Thông tin tài liệu
GOLD A OPARTICLES-PROTEI CO JUGATE STUDIES:
CE TRIFUGATIO A D BI DI G A ALYSIS
HUA G SHU-YI G
ATIO AL U IVERSITY OF SI GAPORE
2012
GOLD A OPARTICLES-PROTEI CO JUGATE STUDIES:
CE TRIFUGATIO A D BI DI G A ALYSIS
HUA G SHU-YI G
(B. E G (HO S.), TU)
A THESIS SUBMITTED FOR THE DEGREE OF
MASTER OF E GI EERI G
DEPARTME T OF CHEMICAL A D BIOMOLECULAR E GI EERI G
ATIO AL U IVERSITY OF SI GAPORE
2012
Acknowledgments
I would like to first thank God for His endless grace and faithfulness in seeing me through this
these three years in NUS.
Secondly, I would like to thank my husband Albert Halim for his support, love and patience
through the stressful times.
Thirdly, I would like to thank my supervisor Dr. Lanry Yung, for his guidance and time
throughout my three years in NUS; for allowing me room to explore and for challenging me to
go beyond my limitations.
Fourthly, I would like to thank my colleagues Deny Hartono, Duong Hoang Hanh Phuoc, Tan
Wei Ling, Seow Nianjia, Harleen Kaur, Fong Kah Ee, Nandhini Elayaperumal, Dai Mengqiao,
Seet Sze Jee, Ma Ying and Jasmine Li for their inspiration and friendships.
Lastly, I would like to thank all the laboratory officers, Li Fengmei, Li Xiang, Yang Liming,
Sylvia Wan, Teo Ai Peng and my friends who have helped and encouraged me in one way or
another.
1
Abstract
Nanoparticles are deemed to be very reactive and possessing enhanced quantum effects due to
their small size. As such, there has been increased research into the field of nanoparticles in the
recent years due to its potential in the biomedical field for purposes such as diagnostics or drug
delivery. This rapid expansion of nanotechnology especially in the biomedical field has raised
some cause for concern because the knowledge of the potential cytotoxicity of these
nanoparticles is still very limited.
Colloidal gold nanoparticles (AuNPs) in particular have gained the interests of many and are
extensively investigated due to its optical properties and relatively inertness. Apart from endpoint in vivo investigations on the cytotoxicity of AuNPs, it is important to understand how
AuNPs interact with the biomolecules and what effects these interactions could have on the local
and overall biological environment. As such, this project has been geared towards investigating
the interactions between AuNPs and two of the most abundant proteins in the human plasma:
serum albumin and fibrinogen.
The AuNP-protein conjugate were characterized by size, zeta potential and the amount of
proteins adsorbed onto each AuNP. The effects of centrifugation (an important equipment when
working with nanoparticles and proteins) on the conjugate were also investigated by varying the
centrifugation parameters. It was found that the AuNP-protein conjugates can almost never be
completely sedimented. However, maximum recovery of the sample can be made by increasing
the centrifugation force and duration. An attempt was also made to investigate the potential
protein conformation change after adsorption onto the AuNP. However, due to limited suitable
analytical methods, this area of investigation could not be completed.
2
Table of contents
Acknowledgments.......................................................................................................................... 1
Abstract .......................................................................................................................................... 2
Table of contents............................................................................................................................ 2
List of figures ................................................................................................................................. 6
List of tables ................................................................................................................................... 7
Chapter 1 Introduction ................................................................................................................. 9
1.1
Background .......................................................................................................................... 9
1.2
Objectives ........................................................................................................................... 12
Chapter 2 Literature Review ..................................................................................................... 13
2.1 Overview of nanoparticles ...................................................................................................... 13
2.2 Toxicity of nanoparticles ......................................................................................................... 19
2.3 Conjugation, size and zeta potential studies............................................................................ 25
2.3.1 UV-vis spectrometry ............................................................................................................ 25
2.3.2 Transmission electron microscopy ....................................................................................... 26
2.3.3 Dynamic light scattering ...................................................................................................... 28
2.4 Centrifugation studies ............................................................................................................. 30
2.4.1 Inductively coupled plasma mass spectrometry ................................................................... 30
2.5 Protein quantification study .................................................................................................... 37
2.5.1 Quantification assays............................................................................................................ 37
2.5.2 Gel electrophoresis ............................................................................................................... 40
2.6 Protein conformation studies................................................................................................... 45
2.6.1 Fourier transform infrared spectrometry .............................................................................. 45
3
2.6.2 Circular dichroism ................................................................................................................ 46
Chapter 3 Materials and methodologies ................................................................................... 49
3.1 Materials .................................................................................................................................. 49
3.2 Procedures ............................................................................................................................... 49
3.2.1 Synthesis of gold nanoparticles (AuNP) .............................................................................. 49
3.2.2 Conjugation of AuNPs with protein ..................................................................................... 51
3.2.3 Size and zeta potential measurement ................................................................................... 51
3.2.4 Centrifugation....................................................................................................................... 52
3.2.5 Acid digestion and inductively coupled plasma mass spectrometry .................................... 53
3.2.6 Gel electrophoresis ............................................................................................................... 54
3.2.7 Circular Dichroism ............................................................................................................... 56
Chapter 4 Results and Discussion .............................................................................................. 57
4.1 Conjugation of AuNPs with proteins ...................................................................................... 57
4.2 Size and zeta potential ............................................................................................................. 59
4.2.1 Transmission electron microscopy ....................................................................................... 59
4.2.2 Dynamic light scattering ...................................................................................................... 60
4.3 Stability study of AuNP-protein conjugates............................................................................ 66
4.4 Centrifugation study on bare AuNPs ...................................................................................... 67
4.4.1 Particle size dependency ...................................................................................................... 68
4.4.2 G-force and time dependency .............................................................................................. 72
4.4.3 Temperature dependency ..................................................................................................... 75
4.4.4 Hydrodynamic size of AuNPs after centrifugation .............................................................. 78
4.5 Centrifugation study on AuNP-protein conjugates ................................................................. 81
4
4.5.1 Protein size dependency ....................................................................................................... 81
4.5.2 Hydrodynamic size of AuNP-protein conjugates after centrifugation ................................. 87
4.6.1 AuNP-bovine serum albumin conjugates ............................................................................. 92
4.6.2 AuNP-fibrinogen conjugates ................................................................................................ 95
4.7 Circular dichroism ................................................................................................................... 96
Chapter 5 Conclusion and Future work ................................................................................. 101
References .................................................................................................................................. 103
5
List of figures
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12
Figure 13
Figure 14
Illustration of a plasma torch with three concentric tubes and the gases that flow through them.
[81]
Structure of coomassie brilliant blue G-250 dye
Characteristic CD curves of secondary structure elements of proteins [95].
UV-vis spectra of AuNPs, BSA and AuNP-BSA conjugates of different initial BSA
concentration. Absorbance wavelength for BSA was 280nm, and for AuNPs at 520nm. AuNPBSA conjugates’ absorbance wavelength was at 525nm. AuNPs used here was 15nm.
UV-vis spectra of AuNPs, Fb and AuNP-Fb conjugates of different initial Fb concentration.
Absorbance wavelength for Fb was 280nm, and for AuNPs at 520nm. AuNP-Fb conjugate
absorbance wavelength was at 525nm. AuNPs used here was 15nm.
Transmission electron microscopy (TEM) images of AuNPs of different size. A. ~15nm
AuNPs; B. ~11nm AuNPs; C. ~5nm AuNPs.
Size (nm) of the AuNP-protein conjugate with increasing protein concentration added to
AuNPs for conjugation. Error bars calculated from the standard deviation of 3 independent
samples are shown.
Zeta potential (mV) of the AuNP-protein conjugate with increasing protein concentration
added to AuNPs for conjugation. Error bars calculated from the standard deviation of 3
independent samples are shown.
Percentage of 3 different sizes of AuNPs remaining in the supernatant after being centrifuged
using 3 different g-force, from 10 minutes up to 60 minutes. All samples here were conducted
at 4˚C. Standard deviation has been included in all data points, but the standard deviations for
the 11nm and 15nm AuNPs are mostly very small (less than 1%) and hence are not apparent in
the data shown.
Percentage of AuNPs remaining in the supernatant after being centrifuged using 3 different gforce, from 10 minutes up to 60 minutes, at 4˚C and 25˚C. A. 15nm AuNPs; B. 11nm AuNPs;
C. 5nm AuNPs.
Percentage of sample remaining in the supernatant after being centrifuged at 4˚C with a gforce of 14900g for 10 to 60 minutes. The graphs show the results for AuNPs before
conjugation, AuNP-BSA and AuNP-Fb conjugates, for 3 different sizes of AuNPs used.
Standard deviation has been included in all data points, but the standard deviations for some of
the samples were very small (less than 1%) and hence are not apparent in the data shown.
Representation picture of the SDS-PAGE gel. Lane 1: protein ladder, lane 2-5: BSA
calibration standards of concentration 25, 50, 90, 125 ug/mL, lane 6-7: sample 1, lane 8-9:
sample 2.
Circular dichroism spectra of BSA, AuNP-BSA conjugate as well as 4 times concentrated
AuNP-BSA conjugates. Excess proteins in the samples were not removed before the CD
measurement.
Circular dichroism spectra of AuNP-BSA conjugate, 4 times concentrated AuNP-BSA
conjugate and 10 times concentrated AuNP-BSA conjugate. Excess proteins in the samples
were removed by centrifugation before the CD measurement.
6
List of tables
Table 1
Table 2
Table 3
Table 4
Table 5
Table 6
Table 7
Table 8
Table 9
Table 10
Table 11
Table 12
Table 13
Table 14
Table 15
Table 16
Table 17
Table 18
Table 19
Table 20
Table 21
Table 22
Table 23
Size of the proteins which is suitable for the percentage of gel used for optimum protein
separation
Centrifugation parameters used for various samples.
Components for the preparation of loading buffer - laemmli buffer.
Component volumes for a 8% resolving gel and a 5% stacking gel.
Components for the preparation of the running buffer – tris-glycine buffer.
Components for the preparation of the staining and de-staining solution.
Results for the size of AuNP-protein conjugate measured using DLS as well as the calculated
number of protein molecule to 1 AuNP at the respective concentrations. Columns 2 & 4: Size
(nm) of the Au P-protein conjugate with increasing protein concentration added to Au Ps for
conjugation. Columns 3 & 5: The number of protein molecule per Au Ps in solution for
conjugation at respective protein concentration.
Zeta potential (mV) the AuNP-protein conjugate with increasing protein concentration added
to AuNPs for conjugation.
The size of the AuNP-BSA conjugates over time from T = 0 to T = 51 days.
Percentage of 15nm AuNPs remaining in the supernatant after centrifugation at different gforce, time and temperature.
Percentage of 11nm AuNPs remaining in the supernatant after centrifugation at different gforce, time and temperature.
Percentage of 5nm AuNPs remaining in the supernatant after centrifugation at different g-force,
time and temperature.
Percentage of 5nm AuNPs remaining in the supernatant after centrifugation at 20000g and 4˚C
for various times.
Percentage of 5nm AuNPs remaining in the supernatant after centrifugation at 15000g and 4˚C
for 90 and 120 minutes.
Size of 15nm AuNPs using DLS after centrifugation at different g-force, time and temperature.
Size of the 11nm AuNPs using DLS after centrifugation at different g-force, time and
temperature.
Size of the 5nm AuNPs using DLS after centrifugation at different g-force, centrifuge time
and temperature.
ercentage of 15nm AuNPs, AuNP-BSA and AuNP-Fb remaining in the supernatant after
centrifugation at different g-force, time and temperature.
Percentage of 11nm AuNPs, AuNP-BSA and AuNP-Fb remaining in the supernatant after
centrifugation at different g-force, time and temperature.
Percentage of 5nm AuNPs, AuNP-BSA and AuNP-Fb remaining in the supernatant after
centrifugation at different g-force, time and temperature.
Size of 15nm AuNP-protein conjugates using DLS after centrifugation at different g-force,
time and temperature.
Size of 11nm AuNP-protein conjugates using DLS after centrifugation at different g-force,
time and temperature.
Size of 5nm AuNP-protein conjugates using DLS after centrifugation at different g-force, time
and temperature.
7
Table 24 Number of BSA or Fb to 1 AuNP for different protein concentrations.
Table 25 The 4 best SDS-PAGE results for different BSA concentration used. The absolute amount of
protein adsorbed, together with the percentage of the BSA added adsorbed and the number of
BSA molecule adsorbed per AuNP are shown. A. [BSA] 50ug/mL, B. [BSA] 200µg/mLand C.
[BSA] 500ug/mL.
Table 26 The 4 best SDS-PAGE results for different Fb concentration used. The absolute amount of
protein adsorbed, together with the percentage of the Fb added adsorbed and the number of Fb
molecule adsorbed per AuNP are shown. A. [Fb] 200ug/mL, B. [Fb] 500ug/mL.
8
Chapter 1 Introduction
1.1 Background
Nanoparticles are becoming increasingly prevalent in the commercial markets due to their
increased use in nanomaterials and nanomedicine. It was estimated that by the year 2020, the
production of nanoparticles will increase to 58000 tons [1]. Nanoparticles, defined by the ASTM
standard definition, are particles that are between 1 nm to 100 nm in length in two or three
dimensions. Materials at this size range exhibit different physical and chemical properties as
compared to their larger counterparts. Bulk gold for example is golden in colour, while gold
nanoparticles (AuNPs) are generally red in colour. Nanoparticles also have an increased relative
surface area and quantum effects, due to their small size. As a particle decreases in size, more
atoms are present on the surface of the particle as compared to the number of atoms inside it [2].
This increased surface area signifies that they would have greater reactivity as compared to a
larger particle of the same material. Nanoparticles therefore may react differently from their
larger counterparts. Similarly, quantum effects become more apparent at the nanoscale level,
leading to better optical, electronic and magnetic properties of the nanoparticles. As a result of
these physiochemical properties, both researchers and commercial companies alike have been
actively improving the functionality of their products, such as cosmetics, clothing, electronics,
building materials, medical diagnostic tools and so on, by exploiting the unique properties of the
nanoparticles.
These products seem beneficial to the consumers as well as the economy at first glance, but, at
the same time, the understanding about the toxicity and in turn health effects of such
nanoparticles are rather incomplete. Because nanotechnology is moving at such a fast pace, the
time from the invention of a product to its commercialization is often too short for an effective
9
and substantial toxicology study to be done. As such, researchers, workers and consumers are
potentially being exposed to hazardous materials that could cause adverse health effects [3]. The
commercialization of nano-containing products could perhaps be moving much faster than the
ability for a full safety evaluation. With such quick advances in nanotechnology, it is appalling
how limited knowledge we have about the interaction between nanoparticles and the biological
systems as well as the potential toxic effects on human health. The sunscreen controversy, for
example, could possibly illustrate how important toxicological studies are. In 2006, the United
States Environmental Protection Agency released findings that titanium dioxide nanoparticles
found in sunscreens could cause brain damage in mice due to the production of reactive
oxidative species in the brain causing oxidative stress [4]. Although it was later found that the
toxic effects were only hazardous if the nanoparticles were absorbed into the skin, but the
incident did create a negative public perception of nanoparticles. These nano-scare incidents
create negative perceptions amongst the general public and could make it difficult for future
genuinely safe nano-products to be well received. Toxicological studies therefore need to catch
up with the quickly advancing nanotechnology to ascertain the safety of commercialized nanoproducts and nanomedicine.
Amongst all the nanoparticles developed, metallic nanoparticles have been receiving a lot more
attention due to their unique properties. Metallic nanoparticles possess unique plasmonic and
fluorescent properties from their bulky counterparts, and are being investigated to be used for
numerous biomedical diagnostic or detection technologies such as molecular imaging [5],
biomarkers and biosensors [6]. Metallic nanoparticles are also being developed as potential drug
or gene carriers, because its size scale mimics that of biomolecules and are thought to be able to
move in and out of cells like biomolecules [7]. It is believed that in utilizing the right
10
nanoparticle material, the solubility, bio-distribution, and release of drugs can be improved [8].
In addition, the drug or gene can be effectively delivered to its target [9-10]. As such, metallic
nanoparticles have sparked vast interests of both researchers and medical professionals due to its
potential to be utilized for biomedical technology.
Colloidal gold nanoparticles (AuNPs) in particular have gained the interests of many and are
extensively investigated due to its optical properties and relatively inertness. The use of AuNPs
for colorimetric detection was first developed by Leuvering et al. [11] and has since been
expanded as a passive or active label for the detection of proteins or DNA [12-14]. They can also
be used as a vehicle to deliver molecules into cells, which has great potentials in the area of
cancer therapy [15-17].
It has now been well accepted that when nanoparticles are exposed to a biological environment,
their first point of interaction is with biomolecules such as proteins which can adsorb on the
surface of the nanoparticles [18-25]. The subsequent cellular responses to the nanoparticles will
reflect that of the adsorbed biomolecules rather than the nanoparticles [18, 20, 23]. It is therefore
important to understand how AuNPs interact with the biomolecules and what effects these
interactions could have on the local and overall biological environment. As a result, this project
is aimed towards investigating and evaluating the toxicity of AuNPs.
11
1.2 Objectives
This project is aimed at investigating the interactions between AuNPs and two of the most
abundant proteins in the human plasma, serum albumin and fibrinogen. The investigation
characterized the physical properties of AuNPs, such as size and zeta potential before and after
the protein adsorption, as well as the amount of proteins that adsorb onto the AuNPs. This
information can help to predict where the AuNPs could be located after being placed in an in
vivo environment, as well as the amount of proteins that could be affected by the adsorption of
proteins onto the AuNPs.
This project also investigated the effects of centrifugation (an important equipment when
working with nanoparticles and proteins) on AuNPs as well as on the AuNP-protein conjugate.
The amount of AuNPs or AuNP-protein conjugate which were not sedimented was quantified
and the presence of any aggregation was after centrifugation was qualified.
Lastly, this project also looked into how the adsorption of proteins onto the AuNPs could affect
the conformation of the protein. Any changes in the conformation of the adsorbed protein may
have an effect on the protein function which could result in a negative health effect.
12
Chapter 2 Literature Review
2.1 Overview of nanoparticles
Nanoparticles are already the next big thing in the scientific world and much research is being
poured into the synthesis of nanoparticles of various shapes, sizes and materials. The term
“nanoparticle” is used to refer to particles that have been engineered and which exhibit sizedependent physiochemical properties [2], unlike ultrafine particles which generally refer to
particles that are produced by processes like combustion, welding or from diesel exhausts. As
such, the engineered nanoparticles are usually homogeneous in size and shape and have specific
physical and chemical properties like electrical, catalytic, magnetic, mechanical, thermal or
imaging properties. These engineered nanomaterials can be grouped into four types, polymeric,
metal-based,
carbon-based and
composites. Popular polymeric
nanoparticles
include
poly(ethylene glycol) (PEG), poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) PLA,
poly(D,L-glycolide) PLG and poly(cyanoacrylate) PCA, while metallic nanoparticles of high
interest include gold, silver, titanium dioxide or ferromagnetic nanoparticles. Quantum dots have
also gained popularity in recent years especially in the biological field due to its non-photobleaching optical property and its small size. Carbon based nanoparticles are made up entirely of
carbon and these include carbon nanotubes, nanocrystalline diamond, fullerene among other
structures. Lastly, composites refer to nanoparticles that are combined with other nanomaterials
or other larger materials. This overview will be focused on a three different nanoparticles
(quantum dots, silver nanoparticles and gold nanoparticles), which are of particular interest for
biomedical purposes.
