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Controlled Evaluation of Silver Nanoparticle Dissolution:
Surface Coating, Size and Temperature Effects
Chang Liu
Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in
partial fulfillment of the requirements for the degree of
Doctor of Philosophy
In
Civil Engineering
Peter J. Vikesland, Chair
Linsey C. Marr
Zhen He
Wei Zhou
February 13, 2020
Blacksburg, VA
Keywords: Nanotechnology, silver nanoparticles, nanosphere lithography, dissolution, atomic
force microscopy, surface functionalization, size effects, temperature effects
Controlled Evaluation of Silver Nanoparticle Dissolution: Surface Coating, Size and
Temperature Effects
Chang Liu
ABSTRACT (academic)
The environmental fate and transport of engineered nanomaterials have been broadly
investigated and evaluated in many published studies. Silver nanoparticles (AgNPs)
represent one of the most widely manufactured nanomaterials. They are currently being
incorporated into a wide range of consumer products due to their purported antimicrobial
properties. However, either the AgNPs themselves or dissolved Ag+ ions has a significant
potential for the environmental release. The safety issues for nanoparticles are continuously
being tested because of their potential danger to the environment and human health. Studies
have explored the toxicity of AgNPs to a variety of organisms and have shown such toxicity
is primarily driven by Ag+ ion release. Dissolution of nanoparticles is an important process
that alters their properties and is a critical step in determining their safety. Therefore,
studying nanoparticles' dissolution can help in the current move towards safer design and
application of nanoparticles. This research endeavor sought to acquire comprehensive
kinetic data of AgNP dissolution to aid in the development of quantitative risk assessments
of AgNP fate.
To evaluate the dissolution process in the absence of nanoparticle aggregation, AgNP
arrays were produced on glass substrates using nanosphere lithography (NSL). Changes in
the size and shape of the prepared AgNP arrays were monitored during the dissolution
process by atomic force microscopy (AFM). The dissolution of AgNP is affected by both
internal and external factors. First, surface coating effects were investigated by using three
different coating agents (BSA, PEG1000, and PEG5000). Capping agent effects
nanoparticle transformation rate by blocking reactants from the nanoparticle surface.
Coatings prevented dissolution to different extents due to the various way they were
attached to the AgNP surface. Evidence for the existence of bonds between the coating
agents and the AgNPs was obtained by surface enhanced Raman spectroscopy. Moreover,
to study the size effects on AgNP dissolution, small, medium, and large sized AgNPs were
used. The surrounding medium and temperature were the two variables that were included
in the size effects study. Relationships were established between medium concentration
and dissolution rate for three different sized AgNP samples. By using the Arrhenius
equation to plot the reaction constant vs. reaction temperature, the activation energy of
AgNPs of different sizes were obtained and compared.
Controlled Evaluation of Silver Nanoparticle Dissolution: Surface Coating, Size and
Temperature Effects
Chang Liu
ABSTRACT (general audience)
Nanomaterials, defined as materials with at least one characteristic dimension less than 100
nm, often have useful attributes that are distinct from the bulk material. The novel physical,
chemical, and biological properties enable the promising applications in various
manufacturing industry. Silver nanoparticles (AgNPs) represent one of the most widely
manufactured nanomaterials and has been used as the antimicrobial agent in a wide range
of consumer products. However, either the AgNPs themselves or dissolved Ag+ ions has a
significant potential for the environmental release. The environmental fate and transport of
AgNPs drawn considerable attentions because of the potential danger to environment and
human health. Dissolution of nanoparticles is an important process that alters their
properties and is a critical step in determining their safety. Ag+ ions migrate from the
nanoparticle surface to the bulk solution when an AgNP dissolves. Studying nanoparticles'
dissolution can help in the current move towards safer design and application of
nanoparticles.
This research aimed to acquire comprehensive kinetic data of AgNP dissolution to aid in
the development of quantitative risk assessments of AgNP fate. AgNP arrays were
produced on glass substrates using nanosphere lithography (NSL) and changes in the size
and shape during the dissolution process were monitored by atomic force microscopy
(AFM). First, surface coating effects were investigated by using three different coating
agents. Coatings prevented dissolution to different extents due to the various way they were
attached to the AgNP surface. Moreover, small, medium, and large sized AgNPs were used
to study the size effects on AgNP dissolution. The surrounding medium concentration and
temperature were the two variables that were included in the size effects study.
v
Acknowledgements
First I would like to thank my advisor Dr. Peter J. Vikesland for his help and support during
my study and research at Virginia Tech. He always gives me valuable suggestions and
inspires to me explore more. He has been very supportive during my pregnancy and
encourages me a lot. I feel so honored to have the opportunity to work in his lab.
I also thank all my committee members, Dr. Linsey C. Marr, Dr. Zhen (Jason) He and Dr.
Wei Zhou, for their comments and help with my dissertation work.
I would like to thank the Nanoscale Characterization and Fabrication Laboratory (NCFL)
and Institute for Critical Technology and Applied Science (ICTAS) at Virginia Tech to
support my research. I thank Stephen McCartney and Dr. Chris Winkler for their assistance
with SEM and TEM. I thank Dr. Erich See, Dr. Hans Robinson, Zhixing He and Meitong
Nie for their assistance with producing silver nanoparticle samples.
I would also like to thank the lab members of our group. They are all so considerate and
friendly. Especially, I thank Dr. Weinan Leng for the help with the experiments and all the
suggestions he gives me regarding the research.
I am deeply grateful to my parents. They are always there for me when I have hard times
and I cannot survive without their support. My special thanks are given to my husband, Dr.
Heyang Yuan. I am lucky to have him in my life, to share the journey and create the future
with him.
vi
Table of Contents
List of Figures ................................................................................................................... vii
List of Tables ..................................................................................................................... ix
1. Introduction ..................................................................................................................... 1
References ........................................................................................................................... 3
2. Controlled Evaluation of the Impacts of Surface Coatings on Silver Nanoparticle
Dissolution Rates ................................................................................................................ 6
2.2 Introduction ................................................................................................................... 7
2.3 Materials and Methods .................................................................................................. 9
2.4 Results and Discussion ............................................................................................... 12
References ......................................................................................................................... 19
3. Controlled Dissolution Kinetics Study of Silver Nanoparticle: the Role of Particle Size
........................................................................................................................................... 36
3.1 Abstract ....................................................................................................................... 36
3.3 Materials and Methods ................................................................................................ 37
3.4 Results and Discussion ............................................................................................... 39
References ......................................................................................................................... 46
3. Environmental Implications and Conclusions .............................................................. 57
References ......................................................................................................................... 59
Appendix A: NanoComposites of Bacterial Cellulose and Metal-Organic Frameworks . 60
Appendix B: Real-Time Monitoring of Ligand Exchange Kinetics on Gold Nanoparticle
Surfaces Enabled by Hot Spot-Normalized Surface-Enhanced Raman Scattering .......... 73
vii
List of Figures
Figure 2-1. (a) Schematic of AgNP array production and coating process, (b) AFM image
of original AgNPs, and (c) AgNP height distribution as measured by AFM. The mean
height of 585 particles was 47.1 with a standard deviation of 1.5 nm. ............................. 25
Figure 2-2. AFM images of uncoated and coated AgNPs measured after 1- and 14-days
dissolution. The left panel is the AFM image for the original AgNPs without coating and
dissolution; on the right side (labeled a-h) are the AFM images for uncoated and coated
AgNPs after 1 and 14 days................................................................................................ 26
Figure 2-3. TEM images of (a, b) uncoated AgNPs and (c,d) BSA coated AgNPs after 12h
dissolution in DI water. ..................................................................................................... 27
Figure 2-4. AFM micrographs and height profiles for NSL-produced AgNP arrays after 1-
and 14-days dissolution experiments. ............................................................................... 28
Figure 2-5. AgNP height distribution as measured by AFM of (a) uncoated, (b) PEG1000
coated, (c) PEG5000 coated and (d) BSA coated samples. For each histogram, about 500
particles were measured. ................................................................................................... 29
Figure 2-6. (a) Mean AgNP height at different times and (b) normalized mean AgNP height
at different times. Inset: Dissolution rates calculated by linear regression for the different
coatings. (At least 486 particles were measured for each specimen to calculate the mean
particle height. Error bars represent the standard deviation for mean heights determined
by AFM for experiments performed in triplicate.) ........................................................... 30
Figure 2-7. Schematic illustration of the interactions between the coating agents and the
AgNP surface. ................................................................................................................... 31
Figure 2-8. (a) Change in the mean AgNP height for different surface coatings as a function
of time and (b) Mean AgNP height for BSA coating solutions of different concentration.
(Error bars represent the standard deviation between mean heights determined by AFM for
experiments performed in triplicate.) ................................................................................ 32
Figure 2-9. (a) Raman spectrum for uncoated and coated AgNP samples after 1 day of
exposure and (b) ratio of peak intensity of 235-1 cm to 76-1 cm during two week
dissolution (Error bars represent the standard deviation between mean ratios determined
by Raman for experiments performed in triplicate.) ......................................................... 33
viii
Figure 2-10. Large area Raman map for uncoated and coated AgNPs at day 0 and day 14.
The map were piloted by integrating the area under peak 235 cm-1 with a width of 150 cm-
1. ........................................................................................................................................ 34
Figure 3-1. SEM, AFM images and height distribution of (a, d, g) small, (b, e, h) medium
and (c, f, i) large AgNPs produced by NSL method. ........................................................ 51
Figure 3-2. Normalized mean AgNP height at different times and data fitted by linear
regression: (a) small, (b) medium and (c) large AgNPs. (The different NaCl concentration
are labeled.) ....................................................................................................................... 52
Figure 3-3. (a) Slopes of the regression lines for dissolution rate as a function of NaCl
concentration. Standard errors are indicated by the error bars. (b) Histogram of dissolution
rates for small, medium and large AgNPs in NaCl solutions with various concentrations.
........................................................................................................................................... 53
Figure 3-4. Normalized mean AgNP height at different times and data fitted by linear
regression: (a) small, (b) medium and (c) large AgNPs. (The different experimental
temperatures are labeled.) ................................................................................................. 54
Figure 3-5. liner regression fitted plot of Ln (rate constant) and T−1 at different
experimental temperature. The unit of rate constant is day-1. .......................................... 55
Figure A-1. SEM images of (a) freeze dried bacterial cellulose, (b) ZIF-8 particles, (c, d)
BC-ZIF nanocomposites, (e) BC-AuNP nanocomposite and (f) BC-AuNP-ZIF
nanocomposite. ................................................................................................................. 68
Figure A-2. XRD spectrum of prepared ZIF-8 from a water system................................ 69
Figure A-3. A-3. Removal rate of MGITC and Rh B adsorption by using BC-ZIF
nanocomposites as the adsorbent. ..................................................................................... 70
Figure A-4. EDS Map of BC-AuNP-ZIF nanocomposite. (a) A map of both Au and Zn, (b)
a map of Au and (c) a map of Zn. ..................................................................................... 71
ix
List of Tables
Table 2-1. Corrected radius and particle volume data for uncoated and coated samples
during two weeks dissolution............................................................................................ 35
Table 3-1. Dissolution percentage and reaction constant of small, medium and large
AgNP at different temperatures. ....................................................................................... 56
Table A-1. Element mass percentage of BC-AuNP-ZIF nanocomposite from 3 different
sample spots. ..................................................................................................................... 72
1
1. Introduction
Nanotechnology has had an increasing impact on the modern scientific research in recent
decades. Nanotechnology deals with small objects with sizes in the 1–100 nm range in at
least one dimension. Nanotechnology enables production of materials of various types at
the nanoscale level and many of these nanomaterials exhibit novel physical, chemical, and
biological properties compared to bulk materials.1 Because of the high surface to volume
ratio and quantum effect, the laws of physics of nanomaterials act in a unique way.
