are there so many studies in the literature reporting
protein multilayer formation on the surface of nanomate-
rials?[14,40–43,59] In some instances, the thickness of the protein
corona is determined in an indirect fashion, and we may specu-
late about the reliability of the method. However, as we have
shown here, even direct sizing methods such as DLS[42,59] may
yield large apparent size increases due to the presence of NP
agglomerates, which can erroneously be interpreted as multi-
layers of adsorbed proteins on individual NPs. Our experiments
with solutions of high ionic strength, notably PBS, a standard
solvent in NP–protein interaction studies, illustrate the effect
of charge screening on NP–protein and NP–NP interactions,
giving rise to protein adsorption-induced NP agglomeration. As
we have shown here, adsorption of only a single Tf molecule per
PS-OSO3H NP can already lead to significant protein-mediated
NP agglomeration. However, if the number of bound proteins
exceeds a certain threshold level, the NP surfaces are com-
pletely passivated against agglomeration, owing to the excellent
colloidal stabilization conveyed by the proteinaceous shell.
Interestingly, the Tf binding curves of PS-COOH NPs and
PS-OSO3H NPs (in 0.05 × PBS) agreed perfectly with a Hill
equation-based sigmoidal model curve, which assumes an equi-
librium process, although Tf adsorption onto PS-OSO3H NPs
was clearly irreversible. Here we have presented a two-step
mechanism of protein adsorption, in which a first equilibrium
binding step is followed by formation of a persistently bound
state via structural changes of the protein. At low protein con-
centration, unfolding and spreading of the polypeptide chain
on the NP surface may give rise to a much enlarged footprint
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and presumably also a volume change due to swelling of the
polypeptide chain. We note that these conformational changes
can introduce uncertainties in quantitative protein corona char-
acterization based on measurements of RH.
To conclude, we have characterized protein corona forma-
tion on PS-NPs using systematic variation of the protein con-
centration and ionic strength of the solvent. We have chosen
Tf and HSA as model proteins because they are often used in
efforts to engineer nanoparticle-based drug carriers.[4] Thus,
we expect that our results and their implications can give guid-
ance to researchers developing NPs for biomedical applications,
where these effects can be exploited to control the properties of
the protein corona by proper choice of the composition (ionic
strength, protein types, and concentration) of the immersion
fluid. To further improve our understanding of the protein
corona, more work will be necessary, including experiments
with more complex biofluids as well as in vivo studies.
4. Experimental Section
Materials: Fluorescent carboxylate-modified and sulfate-modified
polystyrene NPs, PS-COOH NPs (73 nm diameter), and PS-OSO3H NPs
(36 nm diameter), respectively, were obtained from Life Technologies
(Carlsbad, CA). 100 nm diameter PS-OSO3H NPs were purchased
from Sigma-Aldrich (St. Louis, MO). Stock solutions of human Tf and
HSA were prepared in Dulbecco’s PBS (Thermo Fisher Scientific,
Waltham, MA); nondissolved material was removed using Micro Bio-
Spin chromatography columns (Bio-Rad, Hercules, CA). Dilution series
of the proteins in PBS, with concentrations changing by a factor of two
per step, were generated from 1 × 10−3  m stock solutions, covering a
concentration range from 0.06 × 10−9  m to 1 × 10−3  m (i.e., 1 × 10−3  m,
0.5 × 10−3  m, 0.25 × 10−3  m, 125 × 10−6  m, 62.5 × 10−6  m, 31.25 × 10−6  m,
and so on). Concentrations of the Tf and HSA solutions were determined
spectrophotometrically, using extinction coefficients of 8.7 × 104 m−1 cm−1
and 3.5 × 104  m−1 cm−1 at 280 nm, respectively. To decrease the protein
concentration and, inevitably, also the NP concentration in a NP/protein
mixture, the sample was diluted with the appropriate volume of PBS.
To increase the protein concentration, 1–4 µL of a more concentrated
protein solution (chosen to produce the desired concentration jump) was
added to the NP/protein mixture (60 µL) so that the NP concentration
remained essentially constant. All solutions were prepared in LoBind
tubes (Eppendorf, Hamburg, Germany). Using Atto647N-NHS-ester
(ATTO-TEC, Siegen, Germany), dye labeling of Tf was performed as
previously reported.[63] The degree of labeling (dye/protein ratio) was
determined spectrophotometrically as 34.2 ± 0.2%. The solution was
stored at 4 °C in the dark until use.