Quantum dots
13
Quantum dots (QDs) refer to nanocrystals which are mainly made up of elements from group II
and group VI to give compounds such as cadmium selenide (CdSe), cadmium telluride (CdTe) or
cadmium sulfide (CdS). These nanocrystals exhibit fluorescent properties and are semiconductors. QDs have radii which are equal to or smaller than the Bohr radius which thus
accounts for its unique optical properties. QDs are considered more superior than conventional
organic dyes like FITC as they have very broad excitation wavelengths which is tunable
according to its size [26-27]. QDs also have narrower emission spectra compared to the organic
dyes allowing for more fluorescent probes to be resolved at the same time. They are more photostable as they are resistant to photo-bleaching, and they are also generally more resistant to
metabolic and chemical degradation, thus allowing for longer term stability. QDs, however, have
two main disadvantages. The first disadvantage is the “blinking” behavior where there are
intermittent short periods of no emission. This is being gradually overcome by a new generation
of QDs that exhibit no “blinking” effect [28-32]. These non-“blinking” QDs include having
alloyed gradient compositions, having an inorganic shell or the coupling of two coloured QDs as
one to eliminate the “blinking” effect. The second disadvantage of QDs is its biocompatibility
and the possibility of heavy metal toxicity. QDs are typically are made up of a core such as CdSe,
and a shell such as ZnS, which has a large spectral band gap. There are two ways to modify the
surface of QDs to make them more biocompatible. The first way is to perform a ligand exchange,
where the hydrophobic ligands on the QDs are exchanged for hydrophilic bi-functional ligands
or polymer molecules with multiple hydrophilic groups. The second way is to encapsulate the
QDs within a polymer such as a micelle without modifying the surface of the QDs [27, 33]. This
is an important step to avoid the heavy metals from leaching out into the biological environment
causing severe toxicity.
14
Perhaps the most obvious usage of QDs is for visualization purposes. This includes techniques
such as florescence multiplexing, single particle tracking in live cells, fluorescence resonance
energy transfer or FRET, and high throughput screening [33-34]. FRET is a phenomenon that
occurs between a donor and an acceptor fluorophore, and QDs serve as an excellent fluorescence
donor in the case of FRET as demonstrated by Lee et al. A QD-dendrimer-AlexaFluor 488
labeled antibody was used to detect for dopamine, where increased dopamine concentration was
reflected by an increase in the degree of energy transfer [35]. QDs can also be tracked and
visualized for up to several hours, making this a powerful advancement in cell biology as
multiple proteins can be labeled and tracked to monitor their interaction or mobility in the
cellular environment. Simon and coworkers have established a method for labeling live cells
with QDs and have observed their stability for up to three weeks. QDs were first conjugated with
antibodies through avidin or protein G as the linker. These bio-conjugates could then selectively
label the parts of the cells that express the protein of interest, and they were then imaged and
tracked for prolonged periods in the live cells [36]. Similar to AgNPs and AuNPs however, few
focused research are being conducted on the negative impact of QDs in a biological environment.
Silver nanoparticles
Silver nanoparticles (AgNPs) garner a lot of interests as it is well known that silver can act as
both an anti-bacterial and anti-microbial agent. The silver ions can cause the disruption of the
peptidoglycan cell wall of the bacteria and also lysis of the cell membrane. The silver ions can
then bind to both the bacterial ribosomes and DNA, preventing protein synthesis and bacterial
replication. Bulk silver has many limitations due to its known toxicity in humans, but with
AgNPs, the large surface to volume ratio has allowed for a much lower quantity of silver to be
15
used, overcoming the limitation. It is therefore not surprising that AgNPs is by far the
nanoparticle with the most commercialized products. These products range from water treatment
and home appliances to medical devices and treatment [37]. The clothing industry for example
has made use of AgNPs as an anti-bacterial agent to prevent odor-forming bacteria. Household
appliances such as plastic food containers, chopping boards, the interior of refrigerators also
contain AgNPs with the pretext that the food can be preserved longer as microorganisms are
prevented from growing [38]. In the medical sector, silver nanocrystals have already been used
for wound dressings for over ten years in products like ActicoatTM. These products make use of
the anti-microbial properties of silver to allow for longer wear times and have been shown to
promote wound healing. Yang and coworkers for example have observed in their clinical setting
that ActicoatTM (containing silver nanocrystals) remained effective for a longer time period as
compared to other wound dressings containing silver sulfadiazine or silver nitrate [39]. Because
of the promising results AgNPs have shown, many clinical trials are in progress for the treatment
of other more serious skin diseases like burnt wounds, toxic epidermal necrolysis (TEN) and
Steven-Johnson syndrome among others [39-41]. AgNPs can also potentially be used in bone
cements as an anti-bacterial agent. These bone cements are used to hold joint prostheses like
knee and hip replacements in place. Initial investigations have shown significant anti-bacterial
activity and slower biofilm growth [42].
The unique anti-bacterial and anti-microbial properties of AgNPs have opened up numerous
opportunities for its usage and development. The question of concern is then the level of toxicity
it induces and if the toxicity caused is tolerable in the face of its merits. It is known that silver in
its elemental form can cause a high level of toxicity in large doses. A clinical trial in 1998 using
a silver coated silicone heart valve was discontinued after findings that the silver coating affected
16
the normal fibroblast functions [43]. Little is however known about the severity of toxicity that
AgNPs can cause as there have been no definitive reports on this topic.
Gold nanoparticles(Au Ps)
Gold is the most noble of metals and does not tarnish when it is exposed to air as it is relatively
inert and stable. This inert property thus gave very few opportunities for chemists to exploit until
new observation was made that nano-gold, unlike its bulk properties, is extremely reactive.
AuNPs have been used as early as the Vedic period in ancient India for medicinal purposes [44],
and have also been used for a wide variety of other purposes like decoration, Roman glass
making, and so on. Faraday was perhaps the first to present a report on AuNPs in 1857 where he
reported that gold was present in the solution in a “finely divided metallic state” [44]. Following
his report, a steady growth in gold nanotechnology was seen. Another distinguishing journal in
the field of AuNPs was perhaps by Turkevich [45] in 1951, for his report on the synthesis of
colloidal gold which is still being followed now.
AuNPs are generally less reactive and do not oxidize when left exposed to air unlike other metal
nanoparticles. The tunable optical properties based on shape, size and the environment make
AuNPs the obvious choice for applications in the biomedical field. A distinct characteristic of
AuNPs is the strong surface plasmon resonance (SPR). This is caused as a result of light
absorption by the AuNPs due to the coherent oscillation of the conduction band electrons which
is induced by interaction with the electromagnetic field [46]. This optical response of the
nanoparticles can be tuned by the size and shape of the nanoparticles as well as the
environmental conditions the experiment is conducted in. This gives scientists great flexibility
since the size and shape of the AuNPs can be easily manipulated with improved synthesis
17
methods. Synthesis of AuNPs from as small as less than 2 nm to sizes as large as 100nm [45, 4751] and synthesis of gold nanorods of different aspect ratios, nanoplates, nanostars and even
popcorn shaped AuNPs [52-55] have been well developed or are being developed. On top of that,
AuNPs can also be easily functionalized. Scientists can therefore choose almost any type of
AuNPs suitable for their intended application. The only other metal nanoparticle that can
remotely match these advantageous properties of AuNPs is silver nanoparticles.
The main applications of AuNPs in the biomedical field are diagnostics, targeted delivery of
drugs, although polymeric nanoparticles are still the more popular choice for drug delivery and
bio-sensing. These applications arise from AuNPs’ high density, large dielectric constant,
biocompatibility and optical properties [10]. AuNPs are used to label biomolecules such as DNA,
proteins or peptides, or sub-cellular structures for imaging. Labeling can be easily achieved
through surface modifications of AuNPs using thiol, dithiol or amino groups. There are three
ways AuNPs can be utilized in bio-sensing, the first is to detect for aggregation of nanoparticles
after being exposed to biomolecules, the second is to detect the binding of a bio-molecule to the
nanoparticle by measuring a shift in the surface plasmon absorption peak, and lastly to use the
light scattering properties of the nanoparticles for the labeling of biomolecules for imaging.
For AuNPs to be successfully used in the biomedical field, several factors like the size and shape
and surface functionalization need to be suitable in order that the AuNPs can be taken up by the
cells. It has been reported by Chan et al. that under in vitro conditions, 50nm spherical shaped
AuNPs have the greatest uptake as compared to other spherical AuNPs between 14 to 100nm and
nanorods of different aspect ratios [56]. AuNPs have also been conjugated with biomolecules
like proteins or cell penetrating peptides which help AuNPs be internalized more easily. TAT
peptide is a commonly used sequence to enable the AuNPs to be efficiently delivered into the
18
nucleus of the cell [57]. Other important aspects of investigation are the sub-cellular localization
of the AuNPs in vitro and the organ distribution of AuNPs in vivo. These are both for the
purpose of targeted delivery of AuNPs as well as to determine possible toxicity of AuNPs. It has
been found that the organ distribution of AuNPs after an intravenous administration is dependent
on the particle size with small sized particles 10 to 15nm having the widest organ distribution
[58-59].
2.2 Toxicity of nanoparticles
As mentioned in the previous section, nanoparticles are being developed for biomedical purposes,
but there is still a lack of knowledge on the toxicity of these nanoparticles being used. The term
nanotoxicology has been coined to refer to the study of the effects of nanoparticles on living
organisms, and it involves the investigation of properties like the particle size, surface area,
reactivity, dose, and potential toxicity. It has been described as a multidisciplinary science which
spans across chemistry, physics, material science and medicine [60]. This concern for the
potential toxicity of nanoparticles arose from the small particle size and from two independent
findings stating that toxicity of the particles increases as the size becomes smaller. One of the
first studies which indicated the potential toxicity of nanoparticles was by Ferin and coworkers
who worked with titanium dioxide nanoparticles (TiO2) on rat models. They demonstrated that
rats exposed to TiO2 nanoparticles exhibited inflammation in the lungs and that the nanoparticles
had retained in the lungs almost 3 times longer when compared to fine particles. This finding
was followed by a second independent study by Oberdöster et al. who also found increased
inflammation in the lungs when rats were exposed to small sized TiO2 nanoparticles. In addition,
they found that nanoparticles could enter the interstitium more easily compared to fine particles.
19
Oberdöster also suggested that the increased inflammation could be due to the larger surface area
of the nanoparticles and their interaction with the alveolar macrophages and interstitial cells [61].
Following these two studies, nanotoxicology became an area of interest for many researchers.
There have been two main types of toxicity studies commonly carried out. The first is an end
point in vitro study to determine if a particular type and size of nanoparticles causes cell death or
inhibits cell growth, while the second is in vivo studies to examine the distribution of the
nanoparticles in the tissues.
The investigation of the distribution of nanoparticles in tissues is crucial as it reveals which
organs are most susceptible to exposure or accumulation of the nanoparticles which can result in
toxicity effects. The route of the uptake or exposure to the nanoparticles and how they are then
circulated also give scientists a better idea if they are to be used for biomedical purposes. For
example, nanoparticles that are injected could circulate differently from nanoparticles that are
ingested or inhaled. It has been consistently found that small sized nanoparticles when injected
intravenously have greater circulation as compared to larger nanoparticles, and the highest
accumulation of nanoparticles was found in the liver and the spleen [58-59, 62-63]. Sonavane et
al. for example used AuNPs sizes of 15, 50, 100 and 200nm, and found that the 15 and 50nm
AuNPs had higher distribution and were found to have crossed the blood brain barrier as well.
The 200nm AuNPs on the other hand had little translocation [59]. Cho et al. have also found that
not only do the AuNPs accumulate in the liver and spleen, but they are found to be in resident
macrophage populations, indicating possibility of inflammatory responses [63]. These evidences
were supported by other groups where it was found that larger particles were limited in their
distribution as they were mostly taken up into macrophages while smaller particles could evade
recognition and hence were more widely distributed.
20
In vitro investigations are usually focused on one type of cell and the associated effects of
nanoparticles on the particular cell type. This can help to provide a more fundamental
understanding of how some tissue types may be affected by the presence of nanoparticles. Using
a high dosage of nanoparticles on cell models allows for the finding of regulatory toxicity and
threshold markers which can be used to determine the toxic potential of the nanoparticles [64].
By far, AgNPs has the highest number of publications for toxicity studies since it is already
being used in commercial products.
Some basic cell types that research groups tend to focus on includes lung cells, skin cells and
liver cells. Research groups focusing on the lung model research often aim to evaluate how
epithelial cells along the airways or alveoli or macrophages will respond as these are the cells
most likely to be exposed to the nanoparticles. However, there is still little information in this
area, hence no definitive conclusion can be made. AshaRani et al. worked with starch coated
AgNPs and studied its toxicity to normal human lung fibroblast cells and human glioblastoma
cells. They found that there was increased reactive oxygen species (ROS), mitrochondrial and
DNA damage in the cells depending on the concentration of AgNPs. The authors found that the
AgNPs caused a toxic chain reaction starting with the disruption of mitochondrial respiratory
chain which causes production of ROS and disruption of ATP synthesis, and these in turn led to
DNA damage [65]. Oxidative stress of alveolar macrophages when exposed to AgNPs was also
found by Calson et al. [66]. A study on different cell lines exhibiting different sensitivity when
exposed to nanoparticles was reported by Soto and coworkers. They also found that there was a
general decline in cell viability when the alveolar macrophages and lung epithelial cells were
exposed to AgNPs [67]. Many of these findings for the lung model were also reflected in other
cell models. For example, it was also found that there was increased ROS product when a liver
21
epithelial cell line was exposed to AgNPs, resulting in a decrease in cell viability [68]. An
interesting study done by Nel et al. found that the AgNPs induced only low levels of oxidative
stress which resulted in activation of protective mechanisms instead of inflammatory responses
or cell death which is usually caused by high levels of oxidative stress. The group concluded that
AgNPs were therefore not toxic, but rather they stimulated protective responses.
The investigation results shown here are generally reflective of all toxicity studies where
contradicting results reported by different groups could likely be due to the experimental design.
With the fast advancing development of nanomaterials, the toxicological information we have
are still far too scattered and insufficient because there are so many cell types and different
nanomaterials to evaluate. Not only is there a need for more toxicity studies, but the development
and implementation of nanotoxicological protocols to effectively and thoroughly evaluate
nanomaterials is also required. With the accumulation of information, an online collective
database for the results obtained could be set up to collate the information for more effective and
efficient progress in this area.
Apart from determining the toxicity of nanoparticles, there is also a need to get back to the basics
and really understand the reason for any observed toxicity. Nanoparticle interaction with proteins
is a good starting point. As nanoparticles enter a biological environment, their first contact is
with biomolecules, which are more often than not, proteins. It has been shown and there is
growing acceptance that proteins adsorb onto the nanoparticles forming a ‘protein corona’ on the
nanoparticles. This term ‘protein corona’ was first coined by Dawson et al. to describe the ring of
proteins adsorbed around a nanoparticle [19].The type of proteins adsorbed determined the new
identity of the nanoparticles in the biological environment, and the nanoparticles are recognized
by the cells based on this protein corona. Subsequent particle distribution and translocation also
22
depended on the proteins adsorbed on them. A starting point could then be to identify the
proteins that will associate with the particular nanoparticle. This will require information like
binding affinities and stoichiometries, because proteins adsorbed on the particles tend to replace
each other over time due to the Vroman effect, where proteins which move faster will adsorb
first but are subsequently replaced by slower moving proteins with higher affinity for the
nanoparticle.
It was found that nanoparticles with different surface properties attract the binding of different
type of proteins. Nanoparticles with hydrophobic surfaces such as polymeric nanoparticles tend
to attract apolipoproteins as well as albumins. In contrast, inorganic nanoparticles with
hydrophilic surfaces tend to attract proteins such as transferrin or histidine-rich glycoproteins,
which have positively charged domains. A commonly reported phenomenon is that nanoparticles
with hydrophobic surfaces tend to bind more proteins as compared to nanoparticles with
hydrophilic surfaces [69]. Apart from hydrophobicity of the nanoparticle surface, the
nanoparticle surface charge, size as well as curvature are also important factors in determining
protein binding. Gessner et al. for example observed increasing plasma protein adsorption with
increasing surface charge density on the nanoparticles [70]. This makes surface modification and
characterization of nanoparticles an important step in tuning the particles for a specific use. A
separate study made an interesting observation that smaller sized gold nanoparticles of 30 nm
were found to attract more protein binding as compared to their larger counterparts of 50 nm [71].
This is perhaps due to the increased surface area available for binding when particle size is
decreased. A similar observation was made by Lynch et al. when a polymeric nanoparticle was
used [72].
23
This interaction of proteins with nanoparticles is a growing field of interest because adsorption of
the nanoparticles can cause conformational changes, exposure of new epitopes, function
impairment or avidity effects due to close proximity of the same proteins [73]. For example, if
adsorption of fibrinogen on nanoparticles prevents it from carrying out its function, blood
clotting will be inhibited and this could be fatal if the patient has a huge open wound. There has
been little research in this area of protein-nanoparticle interaction partly because there are limited
tools for the analysis of proteins. Tools like circular dichroism and fourier-transform infrared
spectrometry only give very limited information about the conformation of the proteins while
other tools like x-ray crystallography and nuclear magnetic resonance require high expertise and
are time consuming. In addition, x-ray crystallography is not suitable for all samples as it is
dependent upon the sample forming a single crystal. Functional tests are protein dependent and it
could be difficult or expensive to design. Stone and coworkers investigated the interaction
between carbon black nanoparticles and cytokine proteins and how the protein function was
impacted. It was observed that the smaller sized nanoparticles of 14nm caused a decrease in
TNF-α protein function as compared to the larger 260nm microparticles [74]. Many other groups
however only investigated and reported the conformational changes of the protein without
relating it to how the function of the proteins is affected. Gao et al. for example, investigated the
effects of silicon dioxide nanoparticles on cytochrome c, deoxyribonuclease and hemoglobin. It
was found that the secondary structures of the proteins were changed by the presence of the
nanoparticles [75]. Similarly, Roy et al. found that the interaction between AgNPs and lysozyme
resulted in partial unfolding of the α-helix of the protein [76]. However, in both cases, it was not
known how the change in protein conformation affected the function of the proteins. Results of
these changes in protein conformation without the knowledge of how the change affects the
24
function of the protein are not very useful if significant progress is to be made in this area. Few
reports have investigated both these effects together, and this is the gap which needs to be filled.
2.3 Conjugation, size and zeta potential studies
2.3.1 UV-vis spectrometry
UV-vis spectrometry works on the principle that different molecules will absorb ultraviolet (UV)
or visible light of different wavelengths. The UV region is in the range of 190-380nm and the
visible region between 380-750nm. When light energy is absorbed, it can promote an electron
from ground state to a higher orbital. The most energetically favourable transition is for an
electron to move from the highest occupied molecular orbital (HOMO) to the lowest unoccupied
molecular orbital (LUMO). Four electronic transitions are possible, pi to pi*, n to pi*, σ to σ*
and n to σ*. For the UV-vis region, only pi to pi* and n to pi* transitions can be observed as σ to
σ* and n to σ* transitions do not fall within this region.
The working principle of a UV-vis spectrometer is fairly simple; a beam of visible or UV light is
diffracted into monochromatic beams, which are then split into two beams of equal intensity.