Engineered nanomaterials are intentionally produced and designed with physic-chemical
properties appropriate for a specific function.2, 3 The potential applications in consumer
products, medical devices, environmental remediation and nanoelectronics have made
them a promising kind of materials.4-6
Silver nanoparticles (AgNPs) represent one of the most widely manufactured
nanomaterials due to their antibiotic properties which endorse them for various
applications.7 About 30% of consumer products that include engineered nanomaterials
claim to contain AgNPs.7-11 AgNPs can be incorporated into different media and can be
applied in liquid form, in variable colloidal shapes, and also impregnated in solid
materials.1 The size of the silver nanoparticles varies upon the fabrication procedures
employed and typically ranges from 2 nm to several 100 nm. The antimicrobial activities
of silver-containing materials have often been studied in terms of the Ag+ content. The
assumed mechanism of the antimicrobial function is that AgNPs may generate free radicals
and thus trigger cytotoxicity in bacterial cells.12, 13 However, silver nanoparticles have
significant applications in many areas and it is important to study their toxic nature to
understand the risk of using these particles in various applications. The environmental fate
and transport of engineered nanomaterials has been broadly investigated and evaluated in
many published research studies.14-16 The safety issues with nanoparticles are continuously
being tested because of their potential dangers to the environment and human health.
Dissolution of nanoparticles is an important process that alters their properties and is also
a critical step in determining their safety. Therefore, studying nanoparticle dissolution can
help in the current move towards safer design and application of nanoparticles.
2
This research endeavor sought to acquire comprehensive kinetic data of AgNP dissolution
to aid in the development of quantitative risk assessments of AgNP fate. To date, several
analytical techniques have been employed to study nanoparticle dissolution processes
including UV-vis, DLS, TEM and ICP-MS.13, 17-19 However, in these studies the impacts
of particle aggregation are given little attention. Unfortunately, aggregation has many
potential implications on the dissolution process. To evaluate the dissolution process in the
absence of aggregation, AgNP arrays were produced on glass substrates by nanosphere
lithography (NSL) in this project. Changes in the size and shape of the prepared AgNP
arrays were monitored during the dissolution process by atomic force microscopy (AFM).
Nanoparticle dissolution is a dynamic process that is dependent on the particles’ chemical
and surface properties, shape, size, and external factors such as the chemistry of the
surrounding media. 13, 20, 21 In this study we focused on the surface coating and size effects.
Moreover, surrounding medium concentration and temperature are two variables that were
included in the size effects study.
Three chapters follow the introductory chapter. Chapter 2 examines the effects of surface
coating, while PEG and bovine serum albumin (BSA) are used as the coating agents. We
concluded that PEG provides a steric barrier and diminishes the AgNP dissolution rate.
BSA enhance dissolution in the initial phase, but prohibit dissolution in the long term.
Chapter 3 presents the effects of size and temperature on AgNP dissolution. We found that
sodium chloride concentration and temperature have positive effects on AgNP dissolution
rate, while particle size has negative effects. Moreover, the activation energy of larger
AgNPs is higher than for smaller sized particles.
3
References
(1) Devi, G. K.; Suruthi, P.; Veerakumar, R.; Vinoth, S.; Subbaiya, R.; Chozhavendhan,
S., A review on metallic gold and silver nanoparticles. Research Journal of Pharmacy and
Technology 2019, 12, (2), 935-943.
(2) Adeleye, A. S.; Conway, J. R.; Garner, K.; Huang, Y.; Su, Y.; Keller, A. A.,
Engineered nanomaterials for water treatment and remediation: costs, benefits, and
applicability. Chemical Engineering Journal 2016, 286, 640-662.
(3) Babbitt, C. W.; Moore, E. A., Sustainable nanomaterials by design. Nature
Nanotechnology 2018, 13, (8), 621.
(4) Wu, Y.; Pang, H.; Liu, Y.; Wang, X.; Yu, S.; Fu, D.; Chen, J.; Wang, X.,
Environmental remediation of heavy metal ions by novel-nanomaterials: a review.
Environmental Pollution 2019, 246, 608-620.
(5) Haynes, H.; Asmatulu, R., Nanotechnology safety in the aerospace industry. In
Nanotechnology Safety 2013, 85-97.
(6) Habibi, N.; Kamaly, N.; Memic, A.; Shafiee, H., Self-assembled peptide-based
nanostructures: smart nanomaterials toward targeted drug delivery. Nano Today 2016, 11,
(1), 41-60.
(7) Tolaymat, T. M.; El Badawy, A. M.; Genaidy, A.; Scheckel, K. G.; Luxton, T. P.;
Suidan, M., An evidence-based environmental perspective of manufactured silver
nanoparticle in syntheses and applications: a systematic review and critical appraisal of
peer-reviewed scientific papers. Science of the Total Environment 2010, 408, (5), 999-1006.
(8) Graf, C.; Nordmeyer, D.; Sengstock, C.; Ahlberg, S.; Diendorf, J. r.; Raabe, J. r.;
Epple, M.; Koller, M.; Lademann, J. r.; Vogt, A., Shape-Dependent dissolution and cellular
uptake of silver nanoparticles. Langmuir 2018, 34, (4), 1506-1519.
(9) Marambio-Jones, C.; Hoek, E. M., A review of the antibacterial effects of silver
nanomaterials and potential implications for human health and the environment. Journal
of Nanoparticle Research 2010, 12, (5), 1531-1551.
(10) Rai, M.; Yadav, A.; Gade, A., Silver nanoparticles as a new generation of
antimicrobials. Biotechnology Advances 2009, 27, (1), 76-83.
4
(11) Yoksan, R.; Chirachanchai, S., Silver nanoparticle-loaded chitosan–starch based
films: fabrication and evaluation of tensile, barrier and antimicrobial properties. Materials
Science and Engineering: C 2010, 30, (6), 891-897.
(12) Le Ouay, B.; Stellacci, F., Antibacterial activity of silver nanoparticles: a surface
science insight. Nano Today 2015, 10, (3), 339-354.
(13) Misra, S. K.; Dybowska, A.; Berhanu, D.; Luoma, S. N.; Valsami-Jones, E., The
complexity of nanoparticle dissolution and its importance in nanotoxicological studies.
Science of the Total Environment 2012, 438, 225-232.
(14) Dale, A. L.; Casman, E. A.; Lowry, G. V.; Lead, J. R.; Viparelli, E.; Baalousha, M.,
Modeling nanomaterial environmental fate in aquatic systems. Environmental Science &
Technology 2015, 2587-2593.
(15) Li, X.; Lenhart, J. J.; Walker, H. W., Aggregation kinetics and dissolution of coated
silver nanoparticles. Langmuir 2011, 28, (2), 1095-1104.
(16) Echegoyen, Y.; Nerín, C., Nanoparticle release from nano-silver antimicrobial food
containers. Food and Chemical Toxicology 2013, 62, 16-22.
(17) Zook, J. M.; Long, S. E.; Cleveland, D.; Geronimo, C. L. A.; MacCuspie, R. I.,
Measuring silver nanoparticle dissolution in complex biological and environmental
matrices using UV–visible absorbance. Analytical and Bioanalytical Chemistry 2011, 401,
(6), 1993-2002.
(18) Ma, R.; Levard, C.; Marinakos, S. M.; Cheng, Y.; Liu, J.; Michel, F. M.; Brown Jr,
G. E.; Lowry, G. V., Size-controlled dissolution of organic-coated silver nanoparticles.
Environmental Science & Technology 2011, 46, (2), 752-759.
(19) Mitrano, D. M.; Barber, A.; Bednar, A.; Westerhoff, P.; Higgins, C. P.; Ranville, J.
F., Silver nanoparticle characterization using single particle ICP-MS (SP-ICP-MS) and
asymmetrical flow field flow fractionation ICP-MS (AF4-ICP-MS). Journal of Analytical
Atomic Spectrometry 2012, 27, (7), 1131-1142.
(20) Dahle, J. T.; Livi, K.; Arai, Y., Effects of pH and phosphate on CeO2 nanoparticle
dissolution. Chemosphere 2015, 119, 1365-1371.
(21) Ostermeyer, A.-K.; Kostigen Mumuper, C.; Semprini, L.; Radniecki, T., Influence
of bovine serum albumin and alginate on silver nanoparticle dissolution and toxicity to
5
nitrosomonas europaea. Environmental Science & Technology 2013, 47, (24), 14403-
14410.
6
2. Controlled Evaluation of the Impacts of Surface Coatings on Silver Nanoparticle
Dissolution Rates
Chang Liu, Weinan Leng and Peter J. Vikesland*
Department of Civil and Environmental Engineering, Institute of Critical Technology and
Applied Science (ICTAS), and the Center for the Environmental Implications of
Nanotechnology (CEINT), Virginia Tech, 418 Durham Hall, Blacksburg, Virginia,
24061-0246, United States
*Corresponding author. Phone: (540) 231-3568, Email: [email protected]
(This chapter has been published in Environmental science & technology.– Adapted with
permission from Chang Liu et al., Environmental science & technology, 20181. Copy right
(2018) American Chemical Society.)
2.1 Abstract
Silver nanoparticles (AgNPs) are increasingly being incorporated into a range of consumer
products and as such there is significant potential for the environmental release of either
the AgNPs themselves or Ag+ ions. When AgNPs are exposed to environmental systems,
the engineered surface coating can potentially be displaced or covered by naturally
abundant macromolecules. These capping agents, either engineered or incidental,
potentially block reactants from surface sites and can alter nanoparticle transformation
rates. We studied how surface functionalization affects the dissolution of uniform arrays of
AgNPs fabricated by nanosphere lithography (NSL). Bovine serum albumin (BSA) and
two molecular weights of thiolated polyethylene glycol (PEG; 1000 Da and 5000 Da) were
tested as model capping agents. Dissolution experiments were conducted in air-saturated
phosphate buffer containing 550 mM NaCl. Tapping-mode atomic force microscope
(AFM) was used to measure changes in AgNP height over time. The measured dissolution
rate for unfunctionalized AgNPs was 1.69 ± 0.23 nm/d, while the dissolution rates for BSA,
PEG1000, and PEG5000 functionalized samples were 0.39 ± 0.05, 0.20 ± 0.10, and 0.14 ±
0.07 nm/d, respectively. PEG provides a steric barrier restricting mass transfer of reactants
7
to sites on the AgNP surface and thus diminishes the dissolution rate. The effects of BSA,
on the other hand, are more complicated with BSA initially enhancing dissolution, but
providing protection against dissolution over extended time.