Optical Spectroscopy: Absorption spectra were collected on a Cary 100
spectrophotometer (Varian, Palo Alto, CA). Fluorescence excitation and
emission spectra were measured on a Fluorolog-3 spectrofluorometer
(HORIBA Jobin Yvon, Edison, NJ). Samples were kept in quartz ultra-
micro-cuvettes (Hellma, Müllheim, Germany) with a path length
of 0.3 cm. CD spectra were taken on a J-815 circular dichroism (CD)
spectropolarimeter (JASCO Germany, Pfungstadt, Germany).
Dual-Focus Fluorescence Correlation Spectroscopy: Dual-focus FCS
measurements were performed on a confocal microscope (Microtime
200, PicoQuant, Berlin, Germany), as previously described.[34] For
the measurements, solutions of PS NPs and proteins were mixed in
Eppendorf LoBind tubes and, after 15 min, introduced into home-built
sample holders, made from two plasma-cleaned cover slips glued
together by two parallel strips of double-sided adhesive tape, leaving a
narrow channel for the solution in the middle. For each sample, intensity
time traces were recorded for 240 s, from which two autocorrelation and
one cross-correlation curves were calculated and fitted globally to obtain
the diffusion coefficient, D.[34]
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DLS and ζ-Potential Measurements: DLS was employed to determine
RH of the NPs in PBS solutions (pH 7.4, 25 °C) using a Zetasizer
Nano-ZS (Malvern Instruments, Malvern, UK) equipped with a 633 nm
He–Ne laser. Solutions of Tf and HSA were mixed thoroughly with equal
volumes of the PS NP solutions (1.4 × 10−9  m unless noted otherwise,
concentration determined by FCS) and incubated for 15 min before the
DLS measurement. In long-time experiments, the NP/protein mixture
was kept in LoBind tubes between measurements. To measure RH of
PS-OSO3H NPs exposed to a large reservoir of protein solution, 200 µL
PS-OSO3H NP solution mixed with 200 µL protein solution was kept in
a dialysis device (Float-A-Lyzer G2, molecular weight cut off 1000 kDa,
Thermo Fisher) and immersed into 50 mL of protein solution between
measurements. The reported, intensity-based RH values of the NPs are
averages over three individually prepared samples. Each sample was
measured six times, with twelve runs each.
For ζ-potential measurements, samples were centrifuged (30 min,
13 000 ×  g, 20 °C) directly after the DLS measurement, and the NP
pellets were resuspended in dilute PBS and measured in a DTS1070
sample cell on the Zetasizer Nano-ZS (Malvern). For each sample,
10 measurements, each consisting of 10–15 runs, were averaged.
TIRF Imaging and SMLM: For these experiments, a home-built
widefield/TIRF microscope with single-molecule sensitivity was used
as described previously.[64] Experimental details are included as the
Supporting Information.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
The authors thank Dr. Peng Gao and Dr. Youhui Lin for valuable
discussions and technical assistance. This work was supported by
the Helmholtz Association, Program ‘Science and Technology of
Nanosystems’ and the Deutsche Forschungsgemeinschaft, ‘Karlsruhe
School of Optics and Photonics’. R.M. acknowledges support by the
China Scholarship Council (CSC) for a Ph.D. scholarship.
Conflict of Interest
The authors declare no conflict of interest.
Keywords
conformational changes, nanoparticle agglomeration, nanoparticles,
protein adsorption, quantitative fluorescence microscopy
Received: February 22, 2019
Revised: April 8, 2019
Published online: April 25, 2019
[1] E. Roduner, Chem. Soc. Rev. 2006, 35, 583.