One half of the beam will pass through the reference sample while the other half passes through
the sample of interest. The absorption wavelengths and their respective intensities are measured
and compared, and the results are displayed in the form of a graph where the y-axis is the
absorption intensity and the x-axis is the wavelength. One can than deduce the type of structural
groups present in the molecule of interest from the absorption spectrum obtained.
UV-vis spectrometer follows Beer-Lambert’s law which states that A = εbc, where A is the
absorbance with no units, ε is molar absorptivity with units of L mol-1 cm-1, b is the path length
25
of the cuvette the sample is placed in for measurement with units of cm, and c is the
concentration of the sample with units of mol L-1. ε can be defined as the amount of light
absorbed per unit concentration and is a constant value for each specific sample for fixed
concentration. Using Beer’s law, the molar absorptivity of a sample can be calculated from the
measurement taken.
UV-vis spectrometry is not only useful for the determination of the absorptive properties of
molecules, but it can also be used to prove interactions between molecules such as proteins and
nanoparticles by observing at the shifts in the absorbance peak and the intensity of absorbance
[77-79]. For example, AuNPs of 20nm in size exhibit an absorbance peak at 520nm. When it is
successfully conjugated with a protein like bovine serum albumin (BSA), the absorbance peak is
blue-shifted to 525nm. UV-vis spectrometry is thus a useful tool for a first pass analysis since the
measurement is simple and fast and requires no sample preparation for clear liquid samples.
2.3.2 Transmission electron microscopy
Transmission electron microscopy (TEM) differs from light microscopy in that it utilizes a high
energy electron beam instead of light, but both techniques work on the same basic principles.
Light microscopy is limited in their magnification due to the wavelength of light. TEM on the
other hand can allow for atomic scale resolution or samples as small as 10-10m to be visualized as
electrons have a much lower wavelength.
Electrons are emitted from a light source at the top of the microscope, and they travel through a
vacuum column before reaching the electromagnetic lenses which focus the electrons into a thin
beam. The thin beam of electrons then passes through the sample and some electrons are
removed from the beam as they are scattered by the sample. The remainder of the electron beam
26
travels through the sample to a fluorescent screen giving the shadow image of the sample which
can be photographed with a camera. The sample image is a result of an interference pattern
between the transmitted and the diffracted electron beam. The amount of electrons that passes
through the sample is in direct relation with its density. This allows for details such as defects,
grain boundaries or interfaces of the sample image to be visualized as the image varies in
darkness according to the density of the sample at each point.
As powerful as TEM may be, the sample preparation is often a point of limitation especially
when dealing with biological samples. It requires more time and technique as compared to other
optical microscopy. The thickness of TEM samples need to be comparable to the mean free path
of electrons travelling through them. This is often only a few tens or hundreds of nanometers
thick. Sample preparation is specific to the type of sample and the information that is to be
obtained from it. There are several sample preparation techniques such as direct deposition on a
thin TEM substrate such as a copper grid, chemical etching, lithography, electro-polishing and
focused ion beam. Preparation of biological samples is more complicated. They first need to be
fixed using chemicals like formaldehyde or glutaraldehyde before being taken for sectioning
using an ultra-microtome which requires experience and technique. The thinly cut samples can
then be taken for TEM imaging. Imaging of the TEM samples can sometimes be challenging as
well because artifacts need to be identified and separated out from the desired information. This
often requires an experienced eye. These artifacts could have been introduced during the sample
preparation process or it could have resulted when the original form of the sample was changed.
In my investigation, TEM was used to determine the size and consistency of the synthesized
nanoparticles. Aggregated samples can be clearly distinguished from well dispersed single
particles. The results obtained were compared to those obtained from dynamic light scattering
27
(DLS). As TEM measurements do not include surface structures like citrate caps on AuNPs,
while DLS measures the hydrodynamic radius of the nanoparticle, the results obtained from both
techniques are expected to differ up to several nanometers depending on the type of surface
structures. A few nanometers allowance thus has to be given to compensate for the difference in
measuring principle between the two techniques.
2.3.3 Dynamic light scattering
Dynamic light scattering (DLS) is based on two assumptions, the first being that the particles
follow Brownian motion or random walk and the second is that the particles are all spherical.
Brownian motion refers to the random movement of particles which occurs due to the collision
between particles and solvent molecules in the solution. The size of the particles can be
correlated based on the speed of their movement as larger particles move slower while smaller
particles have faster motion. Particles in solution are generally not spherical as it is dynamic and
solvated. Since DLS assumes spherical shaped particles, it thus measures the apparent size of the
particle or a hypothetical hard sphere which is known as the hydrodynamic radius.
According to the semi-classical light scattering theory, the electric field of light causes the
electrons in the particles or molecules to undergo oscillating polarization known as a Doppler
shift when light hits on moving molecules. This causes the wavelength of the initial light source
to be changed resulting in the particles or molecules giving off a secondary source of light and
the scattering of light. Different velocities from particles result in different amount of scattered
light. The change in light intensity can then be correlated to the size of the particle through the
Stokes-Einstein equation: ˞ =
, where ˞ is the hydrodynamic radius of the particle, D is
28
the diffusion coefficient, k is the Botzmann’s constant and η is viscosity. An optical detector is
used to detect the rate of fluctuation of the intensity of scattered light due to moving particles.
A popular DLS machine used is the Zetasizer from Malvern, which essentially consists of two
parallel collimator lenses where the sample solution can be placed between the two lenses, and a
photomultiplier detector. The first collimator lens helps to focus the light beam to the sample
while the second lens helps to filter the amount of scattered light passing through to the
photomultiplier. A laser source is used to pass from one end through the lens and sample, and the
light that is scattered by the sample is detected by a photomultiplier placed at 90 degrees on the
other end. The photomultiplier then converts the light intensity signal into a voltage signal which
is amplified and sent to the computer.
Several factors need to be ascertained prior to the measurement such as the viscosity of the
solvent, as it affects the movement of the particles in it, and the temperature at the time of
measurement as that relates to the viscosity. Other properties which can also affect the accuracy
of results obtained include the ionic strength of the medium, the surface structure of the particles
and particles which are not spherical. Ions in the medium can change the thickness of the electric
double layer around the particles, hence low conducting medium will tend to give a larger
hydrodynamic medium as it forms a thicker double layer around the particles and a highly
conducting medium will give a smaller measured hydrodynamic radius. Surface structures that
protrude out from the particle will result in slower diffusion speed and result in a larger measured
hydrodynamic radius as compared to surface structures that are more compact. As DLS measures
the radius of a hypothetical hard sphere, the measured hydrodynamic radius will tend to reflect
that of the length of the elongated particles rather than the true radius or size.
29
DLS is a well established tool that has been widely used not only for size measurements.
Because of its sensitivity, it can also be used to track changes in the size of particles, and is
therefore useful for the confirmation of the conjugation of molecules or aggregation of particles.
In my investigation, DLS was used to determine coagulation of nanoparticles after centrifugation
as well as aggregation or changes in conjugation after centrifugation of nanoparticle-protein
conjugates. Similar to UV-vis spectrometry, DLS was used to confirm the conjugation between
nanoparticles and proteins and also to measure the size of the synthesized nanoparticles.
2.4 Centrifugation studies
2.4.1 Inductively coupled plasma mass spectrometry
ICP-MS is a spectrometric technique that allows for the detection and quantification of trace
amounts of most of the elements in the periodic table. It is highly sensitive and can detect
concentrations up to the part-per-trillion level. This technique was first introduced in 1983 and
uses high temperature inductively coupled plasma to cause the conversion of atoms into ions
which are then detected by a mass spectrometer. There are six main components which the
sample must go through before a readable signal can be obtained in the computer. The six steps
are sample aerosolization, sample ionization, ion extraction, ion focusing and transmission, ion
separation and signal amplification and counting.
Sample aerosolization
The analysis first begins by introducing the sample into the system. This is achieved by a
peristaltic pump to transfer the sample into a nebulizer which then generates an aerosol to ensure
30
that the sample is efficiently ionized. There are several types of nebulizer such as thermal
nebulizers, hydraulic nebulizers, ultrasonic nebulizers and pneumatic nebulizers with the latter
being the most popular. The pneumatic nebulizer works by using the kinetic energy from a high
velocity gas flow to break the liquid sample up into small droplets. There are two main
configurations for the nebulizer, the cross flow and the concentric nebulizers. The liquid and gas
interacts perpendicularly in the cross flow configuration while for the concentric configuration,
the liquid and the gas interacts concentrically [80]. The cross flow configuration can be used for
samples containing particulates because the distance between the liquid sample and argon gas
tubes are further apart and this helps to reduce clogging problems. However, it is also because of
this distance that makes the cross flow less effective in producing the aerosol. The concentric
configuration demands a cleaner sample, but is more stable and effective in producing the
aerosol [81].
After the aerosol is produced, the droplets are selected in a spray chamber to allow only the small
droplets to proceed to the next phase of the analysis and also to allow a smooth continuous flow
of droplets. There are three types of spray chambers: the double pass, cyclonic and direct spray
chamber with the double pass being the most popular. The horizontal double pass spray chamber
selects the droplets by means of gravity where only the droplets small enough can pass through
the chamber while large droplets fall out by gravity and are collected through a drain tube. The
cyclonic spray chamber on the other uses centrifugal force to select the droplets, where the
tangential flow of the aerosol and the argon gas into the chamber creates a vortex. Large droplets
fall out into the drain tube while the small droplets are carried with the gas stream into the
plasma. The double pass spray chamber is preferred for its higher precision in droplet selection,
however the cyclonic spray chamber has higher sensitivity and lower detection limits.
31
Sample ionization
The heart of the ICP-MS system is the plasma which causes the ionization of elements. The
preferred plasma used for most systems now is the inductively coupled plasma, although the
direct current plasma (DIP) and the microwave-induced plasma (MIP) were used in the early
days. The DIP and the MIP however faced problems such as interference and matrix effects
respectively and were eventually dropped from being used for optical emission and mass
spectrometric studies. The inductively coupled plasma torch is made up of three concentric tubes
made from quartz and has one end placed in the middle of a radio frequency coil which is
coupled to a radio frequency power supply. Three different gases flow through the three tubes,
with the plasma gas, usually argon, flowing through the outermost tube, the auxiliary gas through
the middle tube, and the nebulizer gas carrying the aerosolized sample from the spray chamber
flowing through the innermost tube. An illustration of this setup can be seen in Figure 1.
Figure 1 Illustration of a plasma torch with three concentric tubes and the gases that flow
through them. [81]
32
An inductively coupled plasma discharge needs to be formed before the sample can be ionized.
The RF power is first applied to the RF coil which generates an alternating current creating an
electromagnetic field around the coil. The argon gas is allowed to flow between the outer and
middle tube as shown in Figure 1and a high voltage spark is applied to the gas. This causes some
electrons to be removed from their argon atom which then collides with other atoms to form
more ions. An inductively coupled plasma discharge consisting of argon atoms, argon ions and
electrons is eventually formed. This plasma has four heating regions of different temperature
with the highest temperature reached to be 10000K which is about twice the temperature of the
sun. The aerosol sample that passes through this plasma torch undergoes four physical changes
to become an ion. The sample first undergoes desolvation to strip off the water content before
being vaporized into a gas. It is then atomized and finally ionized by the removal of an electron.
The sample emerges as an ion and is directed into the interface for extraction of the ion.
Ion extraction
This interface is a very important part of the ICP-MS as it connects the sample from the plasma
to the mass spectrometer analyzer. The sample exits from the plasma at very high temperatures
and atmospheric pressure, and the interface has to bring the sample down to near room
temperature and vacuum pressure. The interface is able to achieve this task with two or three
cones of very small orifices. The cones are made of materials such as platinum or nickel, which
has to be tolerable of strong acids since the samples are usually digested with acids prior to the
analysis. Traditionally, two cones are used with the first cone known as the sampler cone. It is in
direct contact with the plasma, has a wider orifice diameter and operates at atmospheric pressure.
The second cone is called the skimmer cone. It has a narrower orifice diameter and operates at
33
very low pressure [81-83]. The two cone interface allows only for a two-step pressure reduction
causing divergence of the ions, hence requiring ion focusing. However, newer designs have
included a three cone configuration, allowing for a three-step pressure reduction. This prevents
substantial divergence of the ion beam and hence the ion focusing lens can be completely
removed. This design also gives better stability in the long term and lower maintenance costs for
the machine. The interface housing is also water cooled and made of either cooper or aluminum
as they can release heat quickly [81]. For the two cones system, the sample ions will move from
the interface towards the optical lens which will focus the ion beam and bring them from the
interface to the mass analyzer.
Ion focusing and transmission
The ion focusing lenses are located between the skimmer cone and the mass analyzer and it is
usually made up of metal plates or rings each carrying a specific voltage. The lenses bring the
ion beam from the interface to the mass analyzer and must be well designed to give low
background noise and in turn a good signal and low detection limit. Millions of ions are
generated in the plasma torch, but only about one out of half a million enters the mass analyzer
[83]. The role of the ion lens is then to ensure that the maximum number of ions can reach the
analyzer for detection and counting. One of the reasons for low efficiency of the ions transmitted
is due to the “space charge” effect where positive ions mutually repel each other hence reducing
the number of ions that can be compressed in the ion beam. Lighter ions are generally lost as
they are deflected away by the heavier ions. This problem is exacerbated by ionized elements
from the salts in the buffer which could be heavier than the analyte ions, resulting in fewer
analyte ions passing through to the analyzer. Another important function of the lenses is that it
34
helps to sort out the ions from particulates, photons and neutral species which could give an
erroneous signal or cause instability if allowed to enter the mass analyzer. The photons and
neutral species in particular cause high background noise and hence affect the detection limit of
the ICP-MS. The particulates on the other hand will tend to deposit on the components in the
machine and cause instability of the system. There are two different ways to get around this
problem. The first is to place a grounded metal plate or disc after the skimmer cone which blocks
the non-ionic species from travelling further. The ions are however able to move around it with
the help of the ion focusing lens. The second method is to place the ion lens off axis or at right
angles to the interface. The non-ionic species coming from the interface will move straight pass
the lenses while the ions will be turned by the ion lenses into the mass analyzer [81].
Ion separation and signal amplification
Once the ions enter the mass analyzer, the singly charged ion of interest is separated from the
rest based on their mass. The mass analyzer is positioned after the ion lens and is maintained at a
vacuum as mentioned previously. There are three commercially available mass analyzers:
quadrupole, time-of-flight (TOF) and magnetic sector, with the quadrupole being the most
commonly used mass analyzer for ICP-MS. The quadrupole was developed in the early 1980s
and consists of four cylindrical metal rods of the same dimensions that are perched parallel to
and at equal distance from the ion beam coming from the ion lens. Two of the rods opposite each
other are connected to a direct current while the other two are connected to a radio frequency
field. The ions coming from the ion lens are allowed to pass through the centre of the quadrupole.
The voltage and radio frequencies applied cause the ions to oscillate, and the magnitude of the
oscillations depends on the mass to charge ratios of the ions. The voltage and radio frequencies
35
can be tuned to select the desired mass to charge ratio. Ions with different mass to charge ratios
exhibit extreme oscillations which will cause them to fall out of the quadrupole, and only the
desired ions will pass through the quadrupole to be counted [82]. As the quadrupole has a very
high scanning rate of more than 5000 atomic mass units (amu) per second, it is able to analyze
many different elements at one time.
Ion counting
Ions that pass through the quadrupole reaches the ion counter and each ion is converted to a
discrete electrical pulse. The number of pulses counted is in direct relation to the number of ions
counted and this can be converted into an absolute concentration by comparing with the
calibration samples measured [82]. ICP-MS is generally used for ultra trace analysis and
therefore uses electron multipliers as the ion detector. Faraday collectors can however be used
for samples with high count rates. The two main electron multipliers used for ICP-MS are the
channel electron multiplier and discrete dynode electron multiplier. The channel electron
multiplier is of a cone shape and the inside of the cone is coated with a semi-conductor material.
The analyte ions enter from one end of the cone which is biased at a negative potential, while the
far end is grounded. When the positive analyte ions enter the cone, they are attracted to the
surface of the cone due to the negative potential. When the ions hit the surface of the cone,
secondary electrons are produced. These electrons will travel further down the cone hitting the
surface as they move along, producing even more electrons. The electrons eventually reach a
preamplifier which detects the electrons and translate it into a pulse which will be counted if the
signal of the pulse is above a certain threshold. This is to distinguish between background level
and the analyte ions of interest [81].
36
The discrete dynode electron multiplier works in a similar manner to the channel electron
multiplier. It uses discrete dynodes for electron multiplication instead of the cone channel, and it
is positioned off centre from the quadrupole. When the ions leave the quadrupole, it follows a
curved path to reach the first dynode. This setup allows for reduced background noise from nonion species and stray radiation. When the ion hits the surface of the dynode, secondary electrons
are produced and these electrons move to hit the next dynode producing more electrons. This
process repeats at each dynode to produce a pulse of electrons which is measured by the
multiplier collector or anode at the end. This discrete dynode electron multiplier is generally
preferred over the channel electron multiplier as it is more sensitive with higher detection
efficiency and lower background feedback [81-82].
2.5 Protein quantification study
2.5.1 Quantification assays
No single protein quantification assay can give an absolutely accurate measurement as each
method has their advantage and limitations. The more commonly used protein quantification
techniques include Bradford assay, bicichininic acid (BCA) assay, Lowry assay and sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Selecting a suitable assay
depends on factors such protein composition, buffer compatibility and presence of detergents or
other compounds that could interfere with the assay.
The Bradford assay [84] is a colorimetric assay and utilizes Coomassie brilliant blue G-250 dye
(CBBG) (structure shown in Figure 2) which binds to the proteins at arginine, tryptophan,
tyrosine, histidine and phenylalanine residues [85]. Under acidic conditions, the normally blue
37
dye turns into its red form and ionizes the proteins, disrupting the protein conformation and
exposing the hydrophobic parts of the proteins. These hydrophobic parts then bind to the nonpolar regions of the dye through Van der Waals forces, while the amine groups on the proteins
form ionic bonds with the dye. Both the hydrophobic and ionic interactions between the proteins
and CBBG stabilize the blue form of the dye, and the amount of the protein-CBBG complex
present can be used to determine the protein concentration by measuring the absorbance
maximum at 595nm. These absorbance measurements can be read using a spectrophotometer,
and the conjugation between the protein and CBBG can be tracked through observing the
shifting of the absorbance peak, as proteins have maximum absorbance at 280nm, while the free
dye at 470nm [86]. Bradford assay is suitable for protein concentrations between 1 to 20ug when
the micro assay is used, or for protein concentrations between 20 to 100ug when the macro assay
is used. Buffer blanks and standard curves need to be performed each time this method is used
for quantification and the measurements have to be taken quickly after the reagents are added as
the assay has a short linear range for the binding response due to precipitation of the protein
caused by the reagents. Bovine serum albumin (BSA) is the most common protein used as the
standard while some others prefer to use immunoglobulin G (IgG) or lysozyme depending on the
sample which is being measured.