2.2 Introduction
Silver nanoparticles (AgNPs) represent one of the most widely used nanomaterials in
commercial and medical products.2-5 A wide range of consumer products such as textiles,
food containers, cosmetics, and medical devices employ AgNPs as antimicrobial agents.6-
8 Unfortunately, this antimicrobial property has the potential to elicit nanotoxicity when
the AgNPs enter the environment.7, 8 AgNP containing products have been shown to release
AgNPs after washing or through direct use and it is expected that the input of AgNPs into
aquatic systems will increase in the coming decades.9 Elevated human and environmental
AgNP exposures raise concerns about potential environmental implications.10-12 Recent
studies have explored the toxicity of AgNPs to a variety of organisms such as plants, algae,
fungi, microorganisms, and human cells.13-15 The negative impacts of AgNPs on the
environment and potentially humans may be long lasting and have been recently
reviewed.12, 16, 17 While all of the mechanisms by which AgNPs elicit a toxic effect remain
unclear,5, 18, 19 it is generally considered that the toxicity of AgNPs is at least partly driven
by Ag+ ion release.17 Even if Ag+ release is only one of many pathways by which AgNPs
elicit toxicity, dissolution remains an important process that alters nanoparticle properties
and is thus a critical aspect of AgNP safety.
Ag+ ions migrate from the nanoparticle surface to the bulk solution when an AgNP
dissolves.20 This dynamic process is dependent on the particles’ chemical and surface
properties, shape, size, and external factors such as the chemistry of the surrounding
media.18 Surface coatings, formed by covering the surface with capping agents, can alter
the dissolution rate. The implications of surface coating on nanoparticle reactivity are
dependent on the identity of the surface coating and the means by which it is attached to
the particle surface.18, 21, 22 When AgNPs enter the environment, pre-engineered surface
layers may be displaced or covered by proteins or other naturally abundant
macromolecules.18, 23 Surface coatings are expected to affect the reactivity of AgNPs in
8
several ways. First, by coordinating with surface atoms, coatings may effectively block
reactants from reaching surface sites and thus slow reaction rates.24 Alternatively, if
organic molecules bind to the metal surface through nucleophilic functional groups, then
they may accelerate oxidation and dissolution.25, 26 It has been hypothesized that reactive
oxygen species formed during AgNP oxidation may be scavenged by organic coatings that
slow the dissolution process by preventing these reactive agents from further oxidizing the
metal surface.24, 27 Finally, surface functionality generally dictates AgNP surface charge,
which in turn affects the local ionic environment near the particle surface and thus may
alter reaction rates. 18
Previous studies have used polyvinylpyrrolidone (PVP)28, 29 and citrate23, 30 as coating
agents and found that these capping agents affect AgNP dissolution. Zong et al.
demonstrated the antimicrobial activity of polyethylene glycol (PEG)-thiol and PVP coated
AgNPs, and observed that smaller PEG-coated particles dissolved faster than larger PEG-
coated particles.31 Li et al. determined that AgNP dissolution was inhibited by coatings of
sodium dodecyl sulfate (SDS) or Tween 80, but not by the initial citrate coating.32
Ostermeyer studied the influence of bovine serum albumin (BSA) and alginate coatings
and found that while BSA prevented NH3-induced dissolution that alginate only weakly
interacted with the AgNP surface and was unable to completely prevent NH3-induced
dissolution.33 To date, several analytical techniques have been employed to study
nanoparticle dissolution processes including UV-vis, DLS, TEM and ICP-MS.18, 22, 34, 35
However, in most of these studies the impacts of particle aggregation were given little
attention. Some prior studies have shown that aggregation increases particle size, and
preserves most of the surface area within the aggregate. Following aggregation the exposed
surface area of the AgNPs is reduced and this decreases the dissolution rate. Thus, it is
important to utilize methods to evaluate the dissolution process in the absence of
aggregation.
Nanosphere lithography (NSL) has been used as a simple and cost-effective technique to
produce metal nanoparticle arrays of controlled shape and size.36, 37 These uniform arrays
of nanoparticles enable controlled evaluation of nanoparticle transformations in the
absence of aggregation.38 In our previous studies, the dissolution and sulfidation of NSL-
produced AgNPs were investigated by atomic force microscopy (AFM). Both shape and
9
height changes were discussed in detail.38, 39 In this contribution, we extend our prior work
and report on the controlled evaluation of how surface coatings affect AgNP dissolution.
NSL was used to produce AgNPs immobilized on glass substrates and then the particles
were functionalized with two different capping agents. BSA and PEG-thiol were chosen as
coating agents due to their favorable binding to the AgNP surface, but differential
interactions with the AgNP surface. BSA is commonly used as a model protein in studies
of nanoparticle-protein interactions.33, 40 Proteins are well known to form “coronas” around
AgNPs in biological media,41, 42 which makes them a highly important class of surface
coating from a toxicological point of view.40 Two different molecular weights of PEG were
chosen to evaluate how the molecular weight of a coating agent affects dissolution.
Specifically, PEG-thiols with molecular weights of 1000 Da and 5000 Da were tested. It is
reported that heavy metals associate with proteins by interacting with thiol groups in
cysteine and acetylcysteine, so this chemical interaction enhances the connection between
a given coating agent and the AgNPs.43, 44 AFM was used to study changes in the
morphology of AgNPs, transmission electron microscopy (TEM) was employed to
investigate crystal structure, and surface enhanced Raman spectroscopy (SERS) was used
to evaluate metal-sulfur interactions.
2.3 Materials and Methods
Materials
Glass coverslips (60 × 24 × 0.15 mm) were purchased from Fisher Scientific. (3-
Mercaptopropyl)-trimethoxysilane and BSA were provided by Sigma-Aldrich. PEG-thiols
(PEG-1000: MW = 1000 Da; PEG-5000: MW = 5000 Da.) were purchased from Nano CS,
Inc. Methanol was purchased from Alfa Aesar. Carboxylated polystyrene spheres were
acquired from Life Technologies. All reagents were analytic purity and were used without
further purification. Deionized (DI) water (>18.2 MΩ-cm) was produced by a Barnstead
water purification system and was used throughout this study. Stainless steel specimen
discs for AFM measurements were purchased from Ted Pella and antimony doped silicon
TESPA-V2 AFM probes were purchased from Bruker.
10
Substrate Production
Glass substrates were cleaned by sequential immersion in RCA1 solution (1 NH4OH:4
H2O2:20 H2O, v:v:v) and then in RCA2 solution (1 HCl:1 H2O2:5 H2O) at 75 °C for 10 min
each.45 The substrates were rinsed with DI water after each cleaning step and then air dried.
To enhance adhesion between deposited silver and the glass, the substrate was thiolated by
immersion in 5% (3-mercaptopropyl)-trimethoxysilane in methanol for 12 h.46 Following
thiolation, glass substrates were rinsed with DI water and stored in methanol until use.
Negatively charged carboxylated polystyrene microspheres with a diameter of 450 nm
were deposited onto cleaned substrates by convective self-assembly (CSA).36 Specifically,
the substrates were held horizontally on a motion stage (Thor Laboratories) below an
angled plate in an airtight container. The space between the substrate and the angled plate
was then set to ≈600 nm. A 4 L aliquot of polystyrene suspension (10% w/v) was placed
between the interspace and the substrate and was then moved at a constant velocity of 0.05
cm/s for 12 cm. The colloidal suspension spread over the substrate and a monolayer of
close-packed spheres formed due to solvent evaporation. Following CSA, electron beam
evaporation (3-kW electron gun, Thermionics) was used to deposit a 45 nm thick layer of
silver metal onto the prepared substrates. Substrates were cut into approximate squares of
≈5 mm2 and the spheres were removed using tape. AgNPs immobilized on glass substrates
were sequentially rinsed with ethanol and DI water for 30 s each.
Coating Treatment
Coating solutions were prepared by dissolving PEG-thiol or BSA in DI water. For BSA,
three different concentrations were prepared with weight: weight ratios of 0.1, 0.5, and 1%.
Coating solutions were transferred to petri dishes and five prepared substrates were
immersed in each solution. The petri dishes were then sealed with sealing film and stored
in the dark for a 12 h coating period. Control experiments determined that this period was
sufficient for complete surface functionalization. The substrates were then rinsed with DI
water and air dried prior to storage in a desiccator.
Nanoparticle Dissolution Experiments
The effects of coating agent identity on AgNP dissolution were evaluated by immersing
prepared substrates in phosphate buffered (1 mM NaH2PO4; 1 mM Na2HPO4) 550 mM
11
NaCl solution with the final pH adjusted to 7.0 ± 0.1 via 0.1 M NaOH addition. 550 mM
is characteristic concentration of seawater and other highly saline solutions.47 Following
the coating treatment the prepared AgNP samples were submerged in 10-mL of NaCl
solution in petri dishes and sealed with Parafilm. The subsequent dissolution experiments
were conducted at room temperature (25 °C) in the dark. To quantify AgNP dissolution
rates, each substrate was removed from solution and dried under N2 after a defined reaction
period. For each reaction time, one specimen was used and then disposed of following an
AFM measurement. To study the effects of coating identity on AgNP dissolution, the
reaction period was set as 0, 1, 2, 4, 7, 10, and 14 days for each coating type. To investigate
how the coating process itself affects dissolution, the coating experiments were conducted
for 0, 1, 2, 4, 7 and 12 h for each coating agent. All AFM images were measured just after
removing the specimen from the reaction solution. The schematic of AgNP array
production and coating process are shown in Figure 2-1a.
Analytical Techniques
Samples were attached to 15 mm stainless steel specimen discs with wax and AFM height
measurements were obtained using a Nanoscope IIIa Multimode AFM (Veeco) equipped
with a J scanner. Antimony doped silicon TESPA-V2 AFM probes were used. The AFM
was operated in tapping mode with a resonant frequency of 260-450 kHz. All images were
acquired at 256 × 256 pixel resolution and a scan rate of 0.5 Hz. For each specimen, 3-5
images were collected at different locations with the scan area of each image set at 5×5
µm2. As discussed elsewhere,38 some parts of the specimen exhibited irregular
morphologies due to defects in the colloid layer, and these portions of the surface were
excluded from measurement. A minimum of 486 particles were measured for each
specimen to calculate the mean particle height. The “Flatten” and “Erase Scan Lines” tools
of the NanoScope software were used to modify the collected images by correcting the
baseline and removing spurious scan lines. The “Particle Analyze” tool was employed to
measure the height of the particles and defects were excluded.
Specimens for TEM measurements were attached to 0.5 mm Ni aperture grids using Loctite
epoxy. An Allied High Tech Multiprep automated polishing system was used to thin the
samples to 5-10 µm. Several diamond lapping films with different particle sizes were used
12
as grinding media. Then the specimens were polished using a Fischione model 1010 ion
mill with an accelerating voltage of 3.5 kV and a beam current of 5 mA. TEM images
were obtained using a JEOL 2100 field thermionic emission TEM. Raman measurements
were obtained on a WITec alpha500R Raman spectrometer using a 785 nm excitation laser.
Raman spectra were collected in a 20 µm × 20 µm image scan using a 100× microscope
objective (N.A. =0.9, Manufacturer: Olympus, model: UIS2 FN26.5) and a laser intensity
of 1.0 mW. The reported spectra were obtained by averaging 2500 scans (integration time
= 30 ms) acquired across the sample area.
2.4 Results and Discussion
Silver was deposited over the carboxylated latex sphere mask by electron beam evaporation
to form AgNP arrays on the glass slides. The typical topography of a NSL-prepared AgNP
array was determined by AFM. As shown in Figure 2-1, defect free domains spanned
several μm2. AFM images revealed that the as produced AgNPs exhibited a truncated
tetrahedral shape (Figure 2-1b), as expected.37, 48 The initial nanoparticle height was
normally distributed with a mean value of 47.1 nm and a standard deviation (SD) of 1.5
nm (Figure 2-1c). Fourteen day dissolution experiments were conducted in air-saturated
phosphate buffer (pH 7.0, 25 °C) containing 550 mM NaCl. The images in Figure 2-2
illustrate how AgNP morphology changes between day 0 (prior to addition of any coating)
and after coating on day 1 and 14. For the uncoated AgNPs, the shape of the AgNPs
changed from triangular to circular after only one day of immersion in NaCl solution
(Figure 2-2a). This phenomenon was not observed for coated AgNP samples (Figures 2-
2b-d). After two weeks, the size of the uncoated AgNPs decreased substantially and there
was obvious loss of individual AgNPs from the glass substrate (Figure 2-2e). In contrast,
the PEG coated AgNPs exhibited no obvious changes in size (Figures 2-2f and 2-2g), while
there was a slight decrease in size for the BSA coated AgNPs (Figure 2-2h). In addition,
TEM measurements were conducted to investigate changes in AgNP morphology during
surface treatment. As shown in Figure 2-3, the uncoated AgNPs become more circular with
and many small pieces of substrate scattered around the initial AgNPs. BSA coated AgNPs
maintain the triangular shape after 12 h surface treatment and the background is very clear
13
with no AgNP fragments. These phenomena are in accordance with the results from the
AFM measurements.
AFM provides a convenient technique to measure the kinetics of AgNP dissolution. The
“Particle Analyze” tool was employed to measure changes in particle height while
excluding defects. Over 450 AgNPs were measured for each specimen to calculate the
mean particle height. Changes in the height of the AgNPs were accurately tracked and the
implications of nanoparticle aggregation were averted. Measured height profiles are shown
in Figure 2-4. Changes in shape are clearly observed in the high magnification AFM
images. The height distribution of each coated and uncoated sample are shown in Figure
2-5. The height of the blank AgNPs was normally distributed which illustrates the
uniformity of the AgNP arrays. Similarly, we observed a uniform distribution for all of the
samples after coating.
The mean height of the original, uncoated AgNPs was 47.1 ± 1.5 nm, but this value
increased to 55.4 ± 1.2 nm after one day reaction with a change in nanoparticle shape. This
growth was only observed for uncoated AgNPs. Previous work by our group has shown
that the AgNP height increases by 6-12 nm during an initial exposure period to solutions
with NaCl >10 mM and that this growth occurs with dissolution at the corners and a
concomitant steepening of the sidewalls.38 Ag0 is oxidized to Ag+ at the bottom edges and
corners of the AgNPs (the anode), while reduction reaction occurs at the top (the
cathode).38, 49, 50 A net flow of silver from the bottom of a nanoparticle to the top is
generated due to the Ag+ concentration gradient until the internal redox gradient is
eliminated. After the initial increase, the mean height of the uncoated AgNPs gradually
decreased to ≈30 nm during the two-week reaction period. Our previous work quantified
dissolved Ag+ in solution using ICP-MS, but with characteristic low Ag+ recoveries.
Furthermore, the measured Ag+ values underestimated the values predicted by the AFM
measurements by up to 40%.38 For these reasons, we did not attempt to conduct parallel
ICP-MS measurements in this work.
The AgNPs with a PEG1000 coating exhibited a mean height that varied between 47.0 ±
0.4 nm and 49.4 ± 0.3 nm during the two-week experimental period. The mean height of
the PEG5000 functionalized sample was 46.3 ± 1.2 nm after the initial coating treatment
14
and the height then fluctuated between 44.3 and 46.0 nm (Figure 2-6). The slight, and
statistically indistinguishable, difference between PEG1000 and PEG5000 may reflect
differences in the initial substrates and we do not attribute this difference to differential
dissolution. If polymer coatings provide a steric barrier to the mass transfer of reactants to
sites on the AgNP surface, then longer polymer chains might have been expected to inhibit
AgNP dissolution to a greater degree than shorter polymers. However, this hypothesis was
not supported by the present data. One possible explanation is that PEG1000 covered the
surface of the AgNPs equally as well as PEG5000. As such, no difference was observed
for these two PEGs with different chain length. The mean height change was very slight
for PEG coated samples and no obvious dissolution was observed. If we were to extend the
reaction period it is possible there would be greater differences. It is notable that there was
a difference in the initial mean heights of the differentially coated samples, (i.e. the initial
mean height of the BSA coated sample was 42.1 ± 0.4 nm which is lower than those of the
PEG coated samples). As discussed later, one possible reason for the difference is that the
coating process has an effect on the height change. Such a hypothesis is discussed vida
infra.
AgNP dissolution is typically modeled using first-order reaction kinetics;11, 22, 27, 51
however, solid-state reactions are dominated by interfacial interactions. Accordingly, the
dissolution process may be more appropriately modeled by assuming that the reaction rate
is proportional to the remaining surface area rather than the remaining mass of solid. If the
solid material is assumed to be a sphere, the rate of change of the particle’s radius is the
linear dissolution rate. This model, generally referred to as the contracting sphere rate
law,11, 38 predicts that the particle’s radius will decrease at a constant rate. Because our
methodology measures the mean height of dissolving AgNPs over time, the dissolution
rate can be determined directly as the slope of a simple linear regression of the AFM time
series data. The applicability of the contracting sphere model was supported by the constant
linear dissolution rates observed herein (Figure 2-6b). By fitting the normalized data with
a linear regression, the dissolution rate constants (k; nm/d) for both the uncoated and coated
agents were determined. The dissolution rate for uncoated AgNPs was 1.69 ± 0.23 nm/d
with a relatively strong correlation (R2 = 0.913). This result is comparable to that obtained
previously (=2.2 nm/d) under similar conditions.38 Following coating with BSA, the
15
dissolution rate decreased to 0.39 ± 0.05 nm/d which suggests the coating layer inhibits
AgNP dissolution. PEG coated samples exhibited a dissolution rate of 0.20 ± 0.10 nm/d
for PEG1000, while it was only 0.14 ± 0.07 nm/d for PEG 5000. The R2 values for these
two agents were <0.5 and the 95% confidence intervals are wide due to the fluctuation in
nanoparticle heights and the fact that no obvious dissolution was observed. To convert the
dissolution rate to a volume percentage, the calculated volumes were investigated. Our
previous work has established a mathematical description of the corroded particle shape.38
The average particle volumes were calculate by using the mean height and corrected mean
radius data. For uncoated AgNPs, obvious shape changes were observed and the radius
decreased from 64.2 nm to 50.0 nm after 14 days. During this period an average ≈61.3%
of the volume of given AgNP dissolved. BSA coated samples dissolved by 15.1% which
is much lower than the uncoated AgNPs. While the triangular shape was maintained to a
large extent, the size changed a little bit which indicated some dissolution. PEG coated
groups protected the samples very well and resulted in little dissolution (3.38% and 5.46%
for PEG-1000 and PEG-5000 coated samples, respectively). SI Table 1The percent
dissolution rates, which were obtained based on the calculated volumes, showed similar
dissolution trend as the dissolution rate in terms of height change. Detailed information on
the calculated volume percentage dissolution may be found in the SI.
Coatings will affect dissolution in the following ways: 1) minimize accessibility or
reactants, 2) engage in nucleophilic dissolution, 3) react with surface derived reactive
oxygen species (ROS), and 4) alter surface charge.18, 21 Of these possibilities we explicitly
tested #1 and #2. Herein, surface coating was employed to improve AgNP chemical
stability. Surface chemistry dependent solubility has been studied extensively for AgNPs,
wherein it is shown that capping agents can considerably alter the dissolution of AgNPs.
Based on the significantly different dissolution rates for the uncoated and coated AgNPs,
we are able to conclude that the presence of an organic surface coating diminishes the
AgNP dissolution rate. BSA irreversibly adsorbs as a monolayer on silver in a side-on
confirmation.52 Assuming the ellipsoidal dimension of BSA (14 nm × 4 nm × 4 nm), 52
each BSA molecule occupies a 16-56 nm2 area on the AgNP surface (see the SI for
calculation details). Under our coating conditions, the AgNP surfaces were saturated by
BSA. The predicted area of each PEG-thiol molecule was 0.22 nm2, 53 and thus the AgNP
16
surfaces were also saturated by PEG-thiol. A key difference between the BSA and PEG-
thiol coating agents is the mechanism by which they interact with the AgNP surface. PEG-
thiol molecules covalently attach to the AgNP through their terminal thiol groups (as shown
in Figure 2-7a), with the long PEG chain extending away from the surface, thus resulting
in a self-assembled monolayer.53 The fact that there was no observed difference in the
dissolution rates for PEG-1000 and PEG-5000 suggests that chain length has minimal
impact on dissolution, at least under our test conditions. In contrast, BSA, while having a
larger footprint, is not expected to as strongly interact with the AgNP surface. BSA has 17
interchain disulfide bonds formed by 34 oxidized cysteines and one free sulfhydryl group
in a reduced cysteine.54 The free sulfhydryl groups are distributed within the BSA molecule
and thus there are expected to be points of greater and lesser contact between BSA and the
surface (Figure 2-7b). The regions of the AgNP surface not directly functionalized by BSA
can be expected to be more unprotected and thus the BSA coated surfaces dissolve more
rapidly than the PEG coated samples.
Because the mean height of the coated samples exhibited some differences immediately
following the coating treatment, we conducted an additional experiment in which we
coated the nanoparticles for different periods of time to investigate how height changes
during the coating process. As shown in Figure 2-8a, the mean height for uncoated AgNPs
increased gradually from 47.8 ± 0.6 nm to 54.0 ± 0.8 nm during this 12 h experiment. In
contrast, the mean height for coated samples decreased to varying extents. There was a
small decrease in height in the first two hours for PEG coated samples, but then only very
slight changes observed in the following 8 hours. This result again shows that PEG coated
AgNPs are very stable.30, 55 PEG is an extremely relevant surface functional group for a
variety of nanoparticles because it renders them biocompatible.55, 56 As discussed
previously the AgNPs should be completed covered by PEG-thiol molecules for both PEG-
1000 and PEG-5000. Compared with PEG coated samples, the BSA coated sample showed
greater height decrease during the coating process. The mean height of the BSA coated
sample was only 40.8 ± 0.38 nm after 12 h treatment. The reason for this decrease is that
the interactions between BSA and AgNPs cause an acceleration of nanoparticle dissolution
in the first hours following exposure.33, 52
17
To further investigate the effects of BSA concentration, three different BSA concentrations
were tested. We observed an increase in the AgNP dissolution rate when the BSA
concentration was increased during coating treatment. The possible explanation is that the
thiol groups present in BSA have a high binding affinity for the surface chemisorbed Ag+.33,
52, 57 AgNPs consist of Ag0 nanoparticles and surface chemisorbed Ag+ in equilibrium with
each other,24, 51 and Ag+ is the only equilibrium product under most conditions in simple
media. It is therefore possible that the binding of Ag+ to BSA makes the NP structure less
stable and and induced further AgNP dissolution. AgNP dissolution is dependent on the
BSA concentration, with excess BSA binding with chemisorbed Ag+ resulting in rapid
dissolution during surface treatment (Figure 2-8b). In general, BSA accelerates AgNP
dissolution during the coating process. Once the BSA molecules fully coat the surface and
form silver-sulfide bonds (as discussed vide infra) further AgNP dissolution is inhibited.
While the mean height of the BSA coated AgNPs was lower than that of the other coated
samples immediately after the coating treatment, the measured dissolution rate of 0.39 ±
0.05 nm/d was much lower than for the uncoated samples.58 Such a result is consistent with
past studies,59 that examined AgNP dissolution in the present of cysteine and found
increased dissolution at the highest cysteine concentration over a few hours. This then lead
to decreased dissolved concentrations for longer reaction times. At the highest used
cysteine concentration (5 μM), dissolved Ag initially increased to 0.95 μM (19% of total
Ag) after 4 h, before decreasing to less than 0.1 mM after 24 h. Ostermeyeret.al33 quantified
AgNP dissolution utilizing UV-vis and found 31%, 12%, 14% 24% and 47% dissolution
after 3 h in the test media containing 0, 10, 40, 150 and 600 ppm BSA, respectively. These
results and our own illustrate that the presence of low concentrations of BSA initially
reduce AgNP dissolution. However, when the BSA concentration is increased, AgNP
dissolution was enhanced by the interaction between the nanoparticles and BSA.
Evidence for the existence of bonds between the coating agents and the AgNPs was
obtained by surface enhanced Raman spectroscopy (Figure 2-9). Prior to exposure and in
the absence of surface coatings, the AgNP substrate exhibits only a weak SERS signal at
235 cm-1 and an artificial spectral peak at 76 cm-1 that arises from the convolution of the
spectral profile of the laser and the transmittance of the notch filter. Following exposure to
all three coating solutions the signal at 235 cm-1 increases in intensity. The presence of this
18
band, which we assign as an enhanced Ag-S vibration mode,60 reveals the presence of a
covalent association between the coating agents and the AgNP surface. To track changes
in the Raman signal intensity, Raman spectra were collected over the course of a
dissolution experiment (Figure 2-9b). After subtracting the SERS background, the ratios
of the intensity of the peaks at 235-1 cm to 76-1 cm were calculated. This normalized ratio
accounts for point-to-point variations in signal intensity and as such enables some
quantitation of the number of Ag-S bonds. The starting ratio for all three coated AgNPs
were around 0.98, and then fluctuate between 0.90-0.98. Decreasing trends were observed
for all of the coated samples. But the declines were slow during the two week period, thus
suggesting the general stability of the coating layer. To probe the spatial heterogeneity of
these covalent interactions, Raman maps were constructed by integrating the area under
the peak at 235 cm-1 (± 75 cm-1). As shown in Figure 2-10a and Figure 2-10e, little signal
was observed for uncoated samples both prior to and following dissolution. In contrast,
AgNP with coatings showed various signal intensities. We note that the pixel size was large
and the AgNP arrays cannot be readily discerned. It is clear, however, that the Ag-S signal
is heterogeneously distributed across the surface.
19
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(52) Wang, X.; Herting, G.; Wallinder, I. O.; Blomberg, E., Adsorption of bovine serum
albumin on silver surfaces enhances the release of silver at pH neutral conditions. Physical
Chemistry Chemical Physics 2015, 17, (28), 18524-18534.
(53) Hinterwirth, H.; Kappel, S.; Waitz, T.; Prohaska, T.; Lindner, W.; Lammerhofer,
M., Quantifying thiol ligand density of self-assembled monolayers on gold nanoparticles
by inductively coupled plasma–mass spectrometry. ACS Nano 2013, 7, (2), 1129-1136.
(54) Siriwardana, K.; Wang, A.; Gadogbe, M.; Collier, W. E.; Fitzkee, N. C.; Zhang, D.,
Studying the effects of cysteine residues on protein interactions with silver nanoparticles.
The Journal of Physical Chemistry C 2015, 119, (5), 2910-2916.
(55) Fernández-López, C.; Mateo-Mateo, C.; Alvarez-Puebla, R. A.; Pérez-Juste, J.;
Pastoriza-Santos, I.; Liz-Marzán, L. M., Highly Controlled Silica Coating of PEG-Capped
Metal Nanoparticles and Preparation of SERS-Encoded Particles†. Langmuir 2009, 25,
(24), 13894-13899.
(56) Niidome, T.; Yamagata, M.; Okamoto, Y.; Akiyama, Y.; Takahashi, H.; Kawano,
T.; Katayama, Y.; Niidome, Y., PEG-modified gold nanorods with a stealth character for
in vivo applications. Journal of Controlled Release 2006, 114, (3), 343-347.
(57) Liu, R.; Sun, F.; Zhang, L.; Zong, W.; Zhao, X.; Wang, L.; Wu, R.; Hao, X.,
Evaluation on the toxicity of nanoAg to bovine serum albumin. Science of the Total
Environment 2009, 407, (13), 4184-4188.
(58) Kaegi, R.; Voegelin, A.; Sinnet, B.; Zuleeg, S.; Hagendorfer, H.; Burkhardt, M.;
Siegrist, H., Behavior of metallic silver nanoparticles in a pilot wastewater treatment plant.
Environmental Science & Technology 2011, 45, (9), 3902-3908.
(59) Sigg, L.; Lindauer, U., Silver nanoparticle dissolution in the presence of ligands
and of hydrogen peroxide. Environmental Pollution 2015, 206, 582-587.
(60) Martina, I.; Wiesinger, R.; Jembrih-Simbürger, D.; Schreiner, M., Micro-Raman
characterization of silver corrosion products: Instrumental set up and reference database.
Raman Spectroscopy 2012, 9, 1-8.
25
Figure 2-1. (a) Schematic of AgNP array production and coating process, (b) AFM image
of original AgNPs, and (c) AgNP height distribution as measured by AFM. The mean
height of 585 particles was 47.1 with a standard deviation of 1.5 nm.
26
Figure 2-2. AFM images of uncoated and coated AgNPs measured after 1- and 14-days
dissolution. The left panel is the AFM image for the original AgNPs without coating and
dissolution; on the right side (labeled a-h) are the AFM images for uncoated and coated
AgNPs after 1 and 14 days.
27
Figure 2-3. TEM images of (a, b) uncoated AgNPs and (c,d) BSA coated AgNPs after
12h dissolution in DI water.
28
Figure 2-4. AFM micrographs and height profiles for NSL-produced AgNP arrays after
1- and 14-days dissolution experiments.
29
Figure 2-5. AgNP height distribution as measured by AFM of (a) uncoated, (b) PEG1000
coated, (c) PEG5000 coated and (d) BSA coated samples. For each histogram, about 500
particles were measured.
30
Figure 2-6. (a) Mean AgNP height at different times and (b) normalized mean AgNP
height at different times. Inset: Dissolution rates calculated by linear regression for the
different coatings. (At least 486 particles were measured for each specimen to calculate
the mean particle height. Error bars represent the standard deviation for mean heights
determined by AFM for experiments performed in triplicate.)
31
Figure 2-7. Schematic illustration of the interactions between the coating agents and the
AgNP surface.
32
Figure 2-8. (a) Change in the mean AgNP height for different surface coatings as a
function of time and (b) Mean AgNP height for BSA coating solutions of different
concentration. (Error bars represent the standard deviation between mean heights
determined by AFM for experiments performed in triplicate.)
33
Figure 2-9. (a) Raman spectrum for uncoated and coated AgNP samples after 1 day of
exposure and (b) ratio of peak intensity of 235-1 cm to 76-1 cm during two week
dissolution (Error bars represent the standard deviation between mean ratios determined
by Raman for experiments performed in triplicate.)
34
Figure 2-10. Large area Raman map for uncoated and coated AgNPs at day 0 and day 14.
The map were piloted by integrating the area under peak 235 cm-1 with a width of 150
cm-1.
35
Table 2-1. Corrected radius and particle volume data for uncoated and coated samples
during two weeks dissolution.
Corrected radius (nm) Particle Volume (nm3) Dissolution
Percentage (%) initial 2-week initial 2-week
Blank 64.18 49.99 456193 176643 61.28
BSA 57.83 56.76 331009 280894 15.14
PEG1000 56.07 55.69 355504 343490 3.38
PEG5000 58.13 57.79 366249 346244 5.46
36
3. Controlled Dissolution Kinetics Study of Silver Nanoparticle: the Role of Particle
Size
3.1 Abstract
Silver nanoparticles (AgNPs) are being widely used in a variety of products due to their
antibiotic properties. There are, however, increasing concerns given to the potential
adverse effects of AgNP release on humans as well as the environment. Dissolution is a
critical step dictating the safety of AgNPs and both internal and external factors impact this
dynamic process. We fabricated small, medium, and large sized AgNPs by nanosphere
lithography (NSL) and electron beam evaporation. The average diameters of the three sized
AgNPs were 44.19 nm, 64.18 nm, and 98.02 nm, respectively. Dissolution experiments
were conducted for each size AgNP in air-saturated phosphate buffer under different NaCl
concentrations and temperatures. Tapping-mode atomic force microscope (AFM) was used
to measure changes in AgNP morphology over time. Higher dissolution rates were
observed with increased NaCl concentrations. A linear relationship was established
between the NaCl concentration and the dissolution rate for three different sized AgNP
samples. Specifically, under the highest NaCl concentration (550mM), the measured
dissolution rates for small, medium, and large AgNPs were, 2.022 ± 0.12 nm, 1.69 ± 0.23
nm and 1.44 ± 0.098 nm per day, respectively. Smaller AgNPs showed higher dissolution
rates than larger AgNPs when the salt concentrations were the same. Moreover, the
dissolution rate of the larger AgNPs is less dependent on the concentration of the NaCl
solution than smaller AgNPs. When we adjusted the temperature for the dissolution
experiments, the results showed that temperature has a positive effect on the dissolution
rate. The Arrhenius equation was used to describe the relationship between the reaction
rate constant and the reaction temperature. The obtained data showed that the activation
energy of larger AgNPs is higher than for smaller sized particles.
3.2 Introduction
Silver nanoparticles (AgNPs) have received increasingly attention as antimicrobial agents
and the concerns about the toxicity of released Ag+ to aqueous systems has been
extensively explored.1-4 Dissolution is a critical factor that determines the safety of metal
37
nanoparticles. During this dynamic process, antimicrobial properties, toxicity and
environmental impacts will be altered.2, 5, 6 Chemical kinetic experiments explore the
factors that affect the reaction rate including the nature of the reactants, their physical state
and surface area, etc. In addition, external factors are worth exploring such as the reaction
medium, temperature, etc.2, 7-9 Studies have shown that the intrinsic properties of
engineered AgNPs such as reactivity, solubility, electrochemical, and optical properties,
are dependent on particle size.10, 11 In studies of the dissolution of AgNP the impacts of
size effects should be considered. It is generally assumed that solubility increases with a
decrease in particle size since small particles are hypothesized to have higher chemical
reactivity and surface energy compared to larger particles of the same material.12-17
However, it is often challenging to experimentally demonstrate such size effects, especially
in studies which the size control involves surface functionalization.13 Moreover,
aggregation of AgNP will lead to an increase in the collective particle size and a decrease
in surface area which may adversely impact the dissolution process.
Nanosphere lithography (NSL) has been used to produce a large variety of well-ordered
periodic nanopatterns, such as nanorings, dots, grids, wires, etc, from a wide variety of
materials on many substrates. 18-20 The advantages of NSL include its simplicity to
implement, low cost, fast fabrication speed, and high-throughput.21-23 NSL provides a good
template for the control of size, shape, and interparticle spacing. Specifically, the formation
of the 2D colloidal crystal mask and the postdeposition processing steps can be controlled
to adjust the in-plane width and out-of-plane height of the produced nanopatterns.23, 24 By
using NSL to fabricate AgNPs on glass, we can exclude factors such as surface coating and
crystallinity differences and simply focus on size effects. To intensively explore size
effects, we adjusted two factors: medium concentration and temperature. AFM provides an
easy and accurate measurement of particle size and morphology.
3.3 Materials and Methods
Substrate production
Glass substrates (Fisher Scientific) were cleaned by RCA solution and rinsed with DI water
(>18.2 MΩ-cm). The cleaned substrates were then thiolated in 5% (3-mercaptopropyl)-
38
trimethoxysilane (Sigma-Aldrich) in methanol (Alda Aesar) for 12 h. Negatively charged
carboxylated polystyrene microspheres (Life Technologies) with diameters of 300, 500,
and 800 nm were deposited onto cleaned substrates by convective self-assembly (CSA) to
obtain three different sized AgNPs. A 5 L aliquot of polystyrene suspension (10% w/v)
was placed between the interspace and the substrate and was moved at a constant velocity
of 0.02 cm/s for 12 cm. A monolayer of close-packed spheres formed due to solvent
evaporation. Following CSA, electron beam evaporation (3-kW electron gun,
Thermionics) was used to deposit silver metal onto the prepared substrates. The thickness
of the silver deposition was set as 30, 50, and 80 nm to obtain AgNPs with different heights.
Substrates were cut into squares of ≈5 mm2 and the spheres were removed using tape.
AgNPs immobilized on glass substrates were sequentially rinsed with ethanol and DI water
for 30 s each.
Nanoparticle dissolution experiments
The prepared AgNP substrates were immersed in phosphate buffered (1 mM NaH2PO4; 1
mM Na2HPO4) NaCl solution and the sealed samples were stored in the dark for a specific
dissolution period. To explore the effects of salt concentration, the concentration of the
NaCl solutions were set at 110, 275, 412, and 550 mM. The temperature of the experiments
were controlled by putting the sealed samples in a water bath in a temperature controlled
environment. Temperature were set at 4 °C (refrigerator), 10 °C (cooler), 25 °C (room
temperature), 37 °C (incubator), 50 °C (incubator). The reaction period was set as 0, 1, 2,
4, 7, 10 and 14 days for each size group. For experiments in which the temperature was
varied from the baseline room temperature of 25 °C a NaCl concentration of 550 mM was
used. To quantify the AgNP dissolution rate, each substrate was removed from solution
and dried. The substrate was then attached to stainless steel specimen discs (Ted Pella) for
AFM measurement. Tapping mode AFM was employed using antimony doped silicon
TESPA-V2 AFM probes (Bruker). All AFM images were measured immediately after
removing the specimen from the reaction solution.
39
3.4 Results and Discussion
AgNPs were obtained by NSL method and the final size of the AgNPs was determined by
the size of nanospheres used in fabrication. Single-layer packed polystyrene nanospheres
were used as masks and silver vapor was deposited between the intersphere voids onto the
substrates. With bigger nanospheres, the gap space was larger and thus larger AgNPs were
produced. Moreover, by adjusting the deposition procedure, different amounts of silver
were deposited and lead to different particle heights. The grey part in Figure 3-1 (left)
represents AgNPs immobilized onto a glass substrate when the nanospheres were removed.
The small, medium, and large AgNPs all exhibit triangular shapes. AFM images showed
consistent morphologies and 3-5 images were collected at different locations with the scan
area of each image set at 5×5 µm2 to obtain the average height. All three kinds of AgNPs
were almost normally distributed which indicates a uniform morphology. This result
indicates that by using different sized nanospheres, we can accurately control the size of
AgNPs produced by NSL. The mean heights were 31.2±1.1 nm, 47.1 ± 1.5 nm, and 73.9 ±
1.3 nm for small, medium, and large AgNPs, respectively.
Dissolution experiments were conducted for each size AgNPs in phosphate buffered NaCl
solution. Previous size dependent dissolution studies mainly focused on the release rate of
dissolved Ag+; however, the size and morphology changes of AgNP are neglected in most
of the studies. The method we used in this study tracked both height and morphology
changes by AFM which provides a more comprehensive understanding of the dissolution
process. To focus on size effects, dissolution rates for each specific NaCl concentration
were compared. The heights of the AgNPs were measured at specific times and mean
heights were calculated to quantitatively explore the changes. After 1 day dissolution in
550 mM NaCl solution, the heights of all three AgNP sizes increased to 37.9 ± 0.8 nm,
55.4 ± 1.2 nm, and 79.2 ± 2.1 nm, respectively. These increases in size are consist with our
previous studies in which the height changes occur due to the net flow of silver ion from
the bottom of a nanoparticle to the top until the internal redox gradient is eliminated.25
Various degrees of increases were also observed for experiment sets with lower NaCl
concentrations with the same mechanism. We used the height following the initial increase
as the starting height for the dissolution experiments. Dissolution rates were calculated
based on the normalized heights and linear fitting regressions.
40
The dissolution rates for each series were calculated for the three different sized AgNPs
with 4 different NaCl conditions (Figure 3-2). The relationships between size, NaCl
concentration, and dissolution rate were explored. For a 2 week duration experiment, the
dissolution rates of the medium sized AgNPs increased from 0.633 ± 0.04 nm/day to 1.69
± 0.23 nm/day when the salt concentrations were increased from 110 to 550 mM. The R-
square values for these 4 groups varied between 0.885 and 0.984 which indicated
reasonable linear regression relationships. For the small AgNPs, over the same period and
the same range of salt concentrations, the dissolution rate increased from 0.777 ± 0.12
nm/day to 2.022 ± 0.12 nm/day. The range of variation for the large AgNPs was from 0.45
± 0.10 nm/day to 1.44 ± 0.098 nm/day. Based on this increase, we conclude that the
dissolution process accelerates with an increase in NaCl concentration. This result
corroborates prior work by our group.25 The ratio of Cl: Ag has an effect on AgNP
dissolution since the formation of various possible silver chloride species is ratio
dependent.9, 26 When AgNP dissolves, Ag+ ions are absorbed at the surface of the AgNP.
With greater Cl- in the solution, Ag+ ions tend to form AgClx(x−1)− species and thus remove
Ag+ away from the particle surface. Moreover, as the Cl- concentration increases, soluble
AgClx(x−1)− species dominate over AgCl(s) and lead to a higher AgNP dissolution rate. 26-
29 So an increase in the concentration of NaCl promotes AgNP dissolution.
Higher dissolution rates were observed for smaller AgNPs with the same NaCl
concentration. With 110 mM NaCl solution, the dissolution rate for small AgNP was 0.777
± 0.12 nm. When the AgNP size was increased to medium size, the rate decreased to 0.633
± 0.04 nm/day. An even lower dissolution rate for large AgNP of 0.45 ± 0.10 nm/day was
obtained under the same conditions. This illustrates that particle size had an inverse impact
on the AgNP dissolution process. To convert the dissolution rate from the measured height
change to a volume change, we calculated corrected particle volumes using our prior
approach.25 Decreased percentage of volume was observed dissolved for bigger AgNPs in
550 mM NaCl solution. The dissolved volume for small AgNPs was 80.38%, while the
data for large AgNPs was 52.21%. If a given mass of metal is progressively subdivided
into smaller particles, then the rate of mass conversion from the metallic to the ionic species
will increase as the particle size decreases due to the increase in surface area. Thus, smaller
particles dissolve more rapidly than larger ones on a per mass basis.30, 31 Dissolution rates
41
for different NaCl concentration groups were also calculated and an increase in dissolved
volume was observed for bigger AgNPs and higher NaCl concentration.
Except for the physical and chemical properties of the solute and solvent, the solubility of
a substance are also depended on the pressure, reaction temperature and presence of other
chemicals in the solution.2 For nanoparticles, their chemical property is size depended, thus
the established thermodynamic relationship can be used to describe the size dependence of
the solubility. The Kelvin equation establishes the relationship between the vapor pressure
of a liquid droplet and its curvature, which is a function of the droplet size. A modified
Kelvin equation (Ostwald-Freundlich relation) can be used to describe the correlation of
particle solubility and its radius.
Sr = Sbulk × exp(2γVm/RT × r)
where, Sr is the solubility of Ag NPs with radius r, Sbulk is the solubility of a flat silver
surface, γ is the surface tension of the particle with radius r (J/m2), Vm is the molar volume
of the particle (m3/ mol), R is the universal gas constant (J/mol⋅K), and T is the temperature
(K).
Although there are debates about the applicability of Kelvin’s theory to particle fluid
interfaces, some studies has verified its applicability experimentally.13 The effect of
particle size on nanoparticle dissolution process can be proved if surface tension is
independent of particle size.32 There is still an ongoing debate on this topic since most
studies conclude that the surface tension of nanoparticles is independent of the particle size.
33, 34 However, others have found an increase in surface tension with increasing particle
radius. 35 If the surface tension remains the same for different sized nanoparticles, a higher
solubility of smaller nanoparticles should be observed. Our results indicate that smaller
AgNPs had higher solubility and the increased dissolution rate was related to particle size.
In prior studies, the fabrication of AgNPs with different size always include surface
functionalization which introduced a second experimental variable besides size. It was thus
difficult to explictly focus on the effects of size. In our study, NSL provided a means to
produce different sized AgNPs without other potentially interferent factors.
To further explore the relationship between NaCl concentration and dissolution rate, linear
regressions were obtained for each size AgNP. The regression equation is shown as:
42
R = A×[NaCl] + B
In these expression, R is the dissolution rate (nm/day) and [NaCl] is the concentration of
NaCl solution in unit of moles/L. The linear regression illustrates that the dissolution rate
is directly related to the concentration of NaCl. The slope of the curve (A value) indicates
how much the dissolution rate is dependent on the concentration of NaCl.
The regression equations for small, medium, and large AgNPs are:
R = (2.84 ± 0.256) × [NaCl] + 0.5375 r2=0.976
R = (2.59 ± 0.277) × [NaCl] + 0.3355 r2=0.966
R = (2.12 ± 0.383) × [NaCl] + 0.1885 r2=0.910
The slope for small AgNPs was 2.84, but decreased to 2.59 for medium AgNPs and to 2.12
for the biggest AgNPs (Figure 3-3a). Using the equation for small AgNPs, when the
concentration of NaCl increased by 1 M, the dissolution rate of small AgNPs increased by
2.84 nm/day. The decrease in the slope with an increase in size indicates that the dissolution
rates were less dependent on the NaCl concentration with an increase in particle size. In
other words, the NaCl concentration has a more obvious influence on the dissolution of
smaller particles than bigger particles. This result can be explained by the difference
between particle surfaces that are active for dissolution reaction.2, 13 For smaller AgNPs,
the exposed surface area per unit mass is higher than for bigger AgNPs, which will lead to
more obvious effects of NaCl concentration. We plotted the histogram for the dissolution
rates of three different sized AgNPs by NaCl concentration (Figure 3-3b). A decease in the
dissolution rates was observed with increased AgNP size, a result consistent with previous
analysis.