[2] B. Pelaz, C. Alexiou, R. A. Alvarez-Puebla, F. Alves, A. M. Andrews,
S. Ashraf, L. P. Balogh, L. Ballerini, A. Bestetti, C. Brendel, S. Bosi,
M. Carril, W. C. Chan, C. Chen, X. Chen, X. Chen, Z. Cheng,
D. Cui, J. Du, C. Dullin, A. Escudero, N. Feliu, M. Gao, M. George,
Y. Gogotsi, A. Grunweller, Z. Gu, N. J. Halas, N. Hampp,
R. K. Hartmann, M. C. Hersam, P. Hunziker, J. Jian, X. Jiang,
P. Jungebluth, P. Kadhiresan, K. Kataoka, A. Khademhosseini,
J. Kopecek, N. A. Kotov, H. F. Krug, D. S. Lee, C. M. Lehr,
Small 2019, 15, 1900974 1900974  (11 of 12)
www.small-journal.com
K. W. Leong, X. J. Liang, M. Ling Lim, L. M. Liz-Marzan, X. Ma,
P. Macchiarini, H. Meng, H. Mohwald, P. Mulvaney, A. E. Nel,
S. Nie, P. Nordlander, T. Okano, J. Oliveira, T. H. Park, R. M. Penner,
M. Prato, V. Puntes, V. M. Rotello, A. Samarakoon, R. E. Schaak,
Y. Shen, S. Sjoqvist, A. G. Skirtach, M. G. Soliman, M. M. Stevens,
H. W. Sung, B. Z. Tang, R. Tietze, B. N. Udugama, J. S. VanEpps,
T. Weil, P. S. Weiss, I. Willner, Y. Wu, L. Yang, Z. Yue, Q. Zhang,
Q. Zhang, X. E. Zhang, Y. Zhao, X. Zhou, W. J. Parak, ACS Nano
2017, 11, 2313.
[3] C. D. Walkey, W. C. Chan, Chem. Soc. Rev. 2012, 41, 2780.
[4] Q. Dai, N. Bertleff-Zieschang, J. A. Braunger, M. Bjornmalm,
C. Cortez-Jugo, F. Caruso, Adv. Healthcare Mater. 2018, 7, 1700575.
[5] A. E. Nel, L. Mädler, D. Velegol, T. Xia, E. M. Hoek,
P. Somasundaran, F. Klaessig, V. Castranova, M. Thompson,
Nat. Mater. 2009, 8, 543.
[6] L. Treuel, K. A. Eslahian, D. Docter, T. Lang, R. Zellner, K. Nienhaus,
G. U. Nienhaus, R. H. Stauber, M. Maskos, Phys. Chem. Chem.
Phys. 2014, 16, 15053.
[7] D. Walczyk, F. B. Bombelli, M. P. Monopoli, I. Lynch, K. A. Dawson,
J. Am. Chem. Soc. 2010, 132, 5761.
[8] G. Caracciolo, O. C. Farokhzad, M. Mahmoudi, Trends Biotechnol.
2017, 35, 257.
[9] M. Rabe, D. Verdes, S. Seeger, Adv. Colloid Interface Sci. 2011, 162,
87.
[10] T. Cedervall, I. Lynch, S. Lindman, T. Berggard, E. Thulin,
H. Nilsson, K. A. Dawson, S. Linse, Proc. Natl. Acad. Sci. USA 2007,
104, 2050.
[11] M. P. Monopoli, C. Aberg, A. Salvati, K. A. Dawson, Nat. Nanotechnol.
2012, 7, 779.
[12] E. Polo, M. Collado, B. Pelaz, P. del Pino, ACS Nano 2017, 11, 2397.
[13] M. Mahmoudi, Trends Biotechnol. 2018, 36, 755.
[14] D. Docter, D. Westmeier, M. Markiewicz, S. Stolte, S. K. Knauer,
R. H. Stauber, Chem. Soc. Rev. 2015, 44, 6094.
[15] C. Rodriguez-Quijada, M. Sanchez-Purra, H. de Puig,
K. Hamad-Schifferli, J. Phys. Chem. B 2018, 122, 2827.
[16] R. Cai, C. Chen, Adv. Mater. 2018, 1805740.
[17] E. Casals, T. Pfaller, A. Duschl, G. J. Oostingh, V. Puntes, ACS Nano
2010, 4, 3623.
[18] P. C. Ke, S. Lin, W. J. Parak, T. P. Davis, F. Caruso, ACS Nano 2017,
11, 11773.