38
Figure 2 Structure of coomass
coomassie brilliant blue G-250 dye
The Lowry assay [87] is suitable for protein concentrations of 10 to 1000µg/mL under alkaline
conditions. This assay consists of two reaction steps. The first step involves the reduction of Cu
(II) to Cu(I) by forming
ming a complex with the peptide bonds of the proteins which is called a Biuret
chromophore which can be stabilized by tartrate. Next, the Cu (I) together with the tyrosine,
tryptophan and cysteine side chains react with the Folin reagent to give an initial unstable
product that is slowly reduced to molybdenum/tungsten blue [85-86].. The concentration of
protein is proportional
ional to the reduced Folin rea
reagent
gent and can be found by measuring the intensity
readings over the range of 500 to 750nm wavelength. This assay however has a major limitation,
and that is it can be interfered by many compounds such as detergents, strong acids or buffers,
agents which chelate
late or reduce the copper among others.
An improved and more reliable assay which was modified from the lowry assay is the BCA
assay [88]. The BCA assay, similarly, works on a colorimetric technique where the colour
changes from green to purple according to the concentration of protein present. The
measurement is taken under alkaline conditions. The BCA assay consists of 5 chemicals,
39
bicinchoninic acid, sodium carbonate, sodium bicarbonate, sodium tartate and cupric sulfate
pentahydrate. Copper (Cu) (II) ions from the cupric sulfate are reduced to Cu (I) ions by the
peptide bonds in the proteins according to the concentration of the proteins in the sample. The
reduced Cu (I) ions chelate with two bicichoninic acid molecules forming a purple-coloured
complex absorbing light at 562nm. This complex is aided by the cysteine, cystine, tyrosine and
tryptophan side chains. The sensitivity of BCA assay can be increased by performing the assay at
higher temperatures of 37˚C to 60˚C. This is because at higher temperatures, the peptide bonds
help to contribute to the colour changes.[89]. Variation caused by different amino acid
composition in the proteins is also reduced when the temperature is elevated, and reaction is
thought to be complete if the assay is incubated at 60˚C, as the absorbance does not increase
much after that. BCA assay is preferred over the Lowry assay as it is not affected by the presence
of buffers, contaminants and detergents as much as the Folin-Ciocalteu reagent in the Lowry
assay. In the BCA assay, Bicinchoninic acid is used to chelate with the Cu (I) ions instead of the
Folin reagent. This is advantageous as this complex is more stable under alkaline conditions.
2.5.2 Gel electrophoresis
The concept of electrophoresis was introduced as early as 1791 by Michael Faraday. The initial
applications for electrophoresis were mainly for small ions or molecules which were then slowly
expanded to include proteins over the nineteenth century. Moving boundary electrophoresis was
amongst the first significant development in the field followed by zone electrophoresis after the
1950s. It was then followed by displacement electrophoresis and eventually led to the
development of the isoelectric focusing and two-dimensional gel electrophoresis methods in the
1960s to 1970s. Initial electrophoresis experiments used gelatin and agar gels, and many
40
applications with agarose gels were developed. These included separation of serum proteins for
diagnostics, quantification methods and immune-electrophoresis [90]. It was not until 1959 that
electrophoresis using polyacrylamide gels were reported [91], and an important publication using
SDS-PAGE technique appeared in the 1967 [92]. These methods have now become some of the
most widely used analytical and preparative techniques which are second nature to researchers in
the biomedical field working with proteins, DNA and other biomolecules [93].
Electrophoresis involves the movement of charged molecules in a medium where an electric
field is applied between an anode and a cathode. The most common medium used for the
separation of biological molecules such as deoxyribonucleic acid, ribonucleic acid or proteins is
gel, which acts as a sieve and an anti-convective medium during electrophoresis. The movement
of molecules in the gel is impeded by the gel either due to its size or charge, hence allowing for
the separation of the molecules. The gel also acts as an anti-convective medium as it suppresses
the thermal convection generated by the electric field which allows the molecules to be preserved
for subsequent analysis. These properties of the gel therefore allow the gel electrophoresis
technique to be used for both analytical and preparative purposes.
Apart from typical protein quantification assays as mentioned above, gel electrophoresis can also
be used as an alternative method for protein determination, especially when experimental
materials exhibit optical properties which interfere with the spectrophotometer used in the
traditional quantification assays. Proteins are usually analyzed either by SDS-PAGE which
denatures the sample or native gel electrophoresis which allows the sample to be used for
subsequent experiments.
Conducting a SDS-PAGE experiment generally consist of five parts – gel preparation, sample
preparation, running of the gel, processing and visualization and lastly the gel analysis. Gel
41
preparation require chemicals like Tris, bis/acrylamide, SDS, ammonium persulfate (APS) and
tetramethylethylenediamine (TEMED), while the sample is prepared using the laemmli buffer
consisting of tris/HCl of pH6.8, SDS, glycerol, beta-mercaptoethanol and bromophenol blue.
Laemmli buffer is added to the samples and heated up to 95˚C for 3 minutes before the samples
are loaded into the gel. The running buffer typically used is a tris-glycine buffer which is made
up of tris, glycine and SDS. The function of each ingredient added is elaborated upon below.
SDS-PAGE utilizes two types of gel, the first is to use a gradient gel and the second is having
two layers of gel. The gradient gel is useful in situations where the proteins to be separated span
over a large molecular weight range, while the two layers of gel, made up of a stacking and a
resolving gel is more commonly used. The stacking gel is typically a large pore gel of about 4%,
and it is prepared with Tris/hydrochloric acid which is about 2 pH units lower than that of the
running buffer. These create an environment which is suitable for Kohlrausch reactions, causing
the proteins to be concentrated into a tight band before entering the resolving gel. This allows for
better protein separation since the proteins are separated based on the distance moved in the gel
according to their sizes. The resolving gel on the other hand typically has a smaller pore size and
is formed using a pH8.8 Tris/hydrochloric acid buffer. The percentage of the resolving gel used
is based on the size of the proteins in the sample and it has to be chosen carefully to allow
optimum separation of the proteins in the sample. A general guide is shown in Table 1 below.
Table 1 Size of the proteins which is suitable for the percentage of gel used for optimum protein
separation
Percentage of gel (%)
Size of proteins (kDa)
8
24 - 205
10
14 - 205
12
14 - 66
42
The gel is formed by cross-linking between two molecules: acrylamide and bisacrylamide. The
percentage of the gel to be formed can be adjusted by the amount of bis/acrylamide added.
Lower percentage gels are used for proteins with larger molecular weights while smaller proteins
require a higher percentage gel for good separation. In the presence of free radicals, the
acrylamide monomers are activated, and together with the cross linking agent bisacrylamide,
form long polymer chains. The polymerization reaction is initiated by APS and TEMED. APS
acts as an initiator by producing free radicals while TEMED accelerates the rate of formation of
these free radicals. The free radicals cause acrylamide monomers free radicals to be produced
which eventually form the long chain polymers.
Tris is usually used as the buffer system for both the running buffer as well as for gel formation
as it is non reactive to most proteins and has a pKa of 8.3 at 20˚C. This makes it suitable for
experiments in the pH range of 7 to 9. SDS is added into both the loading buffer as well as for
the formation of the gel, where it acts as an anionic detergent which denatures the proteins and
applies a negative charge to the proteins, giving all the proteins the same overall charge. This
causes the protein intrinsic charges to become negligible, ensuring that the proteins are separated
solely based on size. The mobility of the proteins thus is a linear function of the logarithm of
their molecular weights. Without SDS, the proteins will migrate and be separated based on their
mass-charge ratio instead.
Glycine is used to increase the resolution of the gel. In the stacking gel which is at pH 6.8,
glycine exists mainly as zwitterions, trailing behind the negatively charged protein molecules
which carry the current. However, in the resolving gel which is at pH 8.8, the glycine ions
deprotonate to form anions and share the load of carrying the current with the protein molecules.
43
The glycine ions also tend to overtake the protein molecules due to its small size producing a
sandwiching effect hence increasing the resolution of the gel.
Beta-2-mercaptoethanol added into the laemmli buffer is a reducing agent and is important for
breaking the disulfide bonds in the proteins hence denaturing the proteins. Denaturation of the
proteins by SDS as well as beta-2-mercaptoethanol is important to ensure that the proteins are
separated according to their sizes and not their shapes. Dithiothreitol can also be added to reduce
the disulphide bonds and to prevent the formation of dimers.
As proteins are usually colourless, bromophenol blue is added into the laemmli buffer to act as
an indicator dye. It runs ahead of the proteins due to its small size and can be used as a gauge for
when the gel run should be stopped to prevent the sample proteins from running out of the gel. It
binds weakly to the proteins and makes it easier to see the samples when they are being loaded
into the wells of the gel. Glycerol is added into the laemmli buffer as a preservative and a
weighing agent. It helps to keep the proteins at a low temperature and also increases the density
of the samples so that it can be weighed down into the wells when being loaded into the gel. This
is important to prevent the loss of samples into the buffer during loading.
Coomassie blue and silver staining are the two common dyes used for visualization of the gel.
Silver staining is a very sensitive procedure which can be used to detect trace amounts of
proteins in gels. It is about fifty times more sensitive than using coomassie blue and can detect a
protein as low as 50ng. The silver staining procedure is however a toxic process due to the use of
formaldehyde and silver nitrate, hence extreme care must be taken when using silver staining.
Coomassie blue staining on the other hand is much simpler to use and more quantitative, but the
trade off is its sensitivity. It is an anionic dye that binds non-specifically to proteins through Van
der Waals forces and ionic interactions between the amine groups of the proteins and the sulfonic
44
acid groups of the dye. Coomassie blue is usually used with methanol and acetic acid to stain the
gel, and the gels are subsequently destained with a methonal/acetic acid solution to remove the
unbound dye.
2.6 Protein conformation studies
2.6.1 Fourier transform infrared spectrometry
Fourier transform infrared (FTIR) spectrometry uses infrared as the name suggests, to obtain the
absorption, emission, photoconductivity or Raman scattering spectrum of samples in any of the
three states, solid, liquid or gas. It is able to acquire data over a wide spectral range unlike
conventional dispersive spectrometers hence making the latter obsolete. Dispersive
spectrometers work on the principle of passing a monochromatic light beam through a sample,
measuring the amount of light absorbed and repeating this process for each wavelength.
FTIR works on a different principle allowing for more information to be obtained at one time. It
utilizes a beam that has many different frequencies, and measures the intensity absorbed by the
sample. This beam is produced by a glowing black body source or a broadband electromagnetic
source which gives the full spectrum of wavelengths for measurement. The beam is modified
using an interferometer to include a different mixture of frequencies before being passed through
the sample to give a data point. This process is repeated many times and all the results obtained
are combined using fourier transform to calculate the intensity absorbed at each wavelength.
Most interferometers usually use beam splitters to modify the beam. The beam is divided into
two and one beam reflects off a flat mirror that is in a fix position while the other beam reflects
off another flat mirror that is movable. The two reflected beams meet back at the beam splitter
45
and enter the sample compartment. The movable mirror constantly changes position to produce a
different mixture of frequencies giving a wide spectrum of resulting signals. The absorption and
transmission data collected are then processed using the Fourier transform algorithm.
The spectrum obtained from FTIR gives a molecular fingerprint of the absorption and
transmission peaks. Each of these peaks represents the frequencies of the vibrations between the
bonds of the atoms in the sample. These peaks are therefore unique as different compounds are
made up of different mixture of atoms, producing unique spectrums known as the molecular
fingerprint. This molecular fingerprint can thus be used to identify unknown samples, to
determine the components in a mixture, or to determine the quality of a sample by testing for
impurities in the sample. In addition to qualitative analysis, the amount of sample present can
also be found based on the size of the peaks obtained in the spectrum. FTIR has numerous
advantageous: short measurement times as all the infrared frequencies can be scanned
simultaneously, samples can be measured easily with good precision and sensitivity, and most
importantly, the samples are not destroyed in the measurement process. A limitation of FTIR
however, is that samples which are optically active will interfere with the measurement and
could give rise to inaccurate results.
2.6.2 Circular dichroism
Circular dichroism (CD) is a spectroscopy method that is useful for the study of conformation or
structural changes of biomolecules like DNA, proteins or small peptides caused by physical
conditions like pH, temperature or ionic strength [94]. CD is not able to give specific
conformation or residues information like the X-ray crystallography or nuclear magnetic
resonance, but it is a first pass technique for the determination of structural changes in proteins
46
or DNA. CD requires little or no sample preparation, and the samples can be measured relatively
fast and the results are easily analyzed. It also has the added advantage where sample
measurements can be taken in their physiological buffers. However, similar to FTIR, it is an
optical tool; hence samples that exhibit optical properties could cause interference and give
inaccurate or erroneous readings.
CD’s working principle utilizes the intra or intermolecular asymmetry or helicity of the
molecular structure. As the name suggests, it works by the measurement of the absorption of
circularly polarized light. Circularly polarized light can be produced when two linearly polarized
light that are oscillating perpendicular to each other and are propagating with a phase difference
of π/2 are superpositioned [95]. When the circularly polarized light passes through a
chromophore sample, it can either be refracted as it passes through the solution, or it can be
absorbed, and the absorption is measured by refractive index, n, or molar extinction coefficient,
ε, respectively. Light passing through an optically active sample absorbs the left and right
circularly polarized light to different extents yielding different molar extinction coefficients. The
difference in the light absorbed is a measure of CD and can be expressed by Beer-Lambert’s law,
∆A = AL – AR = ∆ε.c.l, where c is the concentration, l is the path length, AL is the absorbance of
the left circularly polarized light, AR is the absorbance of the right circularly polarized light and
A the absorbance [94-95]. The differential absorbance is plotted against the wavelength to obtain
a CD spectrum. When a protein sample is measured using CD, the protein electronic structure
gives characteristic spectrums according to the protein secondary structure like α helix, β sheets
or random coil structures. Bands of distinctive shapes in specific regions of the CD are obtained
and the type and amount of secondary structural elements present can be calculated [95]. The
characteristic CD curves for secondary structures of proteins are shown in Figure 3 below.
47
Figure 3 Characteristic CD curves of secondary structure elements of proteins [95].
48
Chapter 3 Materials and methodologies
3.1 Materials
Hydrochloric acid was obtained from Merck and Fisher Scientific, nitric acid was obtained from
Honeywell and Merck, and thrombin was obtained from Sigma-Aldrich and Randox
Laboratories The human fibrinogen was obtained from Merck, SeeBlue® Plus2 Pre-Stained
Standard for gel electrophoresis was obtained from Invitrogen, acrylamide/bis solution was
obtained from Bio-Rad, and the gold standard was obtained from Agilent Technologies
The other chemicals hydrogen tetracholroaurate (III), trisodium citrate, sodium borohydride,
tannic acid, platinum standard, N,N,N′,N′-tetramethylethylenediamine, ammonium persulfate,
glycerol, beta-mercaptoethanol, sodium dodecyl sulphate, bromophenol blue, glycine, tris, were
all obtained from Sigma-Aldrich.
3.2 Procedures
3.2.1 Synthesis of gold nanoparticles (AuNP)
Three different sizes of AuNP were synthesized in our laboratory – 15nm, 11nm and 5nm.
15nm Au Ps synthesis
The 15nm AuNPs was synthesized by the Turkevich method where tetrachloroauric acid
(HAuCl4.3H2O) was reduced by trisodium citrate to give AuNPs. The synthesis started with
adding 100µL of the HAuCl4.3H2O stock solution of 1g/10mL concentration to 95mL of
ultrapure water. This solution was heated up to 105±5˚C using a 3-neck flask immersed in a
49
silicon oil bath with a magnetic stirrer, which was set to 700-1000rpm. When the temperature
stabilized at 105±5˚C, 5mL of 1% trisodium citrate was added to it. The reaction was then
allowed to take place over the next 30 minutes at 105±5˚C, where the solution will turn from
pale yellow to a deep red colour [45]. The solution was then allowed to cool with continued
stirring.
11nm Au Ps synthesis
The synthesis of 11nm AuNPs utilized tannic acid and citric acid as the reducing agents. A 16mL
solution of chloroauric acid of concentration 0.0125g/mL was prepared and heated up to 70±3˚C
using a 3-neck flask immersed in a silicon oil bath with a magnetic stirrer which was set to 7001000rpm. A 4mL reductant solution consisting of 0.2g/mL trisodium citrate with 0.0005g/mL
tannic acid was also prepared and heated up to 70±3˚C in a separate water bath. Both solutions
were heated for 30 min at 70±3˚C before the reductant was added to the choloroauric acid
solution. The solution was allowed to change from colourless to a wine red colour before the
temperature was raised to 115±3˚C and heated for another 15 minutes. The solution remained
dark red and was then allowed to cool with continued stirring.
5nm Au Ps synthesis
The synthesis of 5nm AuNPs utilized sodium borohydride as the reducing agent at room
temperature. 0.6mL of ice cold 0.1M sodium borohydride was added quickly to 20mL solution
containing 2.5x10-4M of chloroauric acid and 2.5×10-4M of trisodium citrate. The solution turned
orange-brown immediately indicating the formation of AuNPs.
50
The synthesized AuNPs were all filtered using Millex GP 0.22um PES membrane filters to
remove large particles as well as particulate impurities before being used or stored at 4˚C. The
AuNPs were characterized using transmission electron microscopy (TEM) and measurements of
more than 100 particles per sample were taken using image J to give the average particle size.
The three different sizes of the particles used were measured to be 15.2±1.4nm, 11.5±1.3nm and
5.3±0.9nm. Representative TEM images of the AuNPs can be found in Figure 6.
3.2.2 Conjugation of AuNPs with protein
Two proteins were used for the experiments, namely bovine serum albumin (BSA) and
fibrinogen (Fb). The two proteins were dissolved in 0.1x phosphate buffer solution (PBS) of
about pH 7.5 as the pI of BSA is 4.7 while that of Fb is 5.5. The different sized AuNPs were first
centrifuged with their respective optimized centrifugation parameters to obtain a concentrated
AuNPs pellet. The protein solution was then added to the AuNPs pellet to make up a volume of
1mL. The AuNPs and protein mixture was then left to stand at 4˚C for at least 15 hrs before it
was used for subsequent investigations.
3.2.3 Size and zeta potential measurement
Size measurement
Dynamic light scattering (DLS) was done using a Malvern Zetasizer NanoZS system as part of
characterizing the AuNP-protein conjugate. A DLS measurement gives the hydrodynamic size of
the particles as well as its size distribution at room temperature and can therefore be used to
determine the size of the conjugate. The AuNPs and AuNP-protein conjugates were first
51
centrifuged to form a pellet and re-suspended in ultrapure water or PBS respectively. The
solutions were then transferred into a disposable polystyrene cuvette for the measurement. Other
then measuring the size of the conjugates, any significant aggregation that could have resulted
from centrifugation and the stability of the conjugates over time could also be determined from
the DLS measurement.
Zeta potential measurement
Similar to the description above, the zeta potential measurement was also done using the
Malvern Zetasizer NanoZS system. The AuNPs or AuNP-protein pellets obtained after
centrifugation was re-suspended in ultrapure water or PBS respectively before being transferred
to a disposable zeta-cell which consisted of two electrodes and a folded capillary where the
sample was held. The zeta-cell was then inserted into the Zetasizer for the measurement to be
taken.
