This document provides details of an individual project investigating the use of protein-bound gold nanoparticles (AuNPs) as biochemical sensors. The student synthesized citrate-capped AuNPs and bound them to proteins including papain and bovine serum albumin (BSA) to test their ability to detect heavy metals like mercury and copper compared to unmodified AuNPs. Detection ability was assessed based on color change intensity, signal-to-noise ratio, limit of detection, and stability over time. The results of the study are presented and discussed regarding the effects of protein coronas on AuNP sensing properties.

1. School of Biological Sciences
BS3010
INDIVIDUAL PROJECT
FULL PROJECT TITLE: Organic Gold: A study of the
effects of the cysteine content of protein-bound gold
nanoparticles in novel biochemical sensing.
STUDENT NAME: Christopher Huckle
STUDENT ID NUMBER: 100740106
RHUL EMAIL ADDRESS: Christopher.Huckle.2012@live.rhul.ac.uk
School of Biological Sciences, Royal Holloway, University of London,
Egham, Surrey TW20 0EX
PROJECT SUPERVISOR: Dr James McEvoy
Wet Project Dry Project
TURNITIN RECEIPT NUMBER:
X
2014-15

2. Contents
Abstract.................................................................................................................................3
1. Introduction......................................................................................................................4
2. Materials and methods.....................................................................................................6
2.1 Materials.......................................................................................................................7
2.2 Instruments...................................................................................................................7
2.3 Preparation of citrate-capped AuNPs...........................................................................7
2.4 Binding proteins to AuNPs...........................................................................................8
2.5 Sensing heavy metal ions..............................................................................................9
3. Results..............................................................................................................................10
3.1 Intensity and S/N ratio................................................................................................10
3.2 Limits of detection......................................................................................................11
3.3 Stability.......................................................................................................................11
3.4 Control experiments....................................................................................................11
4. Discussion........................................................................................................................12
4.1 The effects of protein coronas....................................................................................12
4.2 Selectivity towards mercury.......................................................................................13
4.3 Evaluation...................................................................................................................14
4.4 Alternative implications..............................................................................................15
5. Conclusions.....................................................................................................................16
6. Acknowledgements.........................................................................................................17
7. References.......................................................................................................................18
8. Figures and tables...........................................................................................................20
9. Appendix I – Project diary............................................................................................28
10. Appendix II – Raw data (absorption spectra)
11. Appendix III – Forms A-D and risk assessment
2

3. Abstract
Gold nanoparticles (AuNPs) have been extensively used in many areas of scientific
research for centuries. One area in particular is their use as biochemical sensors to detect
miniscule quantities of material. Many assays and experiments have designed unique
processes involving various methods and chemicals to tailor AuNPs to specific analytes
and improve their detection ability.
It has been suggested that binding papain to AuNPs achieves this via the use of its cysteine
residues and unique interactions between thiol groups and cations. This investigation
sought to determine the efficacy of using papain-bound citrate-capped AuNPs (P-AuNPs)
as more effective sensors. Results were compared with AuNPs alone, bovine serum
albumin (BSA)-bound AuNPs (BSA-AuNPs) with higher cysteine contents, and also
control experiments.
It was observed that AuNPs alone performed as better heavy metal detectors than their
protein-bound counterparts. They induced a more intense colour change, better S/N ratios,
lower limits of detection and remained stable for longer periods of time.
Whilst there is a unique interaction between thiol groups and mercury cations, such an
interaction cannot be achieved via protein-bound AuNPs, seemingly due to steric
hindrance. There are many other sulphur-containing molecules that are cheaper and can
provide better results as AuNP caps. Protein-bound AuNPs may still have many uses,
albeit not as sensors. For instance, their sustained prevention of aggregation at pH
extremes could lead to their two-fold use as non-degradable scaffolds for therapeutic
molecules in drug delivery. Concerning detection of heavy metals however, AuNPs alone
remain the most sensitive method of detection.
3

4. 1. Introduction
Nanotechnology remains an ambitious area of research for the scientific community with
widespread applications and many potential breakthroughs waiting to be discovered.
Ranging from drug delivery to substance detection, nanoparticles will likely play many
biochemical roles in the near-future. Indeed, the use of nanoparticles in the detection of
materials alone is a subject of great interest within forensic, medical and environmental
science groups, to name a few. A method to detect substances in biological samples could
lead to rapid diagnosis and treatment of disease, identification of substances, quality
control for water and food and more.
Many heavy metals play major biological roles at relatively minor concentrations, such as
the iron centre in the metalloprotein haemoglobin. However, ingestion or exposure to
higher concentrations of heavy metals often have a detrimental effect on health, even being
fatal in high enough concentrations. This can be by acting as enzyme inhibitors or reacting
with various biological molecules to produce toxic variations. Therefore, the detection of
such metals, especially in food and water, remains vital.
A ubiquitous method for the detection of miniscule quantities of chemical or biological
material requires the use of simple, inexpensive and sensitive techniques. Gold
nanoparticles have the potential to fulfil these criteria. With the rapid synthesis of small
amounts of sensors, one batch could be applied to dozens or perhaps hundreds of samples
to give clear results.
The use of colloidal gold dates back to antiquity and the details of its initial discovery
remains unknown. With historic uses ranging from stained glass to proposed alchemical
elixirs of life, scientific investigation began with Michael Faraday who synthesised pure
colloidal gold in 1856 (Tweney, 2006). Ever since, AuNPs have been a commonly-used
sensor in many biochemical circumstances.
The most common method of AuNP synthesis – and the one used in this investigation – is
the Turkevich method (Kimling et al., 2006). First used in 1951 (Turkevich et al., 1951),
this process involves reduction of hot, aqueous chloroauric acid (HAuCl4) by citrate to
form small spheres of gold, mere nanometres in diameter. After rapid synthesis, citrate also
4