[19] C. Carrillo-Carrion, M. Carril, W. J. Parak, Curr. Opin. Biotechnol.
2017, 46, 106.
[20] S. Dominguez-Medina, L. Kisley, L. J. Tauzin, A. Hoggard,
B. Shuang, A. S. D. S. Indrasekara, S. S. Chen, L. Y. Wang,
P. J. Derry, A. Liopo, E. R. Zubarev, C. F. Landes, S. Link, ACS Nano
2016, 10, 2103.
[21] C. C. Fleischer, C. K. Payne, Acc. Chem. Res. 2014, 47, 2651.
[22] P. Satzer, F. Svec, G. Sekot, A. Jungbauer, Eng. Life Sci. 2016, 16, 238.
[23] L. Vroman, Semin. Thromb. Hemostasis 1987, 13, 79.
[24] S. Tenzer, D. Docter, J. Kuharev, A. Musyanovych, V. Fetz, R. Hecht,
F. Schlenk, D. Fischer, K. Kiouptsi, C. Reinhardt, K. Landfester,
H. Schild, M. Maskos, S. K. Knauer, R. H. Stauber, Nat.
Nanotechnol. 2013, 8, 772.
[25] F. Barbero, L. Russo, M. Vitali, J. Piella, I. Salvo, M. L. Borrajo,
M. Busquets-Fite, R. Grandori, N. G. Bastus, E. Casals, V. Puntes,
Semin. Immunol. 2017, 34, 52.
[26] L. Guerrini, R. A. Alvarez-Puebla, N. Pazos-Perez, Materials1154,
2018, 11.
[27] J. S. Gebauer, M. Malissek, S. Simon, S. K. Knauer, M. Maskos,
R. H. Stauber, W. Peukert, L. Treuel, Langmuir 2012, 28, 9673.
[28] S. B. Gunnarsson, K. Bernfur, A. Mikkelsen, T. Cedervall, Nanoscale
2018, 10, 4246.
[29] K. Mohr, M. Sommer, G. Baier, S. Schöttler, P. Okwieka, S. Tenzer,
K. Landfester, V. Mailänder, M. Schmidt, R. G. Meyer, J. Nanomed.
Nanotechnol. 2014, 05, 2.
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
[30] C. Röcker, M. Pötzl, F. Zhang, W. J. Parak, G. U. Nienhaus,
Nat. Nanotechnol. 2009, 4, 577.
[31] X. Jiang, S. Weise, M. Hafner, C. Röcker, F. Zhang, W. J. Parak,
G. U. Nienhaus, J. R. Soc. Interface 2010, 7, Suppl 1, S5.
[32] P. Maffre, K. Nienhaus, F. Amin, W. J. Parak, G. U. Nienhaus,
Beilstein J. Nanotechnol. 2011, 2, 374.
[33] C. Loos, T. Syrovets, A. Musyanovych, V. Mailänder, K. Landfester,
G. U. Nienhaus, T. Simmet, Beilstein J. Nanotechnol. 2014, 5,
2403.
[34] G. U. Nienhaus, P. Maffre, K. Nienhaus, Methods Enzymol. 2013,
519, 115.
[35] H. Wang, L. Shang, P. Maffre, S. Hohmann, F. Kirschhöfer,
G. Brenner-Weiss, G. U. Nienhaus, Small 2016, 12, 5836.
[36] R. A. Latour, J. Biomed. Mater. Res., Part A 2015, 103, 949.
[37] L. Shang, G. U. Nienhaus, Acc. Chem. Res. 2017, 50, 387.
[38] P. Maffre, S. Brandholt, K. Nienhaus, L. Shang, W. J. Parak,
G. U. Nienhaus, Beilstein J. Nanotechnol. 2014, 5, 2036.
[39] H. Wang, Y. Lin, K. Nienhaus, G. U. Nienhaus, Wiley Interdiscip.
Rev.: Nanomed. Nanobiotechnol. 2018, 10, e1500.
[40] S. H. D. Lacerda, J. J. Park, C. Meuse, D. Pristinski, M. L. Becker,
A. Karim, J. F. Douglas, ACS Nano 2010, 4, 365.
[41] S. Milani, F. B. Bombelli, A. S. Pitek, K. A. Dawson, J. Rädler,
ACS Nano 2012, 6, 2532.