3.2.4 Centrifugation
A centrifugation study was done to determine the optimal parameters for maximum recovery of
the AuNPs as well as the AuNP-protein conjugate. In the centrifugation study, three g-forces
(8400g, 12000g and 14900g) were employed for centrifugation for 10 to 60 minutes at 4˚C and
25˚C. After centrifugation, the supernatant was carefully removed for the measurement of the
AuNPs concentration using ICP-MS, and the pellet was re-suspended in ultrapure water (or PBS
for AuNP-protein samples) for the size measurement using DLS. The control used in the AuNPs
experiment sets was from the same batch of AuNPs but without centrifugation, and the control
52
used for the AuNP-protein experiment sets was the AuNP-protein conjugates without
centrifugation. Triplicates were conducted for all centrifugation experiments. The optimal
parameters for each sample were determined at the end of this study, and the following
centrifugation parameters were used for all subsequent experiments:
Table 2 Centrifugation parameters used for various samples.
Sample
Centrifugation
speed
Centrifugation
time
Temperature
15nm AuNP
15000g
30 min
4˚C
15nm AuNP - BSA conjugate
15000g
30 min
4˚C
15nm AuNP – Fb conjugate
20000g
30 min
4˚C
3.2.5 Acid digestion and inductively coupled plasma mass spectrometry
AuNPs concentration in the supernatant was determined using inductively coupled plasma mass
spectrometry (ICP-MS). The supernatant of the centrifuged samples were first digested using
aqua regia (3HCl : 1HNO3) and the acid concentration of the sample was reduced to less than 2%
prior to the ICP-MS measurement. ICP-MS was carried out using an Agilent 7500cx system
equipped with a double pass spray chamber and a quadrupole mass analyzer. A platinum internal
standard was added to each sample to eliminate the fluctuations due to preparation steps and
measurement conditions. An external gold standard was prepared for calibration. The intensity of
the gold ions was compared against the calibration curve to obtain the Au concentration. The
intensity readings for all the samples were kept within the range of the calibration curve for
accurate AuNPs concentration analysis. The concentration values were then converted into
percentage values based on the concentration of the control.
53
3.2.6 Gel electrophoresis
Gel electrophoresis was used to determine the amount of protein that was bound onto the AuNPs
based on the method developed by Laemmli [96]. Conjugate samples were prepared 1 day before
and washed twice to remove all excess proteins before the loading buffer was added in a 1:1
ratio. The mixture was then heated at 95˚C for 3 minutes and stored at 4˚C prior to being loaded
onto the gel. The loading buffer used here was the modified laemmli buffer commonly used for
the denaturation and hence desorption of proteins from nanoparticles [97-98]. The components
of the modified laemmli buffer are shown in
Table 3. The glass plates to be used were first washed and wiped clean with 70% ethanol
ensuring that all dust particles were removed before an 8% resolving gel mixture was prepared
and loaded. Once the resolving gel has polymerized, a 5% stacking gel mixture was then
prepared and loaded on top of the resolving gel together with the comb to form the wells in the
gel. All gel mixtures were prepared based on the component volumes shown in Table 4.
Table 3 Components for the preparation of loading buffer - laemmli buffer.
Components
Concentration
Tris-HCl, pH 6.8 (top up to prepared volume)
0.2M
SDS
4%
Glycerol
20%
Beta-mercaptoethanol
12%
Bromophenol blue
0.06%
54
Table 4 Component volumes for a 8% resolving gel and a 5% stacking gel.
Components
For 8% resolving gel
For 5% stacking gel,
Component volume (mL)
Component volume (mL)
Water
9.3
4.1
30% acrylamide mix
5.3
1.0
1.5M Tris (pH 8.8)
5.0
0.0
1.0M Tris (pH6.8)
0.0
0.75
10% SDS
0.2
0.06
10% ammonium persulfate
0.2
0.06
TEMED
0.12
0.006
Total volume
20 mL
6 mL
Once the gel had polymerized, the marker as well as the samples were loaded individually into
each well, and the volume loaded for each well was taken note. The marker used was the
SeeBlue® Plus2 Pre-Stained Standard. The electrophoresis tank was then filled up with the
running buffer – tris-glycine buffer (see buffer components in Table 5). A voltage of 60V was
applied for 30 minutes to allow all the samples to be aligned in between the stacking and the
resolving gel before the voltage was increased to 100V. The samples were then allowed to run
through the gel for about 90 minutes.
Table 5 Components for the preparation of the running buffer – tris-glycine buffer.
Components
Amount for a 1 x solution
Tris base
3.02g
Glycine
18.8g
10% (w/v) SDS, electrohphoresis grade
10mL
DI water
Up to 1 L
55
At the end of the gel run, the gels were removed from the glass plate and placed in air tight
containers where the staining solution was added. The gels were placed on a shaker for staining
for at least 4 hours before they were placed in the de-staining solution overnight. The staining
and de-staining solution components are shown in Table 6. The de-stained gel was then analyzed
using the Syngene gel documentation system to determine the amount of proteins bound onto the
AuNPs based on the relative intensities of the protein bands.
Table 6 Components for the preparation of the staining and de-staining solution.
Components
Concentration for
staining solution
Concentration for destaining solution
Coomassie blue
0.2%
-
Methanol
40%
40%
Glacial acetic acid
10%
10%
D.I water
49.8%
50%
3.2.7 Circular Dichroism
The far-UV CD spectra was measured using a Jasco J810 spectropolarimeter to determine any
change in the conformation of the proteins after being conjugated with AuNPs. Conjugated
samples were prepared 1 day in advance, while controls consisting of protein solutions only were
prepared fresh. The far UV region was scanned between 190 and 300nm with an average of 2
scans. Measurements we conducted at 25˚C with bandwidth of 2nm, resolution of 0.2nm, scan
speed of 50nm/min and time constant of 0.125sec. The final spectra were obtained by subtracting
the buffer contribution from the original protein spectra.
56
Chapter 4 Results and Discussion
4.1 Conjugation of Au Ps with proteins
UV-Vis spectrometry
15nm AuNPs exhibit a characteristic absorbance at 520nm, while that for proteins is at 280nm.
After conjugation with proteins, it was found that the absorbance wavelength for the conjugate
was red-shifted to 525nm. This indicated successful conjugation of the proteins onto the AuNPs.
It can be seen in Figure 4 and Figure 5 that when a low protein concentration of
20µg/mLµg/mLfor conjugation, the protein peak at 280nm was not visible, and the absorbance
intensity was also lower than that for the AuNPs only. However, with increasingly protein
concentration, the protein peak at 280nm started to reappear and the intensity of the peak at
525nm was comparable to that of the AuNPs only.
57
2
AuNP
BSA only
20ug/mL AuNP-BSA
200ug/mL AuNP-BSA
500ug/mL AuNP-BSA
1000ug/mL AuNP-BSA
Absorbance
1.5
1
0.5
0
200
300
400
500
600
700
800
Wavelength (nm)
Figure 4 UV-vis spectra of AuNPs, BSA and AuNP-BSA conjugates of different initial BSA
concentration. Absorbance wavelength for BSA was 280nm, and for AuNPs at 520nm. AuNPBSA conjugates’ absorbance wavelength was at 525nm. AuNPs used here was 15nm.
Figure 5 shows the UV-vis spectrometry measurement for the AuNP-Fb conjugates. Similarly,
when Fb concentration of 20µg/mL was used for conjugation, the Fb peak at 280nm was not
visible, and the absorbance intensity of the peak at 525nm was significantly lower than that for
the AuNPs. However, at higher Fb concentration, the Fb peaks at 280nm were visible, and the
intensity of the peak at 525nm increased with increasingly Fb concentration. This suggested that
as the concentration of Fb was increased, the amount of Fb conjugated to the AuNPs increased as
well. On the other hand, when the concentration of the BSA was increased, the amount of BSA
conjugated to the AuNPs remained the same since the intensity at 525nm stayed the same. These
phenomena were consistent with the other results which will be shown in the subsequent sections
of this report.
58
2
AuNP
Fb only
20ug/mL AuNP-Fb
200ug/mL AuNP-Fb
500ug/mL AuNP-Fb
1000ug/mL AuNP-Fb
Absorbance
1.5
1
0.5
0
200
300
400
500
600
700
800
Wavelength (nm)
Figure 5 UV-vis spectra of AuNPs, Fb and AuNP-Fb conjugates of different initial Fb
concentration. Absorbance wavelength for Fb was 280nm, and for AuNPs at 520nm. AuNP-Fb
conjugate absorbance wavelength was at 525nm. AuNPs used here was 15nm.
4.2 Size and zeta potential
4.2.1 Transmission electron microscopy
The AuNPs were characterized using transmission electron microscopy (TEM). Measurements
of at least 140 particles per sample were taken and the three different sizes of the particles used
were measured to be 15.2±1.4nm, 11.5±1.3nm and 5.3±0.9nm. Representative TEM images of
the AuNPs are shown in Figure 6.
59
A.
B.
C.
50nm
50nm
20nm
Figure 6 Transmission electron microscopy (TEM) images of AuNPs of different size. A.
~15nm AuNPs; B. ~11nm AuNPs; C. ~5nm AuNPs.
4.2.2 Dynamic light scattering
DLS was used for the characterization of the size of the conjugate as well as the size effect of
increasing protein concentration added to AuNPs for conjugation. The measurements of all the
sizes are shown in Figure 7 and Table 7. Table 7 also lists the ratio of protein molecule to AuNP
in each solution. This ratio is found by calculating the number of protein molecules and AuNP in
the 1 mL solution based on their individual molecular weights. DLS measures the hydrodynamic
radius of the particles, hence the measured size would be slightly larger than that found using
TEM as described in the literature review section.
The size of AuNPs alone before conjugation with any protein was 21.8nm, while that for BSA
was 6.5nm and for Fb was 32.9nm. When the BSA concentration added for the conjugation was
increased from 20µg/mL to 1mg/mL, the size of the AuNP-BSA conjugate remained fairly
constant at around 32 ± 2nm. This indicated a monolayer adsorption by the BSA molecules on
the AuNPs since the conjugate size did not increase with increasing BSA concentration added.
This was consistent with what was reported in literature [99-102] as well as the other results
reported here. On the other hand, the size of the AuNP-Fb conjugate increased continuously as
60
the Fb concentration added for conjugation was increased. This seemed to indicate multi-layered
adsorption of the Fb molecules on AuNPs.
140
AuNP-BSA
AuNP-Fb
Size of Au P-protein conjugate
120
100
80
60
40
20
0
0
200
400
600
800
1000
Concentration of protein added to Au P for conjugation (ug/mL)
Figure 7 Size (nm) of the AuNP-protein conjugate with increasing protein concentration added
to AuNPs for conjugation. Error bars calculated from the standard deviation of 3 independent
samples are shown.
The zeta potentials of the samples at different protein concentrations were also measured using
DLS (Figure 8 and Table 8). AuNPs before conjugation with proteins had zeta potential of
approximately 16.8mV. As the proteins were dissolved in PBS of pH 7.5, they carry negative
surface charges. The zeta potentials of BSA and Fb were measured to be -8.6mV and -6.7mV
respectively before conjugation. After AuNPs were conjugated with proteins, the zeta potential
of AuNP-BSA conjugate was approximately -10mV while that for AuNP-Fb was approximately
-6mV. This indicated that the surface charge of the AuNP-protein conjugates were dominated by
61
the type of proteins adsorbed on the AuNPs surface. In addition, the concentration of protein
added for conjugation did not have much effect on the zeta potential of the conjugate.
62
Table 7 Results for the size of AuNP-protein conjugate measured using DLS as well as the
calculated number of protein molecule to 1 AuNP at the respective concentrations. Columns 2 &
4: Size (nm) of the Au P-protein conjugate with increasing protein concentration added to
Au Ps for conjugation. Columns 3 & 5: The number of protein molecule per Au Ps in solution
for conjugation at respective protein concentration.
Sample
Au P-BSA size
(nm)
o. of BSA
molecule to 1
Au P
Au P-Fb size
(nm)
o. of Fb
molecule to 1
Au P
Au Ps
21.8
-
21.8
-
Protein alone
6.5
-
32.9
-
20µg/mL
27.3
60
52.7
12
40µg/mL
28.0
119
58.1
23
60µg/mL
28.6
179
61.1
35
80µg/mL
29.8
239
62.3
46
100µg/mL
29.5
298
63.8
58
120µg/mL
29.6
358
65.7
70
140µg/mL
30.1
418
68.2
81
160µg/mL
30.6
478
68.8
93
180µg/mL
31.2
537
70.9
104
200µg/mL
30.4
597
73.5
116
300µg/mL
33.7
895
73.3
174
400µg/mL
33.3
1194
78.5
232
500µg/mL
33.8
1492
93.7
290
600µg/mL
34.2
1791
93.8
348
700µg/mL
35.4
2089
100.3
406
800µg/mL
32.3
2388
108.1
464
900µg/mL
34.5
2686
104.5
521
1000µg/mL
34.5
2985
126.3
579
63
0
Zeta potential of Au P-protein conjugate
0
200
400
600
800
1000
-5
-10
-15
-20
AuNP-BSA
AuNP-Fb
-25
-30
Concentration of protein added to Au P for conjugation (ug/mL)
Figure 8 Zeta potential (mV) of the AuNP-protein conjugate with increasing protein
concentration added to AuNPs for conjugation. Error bars calculated from the standard deviation
of 3 independent samples are shown.
64
Table 8 Zeta potential (mV) the AuNP-protein conjugate with increasing protein concentration
added to AuNPs for conjugation.
Sample
Au P-BSA zeta
potential (mV)
Au P-Fb zeta
potential (mV)
-16.8
Au Ps
Protein alone
-8.6
-6.7
20 µg/mL
-12.3
-7.6
40 µg/mL
-10.5
-4.6
60 µg/mL
-12.1
-7.0
80 µg/mL
-10.3
-6.6
100 µg/mL
-12.7
-5.4
120 µg/mL
-8.6
-6.4
140 µg/mL
-12.4
-6.2
160 µg/mL
-11.7
-5.6
180 µg/mL
-10.3
-5.7
200 µg/mL
-9.5
-6.1
300 µg/mL
-9.3
-6.2
400 µg/mL
-8.9
-5.7
500 µg/mL
-9.2
-5.7
600 µg/mL
-8.8
-6.5
700 µg/mL
-8.1
-6.2
800 µg/mL
-9.5
-6.5
900 µg/mL
-9.4
-6.2
1000 µg/mL
-9.7
-6.9
65
4.3 Stability study of Au P-protein conjugates
The sizes of the AuNP-BSA conjugates were monitored for more than a month after conjugation
to study their long term stability. The AuNPs and BSA were conjugated as described above and
taken for DLS measurement immediately and for up to 3 hours after conjugation on the first day.
Measurement of the samples was then continued every few days with decreased frequency for up
to one and a half month. The samples were kept at 4˚C in glass containers to minimize the
adsorption of samples onto the surface of the container. The results are shown in Table 9.
Table 9 The size of the AuNP-BSA conjugates over time from T = 0 to T = 51 days.
Au P-BSA conjugates
Au P-BSA conjugates
Time
Size(nm)
Stdev
Time
Size(nm)
Stdev
Immediately
27.7
0.35
10 days
27.3
0.36
15 mins
27.5
0.86
14 days
27.3
0.69
30 mins
27.4
0.79
15 days
27.6
0.42
45 mins
27.4
0.76
16 days
31.1
2.32
1 hr
27.5
0.60
21 days
29.9
0.21
2 hrs
27.6
0.82
22 days
30.2
0.44
3 hrs
27.7
0.66
24 days
29.8
0.53
1 day
27.3
0.76
25 days
29.8
0.69
2 days
29.8
0.67
30 days
29.3
0.33
3 days
27.3
0.99
31 days
29.4
0.76
4 days
27.6
0.51
37 days
29.0
1.11
8 days
26.9
0.64
44 days
29.0
1.17
9 days
26.8
0.55
51 days
30.3
2.83
Analysis of the results found that the AuNP-BSA conjugates did not differ much in size and were
very stable for up to 15 days. After 15 days, the conjugate size increased by about approximately
2 nm up until the 51st day. This 2 nm increase in size could not be attributed to aggregation of the
conjugates, however, it may suggest that there was a general decline in stability of the
conjugates. It could also be observed that the conjugates were more “sticky” after the first two
66
weeks as some could be seen to have adsorbed onto the glass container. It is therefore
recommended that the conjugates be kept for no more than two weeks after its synthesis.
4.4 Centrifugation study on bare Au Ps
Concentration of nanoparticles after synthesis is a necessary processing step before using the
particles for further functionalization or surface decoration, such as conjugation of drugs or
biomolecules. The most common way to purify or concentrate nanoparticles is by centrifugation.
Centrifugation at 7000g – 10000g for 20 to 30 minutes is commonly employed for nanoparticles
of sizes 15 – 20nm [103-106], while nanoparticles coated with biomolecules tend to be
centrifuged at a higher g-force of 10000g – 20000g for up to an hour [56, 75, 107-109]. This has
become a common practice to which researchers give minimal attention. A 100% recovery of
these nanoparticles by centrifugation is usually assumed due to several reasons. First, for
coloured nanoparticles like AuNPs, the supernatant is mostly colourless after centrifuging at high
speeds. Second, it is more convenient to assume total recovery of the nanoparticles to make the
stoichiometric calculation of the subsequent surface conjugation chemistry easier. Total recovery
of the samples is however not achieved in most situations. This project thus investigated
centrifugation related parameters in an effort to determine how each of the parameters influence
the recovery of the samples, as well as to determine if aggressive centrifugation parameters result
in aggregation of the samples. To the best of the author’s knowledge, there is no single study that
evaluates how centrifugation parameters influence the recovery of AuNPs and their conjugates as
well as how it may cause aggregation of the samples.
67
4.4.1 Particle size dependency
Nanoparticle size played an important role in determining the recovery rate of AuNPs after
centrifugation. As the size decreased, it can increasingly be difficult to recover the nanoparticles.
The percentage of AuNPs remaining in the supernatant after centrifugation (Figure 9) showed
that after 60 minutes of centrifugation at 14900g, almost all of the 15nm and 11nm AuNPs could
be recovered, with only 0.07% and 0.56% of the AuNPs left in the supernatant respectively.
However, this was not the case for the 5nm AuNPs. Approximately 54% of the particles
remained in the supernatant after centrifuging at the same condition (see Table 10, Table 11 and
Table 12 for full data). This was due to the small size, and hence high diffusivity of AuNPs
which rendered ineffective sedimentation, even at the centrifugal force of 14900g. The 5nm
AuNPs therefore required a much higher g-force or a longer centrifugation time to enable better
recovery. These results showed that the size of nanoparticles used must be taken into
consideration as smaller particles tend to be more difficult to spin down compared to bigger
particles.
68
100
Percentage %
80
60
40
20
0
0
10
15nm Au P - 8400g
11nm Au P - 8400g
5nm Au P - 8400g
20
30
15nm Au P - 12000g
11nm Au P - 12000g
5nm Au P - 12000g
40
50
60
Time (mins)
15nm Au P - 14900g
11nm Au P - 14900g
5nm Au P - 14900g
Figure 9 Percentage of 3 different sizes of AuNPs remaining in the supernatant after being
centrifuged using 3 different g-force, from 10 minutes up to 60 minutes. All samples here were
conducted at 4˚C. Standard deviation has been included in all data points, but the standard
deviations for the 11nm and 15nm AuNPs are mostly very small (less than 1%) and hence are
not apparent in the data shown.
69
Table 10 Percentage of 15nm AuNPs remaining in the supernatant after centrifugation at
different g-force, time and temperature.