5. caps and stabilises the nanoparticles to prevent aggregation. The negatively charged citrate
molecules repel one another, causing the AuNPs to remain separate.
In terms of detection of materials, AuNPs, akin to other sensors, rely on a specific receptor
element and a transducer element to signal binding. This ensures selective recognition and
binding to analytes as well as an easily-detectable response, often visible to the naked eye.
AuNPs rely on colorimetric detection, undergoing a drastic colour change if an analyte is
introduced (Saha et al., 2012). Individual AuNPs in solution with a diameter between 10-
30nm cause a deep red colour. This is due to a unique plasmon surface resonance where
the electrons absorb blue and green light whilst reflecting mostly red. However, after the
introduction of an analyte which cancels out the negative charge of the citrate, such as
positively-charged heavy metal ions, the AuNPs swiftly aggregate. The assembly of
multidentate complexes between ions and particles greatly alters the plasmon surface
resonance, inducing a near-immediate colour change to blue, shifting to a longer
wavelength (Figure 1).
AuNPs, if synthesised correctly, remain stable for extended periods of time and can be
used as detectors at a moment’s notice by simply combining suitable quantities of sensor
and sample. Despite already having many properties of an excellent sensor, these
properties can be adjusted to suit a specific material. This allows much greater accuracy of
detection. The method of AuNP synthesis itself can be modified to create nanoparticles of
different shapes and sizes, contained within various microenvironments. In addition,
numerous molecules can be bound to the outer-surface of AuNPs. These can act as unique
ligands, only permitting specific binding of substances and altering the physiochemical
characteristics of the AuNPs such as redox potential and plasmon surface resonance.
One such class of materials are proteins. For instance, if an AuNP is capped with an
enzyme, detection is highly specialised towards that enzyme’s substrate, reducing the
chance of a false-positive result. Various studies have reported the results of detection
experiments with different proteins bound to the surface of AuNPs. The levels of success
and efficiency detailed in these studies show great heterogeneity, depending on the
techniques and proteins used. Guo et al. (2011) appear to have developed a novel method
for detection of heavy metals in environmental sciences and clinical toxicology using
papain-bound gold nanoparticles (P-AuNPs). Their claim is that the seven cysteine
5

6. residues within papain allow unique binding between the sulphur groups and various heavy
metal ions, such as mercury and copper. This binding event will allow rapid detection of
heavy metals in solution at concentrations as low as 200nM.
This investigation seeks to test these claims by qualitatively and quantitatively comparing
the detection abilities of AuNPs alone and when bound to proteins of different cysteine
contents. Do papain-bound AuNPs have the greatest detection ability for heavy metal ions
and what precisely is the unique interaction with thiol groups in cysteine residues? Is the
additional cost and time required to synthesise protein-bound AuNPs worth the supposed
greater ability of detection, or can AuNPs alone still give sufficient results?
2. Materials and methods
The majority of materials used and procedures followed are identical to those found in
sections 2.4-2.6 of Guo et al. (2011). Any changes or deviations are shown and explained.
The properties of AuNPs, and therefore their detection ability, largely depend on their size.
Therefore, synthesis requires high precision. According to the Turkevich method (Kimling
et al., 2006), aqueous, pale yellow HAuCl4 is boiled and rapidly stirred before the addition
of sodium citrate. Rapid and uniform addition of citrate is required since the rate of citrate
packaging is directly proportional to the size of the AuNPs.
The solution turns dark blue as the citrate reduces Au3+
ions in solution to Au+
. Three Au+
ions then convert to one Au3+
ion and two Au atoms. These two gold atoms induce
reduction of additional Au+
ions to rapidly form a nucleus of gold atoms, creating a
nanoparticle. Citrate molecules then cap the nanoparticles to prevent aggregation or further
growth. The result is a monodisperse, deep red solution of spherical AuNPs.
Recent studies have also determined that the cause of the brief dark blue colour after the
addition of sodium citrate between the pale yellow HAuCl4 and deep red AuNPs is due to
the formation of intermediate gold nanowires (Pong et al., 2007).
6

7. 2.1 Materials
Hydrochloric and nitric acid were used to synthesise aqua regia. Chloroauric acid
(HAuCl4) was used in citrate reduction to synthesise AuNPs. Two different heavy metal
solutions were used in detection experiments. These were copper(II) sulphate pentahydrate
and mercury(II) nitrate which were dissolved in ultrapure water. Proteins of varying
cysteine content were used to determine the relevance of sulphur in AuNP detection. These
proteins were BSA and papain which were purchased from Sigma-Aldrich.
2.2 Instruments
All measurements and transfer of microlitre quantities of liquids and solutions were carried
out using Gilson pipettes for accuracy. Ehrlenmeyer flasks were used to dissolve
substances for easier observation. Citrate reduction of HAuCl4 was carried out in a fume
cupboard to avoid vapours from the boiling solution. A magnetic stirrer and a beaker were
used to ensure fast pouring and mixing to create small AuNPs of the same size as those
seen in Guo et al. (2011). Absorbance spectra were recorded on a Unicam Heλios β UV-
vis spectrophotometer (Thermo Scientific) at room temperature between 400nm and
750nm. Centrifugation was carried out using a Heraeus Pico desktop centrifuge (Thermo
Scientific).
2.3 Preparation of citrate-capped AuNPs
Aqua regia was synthesised by combining HCl and HNO3 at a 3:1 ratio (150ml HNO3 and
50ml HCl). All glassware was cleaned using aqua regia to remove any contaminating metal
ions and prevent any false-positive results. Afterwards, this glassware was rinsed with
ultrapure water and left to dry.
AuNPs were synthesised via the Turkevich method of citrate reduction of chloroauric acid
(HAuCl4). 50ml of 38.8mM sodium citrate was added to 500ml of boiling, stirred 1mM
HAuCl4. This became the stock of AuNPs and was stored in a dark glass bottle since
AuNPs are light sensitive. In order to determine and compare their size, visible absorption
spectra were obtained. The AuNPs synthesised by Guo et al. (2011), after a 1-in-5 dilution,
had a maximum absorbance of 0.7-0.9 at a peak located at 516-518nm. At the same
dilution, AuNPs synthesised in this investigation had a maximum absorbance of 0.684 at
7