[42] J. Piella, N. G. Bastus, V. Puntes, Bioconjugate Chem. 2017, 28, 88.
[43] D. V. Sotnikov, A. N. Berlina, V. S. Ivanov, A. V. Zherdev,
B. B. Dzantiev, Colloids Surf., B 2019, 173, 557.
[44] C. Gräfe, A. Weidner, M. von der Luhe, C. Bergemann,
F. H. Schacher, J. H. Clement, S. Dutz, Int. J. Biochem. Cell Biol.
2016, 75, 196.
[45] M. P. Monopoli, D. Walczyk, A. Campbell, G. Elia, I. Lynch,
F. Baldelli Bombelli, K. A. Dawson, J. Am. Chem. Soc. 2011, 133,
2525.
[46] L. Zemanova, A. Schenk, G. U. Nienhaus, M. J. Valler, R. Heilker,
Drug Discovery Today 2004, 9, 26.
Small 2019, 15, 1900974 1900974  (12 of 12)
www.small-journal.com
[47] T. L. Moore, L. Rodriguez-Lorenzo, V. Hirsch, S. Balog, D. Urban,
C. Jud, B. Rothen-Rutishauser, M. Lattuada, A. Petri-Fink, Chem.
Soc. Rev. 2015, 44, 6287.
[48] M. E. Soderquist, A. G. Walton, J. Colloid Interface Sci. 1980, 75, 386.
[49] E. Casals, T. Pfaller, A. Duschl, G. J. Oostingh, V. F. Puntes, Small
2011, 7, 3479.
[50] G. U. Nienhaus, J. D. Müller, B. H. McMahon, H. Frauenfelder,
Physica D 1997, 107, 297.
[51] M. Ozboyaci, D. B. Kokh, S. Corni, R. C. Wade, Q. Rev. Biophys.
2016, 49, e4.
[52] R. R. Seigel, P. Harder, R. Dahint, M. Grunze, F. Josse, M. Mrksich,
G. M. Whitesides, Anal. Chem. 1997, 69, 3321.
[53] J. J. Ramsden, J. E. Prenosil, J. Phys. Chem. 1994, 98, 5376.
[54] S. Neupane, Y. X. Pan, S. Takalkar, K. Bentz, J. Farmakes, Y. Xu,
B. C. Chen, G. D. Liu, S. Y. Qian, Z. Y. Yang, J. Phys. Chem. C 2017,
121, 1377.
[55] J. S. Weltz, D. K. Schwartz, J. L. Kaar, ACS Nano 2016, 10, 730.
[56] D. Marruecos, D. Schwartz, J. Kaar, Curr. Opin. Colloid Interface Sci.
2018, 38, 45.
[57] S. Pujals, N. Feiner-Gracia, P. Delcanale, I. Voets, L. Albertazzi,
Nat. Rev. Chem. 2019, 3, 68.
[58] K. Nienhaus, G. U. Nienhaus, J. Mol. Biol. 2016, 428, 308.
[59] S. Goy-Lopez, J. Juarez, M. Alatorre-Meda, E. Casals, V. F. Puntes,
P. Taboada, V. Mosquera, Langmuir 2012, 28, 9113.
[60] M. Waghmare, B. Khade, P. Chaudhari, P. Dongre, J. Nanopart. Res.
2018, 20, 185.
[61] Y. Klapper, P. Maffre, L. Shang, K. N. Ekdahl, B. Nilsson, S. Hettler,
M. Dries, D. Gerthsen, G. U. Nienhaus, Nanoscale 2015, 7, 9980.
[62] B. Pelaz, P. del Pino, P. Maffre, R. Hartmann, M. Gallego,
S. Rivera-Fernandez, J. M. de la Fuente, G. U. Nienhaus, W. J. Parak,
ACS Nano 2015, 9, 6996.
[63] L. Shang, P. Gao, H. Wang, R. Popescu, D. Gerthsen,
G. U. Nienhaus, Chem. Sci. 2017, 8, 2396.
[64] Y. Li, L. Shang, G. U. Nienhaus, Nanoscale 2016, 8, 7423.
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim