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: pvikes@vt.edu

(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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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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Multiple-patterning nanosphere lithography for fabricating periodic three-dimensional

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gold co-patterned surfaces via nanosphere lithography. Journal of Colloid and Interface

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nanostructures: state-of-the-art and potential. Nanotechnology 2015, 26, (4), 042001.

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Brown Jr, G. E., Effect of chloride on the dissolution rate of silver nanoparticles and

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active silver from nanosilver surfaces. ACS Nano 2010, 4, (11), 6903-6913.

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Akbarzadeh, A., A review on potential role of silver nanoparticles and possible

mechanisms of their actions on bacteria. Drug Research 2017, 11, (02), 70-76.

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typhimurium: role of inner membrane dysfunction. Current Microbiology 2017, 74, (6),

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coating and environmental conditions on the fate and transport of silver nanoparticles in

the aquatic environment. Science of the Total Environment 2016, 568, 95-106.

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(45) Zhang, W.; Yao, Y.; Sullivan, N.; Chen, Y., Modeling the primary size effects of

citrate-coated silver nanoparticles on their ion release kinetics. Environmental Science &

Technology 2011, 45, (10), 4422-4428.

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amorphous silicon nanoparticles. Nanoscale 2014, 6, (3), 1286-1294.

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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Science of the Total Environment 2012, 438, 225-232.

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

References

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engineering applications of nanocellulose-based nanocomposites. Environmental Science:

Nano 2014, 1, (4), 302-316.

(2) Wang, J.; Tavakoli, J.; Tang, Y., Bacterial cellulose production, properties and

applications with different culture methods–A review. Carbohydrate Polymers 2019.

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J., Review of nanocellulose for sustainable future materials. International Journal of

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packaging field: A review. Packaging Technology and Science 2015, 28, (6), 475-508.

(5) Cacicedo, M. L.; Castro, M. C.; Servetas, I.; Bosnea, L.; Boura, K.; Tsafrakidou,

P.; Dima, A.; Terpou, A.; Koutinas, A.; Castro, G. R., Progress in bacterial cellulose

matrices for biotechnological applications. Bioresource Technology 2016, 213, 172-180.

(6) Stumpf, T. R.; Yang, X.; Zhang, J.; Cao, X., In situ and ex situ modifications of

bacterial cellulose for applications in tissue engineering. Materials Science and

Engineering: C 2018, 82, 372-383.

(7) Khan, N. A.; Hasan, Z.; Jhung, S. H., Adsorptive removal of hazardous materials

using metal-organic frameworks (MOFs): a review. Journal of Hazardous Materials 2013,

244, 444-456.

(8) Stock, N.; Biswas, S., Synthesis of metal-organic frameworks (MOFs): routes to

various MOF topologies, morphologies, and composites. Chemical Reviews 2011, 112, (2),

933-969.

(9) Alhamami, M.; Doan, H.; Cheng, C.-H., A review on breathing behaviors of metal-

organic-frameworks (MOFs) for gas adsorption. Materials 2014, 7, (4), 3198-3250.

(10) Jian, M.; Liu, B.; Zhang, G.; Liu, R.; Zhang, X., Adsorptive removal of arsenic

from aqueous solution by zeolitic imidazolate framework-8 (ZIF-8) nanoparticles. Colloids

and Surfaces A: Physicochemical and Engineering Aspects 2015, 465, 67-76.

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(12) Lu, G.; Farha, O. K.; Zhang, W.; Huo, F.; Hupp, J. T., Engineering ZIF‐8 Thin

Films for Hybrid MOF‐Based Devices. Advanced Materials 2012, 24, (29), 3970-3974.

(13) Yin, N.; Wang, K.; Wang, L.; Li, Z., Amino-functionalized MOFs combining

ceramic membrane ultrafiltration for Pb (II) removal. Chemical Engineering Journal 2016,

306, 619-628.

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G., From metal–organic framework (MOF) to MOF–polymer composite membrane:

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992.

(15) Zhu, H.; Yang, X.; Cranston, E. D.; Zhu, S., Flexible and Porous Nanocellulose

Aerogels with High Loadings of Metal–Organic‐Framework Particles for Separations

Applications. Advanced Materials 2016, 28, (35), 7652-7657.

(16) Zhu, L.; Zong, L.; Wu, X.; Li, M.; Wang, H.; You, J.; Li, C., Shapeable fibrous

aerogels of metal–organic-frameworks templated with nanocellulose for rapid and large-

capacity adsorption. ACS Nano 2018, 12, (5), 4462-4468.

(17) Wu, Y. n.; Zhou, M.; Li, S.; Li, Z.; Li, J.; Wu, B.; Li, G.; Li, F.; Guan, X., Magnetic

Metal–Organic Frameworks: γ‐Fe2O3@ MOFs via Confined In Situ Pyrolysis Method for

Drug Delivery. Small 2014, 10, (14), 2927-2936.

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surfaces enabled by hot spot-normalized surface-enhanced Raman scattering.

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.