The antibacterial property of AgNP is mainly driven by Ag+ release.4, 14 Previous studies
reported that AgNP infiltrate the bacterial cell wall by anchoring to the surface and thus
effect the physical properties of the bacterial membrane. The membrane damage can result
in cellular contents leakage and may cause bacterial death.36-38 Surface area of the
nanomaterials is one of the vital factors that have effects on the AgNP activity against
microbes. The higher surface area of smaller nanoparticles makes it easier to contact with
the cytoplasm than bigger nanoparticles.36 On the other hand, smaller AgNPs contain more
43
surface atoms than bigger AgNPs and the greater thermodynamic driving force towards
oxidation of the smaller AgNP can lead to greater dissolution. There is thus a conflict
between the antimicrobial property and the dissolution rate. Smaller particles have more
surface area available for the reaction and thus exhibit more rapid dissolution. As for the
antibiotic properties, higher surface area is favored due to higher efficiency of killing the
bacteria.39, 40 Attention should therefore be given in the future design of incorporation of
AgNP in customer products to address both antibacterial efficiency and dissolution rate.
The use of AgNP containing products likely occurs in a variety of environmental
conditions. 9, 41-43 Temperature is one external factor that is worth studying for the
dissolution process since reaction rates are related to it.2, 26 Chemical reactions only take
place when the reactant molecules, atoms, or ions collide. According to the collision model,
more than a certain amount of kinetic energy is required and the reactant should be in the
proper orientation.44 To speed up the reaction rate, the number of the very energetic
molecules present at any particular instant, which with energies equal to or greater than the
activation energy, should be enhanced. If we heat a reaction system, the atoms move faster
and thus they collide more frequently. As a consequence, the reaction rate will be increased.
In this study, we controlled the experimental temperature over the range between 4 and 50
°C. This range reflects cold storage, room temperature, biological growth conditions (e.g.,
37 °C), and elevated temperatures (50 °C) that might be found under adverse environmental
conditions. All temperature controlled experiments were conducted using a 550 mM NaCl
solution to maximize the salt induced dissolution rate.
The dissolution process was tracked for two weeks and different degrees of dissolution
were observed (Figure 3-4). At 4°C, the dissolution rates for small, medium, and large
AgNPs were 1.31 ± 0.11, 1.02 ± 0.09 and 0.73 ± 0.09 nm/day, respectively. At the highest
experimental temperature of 50 °C, the dissolution rate increased to 2.65 ± 0.25, 2.46± 0.34
and 2.34 ± 0.23 nm/day. As expected, an increase in temperature led to an increased degree
of dissolution. The dissolution of the AgNPs takes place more slowly and to a lesser degree
at the lower temperature. As for small AgNPs, the mean height decreased from 35.98 nm
to 1.39 nm after 14 days at 50 °C, which indicates only a small portion of the AgNPs
remained. By using the corrected volume equation to covert the height change to volume
change, 98.95% volume of small AgNPs was dissolved under this experimental condition.
44
For the large AgNPs, the size decreased from 78.5 nm to 44.98 nm during the 2 week
dissolution process and the volume decrease was 79.89%. When the temperature of a
reaction system increases, the average kinetic energy of each component is increased. The
components move faster and collide with each other more frequently in a given amount of
time. The increased activity results in higher energy or collision force and thus lead to the
end products more quickly.
The Arrhenius equation describes the temperature dependence of reaction rates.
k = A 𝑒−𝐸𝑎𝑅 𝑇
where k is the rate constant, A is the pre-exponential factor and a constant for each
chemical reaction, Ea is the activation energy for the reaction, T is the temperature
in Kelvin, and R is the universal gas constant.
To explore the temperature dependence, this equation can be converted to:
In k = In A - 𝐸𝑎
𝑅 1
𝑇
A plot of ln k vs. T−1 gives a straight line and the slope value is equal to negative Ea/R.
AgNPs may considered as soluble reactants due to their small sizes and first-order kinetics
model can be used to describe the dissolution reaction.15, 45
Ct=C0 (1-𝑒−𝑘𝑡)
where Ct is the concentration at time t (d), C0 is the concentration when AgNPs are
completely dissolved, and k (d-1) is the mass-based first-order rate constant. Assuming that
the substrates are completely uniform with 10 particles/μm2 for medium sized AgNP
sample. Based on the measured AgNP volume change, we predicted the concentrations
required to fit this pseudo-first order kinetic model. When we plot t vs. In(1- Ct/Co), the
value of the slope is the rate constant for this specific experiment setting. For medium sized
AgNP, the obtained rate constant increased from 0.023 to 0.143 day-1 as the temperature
increased from 4 to 50 °C. In the next step, negative Ea/R values for three different sized
AgNPs were obtained by fitting ln k vs. T−1 (Figure 3-5). The values were 2811.8 ± 316.6,
3166.8 ± 387.3 and 3515.7 ± 312.1, respectively. Thus the activation energies for small,
medium and large AgNP were 23.37 ± 2.63, 26.32 ± 3.22 and 29.22 ± 2.59 kJ, respectively.
45
Previous studies have observed a reduction in the activation energy for nanoparticles with
decreased size33, 34, 46 and our results show the same trend. For smaller nanoparticles, more
surface atoms are available for the reaction and thus less energy is required to overcome
the transition state. This result indicates that the surface energy of smaller nanoparticles
are higher and they dissolve more with the same experimental condition as shown in the
previous dissolution part.
In this work, we concluded the linear relationship between the NaCl concentration and the
dissolution rate for three different sized AgNP samples. Smaller AgNPs showed higher
dissolution rates than larger AgNPs when the salt concentrations were the same, which
indicates that particle size has a negative effect on dissolution. Moreover, the dissolution
rate of the larger AgNPs is less dependent on the concentration of the NaCl solution than
smaller AgNPs. The experiments also showed that temperature has a positive effect on the
dissolution rate. And the fitted data based on Arrhenius equation illustrates that the
activation energy of larger AgNPs is higher than for smaller sized particles.
46
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51
Figure 3-1. SEM, AFM images and height distribution of (a, d, g) small, (b, e, h) medium
and (c, f, i) large AgNPs produced by NSL method.
52
Figure 3-2. Normalized mean AgNP height at different times and data fitted by linear
regression: (a) small, (b) medium and (c) large AgNPs. (The different NaCl concentration
are labeled.)
53
Figure 3-3. (a) Slopes of the regression lines for dissolution rate as a function of NaCl
concentration. Standard errors are indicated by the error bars. (b) Histogram of
dissolution rates for small, medium and large AgNPs in NaCl solutions with various
concentrations.
54
Figure 3-4. Normalized mean AgNP height at different times and data fitted by linear
regression: (a) small, (b) medium and (c) large AgNPs. (The different experimental
temperatures are labeled.)
55
Figure 3-5. liner regression fitted plot of Ln (rate constant) and T−1 at different
experimental temperature. The unit of rate constant is day-1.
56
Table 3-1. Dissolution percentage and reaction constant of small, medium and large
AgNP at different temperatures.
Temperature 4 10 25 37 50
% k % k % k % k % k
Small AgNP 40.61 0.053 64.26 0.090 80.38 0.119 92.10 0.185 98.95 0.250
Medium AgNP 25.09 0.023 40.54 0.041 61.02 0.066 72.06 0.081 89.67 0.143
Large AgNP 18.22 0.017 33.97 0.032 52.21 0.048 61.88 0.080 79.89 0.121
57
3. Environmental Implications and Conclusions
The market for nanoparticle incorporating materials has been developed in the past decades
and the number of potential applications have been extended. To take better advantage of
this kind of material, concerns about their safety have drawn considerable attention. The
safety issues with nanoparticles are continuously being tested because of their potential
dangers for environmental and human health. Previous studies reported the toxicity of
AgNPs to a variety of organs including the brain, liver, lung, vascular system and
reproductive organs.1, 2 The dissolution of nanoparticles is an important process that alters
their properties and is also a critical step in determining their safety.3 Therefore, studying
nanoparticle dissolution can help in the current move towards safer design and application
of nanoparticles.
In this work, the uniform arrays of nanoparticles enabled the controlled evaluation of
nanoparticle dissolution in the absence of aggregation. We tracked the height changes of
AgNPs fabricated on glass slides. The controlled evaluation of height changes were
obtained by AFM measurements. The effects of surface coating and size on nanoparticle
dissolution were illustrated in the absence of aggregation. Information obtained from this
study demonstrated different coating agents exhibit various remissions for the dissolution
process. Specifically, a PEG coating prevented AgNP dissolution in two weeks which lead
almost no Ag+ released to the solution. For BSA coated samples, slight changes were
observed for the shape and height. The results of this work will provide guide to the
engineer of environmental friendly nanomaterials and to predict the environmental impacts
of coated NPs. For the study of size effects, results showed that smaller AgNPs dissolved
more with the same medium concentration which indicates less stability. With increased
salt concentration, the dissolution rates for each size AgNPs increased and the smaller
AgNPs were easier to be effected by the external environment. When the temperature of
the reaction system was increased, the dissolution rates grew to different extents. By fitting
the reaction constant with temperature for small, medium, and large AgNPs a higher
activation energy was observed for bigger AgNPs. On the other hand, smaller AgNPs with
higher surface area could have more efficiency for the antibiotic property. These two
aspects are in conflict and future fabrication and applications should consider both. Overall,
the results from the research provide information about the kinetics data of AgNP
58
dissolution under various circumstances. Several effect factors were included and could
provide guide for future studied regarding this topic.
59
References
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60
Appendix A: NanoComposites of Bacterial Cellulose and Metal-Organic Frameworks
Introduction
Bacterial cellulose (BC) is a three-dimensional hydrophilic biopolymer produced by
certain types of bacteria and has unique properties including ultra-fine fiber network, high
water holding capacity, high mechanical strength, and biocompatibility.1, 2 Moreover, the
foldability in situ and cost efficient merits make it applicable as commercial biomaterial.
Varied applications of BC include food packing, transparent coatings or films,
biomaterials, artificial medical materials and scaffolds for tissue engineering.3-5 Static,
shaking, and bioreactor cultures are common methods to obtain BC and the bacterium G.
xylinum has shown the highest production rate of BC among all bacteria types. This aerobic
bacterial strain can transform glucose and other organic substrates into cellulose within a
few days.2 The static culture method is a relatively simple technique; therefore, it is the
most commonly used method to produce BC in lab scale. BC has abundance of hydroxyl
functional groups which is the same chemical structure as cellulose. It can be
functionalized by in situ and ex situ methods and used as scaffolds.6 The culture conditions
are modified by the use of additives or reinforcement materials for in situ method. While
ex situ method is to obtain BC first and conduct the modification as the following
procedure. Due to the porous structure and hydroxyl groups, it is a favorable substrate to
load with other nanomaterials.