8. 521nm. Therefore, these AuNPs were marginally larger than those in Guo et al. (2011).
However, this difference in size is negligible.
After comparing the size of AuNPs synthesised in this investigation with those in Guo et
al. (2011), further calculations were carried out to precisely determine the concentration of
the AuNPs. Assuming the AuNPs were spheres and taking their extinction coefficient as
3.1 x 108
…
Concentration = absorbance / extinction coefficient
= 0.684 / 3.1 x 108
= 2.206 x 10-9
Mol/L in the 1-in-5 dilution.
The final concentration of the undiluted sample was 1.103 x 10-8
Mol/L, or 11.03 mMol/μl.
2.4 Binding proteins to AuNPs
Solid papain and BSA were dissolved in ultrapure water to give concentrations of 10-5
M.
Excess amounts of each protein solution was then added to 1ml AuNP solutions. These
were shaken for 30 minutes before being left to stand for 24 hours, ensuring successful
binding. No further steps to adsorb proteins onto the surface of the AuNPs were required.
This is because the proteins bind non-covalently to the citrate caps via electrostatic
interactions (Brewer et al., 2005). All aqueous proteins and protein-bound AuNP solutions
were kept in a fridge and solid BSA and papain were kept in a freezer to maintain stability
for longer periods of time. The protein-bound AuNP solutions were then centrifuged at
10,000x speed for 20 minutes before the supernatant was removed and the pellet
resuspended. This was repeated twice more to remove all unbound protein.
Problems arose during centrifugation of protein-bound AuNPs. Initially, spinning samples
at 14,000x for 20 minutes even once induced too much pelleting. This both separated some
gold from the citrate and made it difficult to resuspend the AuNPs in water without altering
the concentration. Therefore, they were centrifuged at a lower speed of 10,000x.
Similarly to Guo et al. (2011), the protein-bound AuNPs had a purple colour rather than
the deep red colour of AuNPs alone (Figure 2).
8

9. 2.5 Sensing heavy metal ions
2.5.1 Comparing intensity and S/N ratio
In each experiment, 500μl metal solution was added to 500μl AuNP solution to induce
aggregation and detection, resulting in a colour change. The intensity of the colour change
was first observed by the naked eye for a quick comparison. Next, the visible absorption
spectra between 400nm and 700nm were obtained. The maximum absorbance and the peak
wavelength were recorded.
Comparisons between absorbances, wavelengths and colour intensities were drawn from
combinations different proteins adsorbed to the AuNPs (if any) and different metal ions
detected. This allowed further comparisons to be drawn concerning intensities of colour
changes as the signalling event and S/N ratios. These experiments were also repeated to
gain a mean value and exclude any anomalies.
2.5.2 Comparing limits of detection
Measuring limits of detection was carried out as above but with serial dilutions of metal
solutions to detect the minimum concentration required for the AuNPs and protein-bound
AuNPs to be able to detect the ions. The lowest concentration of heavy metal solution that
was able to induce a colour change was measured as the limit of detection. Since 500μl of
both AuNP and metal solutions were mixed together, any dilutions of metal solutions
would be doubled to calculate the final concentration. For instance, if 500μl 0.1M metal
solution was added to 500μl AuNP solution, this would give a final concentration of
0.05M.
2.5.3 Comparing relative stabilities
Investigations were also carried out to determine how stable different versions of AuNPs
were in solution. Stocks of both AuNPs alone and protein-bound AuNP solutions were
kept in a fridge for one month before being taken out and used in detection experiments
identical to those measuring intensity. Results were then also compared with those
obtained from the original intensity experiments in section 2.5.1.
2.5.4 Control experiments
9

10. Additionally, two sets of control experiments were carried out. For the first set, 500μl of
each metal solution was added to 500μl of each protein solution in the complete absence of
AuNPs. Secondly, 500μl ultrapure water was added to 500μl of each type of AuNP
solution. Any observable colour changes were noted and their maximum absorbances and
peak wavelengths were measured.
3. Results
3.1 Intensity and S/N ratio
In all experiments with all materials, the addition of heavy metal ions to AuNPs, protein-
bound or otherwise, induced a colour change from red to blue as well as a shift in
maximum absorbance and peak wavelength (Table 1). The intensity of the colour change
as well as the amount of difference between the maximum absorbances and peak
wavelength was heterogeneous, depending on the particular metal ion used and if there was
any protein capping the AuNPs. In addition, band broadening was observed after addition
of copper or mercury.
AuNPs alone showed the greatest intensity in their colour change from red to blue, giving
greater differences between peak wavelengths before and after addition of metal ions
(>10nm). Whilst papain-bound AuNPs did exhibit changes in colour, absorbance and
wavelength after addition of heavy metals, these were all to a lesser extent than AuNPs
alone. Here, all differences in peak wavelength were <10nm. BSA-AuNPs provided the
poorest detection with the least intense colour changes, if any. In fact, in experiments with
copper, no colour change was observed at all. As a result, no further experiments were
carried out on BSA-AuNPs since they had already proven to be extremely poor detectors.
Furthermore, the adsorption of proteins onto the surface of the AuNPs gave the protein-
bound AuNP solutions a lower maximum absorbance (a paler solution) in all cases (Figure
2). This, if anything, reduced the S/N ratio significantly since any changes in maximum
absorbance or peak wavelength would be more difficult to determine.
Interestingly, whilst both copper and mercury were visibly detected, the signalling event to
indicate this detection manifested in different ways (Figure 3). Copper induced a rapid
10