%
8400g
12000g
14900g
4˚C
10 min
37.97
35.58
26.72
20 min
10.00
6.80
3.55
30 min
2.28
0.63
0.22
40 min
0.33
0.42
0.10
50 min
0.07
0.10
0.02
60 min
0.03
0.03
0.07
25˚C
10 min
41.66
27.26
14.89
20 min
21.53
9.22
6.33
30 min
11.61
4.54
1.71
40 min
7.58
2.40
1.16
50 min
6.01
1.75
0.65
60 min
4.21
1.36
0.43
70
Table 11 Percentage of 11nm AuNPs remaining in the supernatant after centrifugation at
different g-force, time and temperature.
%
8400g
12000g
14900g
4˚C
10 min
74.15
58.84
55.00
20 min
49.22
26.35
18.34
30 min
31.57
8.45
5.04
40 min
18.84
2.96
1.44
50 min
9.73
0.76
0.76
60 min
2.24
0.45
0.56
25˚C
10 min
66.81
47.24
22.33
20 min
41.80
19.85
11.56
30 min
31.70
11.15
7.42
40 min
27.05
7.58
6.70
50 min
20.46
6.26
6.36
60 min
18.52
5.47
3.98
71
Table 12 Percentage of 5nm AuNPs remaining in the supernatant after centrifugation at different
g-force, time and temperature.
%
8400g
12000g
14900g
4˚C
10 min
82.74
81.56
85.90
20 min
84.35
75.90
81.61
30 min
83.91
71.10
73.04
40 min
80.47
68.41
65.28
50 min
79.79
64.47
60.24
60 min
76.58
60.38
54.01
25˚C
10 min
85.26
83.88
86.16
20 min
89.52
78.14
81.53
30 min
89.53
80.60
81.09
40 min
86.50
82.99
78.77
50 min
85.21
74.06
78.37
60 min
83.46
71.90
79.05
4.4.2 G-force and time dependency
It can be observed in Figure 9 that utilizing a higher g-force resulted in higher AuNPs recovery.
This was especially significant at shorter centrifugation time. After 20 minutes of centrifugation
at 8400g, approximately 10% of the 15nm AuNPs remained in the supernatant. But at 14900g,
only 3.5% remained in the supernatant. Similarly, for the 11nm AuNPs, after centrifuging for 20
minutes at 8400g, 49% of particles still remained in the supernatant while only 18% remained
unrecovered at 14900g. This trend was not obvious with the 5nm AuNPs since 20 minutes was
too short to recover substantial amounts of particles at any of the three g-forces used. The g-force
dependency exhibited a more significant effect at a longer centrifugation time for the 5nm
72
AuNPs. After centrifuging for 60 minutes, the amount of particles remaining in the supernatant
dropped from 76% to 54% when the g-force increased from 8400g to 14900g. Despite this, the
amount of 5nm AuNPs recovered was still less than 50%. A larger g-force was attempted to
increase the particle recovery. At 20000 g-force (data shown in Table 13 below) a better
recovery of 5nm AuNPs was obtained, with 21% of the AuNPs remaining in the supernatant
after centrifugation for 60 minutes.
Table 13 Percentage of 5nm AuNPs remaining in the supernatant after centrifugation at 20000g
and 4˚C for various times.
Time
Average %
10 min
96.7
20 min
74.7
30 min
41.7
40 min
49.5
50 min
25.5
60 min
21.2
In our investigation of the centrifugation time used, the amount of 15nm AuNPs remaining in the
supernatant at 14900g g-force dropped from 26% to 0.07% when the centrifugation time
increased from 10 to 60 minutes (Figure 9). While at 8400g, the amount of AuNPs in the
supernatant dropped from 38% to 0.03% when the centrifugation time increased from 10 to 60
minutes. It appeared that at short centrifugation time (10 or 20 minutes), even the high g-force of
14900g was not sufficient to recover all the 15nm AuNPs. However, the amount of AuNPs
recovered was close to 100% at 8400g or 14900g when the centrifugation time increased to 40
minutes or higher. Hence it was possible to make up for the usage of a lower g-force by
increasing the centrifugation time. This trend was also observed for the 11nm AuNPs when the
73
centrifugation time increased. At 14900g, almost complete recovery of AuNPs was observed
after centrifugation for 50 or 60 minutes, but a full 60 minutes was required to maximize the
recovery at 8400g. In addition, the first 30 minutes of centrifugation was found to be crucial for
recovering majority of the 15nm and 11nm AuNPs. Apart from the 11 nm AuNPs centrifuged at
8400g, the amount of particles remained in the supernatant was less than 10% when the
centrifugation time reached 30 minutes. Hence it was most preferable to centrifuge at least 30
minutes or longer to recover most of the AuNPs.
The minimal centrifugation time of 30 minutes was not applicable to the 5nm AuNPs. In this
case, even increasing the centrifugation time to 60 minutes only exhibited marginal effect in
particle recovery. As mentioned, the 5nm AuNPs required a high g-force for better recovery. At
8400g, the amount of AuNPs in the supernatant remained almost the same (~80%) after 10 or 60
minutes centrifugation time. Only when the g-force increased to 14900g was there a more
obvious effect of the centrifugation time, with the amount of AuNPs in the supernatant being
54% after 60 minutes. However, 50% of the particles were still not recovered. Longer
centrifugation time of 120 minutes was attempted (date shown in Table 14 below) but there was
still ~32% of AuNPs unrecovered.
Table 14 Percentage of 5nm AuNPs remaining in the supernatant after centrifugation at 15000g
and 4˚C for 90 and 120 minutes.
Time
Average %
90 min
31.8
120 min
32.2
Depending on the purpose for the recovery of the nanoparticles, various centrifugation g-forces
and time have been used by different research groups. For example, for electron microscopy
74
sample preparation, a high recovery yield of nanoparticles is not necessary and a less rigorous
centrifugation can be used [110]. However, in the event of surface modification of the particles,
it is preferable to recover as much precious sample as possible. This can be done by using a high
g-force and long centrifugation time, or by centrifuging the sample a few times using a lower gforce and a shorter time [110-111].
4.4.3 Temperature dependency
The effect of centrifugation temperature on AuNPs recovery was investigated at 4˚C and 25˚C
(Figure 10). In general, centrifuging at 4˚C yielded higher AuNPs recovery compared with the
case at 25˚C. For 15nm AuNPs, this difference may not be significant (Figure 10A). After
centrifuging for 60 minutes at 14900g, only 0.07% and 0.43% of AuNPs remained in the
supernatant at 4˚C and 25˚C respectively, which was relatively insignificant. The temperature
effect was slightly more pronounced at 8400g, where 0.03% and 4.2% of AuNPs remained in the
supernatant at 4˚C and 25˚C respectively after 60 minutes centrifugation. The temperature effect
was even more evident for the 11nm AuNPs (Figure 10B). At 8400 g and 60 minutes
centrifugation, 2.2% AuNPs was found in the supernatant at 4˚C, while 19% of AuNPs remained
in the supernatant at 25˚C. As expected, this difference again was decreased at higher g-forces of
14900g (0.6% and 4.0% AuNPs in supernatant at 4˚C and 25˚C respectively). The same trend
could be observed for the 5nm AuNPs (Figure 10C). At 14900g and 60 minutes centrifugation
time, 54% AuNPs was found in the supernatant at 4˚C and 79% of AuNPs remained in the
supernatant at 25˚C. The temperature effect at 8400g and 12000g was not very obvious for the
5nm AuNPs, possibly due to the already inefficient particle recovery at the low g-forces and
large standard deviation among different batches of runs.
75
100
8400g, 4C
12000g, 4C
14900g, 4C
8400g, 25C
12000g, 25C
14900g, 25C
A.
Percentage %
80
60
40
20
0
0
10
20
30
40
Time (mins)
100
60
8400g, 4C
12000g, 4C
14900g, 4C
8400g, 25C
12000g, 25C
14900g, 25C
B.
80
Percentage %
50
60
40
20
0
0
10
20
30
Time (mins)
40
50
60
76
100
C.
Percentage %
80
60
40
8400g, 4C
12000g, 4C
14900g, 4C
8400g, 25C
12000g, 25C
14900g, 25C
20
0
0
10
20
30
40
Time (mins)
50
60
Figure 10 Percentage of AuNPs remaining in the supernatant after being centrifuged using 3
different g-force, from 10 minutes up to 60 minutes, at 4˚C and 25˚C. A. 15nm AuNPs; B. 11nm
AuNPs; C. 5nm AuNPs.
A lower recovery of AuNPs at higher temperature may be attributed to the kinetic energy of the
particles. At 25˚C, the AuNPs possess more kinetic energy; hence a higher g-force was required
to overcome the particle Brownian motion to sediment the AuNPs. However, at lower
temperature, the Brownian effect became weaker and the same centrifugal force was sufficient to
sediment the particles, resulting in a higher recovery. A study done by Colvin and co-wrokers
reported the same phenomenon and attributed it to decreased solubility of the nanoparticles or
possible aggregation at lower temperatures [112]. However, from our dynamic light scattering
measurements (see next section), no significant AuNPs aggregation was found and thus the
argument of diffusion versus sedimentation is believed to be more appropriate in explaining the
temperature effect.
77
From the data presented here, a low temperature for centrifugation would be preferred for higher
recovery of nanoparticles. However, one would have to consider the suitability of using low
temperature centrifugation for the type of nanoparticles being used. For example, if the
aggregation of nanoparticles is serious under low temperature conditions and adversely affects
the use of particles in the subsequent experiments, then a higher temperature may only be chosen.
4.4.4 Hydrodynamic size of AuNPs after centrifugation
The hydrodynamic size of the AuNPs after centrifugation was measured using DLS to
investigate if any significant aggregation was resulted from centrifugation. The values obtained
were compared against the AuNPs sample without centrifugation (control). Based on the analysis
shown in Table 15, Table 16 and Table 17, there was no significant aggregation caused by
centrifugation. The sizes of all the three different AuNPs remained approximately the same after
being centrifuged for 60 minutes compared with the respective control. Increasing g-forces from
8400g to 14900g also did not exhibit any substantial particle aggregation in all three sizes of
AuNPs. It should take note that as DLS measured the particle hydrodynamic size, it typically
gives a larger size than the measurement using TEM. Although this investigation showed that
there was no significant aggregation of AuNPs, however, this may not hold true for other types
of nanoparticles as the stability may differ. For example, weakly charged particles would tend to
aggregate more easily and hence cannot be centrifuged at a high g-force unless they are first
capped with a stabilizing agent such as surfactants [56, 113] or coated with proteins [111]. Hence
centrifugation conditions depend on the stability of the nanoparticles of interest and should be
optimized accordingly.
78
Table 15 Size of 15nm AuNPs using DLS after centrifugation at different g-force, time and
temperature.
Size (nm)
8400g
12000g
14900g
4˚C
Control
21.1
19.0
19.0
10 min
22.2
18.1
19.9
20 min
22.2
18.6
19.6
30 min
23.0
19.3
20.1
40 min
23.1
19.9
19.5
50 min
22.2
19.6
19.9
60 min
23.6
19.8
20.2
25˚C
Control
19.0
19.0
19.7
10 min
20.7
20.9
23.8
20 min
20.5
21.5
24.2
30 min
20.7
21.7
23.45
40 min
21.6
22.5
23.2
50 min
21.5
21.5
24.6
60 min
20.8
22.2
23.4
79
Table 16 Size of the 11nm AuNPs using DLS after centrifugation at different g-force, time and
temperature.
Size (nm)
8400g
12000g
14900g
4˚C
Control
15.6
15.3
13.6
10 min
17.7
18.9
14.9
20 min
17.7
18.1
14.6
30 min
18.8
18.3
15.2
40 min
18.2
17.8
15.0
50 min
17.7
17.3
18.3
60 min
17.6
17.8
15.4
25˚C
Control
19.2
21.2
18.0
10 min
22.8
21.4
23.0
20 min
21.8
18.8
22.4
30 min
22.7
20.9
16.4
40 min
21.8
20.6
22.6
50 min
21.7
20.4
21.5
60 min
22.6
21.3
21.4
80
Table 17 Size of the 5nm AuNPs using DLS after centrifugation at different g-force, centrifuge
time and temperature.
Size (nm)
8400g
12000g
14900g
4˚C
Control
10.8
11.4
10.5
10 min
9.7
7.6
9.8
20 min
10.3
11.2
12.6
30 min
9.3
14.6
12.6
40 min
12.1
11.1
10.0
50 min
10.6
8.3
10.2
60 min
11.3
11.2
10.2
25˚C
Control
10.93
10.8
10.8
10 min
10.94
10.9
11.3
20 min
12.45
10.6
10.7
30 min
10.52
10.4
9.7
40 min
8.66
9.6
10.8
50 min
10.31
10.6
10.4
60 min
13.07
9.7
9.3
4.5 Centrifugation study on Au P-protein conjugates
4.5.1 Protein size dependency
In the previous section, it was illustrated that the recovery of particles after centrifugation was
dependent upon on the particle size. Here, it can be shown that molecules conjugated to the
particles also influence the effective recovery of samples. BSA and Fb were chosen for this
investigation due to their differing molecular weights where BSA is 67kDa, while Fb is 340kDa.
This allowed us to observe how the size of the molecule conjugated to the particles could
81
influence the recovery of the samples. Figure 11 shows the amount of sample left in the
supernatant after centrifugation at 4˚C with a g-force of 14900 for 60 minutes for bare AuNPs,
AuNP-BSA and AuNP-Fb. It can be observed (full data in Table 18, Table 19 and Table 20)that
the recovery of the samples at each time interval was the highest for the AuNPs without any
conjugation. This was due to the compact nature, or the density of the AuNPs making it easier to
sediment.
It could be observed that for the 15nm and 11nm AuNP-protein conjugated samples at long
centrifugation time of more than 30 minutes, recovery was significantly better for the AuNPBSA samples as compared to that of the AuNP-Fb samples. For example, at centrifugation time
of 60 minutes, recovery of the 15nm AuNP-BSA conjugates was 99.999% while that for 15nm
AuNP-Fb was 99.6%. Similarly, for the 11nm AuNP-BSA conjugates, the recovery was 99.8%
while for the 11nm AuNP-Fb, it was 99.4% after 60 minutes of centrifugation. This trend could
be attributed to the protein conjugated onto the AuNPs. Although Fb is bigger than BSA in size,
but the volume to mass ratio of Fb could be bigger than BSA. This could therefore affect the
drag force of the conjugate making it more difficult to sediment.
5nm AuNP-protein conjugates on the other hand exhibited a contradicting phenomenon from that
for the 15nm and 11nm AuNP-protein conjugates. From Figure 11 it can be observed that the
5nm AuNP-Fb conjugates had significantly higher recovery for all time points as compared to
that of 5nm AuNP-BSA. This different behavior of 5nm AuNP-BSA and AuNP-Fb could be due
to the small sized AuNPs. From DLS measurements (Table 7), Fb was about 33nm in size while
BSA was about 6.5nm. As the Fb is very much bigger the 5nm AuNPs, and the BSA was similar
in size to the AuNPs, AuNP-protein conjugates could have been exhibiting the physical
properties of the protein rather than that of the AuNPs. As such, the conjugates with Fb were
82
easier to sediment compared to that with the BSA because Fb is a much bigger protein compared
to BSA. This could have resulted in the observations made for the experimental sets conducted
using the 5nm AuNPs.
100
Percentage %
80
60
40
20
0
0
15nm Au P
11nm Au P
5nm Au P
10
20
30
15nm Au P-BSA
11nm Au P-BSA
5nm Au P-BSA
40
50
15nm Au P-Fb
11nm Au P-Fb
5nm Au P-Fb
60
Time (mins)
Figure 11 Percentage of sample remaining in the supernatant after being centrifuged at 4˚C with
a g-force of 14900g for 10 to 60 minutes. The graphs show the results for AuNPs before
conjugation, AuNP-BSA and AuNP-Fb conjugates, for 3 different sizes of AuNPs used.
Standard deviation has been included in all data points, but the standard deviations for some of
the samples were very small (less than 1%) and hence are not apparent in the data shown.
In general, it was easier to sediment the AuNPs with no protein conjugates as the AuNPs alone
were denser. For bigger sized AuNPs, the properties of the AuNPs were dominant when
centrifuging the AuNP-protein conjugates and the ease of sedimentation were as follows –
AuNPs > AuNP-BSA > AuNP-Fb. However, the proteins had a more dominant role when
83
smaller AuNPs were used, resulting in a different trend observed. The other centrifugation
parameters affected the conjugates in a similar fashion as those of the bare AuNPs. Higher
recovery was observed when centrifugation was carried out at 4˚C as compared to 25˚C, and a
higher g-force also yielded better recovery of the samples. In addition, centrifugation times of at
least 30 minutes gave much higher recovery. For example, in Table 18, the AuNP-Fb conjugates
centrifuged at 14900g, 4˚C, 13.45% of the sample was left in the supernatant after 20 min, while
only 4.99% of the sample was left after 30 minutes of centrifugation. It is however recommended
that centrifugation of 60 minutes is utilized for maximal recovery.
84
Table 18 Percentage of 15nm AuNPs, AuNP-BSA and AuNP-Fb remaining in the supernatant
after centrifugation at different g-force, time and temperature.
%
8400g
12000g
14900g
4˚C
Time
(mins)
Au
Ps
Au PBSA
Au PFB
Au
Ps
Au PBSA
Au PFB
Au
Ps
Au PBSA
Au PFB
10
38.85
55.83
44.10
35.08
46.02
46.15
28.26
35.14
38.82
20
10.12
24.87
22.67
6.71
14.76
22.27
3.70
4.67
13.45
30
2.39
8.28
13.03
0.62
11.48
12.07
0.22
0.35
4.99
40
0.35
2.75
6.08
0.42
4.19
4.77
0.10
0.26
1.84
50
0.08
1.15
3.82
0.11
1.68
2.39
0.02
0.07
0.85
60
0.03
0.76
1.62
0.03
1.77
1.42
0.07
0.001
0.41
25˚C
10
41.80
45.72
41.70
27.78
57.27
42.05
14.84
17.84
31.65
20
21.25
20.76
24.94
9.28
30.33
23.91
6.27
6.26
17.55
30
11.56
12.49
16.17
4.47
12.64
14.08
1.70
2.25
12.00
40
7.69
7.04
13.19
2.41
6.50
10.32
1.16
1.10
7.86
50
6.00
3.75
10.14
1.76
3.99
7.94
0.67
0.50
6.02
60
4.19
1.59
8.18
1.39
2.90
6.32
0.43
0.37
4.66
85
Table 19 Percentage of 11nm AuNPs, AuNP-BSA and AuNP-Fb remaining in the supernatant
after centrifugation at different g-force, time and temperature.
%
8400g
12000g
14900g
4˚C
Time
(mins)
Au
Ps
Au PBSA
Au PFB
Au
Ps
Au PBSA
Au PFB
Au
Ps
Au PBSA
Au PFB
10
71.67
86.89
67.26
57.48
76.10
49.00
49.38
73.93
54.57
20
47.77
59.19
48.55
26.26
39.85
23.23
15.55
34.17
23.41
30
30.53
36.98
28.54
8.28
17.81
10.04
4.10
8.76
10.29
40
18.20
22.43
20.53
2.89
7.79
7.69
1.19
0.81
5.50
50
9.35
13.18
16.23
0.74
2.36
3.59
0.61
0.62
3.08
60
2.16
7.38
10.94
0.44
0.98
2.25
0.48
0.20
0.64
25˚C
10
66.81
66.52
60.32
40.08
49.48
44.89
22.93
62.83
29.70
20
41.80
51.57
43.13
17.72
32.96
26.20
11.12
44.41
21.74
30
31.70
39.23
35.59
10.65
22.62
18.82
6.89
19.18
18.48
40
27.05
31.48
31.68
7.63
18.73
15.40
5.79
11.47
14.81
50
20.46
27.26
28.26
6.11
14.11
11.75
5.05
8.02
12.32
60
18.52
23.90
26.07
5.32
12.87
9.61
3.13
7.46
9.66
86
Table 20 Percentage of 5nm AuNPs, AuNP-BSA and AuNP-Fb remaining in the supernatant
after centrifugation at different g-force, time and temperature.
