1. Impacts of Gold Nanoparticle Charge and Ligand Type on Surface Binding and
Toxicity on Gram-Positive Bacteria
Hilena Frew1, Lyle Nyberg1, Ian Gunsolus2, Katie Hurley2, Autumn Qiu2, Kyle Johnson2,
Christy Haynes2, Vivian Feng1
1 Chemistry Department, Augsburg College; 2 Department of Chemistry, University of Minnesota
Conclusions and Outlook
• Bacteria play various vital roles in the ecosystem, including
nutrient cycling and environmental remediation. At the bottom
of the food chain, they also become an important entry point
for nanomaterials to potentially interact with higher-trophic-
level organisms.
• The environmentally relevant gram positive bacterium,
Bacillus subtilis, was chosen for this study.
This bacterium model allows us to :
• Carry out studies parallel to the gram negative
bacterium Shewanella oneidensis
• Illustrate the importance of cell surface structure as well as
NP surface properties in NP-biological interaction.
The 4 nm gold nanoparticles are functionalized with the
following surface chemistries.
Background
MPA-AuNP
(3-mercaptopropionic acid)
MPNH2-AuNPs
(3-mercaptopropylamine)
PAH-AuNPs
(poly(allylamine hydrochloride))
Gram (-)Gram (+)
AuNP Characterizations
• AuNPs were characterized with a variety of methods,
which was critical for optimal interpretation of
nanoparticle/cell interaction.
MPA-AuNP MPNH2-AuNPs PAH-AuNPs
• NSF Center for Chemical Innovation Program: The
Center for Sustainable Nanotechnology (CHE-1240151)
• Lindstrom Fund and Augsburg URGO Office
• Haynes Research lab at University of Minnesota
• Ariane Vartanian, Sam Lohse, Lisa Jacob and Prof.
Cathy Murphy at University of Illinois
• Marco Torelli and Prof. Robert Hamers at University of
Wisconsin
• While major damage to the membranes was observed, there
was no internalization of the AuNPs.
• Bacteria-NP interactions greatly determined by electrostatic
interactions.
• Polyelectrolytic PAH-AuNPs exhibits the highest cell
surface binding, cell damage, and toxicity to both bacteria.
• Differences in cell wall structures results in different
surface binding of NPs.
Bacterial Viability
• Bacillus viability is more impacted by cationic NPs than Shewanella
• PAH-AuNPs are more toxic than MPNH2-AuNPs to both bacteria
• Respirometry: a growth assay to
measure the rate of oxygen uptake to
assess bacterial growth
• Colony counting assays: examine the concentration-dependent toxic
effect of the AuNPs
0 2 0 4 0
0 . 0
0 . 5
1 . 0
T i m e ( h )
NormalizedO2
Uptake
0 2 0 4 0
0 . 0
0 . 5
1 . 0
T i m e ( h )
NormalizedO2
Uptake
Shewanella Bacillus
• Negatively charged MPA-AuNPs have
minimal toxic effect on both bacteria
5 ppm MPA-AuNP
• MPA-AuNP led to minimal cell damage and NP association.
• MPNH2-AuNP resulted in some surface association and cell-
wall damage.
• PAH-AuNP showed an significant cell surface interaction
and cell lysis, and revealed strong interactions with
fragmented outer lipid membranes.
(a, d): 5 µg/mL
MPA-
AuNP
(b, e): 5 µg/mL
MPNH2-AuNP
(c, f): 0.5 µg/mL
PAH-
AuNP
Yellow arrows: lysed
cells
White arrows: NP to cell
surface association
Red arrows: lipid bilayer
structures.
Yellow outline: spilled
cytoplasm
Left: Comparison of
Shewanella and
Bacillus cell-NP
association at 5 µg/mL
AuNPs
Right: Concentration-dependence
of PAH-AuNPs surface association
to Bacillus and Shewanella.
• Drastically different
concentration -dependent
binding profile
• Similar % association led to
different viability for the two
bacteria
• Determining the mechanism of interactions between
Bacillus and PAH-AuNPs will help with design of future
nanomaterials with reduced environmental impact.
• Building model cell wall for gram positive bacteria will
allow us to focus on NP-cell wall interactions
• Mutant strains of Bacillus with defected teichoic acids
will be used to identify the cell surface component
responsible for interacting with NPs.
unstained Bacillus
AuNP-attached Bacillus
SYTO 9-stained Bacillus
AuNP-attached and
SYTO 9-stained Bacillus
Shewanella
Bacillus
Bacillus
Shewanella
Control
With AuNP
Bacillus
Shewanella
TEMFlow Cytometry
Acknowledgements