11. colour change from red to blue, leaving a transparent solution as before. Mercury on the
other hand induced a rapid colour change from red to purple and gave a more visible
aggregation with the mercury-AuNP complexes precipitating out of solution. This can
easily be seen in the jagged absorption spectrum taken immediately after addition of
mercury. After the precipitate settled at the bottom of the cuvette, a second spectrum was
taken and the peak was smoother with a lower maximum absorbance, but at the same peak
wavelength. Both of these stark colour changes showed excellent S/N ratios but were more
notable in experiments with AuNPs alone.
3.2 Limits of detection
Serial dilutions of copper and mercury allowed an ‘intensity scale’ to be made for simple
observation of the limits of detection or ‘cut-off point’ (Figure 4, Table 2) For AuNPs
alone, limits of detection for both copper and mercury were similar but not identical. The
most dilute solutions able to induce a colour change were 0.003M for copper and 0.004M
for mercury, giving final concentrations of 0.0015M and 0.002M respectively, or 1.5mM
and 2mM. P-AuNPs on the other had higher limits of detection in relation to both copper
and mercury, both of which were at 2.5mM.
3.3 Stability
Concerning solutions of AuNPs alone, no significant differences in results were seen after
measuring intensity of colour changes in relation to both copper and mercury after one
month compared to identical experiments carried out mere days after synthesis. On the
other hand, P-AuNP solutions left in the fridge for one month did not undergo any colour
changes after being mixed with either copper or mercury. This showed that the P-AuNPs
could not remain stable for this length of time and had either degraded or all AuNPs had
aggregated, preventing any detection whatsoever.
3.4 Control experiments
No significant changes in colour, absorbance or wavelength were measured in any control
experiments (Figure 5).
4. Discussion
11

12. 4.1 The effects of protein coronas
In the control experiments, the addition of each heavy metal solution alone to each protein
induced no significant change in colour, absorbance or wavelength. In comparison to other
data, this confirmed that it was indeed the combination of AuNPs and heavy metals that
caused any and all colour changes. Therefore, any significant differences between data
obtained from the same stock of AuNPs, protein-bound or otherwise, were either due to
anomalies (none noted) or differences in heavy metal or protein content.
All protein-bound AuNPs were seen to have lower colour change intensities and S/N
ratios, higher limits of detection and less stability. Interestingly, BSA-AuNPs, which had
the highest cysteine content, were the poorest detectors relating to all of these criteria.
These results could indicate that higher cysteine content, far from providing better
detection, actually hinders detection. There are two possible reasons why P-AuNP and
BSA-AuNP solutions synthesised in this experiment had a purple colour rather than the
original red. The more likely reason is is due to the fact that the cysteine residues – and
also probably many other positively-charged amino acids – already induced aggregation of
the AuNPs. This has been confirmed in recent experiments where isolated and purified
cysteine separate from a polypeptide chain was detected by aggregating AuNPs
(Jongjinakool et al., 2014). As a result, any further additions of positively-charged
molecules such as metal cations did induce a minimal further colour change, but the
AuNPs were already aggregated. An alternative but less plausible explanation is that the
proteins altered the plasmon surface resonance of the AuNPs to provide a different colour
but did not induce aggregation, instead providing greater stability, even at higher ionic
strengths to make aggregation and therefore detection more difficult.
The low S/N ratios of P-AuNPs and BSA-AuNPs made it more difficult to determine via
observation alone if a signalling event had even occurred in response to detection of metal
ions and aggregation. This was particularly noticeable in limits of detection experiments.
AuNPs alone gave a remarkably distinguishable colour change right up to the ‘cut off
point’ beyond which the concentration was too low to induce aggregation. P-AuNPs on the
other hand provided far less noticeable colour changes. These S/N ratios become less
applicable when comparing absorption spectra or even images taken from microscopy
since any differences in absorbance and peak wavelength before and after addition of an
12

13. analyte can be more easily seen. However, the ability to provide a quick visual comparison
should be a hallmark of a good sensor with more advanced analysis techniques such as
absorption spectra only being taken if needed, such as to distinguish between two samples
with miniscule differences in concentration.
Data obtained in this investigation appear to refute the hypothesis that AuNPs capped with
papain have a greater detection ability for heavy metals than AuNPs alone due to their
cysteine content. AuNPs remain stable for far longer periods of time than P-AuNPs despite
methods of storage to prevent degradation. Proteins degrade in a matter of weeks within
solution. To remain stable and usable for longer, various inhibitors of degradation or
buffers would need to be added, complicating synthesis further (Simpson, 2010).
The poorer detection abilities of all protein-bound AuNPs were not due to inadvertent
dilution or contamination from centrifugation during preparation. This is because the pellet
was carefully and fully re-suspended in ultrapure water in all cases to ensure that no
dilution occurred and any supernatant that was removed was merely unbound protein.
Indeed, initial experiments with too much pelleting were altered to prevent this. In
addition, Guo et al. (2011) insist that centrifugation is a necessary step in P-AuNP
preparation, even going as far as to say that detection of mercury is impossible without this
step.
4.2 Selectivity towards mercury
It was interesting to note the somewhat different signalling events between copper and
mercury. Addition of copper initially gave a blue, transparent solution but mercury gave a
purple precipitate. After several minutes however, a visible precipitate was seen at the
bottom of the copper-AuNP solution with the colour remaining blue. These results indicate
that Hg2+
is able to induce a greater level of aggregation amongst AuNPs. This difference
in signalling is not due to differences in concentration or charge. Adding the same
concentrations of mercury and copper ions – both of which had a +2 charge – to AuNPs
gave rise to different signalling events. Therefore, there must be another factor which
causes this difference. Further investigations into AuNP interactions with different
elements are required.
13

14. Concerning this, Guo et al. (2011) suggested that mercury ions are more thiophilic. This is
indeed true since experiments with dithioerythritol, a sulphuric compound, bound to
AuNPs showed greater sensitivity and selectivity towards Hg2+
alone, even in the presence
of other cations (Kim et al., 2010). This concept appears to be the main basis for the
hypothesis of cysteine residues interacting with mercury in a unique way in protein-bound
AuNP colorimetric detection. Similar results were also obtained in Guo et al. (2011).
However, in this investigation, greater aggregation was also seen upon addition of mercury
in experiments where papain and thus all thiol groups were absent. Furthermore, P-AuNPs,
despite containing cysteine residues and thiol groups, do not appear to be able to produce
the same results as dithioerythritol according to the data obtained in this investigation.
Even if it was suggested that the purple colour of P-AuNPs was not due to premature and
unwanted aggregation before addition of metal, it would appear that the large tertiary
structures of papain and BSA shield the cysteine residues from interacting with Hg2+
, thus
preventing aggregation. Whilst papain may contain seven cysteine residues, perhaps a
greater number than many other proteins, these are only seven amino acids amongst many
others in the polypeptide chain folded into a unique conformation. Indeed, papain has six
cysteine residues acting as disulphide bridges so they are not exposed for binding. The
remaining residue is at the centre of the enzyme in the active site, making it difficult to
access (UniProtKB, 1986). This explains why no unique interaction between thiol groups
and Hg2+
ions was seen in this investigation with P-AuNPs. Dithioerythritol on the other
hand is a far smaller molecule with exposed thiol groups that can easily bind to Hg2+
(Figure 6)
The results in Guo et al. (2011) and Kim et al. (2010) indicate that thiol groups only
interact with mercury ions and no aggregation is seen in the presence of other cations. This
would explain the poorer detection abilities of P-AuNPs and BSA-AuNPs in relation to
copper in particular. A protein corona preventing aggregation at high ionic strengths would
already act as a poorer detector, but ensuring that this protein is specifically tailored
towards a different cation, namely Hg2+
, further reinforces this.
4.3 Evaluation
14