%
8400g
12000g
14900g
4˚C
Time
(mins)
Au
Ps
Au PBSA
Au PFB
Au
Ps
Au PBSA
Au PFB
Au
Ps
Au PBSA
Au PFB
10
82.74
86.78
90.87
79.26
102.41
85.78
68.87
98.17
69.86
20
84.35
74.16
82.70
77.87
96.66
75.63
60.96
90.75
61.82
30
83.91
73.10
72.28
77.49
93.46
61.78
54.89
88.11
59.87
40
80.47
71.39
66.98
76.15
90.24
56.94
51.33
83.44
57.93
50
79.79
68.10
55.27
73.92
85.96
47.31
46.34
79.90
47.59
60
76.58
59.34
55.77
69.37
82.16
48.29
41.91
70.66
45.77
25˚C
10
86.60
96.38
91.25
97.48
83.18
72.59
96.29
97.58
77.95
20
86.50
90.88
85.28
98.86
89.86
72.57
92.96
97.17
64.22
30
87.02
86.51
77.17
99.01
88.86
59.59
93.18
100.93
62.04
40
87.95
89.82
69.57
96.56
96.20
58.00
92.60
97.46
62.32
50
86.68
85.58
70.53
95.17
97.27
59.17
93.13
100.51
62.94
60
86.25
88.23
69.93
95.99
87.41
53.75
90.31
97.83
58.77
4.5.2 Hydrodynamic size of AuNP-protein conjugates after centrifugation
The sizes of the AuNP-protein conjugates were measured after centrifugation where excess
proteins in the solution were removed. This is to determine if any aggregation or desorption of
the protein had resulted from centrifugation. The sizes of the samples were compared with the
AuNP-protein control where the excess proteins were not yet removed by centrifugation. All the
results are illustrated in Table 21, Table 22 and Table 23. The size of the 15nm and 11nm AuNPprotein conjugates were generally not affected after centrifugation at the various g-forces and
87
centrifugation time. The temperature at which centrifugation took place also did not have an
effect on the sample. The 5nm AuNP-protein samples, however, saw some increase in the size of
the conjugates as centrifugation time increased when centrifugation took place at 25˚C. This
could indicate some aggregation of the samples after centrifugation. These results did not come
as a surprise as preliminary experiments (data not shown) did suggest that the 5nm AuNPs were
less stable compared to the 11nm and 15nm AuNPs. The aggregation experienced after
centrifugation however was not severe, and with proper and careful treatment of the conjugate
samples, as well as suitable centrifugation conditions, the aggregation can be avoided.
88
Table 21 Size of 15nm AuNP-protein conjugates using DLS after centrifugation at different gforce, time and temperature.
Size (nm)
8400g
12000g
14900g
4˚C
Time
(mins)
Au Ps
Au PBSA
Au P
-FB
Au Ps
Au PBSA
Au P
-FB
Au Ps
Au PBSA
Au P
-FB
Control
19.7
33.9
74.0
17.9
33.9
70.0
17.9
33.0
75.0
10
24.4
33.4
82.6
20.1
33.2
75.4
18.7
34.2
78.6
20
21.0
32.1
80.7
17.7
32.2
73.6
18.7
33.5
77.0
30
23.8
32.5
76.6
18.0
35.1
70.7
19.2
34.7
72.8
40
22.0
31.5
80.1
17.5
33.3
71.6
18.2
33.5
76.1
50
21.4
30.9
78.6
19.1
35.1
71.2
19.0
35.2
73.5
60
22.1
36.1
77.3
18.9
35.5
71.8
19.3
35.9
73.5
25˚C
Control
17.7
33.9
71.9
17.7
33.4
75.4
17.8
33.0
73.2
10
19.7
35.4
80.3
19.0
33.7
81.3
21.4
32.3
78.6
20
18.9
33.0
80.1
18.9
33.5
77.8
21.2
37.7
76.9
30
18.2
33.3
80.0
19.3
34.3
79.7
20.2
36.9
75.7
40
18.7
35.1
82.3
19.1
34.5
77.6
20.7
35.6
75.3
50
19.0
35.5
78.6
18.4
35.9
79.5
19.5
36.0
78.2
60
18.3
34.9
79.6
18.9
36.0
79.7
19.7
36.5
79.6
89
Table 22 Size of 11nm AuNP-protein conjugates using DLS after centrifugation at different gforce, time and temperature.
Size (nm)
8400g
12000g
14900g
4˚C
Time
(mins)
Au
Ps
Au PBSA
Au PFB
Au
Ps
Au PBSA
Au PFB
Au
Ps
Au PBSA
Au PFB
Control
15.58
30.0
83.49
15.25
27.0
82.79
13.64
27.5
77.20
10
17.68
27.4
95.44
18.90
28.5
97.88
14.90
26.4
84.70
20
17.68
30.3
88.78
18.05
29.9
92.99
14.63
27.7
81.12
30
18.83
32.0
89.58
18.26
30.1
87.06
15.15
28.5
77.98
40
18.18
33.5
87.94
17.83
28.8
88.80
15.01
27.4
77.86
50
17.69
32.3
83.40
17.34
28.3
83.95
18.29
29.9
80.62
60
17.64
28.0
86.14
17.83
29.6
87.21
15.39
28.7
74.54
25˚C
Control
19.22
26.1
76.62
21.15
26.0
82.49
18.02
25.5
77.32
10
22.78
28.1
92.55
21.41
30.9
88.47
22.99
26.0
93.41
20
21.79
27.8
89.69
18.80
31.1
88.89
22.40
26.9
87.55
30
22.65
28.0
85.71
20.88
30.4
88.52
16.43
27.2
87.40
40
21.83
28.6
86.89
20.61
30.0
87.15
22.60
26.4
86.54
50
21.68
28.6
88.07
20.38
31.5
92.72
21.52
26.7
85.37
60
22.60
28.8
86.94
21.27
31.1
93.01
21.40
27.8
87.64
90
Table 23 Size of 5nm AuNP-protein conjugates using DLS after centrifugation at different gforce, time and temperature.
Size
(nm)
8400g
12000g
14900g
4˚C
Time
(mins)
Au P
Au PBSA
Au PFB
Au P
Au PBSA
Au PFB
Control
10.75
35.9
10
9.65
20
Au P- Au PBSA
FB
Au P
77.11
11.37
22.5
82.45
10.53
14.3
82.91
17.0
46.79
7.62
30.1
96.81
9.76
28.7
74.98
10.31
31.1
69.27
11.21
25.8
80.74
12.63
27.8
94.20
30
9.32
18.9
80.18
21.86
25.8
92.02
12.56
33.1
97.33
40
12.12
20.9
61.73
11.09
25.8
100.29
9.99
22.0
103.18
50
10.62
32.3
67.94
8.31
27.3
89.51
10.15
27.7
98.68
60
11.34
34.5
82.22
11.20
24.3
82.28
10.18
26.0
90.29
25˚C
Control
10.93
16.7
83.20
10.79
17.9
80.82
10.79
19.2
87.11
10
10.94
14.0
45.26
10.87
10.1
88.48
11.26
21.8
69.93
20
12.45
19.9
62.57
10.55
16.8
92.50
10.69
24.7
80.15
30
14.96
20.4
81.76
10.38
12.5
88.72
9.66
20.0
107.70
40
8.63
22.1
98.28
9.56
16.0
100.30
10.79
25.1
122.60
50
14.68
23.7
109.32
10.60
17.3
96.20
10.41
24.1
132.40
60
19.43
14.7
108.60
9.66
25.6
100.26
9.32
25.0
91.84
4.6 Gel electrophoresis
SDS-PAGE was conducted using 15nm AuNPs for the AuNP-BSA and AuNP-Fb conjugates of
different protein concentration. BSA of 50ug/mL, 200µg/mLand 500µg/mLconcentrations was
used while Fb of 200ug/mL, 500µg/mLand 1mg/mL concentrations were used. Table 24 shows
91
the theoretical number of protein there is to 1 AuNP for each of the protein concentration used. It
can be safely assumed that the proteins were in excess for each of the concentrations used.
Table 24 Number of BSA or Fb to 1 AuNP for different protein concentrations.
[protein]
ug/mL
o. of BSA to 1
Au P
o. of Fb to 1
Au P
50
149
29
200
597
116
500
1492
290
1000
2985
579
4.6.1 AuNP-bovine serum albumin conjugates
Prior to the SDS-PAGE, the conjugated samples were washed twice by the centrifugation
method to remove excess proteins and to collect the sample into pellet form for loading onto the
gel. SDS-PAGE for the two different conjugate samples were ran separately. Figure 12 shows a
representative gel for AuNP-BSA with BSA concentration of 200µg/mL. The protein ladder was
loaded into the first lane of the gel. This ladder was added into every gel run to ensure that the
protein observed was BSA and that there were no contamination by other proteins. The next four
lanes contain the BSA calibration standard of concentrations 25, 50, 90 and 125µg/mL. The
intensities of these four standards on the gel were used to form a calibration line to determine the
relative intensity of the samples loaded onto the same gel. All the samples collected into a pellet
form by centrifugation were loaded into the gel for the determination of the absolute amount of
protein conjugated to the AuNPs. As one lane in the gel could only hold up to 25ul in volume,
two lanes had to be used for the loading of a single sample, and the absolute amount of protein
was later taken as the summation of the amount of protein collected from both lanes.
92
Figure 12 Representation picture of the SDS-PAGE gel. Lane 1: protein ladder, lane 2-5: BSA
calibration standards of concentration 25, 50, 90, 125 ug/mL, lane 6-7: sample 1, lane 8-9:
sample 2.
From previous data presented for the size of the conjugates, it was observed that BSA formed a
monolayer on the surface of the AuNPs and hence the size of the conjugate stays about the same
even with an increase in BSA added for conjugation. The SDS-PAGE results obtained here
complemented our findings from DLS and the results are shown in Table 25. On the average,
about 7 BSA molecules were adsorbed onto one 15nm AuNPs even in exceedingly excess BSA
concentrations. The concentration of AuNPs used for the calculation was based on an average
concentration obtained from the previous ICP-MS results.
93
Table 25 The 4 best SDS-PAGE results for different BSA concentration used. The absolute
amount of protein adsorbed, together with the percentage of the BSA added adsorbed and the
number of BSA molecule adsorbed per AuNP are shown. A. [BSA] 50ug/mL, B. [BSA]
200µg/mLand C. [BSA] 500ug/mL.
A.
[BSA] = 50µg/mL
Run
Amt of protein
adsorbed [µg]
% of BSA
adsorbed
O. of BSA
molecule per
Au P
1
2.8
5.6
8
2
1.8
3.5
5
3
1.6
3.3
5
4
2.4
4.8
7
Average
2.1
4.3
6
B.
[BSA] = 200µg/mL
Run
Amt of protein
adsorbed [µg]
% of BSA
adsorbed
O. of BSA
molecule per
Au P
1
2.7
1.1
8
2
2.3
1.2
7
3
2.2
1.1
7
4
1.9
1.0
6
Average
2.3
1.1
7
94
C.
[BSA] = 500µg/mL
Run
Amt of protein
adsorbed [µg]
% of BSA
adsorbed
O. of BSA
molecule per
Au P
1
2.9
0.6
9
2
2.7
0.5
8
3
2.3
0.5
7
4
2.3
0.5
7
Average
2.5
0.5
8
4.6.2 AuNP-fibrinogen conjugates
The AuNP-fibrinogen conjugates exhibited a different phenomenon as compared to the AuNPBSA conjugates, but the results obtained for SDS-PAGE (shown in Table 26) is in conjunction
with the results from DLS. It can be observed that unlike the AuNP-BSA conjugates, the number
of Fb molecules that conjugated with a single AuNP increases as the concentration of Fb added
was increased. This suggests that the Fb formed a multilayer around the AuNPs. The functional
study for fibrinogen is thus crucial to complement these results because if the adsorption of
fibrinogen onto AuNPs disrupts the protein’s function, it can result in blood being unable to clot.
Using high concentrations of AuNPs can then be toxic to the organism as large amounts of
fibrinogen will become unavailable due to adsorption on the nanoparticles. Two other crucial
factors which require investigation is the affinity of fibrinogen for AuNPs, and if any change in
conformation or function of Fb are reversible. If the fibrinogen has lower affinity for AuNPs
compared to other proteins, they will only be adsorbed temporarily on the AuNPs, and reversible
conformation or functional changes will allow the fibrinogen to revert back to its native
conformation to carry out its required functions.
95
Table 26 The 4 best SDS-PAGE results for different Fb concentration used. The absolute
amount of protein adsorbed, together with the percentage of the Fb added adsorbed and the
number of Fb molecule adsorbed per AuNP are shown. A. [Fb] 200ug/mL, B. [Fb] 500ug/mL.
[Fb] = 200µg/mL
Run
Amt of
protein
adsorbed [µg]
% of BSA
adsorbed
O. of Fb
molecule
per Au P
1
13
6.5
8
2
16.1
8.1
9
3
14.1
7.1
8
4
14
7
8
Average
14.3
7.2
8
[Fb] = 500µg/mL
Run
Amt of
protein
adsorbed [µg]
% of BSA
adsorbed
O. of Fb
molecule
per Au P
1
25
5
14
2
24.8
5
14
3
21.6
4.3
13
4
24
4.8
14
Average
23.9
4.8
14
4.7 Circular dichroism
Circular dichroism was utilized to determine any changes in the secondary structure of the
proteins after conjugation with AuNPs. Changes in conformation would then need to be further
investigated to determine if the function of the proteins were affected which could result in
detrimental effects. Circular dichroism measurements were based on spectrophotometry
96
technique, and the samples were measured from 190nm to 300nm wavelengths. AuNPs, however,
exhibit strong absorption in this region and was found to interfere with the measurements. Figure
13 below illustrates three samples measured; the first was BSA alone, the second was AuNPBSA conjugate of 1 time concentration and the third was AuNP-BSA conjugates concentrated 4
times. The excess proteins in the two conjugate samples here were not removed. The BSA
sample’s results showed a typical curve that has a high percentage of alpha-helix which is similar
to literature. The conjugate samples showed similar curves with decreased intensity. However,
because the excess proteins in the solutions were very high, the results obtained could reflect that
of the excess proteins and not the conjugated proteins. The decrease in intensity could have been
attributed to the presence of AuNPs. A second set of samples were therefore employed where the
excess proteins were removed as shown in Figure 14.
97
100
BSA
AuNP-BSA
AuNP-BSA x4
80
60
CD (mdeg)
40
20
Wavelength (nm)
0
190
210
230
250
270
290
-20
-40
-60
Figure 13 Circular dichroism spectra of BSA, AuNP-BSA conjugate as well as 4 times
concentrated AuNP-BSA conjugates. Excess proteins in the samples were not removed before
the CD measurement.
98
20
AuNP-BSA washed
AuNP-BSA x4 washed
15
AuNP-BSA x10 washed
CD (mdeg)
10
5
Wavelength (nm)
0
190
210
230
250
270
290
-5
-10
-15
-20
Figure 14 Circular dichroism spectra of AuNP-BSA conjugate, 4 times concentrated AuNP-BSA
conjugate and 10 times concentrated AuNP-BSA conjugate. Excess proteins in the samples were
removed by centrifugation before the CD measurement.
It can be observed in Figure 14 that the characteristic curve for BSA is no longer differentiable
when the excess proteins in the conjugate solution were removed. At the AuNP-BSA
concentration of 1 time, the amount of protein present could have been too little for it to be
reflected in the spectrometry measurement, the concentration of the conjugate was thus increased
up to ten times. The spectra at 10 times concentration of AuNP-BSA however still had very low
intensity, and the characteristic curve was still not differentiable. Further increase in the
conjugate concentration was not possible as the conjugates started to aggregate after
centrifugation. The presence of AuNPs in the solution interfered with the measurement and
hence this method was subsequently abandoned as no meaningful results could be derived. This
99
was similar to the investigation using FTIR (data not shown), where no meaningful data could be
derived as the presence of AuNPs interfered with the measurement.
100
Chapter 5 Conclusion and Future work
With the increasing use of nanoparticles for biomedical related purposes, the number of toxicity
studies should increase proportionally as well. Endpoint studies to determine if the particular
nanoparticle exerts cytotoxic effect on cells are important for evaluation purposes. The
fundamental interactions between nanoparticles and biomolecules also need to be thoroughly
studied to fully understand what is going on in the sub-cellular level. Having this knowledge and
understanding will aid in future endeavors when different modifications of nanoparticles are to
be used. Also, any possible long term effects that the nanoparticles could have can also be
predicted if we understand these fundamentals.
The results presented here are only but a preliminary investigation, where the characterization of
AuNP-protein conjugates was studied. The important points that we have learned through the
investigations are:
a) How the size of the AuNP-protein conjugate varies with the concentration of protein
added as well as the amount of proteins conjugated on the AuNPs,
b) The zeta potential of the AuNPs versus the AuNP-protein conjugates,
c) The stability of the AuNP-protein conjugates over time ,
d) BSA forms a monolayer on AuNPs, while Fb forms multi-layers on AuNPs,
e) The centrifugation parameters for effective recovery of AuNPs and AuNP-protein
conjugates.
Because of the unique properties of AuNPs, the study met with a number of predicaments due to
the limited analysis methods that can be used. The possible conformation change of proteins
adsorbed onto AuNPs has yet to be established, and functional changes of the proteins also have
yet to be investigated. These should be the immediate steps that are to be taken to further this
101
work. The effect that conjugated proteins have on the properties of the AuNPs such as their
reactivity or optical properties should also be investigated.
Other crucial factors for investigation include the affinity of proteins for AuNPs, and whether the
changes in conformation or function (if any) are reversible. Steps can also be taken to further the
work that was started by Dawson et al. [19] to fully map out the proteins that adsorb on the
nanoparticles when exposed to human plasma, as well as to determine the binding affinities of
these proteins. The impact of the conjugation of nanoparticles to each adsorbed protein can then
be evaluated to determine if the function of the proteins are impeded by the conjugation. These
evaluations should also include chronic toxicity as well as genotoxicity testing.
Further steps can then be taken to follow the uptake and clearance of nanoparticles from cells.
Factors such as how the nanoparticles are taken up by the cells as well as the location and
duration of the accumulation of nanoparticles in the cells can be the objectives of such studies.
These studies will then give us a better insight into whether the introduction of these foreign
materials into our human bodies is truly warranted or the risks involved outweigh the benefits.
102
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
A. D. Maynard. Woodrow Wilson International Center for Scholars: Washington DC, 2006.
S. Lanone, J. Boczkowski; Current Molecular Medicine; 2006; 6; 651-663.
T. Xia, N. Li, A. E. Nel. 2009, vol. 30, pp 137-150.
T. C. Long, N. Saleh, R. D. Tilton, G. V. Lowry, B. Veronesi; Environmental Science and Technology;
2006; 40; 4346-4352.