Metal−organic frameworks (MOFs) are nanoporous materials that consist of metal centers
connected by various organic ligands to form highly regular networks.7, 8 MOFs have
specific pore apertures tunable by three-dimensional coordination networks with high
crystallinity, high porosities, large specific surface areas, tunable pore functionality and
thermal stability.8, 9 These distinct features make MOFs effective for applications such as
gas storage, separation, catalysis, water purification and drug delivery.8, 10-12 MOFs have
mostly been synthesized in the form of bulk powders or colloidal crystals due to its
crystalline nature. Studies have focused on the fabrication of supported MOF materials to
future explore the potential applications.12-15 Integrating MOFs into matrices or on
scaffolds could make their handling, deployment, and regeneration easier. Furthermore,
the chemical and physical properties of these composite materials are often show
61
improvement. In situ solvothermal growth/deposition from precursor solutions is one of
the most widely used methods to get MOF deposition. 16, 17 The fibrous macroporous
structure of BC makes it a great supported material for the growth/loading of guest MOF
particles. The mass diffusion within the composite membrane may also be improved, which
is more favorable for sorption and catalysis applications. Here, we report the in situ
deposition of MOFs on BC to obtain high-performance adsorptive materials. Moreover,
gold nanoparticles were included in the system to obtain SERS signals.
Method and material
BC Synthesis
Gluconacetobacter Xylinus was used to produce bacterial nanocellulose in ATCC 459
media. Specifically, 40 g of fructose, 5 g of yeast extract and 12.5 g of CaCO3 were
dissolved in 1000 mL of DI water. The media was autoclaved at 121 oC for 15 min and
then cooled down for use. 1 ml of G. Xylinus and 150 mL of the fresh media were mixed
in a cell culture flask with a vented cap. This was then transferred to an incubator and the
temperature was set at 30 oC. For humidity control purposes, an autoclaved open container
with soapy water was placed at the bottom of the incubator. A BC pellicle was formed on
the surface of the cell culture flask after 3 days. The pellicle was shaken vigorously to
extract the bacteria from the pellicle to the media, and then transfer the bacteria enriched
media to centrifuge tubes which were saved as pre-culture for future use. When the pre-
culture was obtained, mix 2 ml of the prepared pre-culture with 300 ml of fresh ATCC 459
media and then cultivate the mixture at 30 oC with humidity in petri dishes. After 10 days
growth, the obtained BC pellicles were removed from the petri dishes and washed
thoroughly with 0.5 M NaOH solution for 5 days. The NaOH solution needed to be
refreshed regularly to remove the remaining bacteria in the pellicles. The pellicles were
then washed with DI water for 7 days until the pH was neutral.
MOF Deposition
Zinc precursor solution was made by mixing 0.37 g of Zn(NO3)2·6H2O with 25 ml of H2O with
stirring until complete dissolution. Then organic precursor solution was obtained by adding 0.81
62
g of 2-methylimidazole into 25 ml of H2O with stirring until complete dissolution. Immerse
the BC in the Zn(NO3)2·6H2O solution for 12 h and then add 2-methylimidazole solution. The
mixture was mixed under stirring for 10 minutes.18 After the synthesis process, the BC-MOF
samples were taken out of the precursor solution and washed by the solvent used during the
synthesis three times. The obtained substrates were dried under vacuum at 150 °C for 24 h for
further use.
Au Nanoparticle Loading
BC were first cut into small pieces (1 cm × 1 cm) and eight pieces of BC were incubated in
1.4 mL HAuCl4 solution (30 mM) and vortexed for 30 s. 100 mL of 1.2 mM Na3Cit
solution was boiled and the BC- HAuCl4 was then transferred into it. Keep the mixture
boiling for 1.5 h to form AuNP on the BC. The obtained AuNP/BC nanocomposite was
rinsed with DI water after preparation.19
To load ZIF-8 on the AuNP/BC, the AuNP/BC were immersed in zinc solution for 12 h
and then mixed with 2-methylimidazole solution to form ZIF-8 particles. The
nanocomposites were washed by DI water and then stored in DI water for future use.
Analytical Techniques
Scanning electron microscopy (SEM) images were obtained by using a LEO (Zeiss) 1550
Schottky field-emission SEM. Energy dispersive X-ray spectroscopy (EDS) measurements
was performed by an FEI Quanta 600 FEG environmental SEM equipped with a Bruker
EDS. Nitrogen adsorption−desorption measurements were conducted with Autosorb
Quantachrome 1MP analyzer at 77 K.
Results and discussion
After 10 days growth, the obtained BC pellicles were thick and showed a transparent color
after washing. BC pellicles were kept in DI water until use and have a high content of water
in the space between the fibers. By using a freeze dryer, the BC was first frozen and the ice
was then removed by sublimation under low pressure. The freeze dried BC as shown in
Figure A-1 (a) are 3D networks with high porosity. The abundant space between the fibers
provide numerous locations for other nanoparticles to be loaded and the hydroxyl groups
63
in BC makes it applicable for various chemical reactions. Blank ZIF-8 were synthesized
by mixing Zn(NO3)2·6H2O solution and 2-methylimidazole solution. The particles were
formed immediately after mixing which indicates rapid nucleation. The great advantage of
the water synthesis system is that it is compatible with many materials. Figure A-1 (b)
shows the uniform ZIF-8 particles. The XRD patterns (Figure A-2) of the synthesized
samples confirmed the ZIF-8 structure.18 To investigate the surface area, nitrogen
adsorption and desorption experiments were conducted. The BC was a macroporous
material with a specific surface of 69.94 m2 g−1, which is attributed to the external fiber
surface. The ZIF-8 particles obtained in this study has a surface area of 988.05 m2 g−1.
Figure A-1 (c, d) show the morphology of BC-ZIF and we can see the BC fibers were
covered by ZIF-8 particles. ZIF-8 particles were attached to BC fibers throughout the
surface and inside the space between fibers. The particle growth process was effected by
the existence of nanofibers in the precursor solution. For this reason, the ZIF-8 particles in
BC-ZIF samples were not as uniform as the blank ZIF-8. The high surface area makes the
BC-ZIF nanocomposite a great material for adsorption and here in we conducted dye
removal experiments using malachite green (MGITC) and Rhodamine B (Rh B). As shown
in Figure A-3, the adsorption was rapid in the first hour with about 40% removal of MGITC
and 63% of Rh B. At the end of 6 hours, the removal was 50% and 80%, respectively.
Normally when MOFs were used as adsorbent, the particles and solution were mixed and
filtration was needed to obtain a clear liquid.7, 20 When the BC-MOF nanocomposite was
used, the solution after adsorption was already purified when the composites sank to the
bottom. An adsorption column is an option to enable the BC-MOFs nanocomposites to be
used in the treatment of various aqueous samples. The application of most functionalized
mesoporous materials is limited by the powder form since they are difficult to collect. By
incorporating the nanoparticles into the BC scaffold makes the MOF composites more
suitable to be applied in adsorption and recycling.
Our previous studies have used BC-AuNP as a SERS substrate to produce hot spots and
detect chemical compounds in aqueous systems. 21, 22 So we introduced AuNP into the BC-
ZIF with the aim is to take advantage of the SERS application. AuNP was first deposited
on BC by using HAuCl4 and Na3Cit. The formed AuNPs were observed to be spread over
the fibers with sizes less than 100 nm. Following AuNP deposition, ZIF-8 was synthesized
64
by using the same precursor solution as was used to produce the BC-ZIF composite. A
small amount of AuNPs were detached during the ZIF-8 synthesis, but the majority of the
AuNPs were maintained. The loading capacity of ZIF-8 is quite high as shown in Figure
A-1 (f) and the surface of BC was covered by ZIF-8 particles. By taking the EDS
measurement of the BC-AuNP-ZIF nanocomposite, the EDS maps (Figure A-4) show the
homogeneous distribution of Au and Zn. The single spot EDS measurements provided the
information about the mass percentage of carbon, gold and zinc. The average value of three
spots for the three elements was 12.79%, 37.37% and 34.31%.
Future Work
The BC-AuNP-ZIF nanocomposites were successfully produced in this study and some
initial characterization measurements were conducted. Future work will focus on the
application of this nanocomposite. Because ZIF-8 provides a high surface area and should
be a perfect material for adsorption, it could be used to collect the chemical compound and
then SERS will be used to detect it.23, 24 Low detection limit are expected to be obtained.
65
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P.; Dima, A.; Terpou, A.; Koutinas, A.; Castro, G. R., Progress in bacterial cellulose
matrices for biotechnological applications. Bioresource Technology 2016, 213, 172-180.
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Environmental Science & Technology 2018, 53, (2), 575-585.
67
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68
Figure A-1. SEM images of (a) freeze dried bacterial cellulose, (b) ZIF-8 particles, (c, d)
BC-ZIF nanocomposites, (e) BC-AuNP nanocomposite and (f) BC-AuNP-ZIF
nanocomposite.
69
Figure A-2. XRD spectrum of prepared ZIF-8 from a water system.
70
Figure A-3. A-3. Removal rate of MGITC and Rh B adsorption by using BC-ZIF
nanocomposites as the adsorbent.
71
Figure A-4. EDS Map of BC-AuNP-ZIF nanocomposite. (a) A map of both Au and Zn,
(b) a map of Au and (c) a map of Zn.
72
Table A-1. Element mass percentage of BC-AuNP-ZIF nanocomposite from 3 different
sample spots.
Mass
percentage (%)
C N O Zn Au
Spot 1 12.42 11.31 5.40 30.53 40.34
Spot 2 13.32 9.87 3.79 36.88 36.14
Spot 3 12.62 12.52 3.71 35.52 35.61
Mean 12.79 11.23 4.30 34.31 37.37
73
Appendix B: Real-Time Monitoring of Ligand Exchange Kinetics on Gold
Nanoparticle Surfaces Enabled by Hot Spot-Normalized Surface-Enhanced Raman
Scattering
Reprinted with permission from Haoran Wei, Weinan Leng, Junyeob Song, Chang Liu,
Marjorie R. Willner, Qishen Huang, Wei Zhou, and Peter J. Vikesland. Real-Time
Monitoring of Ligand Exchange Kinetics on Gold Nanoparticle Surfaces Enabled by Hot
Spot-Normalized Surface-Enhanced Raman Scattering. Environmental Science &
Technology, 2018, 53(2): 575-585. Copyright 2018 American Chemical Society.
ABSTRACT:
Nanoparticle surface coatings dictate their fate, transport, and bioavailability. We used a
gold nanoparticle−bacterial cellulose substrate and “hot spot”-normalized surface-
enhanced Raman scattering (HSNSERS) to achieve in situ and real-time monitoring of
ligand exchange reactions on the gold surface. This approach enables semi quantitative
determination of citrate surface coverage. Following exposure of the citrate-coated
nanoparticles to a suite of guest ligands (thiolates, amines, carboxylates, inorganic ions,
and proteins), the guest ligand signal exhibited first-order growth kinetics, while the
desorption mediated decay of the citrate signal followed a first-order model. Guest ligand
functional group chemistry dictated the kinetics of citrate desorption, while the guest ligand
concentration played only a minor role. Thiolates and BSA were more efficient at ligand
exchange than amine-containing chemicals, carboxylate-containing chemicals, and
inorganic salts due to their higher binding energies with the AuNP surface. Amine-
containing molecules over coated rather than displaced the citrate layer via electrostatic
interaction. Citrate exhibited low resistance to replacement at high surface coverages, but
higher resistance at lower coverage, thus suggesting a transformation of the citrate-binding
mode during desorption. High resistance to replacement in stream water suggests that the
role of surface adsorbed citrate in nanomaterial fate and transport must be better
understood.