15. Greater precision in determining the size of AuNPs could have been achieved via the use
of transmission electron microscopy to directly observe their size. However, such a
microscope was unavailable so measuring the maximum absorption was a good proxy.
Through this, it was determined that the AuNPs synthesised in this investigation were
slightly larger than those in Guo et al. (2011). This was likely due to the fact that AuNP
synthesis requires high precision and speed when adding sodium citrate. During this
process, a few drops of sodium citrate fell outside of the flask and evaporated on the hot
plate, causing slightly slower pouring and less citrate reduction. However, this
investigation sought to determine the efficacy of protein coronas and their cysteine
contents on the detection abilities of AuNPs. A miniscule difference in AuNP size would
not have altered the results from this in any significant way.
There were a number of alterations made to the methods outlined in Guo et al. (2011) as
outlined in Section 2. However, these were made due to lack of resources which were
available to the original methods and alternatives were used. None of these resulted in less
reliable data.
All copper salts that could have been used to create a copper ion solution, including copper
sulphate, have a light blue colour when dissolved in water. This potentially could have
made it more difficult to observe any colour change from red to blue. However,
comparisons with the control experiments with copper sulphate solution or AuNPs alone
made observations of any significant changes possible. This was particularly useful in
determining S/N ratios to see if the red-blue colour change of the AuNPs in all experiments
could contrast strongly enough with the lighter blue of the copper. Furthermore, serial
dilutions of copper sulphate solution became paler, making limits of detection experiments
easier to observe.
4.4 Alternative implications
Protein-bound AuNPs could become specifically tailored towards certain biological
environments. By choosing a protein suitable for a certain tissue or even subcellular
location, this could improve uptake into cells, prevent degradation and reduce harmful side
effects (Saptarshi et al., 2013). Since there is ongoing research into the potential toxicity of
AuNPs, protective capping with protein coronas could solve this problem (Alkilany &
Murphy, 2010). These have far-reaching implications, such as a protein-capped AuNP
15

16. acting as a novel vehicle for drug delivery in nanomedicine. Amino acids in a protein could
provide a suitable scaffold upon which various therapeutic molecules could be attached.
Indeed, numerous groups are currently undertaking to build a library of protein corona
‘fingerprints’, cataloguing their synthesis and biological responses to their presence
(Walkey et al., 2014). In relation to this investigation, serum albumin is an abundant
protein within the bloodstream. Extraction and purification of albumin for use in AuNP
capping before injection could act as a suitable carrier molecule within the circulatory
system which remains undetected by the host’s immune system, preventing premature
degradation. Countless other materials have been hypothesised and investigated as AuNP
ligands to target specific subcellular locations (Zeng et al., 2011).
5. Conclusions
Through near-identical materials and methods, the results of Guo et al. (2011) could not be
replicated with papain or BSA. Rapid aggregation of AuNPs alone in response to the
presence of heavy metal ions provide a more intense colour change in response to lower
concentrations of analyte without being capped with proteins. This investigation has shown
that AuNPs alone are sufficient as detectors of heavy metal ions in environmental sciences
and clinical toxicology. Furthermore, they are a more cost-effective technique than P-
AuNPs, requiring less time and materials to synthesise and remaining stable and usable for
longer. It appears that Guo et al. (2011) attempted to create a novel method of heavy metal
detection through unique sulphur-cation interactions via the cysteine residues present in
papain. Indeed, they concluded that this method shows excellent selectivity towards
mercury with less or no aggregation in the presence of many different heavy metal cations.
However, the cysteines and other cationic residues in papain and BSA induced the AuNPs
to aggregate before the heavy metal solutions were added, negating any worthwhile
detection ability. It appears nonsensical to attempt to functionalise AuNPs with a substance
that induces an identical effect to that which is being detected.
All in all, whilst adsorption of various materials onto the outer-surfaces of AuNPs are a
viable method of detection tailored towards specific analytes, it appears that the cysteine
content of a protein ligand has no effect on heavy metal binding with a negative impact on
detection ability of AuNPs. This is likely also due to the fact that the cysteine residues in
16

17. proteins such as papain and BSA are either not exposed or not free for binding.
Experiments with other sulphur-containing AuNP coronas such as dithioerythritol indicate
that there is indeed a unique interaction between thiol groups and Hg2+
ions.
Since the use of AuNPs as biochemical sensors is still evolving, more thorough
experimentation using more advanced techniques and understanding of sulphur-metal ion
interactions is needed. Certain protein coronas do appear to prevent the aggregation of
AuNPs to a greater extent, even at higher ionic strengths in solution. However, this, if
anything, hinders the efficacy of their use as heavy metal detectors. This does not
completely negate any use of proteins alongside AuNPs however. For instance, the use of
enzyme coronas to tailor AuNPs to metabolic substrates remains a logical concept. Still,
selectivity towards certain cations can be achieved through capping with different sulphur-
containing molecules such as dithioerythritol. In addition to the fact that P-AuNPs have
poorer detection abilities, papain is more expensive than many other molecules with thiol
groups.
Nevertheless, the results obtained in this investigation do not merely refute protein-bound
AuNP methods of detection via cysteine residues. The sustained prevention of AuNP
aggregation via protein coronas has many far-reaching implications for a wide variety of
therapeutic pathways, including drug delivery, tumour detection and combatting
pathogens. Since many of the interactions of protein-bound AuNPs in vivo are not yet fully
understood, new research needs to be carried out in this area to ensure safe and successful
delivery of AuNPs and their attached molecules to the correct location in the body. They
would need to surpass any biological barriers, pH extremes and enzymatic degradation.
Once this is understood, the potential to deliver therapeutic molecules to less easily-
accessible regions of the body whilst targeting specific toxins, pathogens, tumour cells or
even altered gene expression could be a major breakthrough in medicine.
6. Acknowledgements
I would like to first of all thank my supervisor Dr James McEvoy for supporting me
throughout this project, helping to ensure my safety and understanding. I would also like to
thank the laboratory technician staff at the RHUL School of Biological Sciences for
17