X. H. Huang, I. H. El-Sayed, W. Qian, M. A. El-Sayed; J. Am. Chem. Soc.; 2006; 128; 2115-2120.
G. J. Nusz, A. C. Curry, S. M. Marinakos, A. Wax, A. Chilkoti; Nano Lett.; 2009; 3; 795–806.
P. Aggarwal, J. B. Hall, C. B. McLeland, M. A. Dobrovolskaia, S. E. McNeil; Adv. Drug Deliver Rev.;
2009; 61; 428-437.
J. L. West, N. J. Halas; Curr. Opin. Biotechnol.; 2000; 11; 215-217.
S. Goodwin, C. Peterson, C. Hoh, C. Bittner; J. Magn. Magn. Mate; 1999; 194; 132-139.
F. Sonvico, C. Dubernet, P. Colombo, P. Couvreur; Curr. Pharm. Design; 2005; 11; 2091-2105.
J. H. W. Leuvering, P. J. H. M. Thal, M. Van Der Waart, A. H. W. M. Schuurs; J. Immunoass.
Immunoch.; 1980; 1; 77-91.
T. A. Taton, C. A. Mirkin, R. L. Letsinger; Science; 2000; 289; 1757-1760.
N. L. Rosi, D. A. Giljohann, C. S. Thaxton, A. K. R. Lytton-Jean, M. S. Han, C. A. Mirkin; Science;
2006; 312; 1027-1030.
S. Kumar, N. Harrison, R. Richards-Kortum, K. Sokolov; Nano Lett.; 2007; 7; 1338-1343.
A. W. H. Lin, N. A. Lewinski, J. L. West, N. J. Halas, R. A. Drezek; J. Biomed. Opt.; 2005; 10.
D. P. O'Neal, L. R. Hirsch, N. J. Halas, J. D. Payne, J. L. West; Cancer Lett.; 2004; 209; 171-176.
S. K. Libutti, G. F. Paciotti, A. A. Byrnes, H. R. Alexander Jr, W. E. Gannon, M. Walker, G. D. Seidel,
N. Yuldasheva, L. Tamarkin; Clinical Cancer Research; 2010; 16; 6139-6149.
I. Lynch, K. A. Dawson, S. Linse; Science's STKE : signal transduction knowledge environment;
2006; 2006.
T. Cedervall, I. Lynch, S. Lindman, T. Berggård, E. Thulin, H. Nilsson, K. A. Dawson, S. Linse;
Proceedings of the National Academy of Sciences of the United States of America; 2007; 104;
2050-2055.
W. Norde, J. Lyklema; Journal of biomaterials science. Polymer edition; 1991; 2; 183-202.
W. Norde, D. Gags; Langmuir; 2004; 20; 4162-4167.
L. T. Allen, M. Tosetto, I. S. Miller, D. P. O'Connor, S. C. Penney, I. Lynch, A. K. Keenan, S. R.
Pennington, K. A. Dawson, W. M. Gallagher; Biomaterials; 2006; 27; 3096-3108.
J. J. Gray; Current Opinion in Structural Biology; 2004; 14; 110-115.
C. J. Wilson, R. E. Clegg, D. I. Leavesley, M. J. Pearcy; Tissue Engineering; 2005; 11; 1-18.
M. F. M. Engel, A. J. W. G. Visser, C. P. M. Van Mierlo; Proceedings of the National Academy of
Sciences of the United States of America; 2004; 101; 11316-11321.
T. Kurkina, K. Balasubramanian; Cellular and Molecular Life Sciences; 2011; 1-16.
Z. S. Lu, C. M. Li; Current Medicinal Chemistry; 2011; 18; 3516-3528.
S. F. Lee, M. A. Osborne; ChemPhysChem; 2009; 10; 2174-2191.
U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke, T. Nann; Nature Methods; 2008;
5; 763-775.
103
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
D. Ratchford, K. Dziatkowski, T. Hartsfield, X. Li, Y. Gao, Z. Tang; Journal of Applied Physics; 2011;
109.
J. Vela, H. Htoon, Y. Chen, Y. S. Park, Y. Ghosh, P. M. Goodwin, J. H. Werner, N. P. Wells, J. L.
Casson, J. A. Hollingsworth; Journal of Biophotonics; 2010; 3; 706-717.
G. Ruan, J. O. Winter; Nano Letters; 2011; 11; 941-945.
M. M. Barroso; Journal of Histochemistry and Cytochemistry; 2011; 59; 237-251.
A. M. Derfus, W. C. W. Chan, S. N. Bhatia; Advanced Materials; 2004; 16; 961-966.
K. R. Lee, I. J. Kang; Ultramicroscopy; 2009; 109; 894-898.
J. K. Jaiswal, H. Mattoussi, J. M. Mauro, S. M. Simon; Nature Biotechnology; 2003; 21; 47-51.
T. M. Tolaymat, A. M. El Badawy, A. Genaidy, K. G. Scheckel, T. P. Luxton, M. Suidan; Science of
the Total Environment; 2010; 408; 999-1006.
Q. Chaudhry, M. Scotter, J. Blackburn, B. Ross, A. Boxall, L. Castle, R. Aitken, R. Watkins; Food
Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment;
2008; 25; 241-258.
J. Y. Yang, C. Y. Huang, S. S. Chuang, C. C. Chen; Burns; 2007; 33; 793-797.
J. Chen, C. M. Han, X. W. Lin, Z. J. Tang, S. J. Su; Zhonghua wai ke za zhi [Chinese journal of
surgery]; 2006; 44; 50-52.
J. Asz, D. Asz, R. Moushey, J. Seigel, S. B. Mallory, R. P. Foglia; Journal of Pediatric Surgery; 2006;
41; e9-e12.
V. Alt, T. Bechert, P. Steinrücke, M. Wagener, P. Seidel, E. Dingeldein, E. Domann, R. Schnettler;
Biomaterials; 2004; 25; 4383-4391.
G. L. Grunkemeier, R. Jin, A. Starr; Annals of Thoracic Surgery; 2006; 82; 776-780.
G. J. Hutchings, M. Brust, H. Schmidbaur; Chemical Society Reviews; 2008; 37; 1759-1765.
J. Turkevich, P. C. Stevenson, J. Hillier; Discuss. Faraday Soc.; 1951; 55.
C. Noguez; Journal of Physical Chemistry C; 2007; 111; 3606-3619.
M. Iwamoto, K. Kuroda, J. Kanzow, S. Hayashi, F. Faupel; Advanced Powder Technology; 2005; 16;
137-144.
G. Frens; Kolloid-Zeitschrift & Zeitschrift für Polymere; 1972; 250; 736-741.
M. M. Maye, W. Zheng, F. L. Leibowitz, N. K. Ly, C. J. Zhong; Langmuir; 2000; 16; 490-497.
Y. Sun, Y. Xia; Science; 2002; 298; 2176-2179.
A. Henglein, D. Meisel; Langmuir; 1998; 14; 7392-7396.
M. Grzelczak, J. Pérez-Juste, P. Mulvaney, L. M. Liz-Marzán; Chemical Society Reviews; 2008; 37;
1783-1791.
C. J. Murphy; Science; 2002; 298; 2139-2141.
C. H. Kuo, T. F. Chiang, L. J. Chen, M. H. Huang; Langmuir; 2004; 20; 7820-7824.
T. K. Sau, C. J. Murphy; Journal of the American Chemical Society; 2004; 126; 8648-8649.
B. D. Chithrani, A. A. Ghazani, W. C. W. Chan; Nano Lett.; 2006; 6; 662-668.
J. M. De La Fuente, C. C. Berry; Bioconjugate Chemistry; 2005; 16; 1176-1180.
W. H. De Jong, W. I. Hagens, P. Krystek, M. C. Burger, A. J. A. M. Sips, R. E. Geertsma;
Biomaterials; 2008; 29; 1912-1919.
G. Sonavane, K. Tomoda, K. Makino; Colloids and Surfaces B: Biointerfaces; 2008; 66; 274-280.
104
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
K. Donaldson, C. L. Tran; Mutation Research - Fundamental and Molecular Mechanisms of
Mutagenesis; 2004; 553; 5-9.
G. Oberdorster, J. Ferin, P. E. Morrow; Experimental Lung Research; 1992; 18; 87-104.
M. Semmler-Behnke, W. G. Kreyling, J. Lipka, S. Fertsch, A. Wenk, S. Takenaka, G. Schmid, W.
Brandau; Small; 2008; 4; 2108-2111.
W. S. Cho, M. Cho, J. Jeong, M. Choi, H. Y. Cho, B. S. Han, S. H. Kim, H. O. Kim, Y. T. Lim, B. H.
Chung; Toxicology and Applied Pharmacology; 2009; 236; 16-24.
H. J. Johnston, G. Hutchison, F. M. Christensen, S. Peters, S. Hankin, V. Stone; Critical Reviews in
Toxicology; 2010; 40; 328-346.
P. V. AshaRani, G. L. K. Mun, M. P. Hande, S. Valiyaveettil; ACS Nano; 2009; 3; 279-290.
C. Carlson, S. M. Hussein, A. M. Schrand, L. K. Braydich-Stolle, K. L. Hess, R. L. Jones, J. J. Schlager;
Journal of Physical Chemistry B; 2008; 112; 13608-13619.
K. Soto, K. M. Garza, L. E. Murr; Acta Biomaterialia; 2007; 3; 351-358.
S. M. Hussain, K. L. Hess, J. M. Gearhart, K. T. Geiss, J. J. Schlager; Toxicology in Vitro; 2005; 19;
975-983.
P. P. Karmali, D. Simberg; Expert Opinion on Drug Delivery; 2011; 8; 343-357.
A. Gessner, A. Lieske, B. R. Paulke, R. H. Müller; European Journal of Pharmaceutics and
Biopharmaceutics; 2001; 54; 165-170.
M. A. Dobrovolskaia, A. K. Patri, J. Zheng, J. D. Clogston, N. Ayub, P. Aggarwal, B. W. Neun, J. B.
Hall, S. E. McNeil; Nanomedicine: Nanotechnology, Biology, and Medicine; 2009; 5; 106-117.
I. Lynch, T. Cedervall, M. Lundqvist, C. Cabaleiro-Lago, S. Linse, K. A. Dawson; Advances in Colloid
and Interface Science; 2007; 134-135; 167-174.
M. Lundqvist, J. Stigler, G. Elia, I. Lynch, T. Cedervall, K. A. Dawson; Proceedings of the National
Academy of Sciences of the United States of America; 2008; 105; 14265-14270.
D. M. Brown, C. Dickson, P. Duncan, F. Al-Attili, V. Stone; Nanotechnology; 2010; 21.
Z. Xu, S. L. Wang, H. W. Gao; J. Hazard. Mater.; 2010.
G. Chandra, K. S. Ghosh, S. Dasgupta, A. Roy; International Journal of Biological Macromolecules;
2010; 47; 361-365.
E. Casals, T. Pfaller, A. Duschl, G. J. Oostingh, V. Puntes; ACS Nano; 2010; 4; 3623-3632.
Q. Yang, J. Liang, H. Han; Journal of Physical Chemistry B; 2009; 113; 10454-10458.
J. J. Zhou, S. Y. Wang, S. Gunasekaran; Journal of Food Science; 2009; 74; N50-N56.
J. Mora, S. Maestre, V. Hernandis, J. L. Todolí; TrAC - Trends in Analytical Chemistry; 2003; 22;
123-132.
R. Thomas; Spectroscopy; 2002; 17; 36-41.
K. L. Linge, K. E. Jarvis; Geostandards and Geoanalytical Research; 2009; 33; 445-467.
H. Niu, R. S. Houk; Spectrochimica Acta - Part B Atomic Spectroscopy; 1996; 51; 779-815.
M. M. Bradford; Analytical Biochemistry; 1976; 72; 248-254.
B. J. Olson, J. Markwell; Current protocols in protein science; 2007.
C. M. Stoscheck; Methods in Enzymology; 1990; 182; 50-68.
O. H. Lowry, N. J. Rosebrough, A. L. Farr, R. J. Randall; The Journal of biological chemistry; 1951;
193; 265-275.
P. K. Smith, R. I. Krohn, G. T. Hermanson; Analytical Biochemistry; 1985; 150; 76-85.
105
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
K. J. Wiechelman, R. D. Braun, J. D. Fitzpatrick; Analytical Biochemistry; 1988; 175; 231-237.
O. Vesterberg; Electrophoresis; 1993; 14; 1243-1249.
S. Raymond, L. Weintraub; Science; 1959; 130; 711.
A. L. Shapiro, E. Viñuela, J. V. Maizel Jr; Biochemical and Biophysical Research Communications;
1967; 28; 815-820.
O. Vesterberg; Journal of Chromatography A; 1989; 480; 3-19.
V. I. Dodero, Z. B. Quirolo, M. A. Sequeira; Frontiers in Bioscience; 2011; 16; 61-73.
B. M. Bulheller, A. Rodger, J. D. Hirst; Physical Chemistry Chemical Physics; 2007; 9; 2020-2035.
U. K. Laemmli; Nature; 1970; 227; 680-685.
I. García, J. Gallo, N. Genicio, D. Padro, S. Penadés; Bioconjugate Chemistry; 2011; 22; 264-273.
K. V. Singh, J. Kaur, G. C. Varshney, M. Raje, C. R. Suri; Bioconjugate Chemistry; 2004; 15; 168173.
C. Röcker, M. Pötzl, F. Zhang, W. J. Parak, G. U. Nienhaus; Nature Nanotechnology; 2009; 4; 577580.
V. B. C. Figueira, J. P. Jones; Journal of Colloid and Interface Science; 2008; 325; 107-113.
K. Aslan, P. Holley, C. D. Geddes; Journal of Materials Chemistry; 2006; 16; 2846-2852.
X. C. Shen, Q. Yuan, H. Liang, H. G. Yan, X. W. He; Science in China Series B-Chemistry; 2003; 46;
387-398.
L. Jiang, J. Guan, L. Zhao, J. Li, W. Yang; Colloid Surf. A-Physicochem. Eng. Asp.; 2009; 346; 216220.
J. Guan, L. Jiang, L. Zhao, J. Li, W. Yang; Colloid Surf. A-Physicochem. Eng. Asp.; 2008; 325; 194197.
S. K. Balasubramanian, J. Jittiwat, J. Manikandan, C. N. Ong, L. E. Yu, W. Y. Ong; Biomaterials;
2010; 31; 2034-2042.
Y. Qu, X. Lü; Biomed. Mater.; 2009; 4.
P. Hazarika, T. Giorgi, M. Reibner, B. Ceyhan, C. M. Niemeyer; Methods in molecular biology;
2004; 283; 295-304.
W. Jiang, B. Y. S. Kim, J. T. Rutka, W. C. W. Chan; Nat. Nanotechnol.; 2008; 3; 145-150.
A. Ravindran, A. Singh, A. M. Raichur, N. Chandrasekaran, A. Mukherjee; Colloid Surf. BBiointerfaces; 2010; 76; 32-37.
W. J. Qi, C. Z. Huang, L. Q. Chen; Talanta; 2010; 80; 1400-1405.
G. Wei, C. Pan, J. Reichert, K. D. Jandt; Carbon; 2010; 48; 645-653.
J. A. Jamison, K. M. Krueger, J. T. Mayo, C. T. Yavuz, J. J. Redden, V. L. Colvin; Nanotechnology;
2009; 20.
P. Christian, M. Bromfield; J. Mater. Chem.; 2010; 20; 1135-1139.
106
[...]... amount of proteins that could be affected by the adsorption of proteins onto the AuNPs This project also investigated the effects of centrifugation (an important equipment when working with nanoparticles and proteins) on AuNPs as well as on the AuNP -protein conjugate The amount of AuNPs or AuNP -protein conjugate which were not sedimented was quantified and the presence of any aggregation was after centrifugation. .. with proteins is a good starting point As nanoparticles enter a biological environment, their first contact is with biomolecules, which are more often than not, proteins It has been shown and there is growing acceptance that proteins adsorb onto the nanoparticles forming a protein corona’ on the nanoparticles This term protein corona’ was first coined by Dawson et al to describe the ring of proteins... sample forming a single crystal Functional tests are protein dependent and it could be difficult or expensive to design Stone and coworkers investigated the interaction between carbon black nanoparticles and cytokine proteins and how the protein function was impacted It was observed that the smaller sized nanoparticles of 14nm caused a decrease in TNF-α protein function as compared to the larger 260nm... around a nanoparticle [19].The type of proteins adsorbed determined the new identity of the nanoparticles in the biological environment, and the nanoparticles are recognized by the cells based on this protein corona Subsequent particle distribution and translocation also 22 depended on the proteins adsorbed on them A starting point could then be to identify the proteins that will associate with the... binding of different type of proteins Nanoparticles with hydrophobic surfaces such as polymeric nanoparticles tend to attract apolipoproteins as well as albumins In contrast, inorganic nanoparticles with hydrophilic surfaces tend to attract proteins such as transferrin or histidine-rich glycoproteins, which have positively charged domains A commonly reported phenomenon is that nanoparticles with hydrophobic... avidin or protein G as the linker These bio-conjugates could then selectively label the parts of the cells that express the protein of interest, and they were then imaged and tracked for prolonged periods in the live cells [36] Similar to AgNPs and AuNPs however, few focused research are being conducted on the negative impact of QDs in a biological environment Silver nanoparticles Silver nanoparticles. .. the conjugation between nanoparticles and proteins and also to measure the size of the synthesized nanoparticles 2.4 Centrifugation studies 2.4.1 Inductively coupled plasma mass spectrometry ICP-MS is a spectrometric technique that allows for the detection and quantification of trace amounts of most of the elements in the periodic table It is highly sensitive and can detect concentrations up to the... composites refer to nanoparticles that are combined with other nanomaterials or other larger materials This overview will be focused on a three different nanoparticles (quantum dots, silver nanoparticles and gold nanoparticles) , which are of particular interest for biomedical purposes Quantum dots 13 Quantum dots (QDs) refer to nanocrystals which are mainly made up of elements from group II and group VI to... and nanomedicine Amongst all the nanoparticles developed, metallic nanoparticles have been receiving a lot more attention due to their unique properties Metallic nanoparticles possess unique plasmonic and fluorescent properties from their bulky counterparts, and are being investigated to be used for numerous biomedical diagnostic or detection technologies such as molecular imaging [5], biomarkers and. .. physics, material science and medicine [60] This concern for the potential toxicity of nanoparticles arose from the small particle size and from two independent findings stating that toxicity of the particles increases as the size becomes smaller One of the first studies which indicated the potential toxicity of nanoparticles was by Ferin and coworkers who worked with titanium dioxide nanoparticles (TiO2) ... after centrifugation at 15000g and 4˚C for 90 and 120 minutes Size of 15nm AuNPs using DLS after centrifugation at different g-force, time and temperature Size of the 11nm AuNPs using DLS after centrifugation. .. g-force, time and temperature Size of 11nm AuNP-protein conjugates using DLS after centrifugation at different g-force, time and temperature Size of 5nm AuNP-protein conjugates using DLS after centrifugation. .. AuNPs, AuNP-BSA and AuNP-Fb remaining in the supernatant after centrifugation at different g-force, time and temperature Size of 15nm AuNP-protein conjugates using DLS after centrifugation at
Ngày đăng: 07/10/2015, 10:10
Xem thêm: Gold nanoparticles protein conjugate studies centrifugation and binding studies