18. providing access to equipment and materials needed. Finally, I’d like to thank my
Heavenly Father for everything He has done for me over the past two terms during the
entirety of this project. The glory belongs to Him.
7. References
Alkilany, A.M. & Murphy, C.J. (2010) Toxicity and cellular uptake of gold nanoparticles:
what have we learned so far? Journal of Nanoparticle Research, 12(7): 2313-2333. doi:
10.1007/s11051-010-9911-8. [Accessed 24th
February 2015]
18

19. Brewer, S.H., Glomm, W.R., Johnson, M.C., Knag, M.K. and Franzen, S. (2005) Probing
BSA binding to citrate-coated gold nanoparticles and surfaces. ACS Journal of Surfaces
and Colloids, 21(20): 9303-9307. doi: 10.1021/la050588t. [Accessed 17th
October 2014]
Guo, Y., Wang, Z., Qu, W., Shao, H. and Jiang, X. (2011) Colorimetric detection of
mercury, lead and copper ions simultaneously using protein-functionalized gold
nanoparticles. Biosensors & Bioelectronics, 26(10): 4064-4069. doi:
10.1016/j.bios.2011.03.033. [Accessed 1st
October 2014]
Jongjinakool, S., Palasak, K., Bousod, N. and Teepoo, S. (2014) Gold Nanoparticles-based
Colorimetric Sensor for Cysteine Detection. Energy Procedia, 56: 10-18. doi:
10.1016/j.egypro.2014.07.126. [Accessed 4th
January 2015]
Kim, Y.R., Mahajan, R.K., Kim, J.S. and Kim, H. (2010) Highly sensitive gold
nanoparticle-based colorimetric sensing of mercury(II) through simple ligand exchange
reaction in aqueous media. Applied Materials & Interfaces, 2(1): 292-295. doi:
10.1021/am9006963. [Accessed 20th
November 2014]
Kimling, J., Maier, M., Okenve, B., Kotaidis, V., Ballot, H. and Plech, A. (2006)
Turkevich Method for Gold Nanoparticle Synthesis Revisited. Journal of Physical
Chemistry B, 110(32): 15700-15707. doi: 0.1021/jp061667w. [Accessed 14th
November
2014]
Pong, B.K., Elim, H.I., Chong, J.X., Ji, W., Trout, B.L. and Lee, J.Y. (2007) New Insights
on the Nanoparticle Growth Mechanism in the Citrate Reduction of Gold(III) Salt:
Formation of the Au Nanowire Intermediate and its Nonlinear Optical Properties. Journal
of Physical Chemistry C, 111(17): 6281-6287. doi: 10.1021/jp068666o. [Accessed 17th
November 2014]
Saha, K., Agasti, S.S., Kim, C., Li, X. and Rotello, V.M. (2012) Gold nanoparticles in
chemical and biological sensing. Clinical Reviews, 112(5): 2739-2779. doi:
10.1021/cr2001178. [Accessed 10th
October 2014]
Saptarshi, S.R., Duschl, A. and Lopata, A.L. (2013) Interaction of nanoparticles with
proteins: relation to bio-reactivity of the nanoparticle. Journal of Nanobiotechnology,
11(1): 26. doi: 10.1186/1477-3155-11-26. [Accessed 27th
January 2015]
19

20. Simpson, R.J. (2010) Stabilization of proteins for storage. Cold Spring Harbor Protocols,
2010(5): pdb.top79. doi: 10.1101/pdb.top79. [Accessed 27th
January 2015]
Turkevich, J., Stevenson, P.C. and Hillier, J. (1951) A study of the nucleation and growth
processes in the synthesis of colloidal gold. Discussions of the Faraday Society, 11: 55-75.
doi: 10.1039/DF9511100055. [Accessed 14th
November 2014]
Tweney, R.D. (2006) Discovering Discovery: How Faraday Found the First Metallic
Colloid. Perspectives on Science, 14(1): 97-121. doi: 10.1162/posc.2006.14.1.97.
[Accessed 19th
December 2014]
UniProtKB (1986) P00784 – PAPA1_CARPA (Papain). Available:
http://www.uniprot.org/uniprot/P00784. Last updated: 26th
November 2014. [Accessed 4th
March 2015]
Walkey, C.D., Olsen, J.B., Song, F., Liu, R., Guo, H., Wesley, D., Olsen, H., Cohen, Y.,
Emili, A. and Chan, W.C.W. (2014) Protein corona fingerprinting predicts the cellular
interaction of gold and silver nanoparticles. ACS Nano, 8(3): 2439-2455. doi:
10.1021/nn406018q. [Accessed 22nd
February 2015]
Zeng, S., Yong, K.T., Roy, I., Dinh, X.Q., Yu, X. and Luan, F. (2011) A Review on
Functionalized Gold Nanoparticles for Biosensing Applications. Plasmonics, 6(3): 491-
506. doi: 10.1007/s11468-011-9228-1. [Accessed 17th
December 2014]
8. Figures and tables
20

21. Figure 1 – The aggregation of AuNPs in response to the presence of cations, displacing
negative charges of the citrate caps and causing multidentate complexes to form.
The aggregation of AuNPs alters their plasmon surface resonance, causing the solution to
change colour from red to blue (taken from Saha et al., 2012).
21

22. Figure 2 – A visual comparison of the colours of AuNPs, P-AuNPs and BSA-AuNPs before
addition of copper or mercury.
P-AuNPs and BSA-AuNPs not only gave paler solutions than AuNPs alone, but also a
different purple colour due to aggregation.
22
AuNPs P-AuNPs BSA-AuNPs

23. Figure 3 – A visual comparison of the signalling events of AuNPs in relation to copper and
mercury ions.
Addition of copper initially gave a blue solution which led to a
precipitate forming after several minutes which settled at the bottom of the cuvette.
Mercury on the other hand gave a purple solution with a precipitate forming immediately.
Figure 4 – Limits of detection experiments concerning AuNPs and P-AuNPs in the
presence of copper and mercury, with AuNPs and P-AuNPs alone for comparison.
23
AuNPs + copper
AuNPs + copper
after several
minutes
AuNPs + mercury
A

24. Serial
dilutions
of 500μl copper(II) sulphate (A) or mercury(II) nitrate (B) were added to 500μl AuNPsto
determine the lowest concentration able to induce a colour change. Since equal volumes of
AuNPs and metal solutions were mixed together, any concentrations of metal solutions
were halved to give the final limiting concentrations. 50mM of mercury was unavailable
since mercury(II) nitrate solution was created at a lower concentration.
Figure 5 – A visual comparison of the colours observed in control experiments.
24
50mM 5mM 2.5mM 2mM 1.5mM 1mM 500μM
B 50mM 5mM 2.5mM 2mM 1.5mM 1mM 500μM
Unavailable

25. It can be seen that addition of water to AuNPs did not induce a colour change but merely
diluted the solution, making the deep red colour somewhat paler. Addition of papain or
BSA to heavy metals also does not induce a colour change. This shows that it is indeed the
combination of AuNPs and heavy metal ions inducing aggregation that causes any colour
changes observed in this investigation.
25
AuNPs alone AuNPs + H2O Protein + metal

26. Figure 6 – The use of dithioerythritol-bound AuNPs (DTET-AuNPs) to selectively detect
and signal the presence of Hg2+
ions in solution.
The exposed thiol groups of dithioerythritol act as unique binding sites for Hg2+
cations,
inducing aggregation of the AuNPs and a colour change from red to blue (taken from Kim
et al., 2010).
26

27. Table 1 – Maximum absorbance and peak wavelength values obtained from intensity and
S/N ratio experiments with 500μl AuNPs, P-AuNPs and BSA-AuNPs mixed with 500μl
0.01M copper(II) sulphate and mercury(II) nitrate. Comparisons with nanoparticle
solutions alone and control experiments with water are also shown.
It can be seen that addition of water to each nanoparticle solution did not induce a colour
change (shift in peak wavelength), merely a dilution and a more pale solution (decrease in
maximum absorbance). AuNPs alone showed the greatest differences between maximum
absorbances, peak wavelengths and colour change intensities upon addition of heavy
metal ions, followed by P-AuNPs and lastly BSA-AuNPs.
Table 2 – Concentrations of copper(II) sulphate and mercury(II) nitrate solutions used in
limits of detection experiments with AuNPs and P-AuNPs to determine the lowest possible
concentration able to induce a colour change. The concentration ‘cut off points’ are
shown in bold.
27
Type of
nanoparticles
Mixed
with
Maximum
absorbance
Peak
wavelength
(nm)
Observable colour
change
AuNPs Nothing 2.432 520.0 n/a
AuNPs H2O 1.231 520.0 Red  paler red
AuNPs Cu2+
~0.750 None (flat) Red  very pale
blue
AuNPs Hg2+
2.077 540.0 Red  purple
P-AuNPs Nothing 1.700 555.0 n/a
P-AuNPs H2O 0.795 557.0 Purple  paler
purple
P-AuNPs Cu2+
0.980 562.0 Purple  paler
purple
P-AuNPs Hg2+
1.104 564.0 Purple  paler
purple
BSA-AuNPs Nothing 0.450 553.0 n/a
BSA-AuNPs H2O 0.245 553.0 Very pale purple
 even paler
purple
BSA-AuNPs Cu2+
0.277 552.0 None
BSA-AuNPs Hg2+
0.535 None (flat) Very pale purple
 colourless

28. In all cases, it appears that copper and mercury ions have similar limits of detection,
although not identical. AuNPs appear to have lower limits of detection than P-AuNPs,
being able to detect lower concentrations of metal ions, as shown by a visible colour
change.
9. Appendix I – Project diary
Date
Time Hours Description
12/06/14 13:00-14:00 1 Project allocation. Email sent to supervisor.
29/09/14 16:00-16:30 0.5
First meeting with supervisor. Overview of gold
nanoparticles (AuNPs). Discussion of potential project
designs.
28
Mixture Dilution of metal
solution
Final concentration
of metal ions
Colour change?
AuNPs + copper
0.1M (100mM) 0.05M (50mM) Yes
0.01M (10mM) 0.005M (5mM) Yes
0.005M (5mM) 0.0025M (2.5mM) Yes
0.004M (4mM) 0.002M (2mM) Yes
0.003M (3mM) 0.0015M (1.5mM) Yes
0.002M (2mM) 0.001M (1mM) No
0.001M (1mM) 0.0005 (500μM) No
AuNPs + mercury
0.01M (10mM) 0.005M (5mM) Yes
0.005M (5mM) 0.0025M (2.5mM) Yes
0.004M (4mM) 0.002M (2mM) Yes
0.003M (3mM) 0.0015M (1.5mM) No
0.002M (2mM) 0.001M (1mM) No
0.001M (1mM) 0.0005 (500μM) No
P-AuNPs + copper
0.1M (100mM) 0.05M (50mM) Yes
0.01M (10mM) 0.005M (5mM) Yes
0.005M (5mM) 0.0025M (2.5mM) Yes
0.004M (4mM) 0.002M (2mM) No
0.003M (3mM) 0.0015M (1.5mM) No
0.002M (2mM) 0.001M (1mM) No
0.001M (1mM) 0.0005 (500μM) No
P-AuNPs +
mercury
0.01M (10mM) 0.005M (5mM) Yes
0.005M (5mM) 0.0025M (2.5mM) Yes
0.004M (4mM) 0.002M (2mM) No
0.003M (3mM) 0.0015M (1.5mM) No
0.002M (2mM) 0.001M (1mM) No
0.001M (1mM) 0.0005 (500μM) No

29. 29/09/14 16:00-17:00 1
Beginning of research and email correspondence with
supervisor to determine project design before end of
week.
01/10/14 15:00-16:00 1
Installed and experimented with protein visualisation
software (Discovery Studio)
09/10/14 14:30-14:45 0.25
Final discussion of project design. Filled in risk
assessment. Organised first practical work.
10/10/14 13:30-16:30 3
Preparation of aqua regia. Cleaned glassware.
Synthesised AuNPs and capped with citrate. Stored for
future use.
15/10/14 14:00-16:30 2.5
Absorbance spectrum scanning to determine size and
concentration of AuNPs. Introduction to non-covalent
binding with proteins using BSA.
17/10/14 12:00-17:00 5
Write-up of initial method covering qualitative and
quantitative analysis of AuNP detection efficiencies.
Created P-AuNP solution, left to stand in fridge.
20/10/14 14:00-17:00 3
Attempted to centrifuge and resuspend pellet. Difficulty
in doing so due to pellet sticking to side of tube. Any
attempts to remove would alter concentration.
23/10/14 11:00-11:30 0.5
Met with supervisor to discuss experiments with copper,
various heavy metals and work-around of pellet
resuspension.
23/10/14 13:00-15:00 2
Initial mixing and shaking of P-AuNP and BSA-AuNP
solutions. Left to stand for 24 hours.
24/10/14 15:30-16:30 1
Second (successful) attempt at centrifugation to
synthesise P-AuNPs and BSA-AuNPs.
04/11/14 14:00-14:30 0.5
Met with supervisor to discuss creation of mercury(II)
nitrate solution.
05/11/14 12:00-16:00 4
Intensity and S/N ratio experiments with all types of
AuNPs using copper.
06/11/14 13:30-16:30 3
Continuation of intensity and S/N ratio experiments
using copper.
07/11/14 11:30-15:00 3.5
Synthesis of more P-AuNPs. Left a stock in the fridge
for 1 month for stability experiments. Continued
intensity and S/N ratio experiments using copper.
10/11/14 14:00-17:00 3 Control experiments and finished intensity and S/N ratio
29

30. experiments using copper.
14/11/14 14:00-16:00 2 Research into AuNP synthesis.
17/11/14 14:00-16:00 2
Library session. Began first draft of write-up. Organised
front page, contents page and overall format (e.g. page
numbers).
20/11/14 12:00-15:00 3 Continuation of first draft.
07/12/14 14:00-15:00 1 Stability experiments with month-old P-AuNP solution.
10/12/14 13:00-14:00 1 Mid-project feedback.
17/12/14 12:00-15:00 3 Research into alternative uses for AuNPs.
19/12/14 11:00-12:00 1 Research into history of AuNPs.
29/12/14 16:00-20:00 4 Write-up of materials and methods from notes.
30/12/14 16:00-22:00 6
Edited materials and methods. Finished introduction and
began write-up of results.
04/01/15 16:00-20:00 4
Investigation into purple colour of protein-bound
AuNPs for discussion section
13/01/15 12:00-17:00 5
Intensity and S/N ratio experiments with all types of
AuNPs using mercury.
14/01/15 13:30-15:30 2
Began limits of detection experiments with AuNPs +
copper/mercury.
15/01/15 13:00-16:00 3
Finished limits of detection experiments with AuNPs +
copper/mercury and P-AuNPs + copper/mercury.
27/01/15 16:00-18:00 2 Continuation of write-up.
05/02/15 12:00-16:00 4 More data required. Repeated previous experiments.
06/02/15 12:00-16:30 4.5 More data required. Repeated previous experiments.
07/02/15 17:00-21:00 4 Continuation of write-up.
07/02/15 14:30-17:30 3 Continuation of write-up.
08/02/15 15:00-18:00 3 Continuation of write-up.
22/02/15 13:00-16:00 3
Further research into therapeutic uses for AuNPs for
discussion section and conclusions.
24/02/15 13:00-16:00 2
Further research into therapeutic uses for AuNPs for
discussion section and conclusions.
26/02/15 12:00-17:00 5 Additional experimental work
30

31. 27/02/15 12:00-17:00 5 Additional experimental work
27/02/15 17:00-20:00 3 Continuation of write-up.
02/03/15 19:30-22:00 2.5 Continuation of write-up.
04/03/15 17:00-20:00 3
In-depth investigation of papain structure relating to
cysteine.
05/03/15 16:00-20:00 4 Continuation of write-up.
07/03/15 16:30-20:30 4 Continuation of write-up.
08/03/15 16:30-19:30 3 Continuation of write-up.
09/03/15 12:00-15:00 3 Continuation of write-up.
09/03/15 17:50-21:00 3.5 Continuation of write-up.
10/03/15 15:30-19:30 4 Continuation of write-up.
11/03/15 13:00-16:00 3 More data required. Repeated previous experiments.
12/03/15 13:00-16:00 3 More data required. Repeated previous experiments.
17/03/15 13:30-16:30 3 Continuation of write-up.
18/03/15 15:00-17:00 2 Continuation of write-up.
18/03/15 18:00-22:00 4 Finished conclusion and abstract. Formatted figures.
19/03/15 15:30-18:30 3 Continuation of write-up.
20/03/15 15:00-17:00 2 More data required. Repeated previous experiments.
21/03/15 18:00-22:00 4 Continuation of write-up.
22/03/15 14:00-18:30 4.5 Continuation of write-up.
23/03/15 15:00-20:00 5 Continuation of write-up.
24/03/15 15:30-22:00 6.5
Tidied laboratory space. Final stages of write-up and
editing.
25/03/15 10:00-14:00 4
Signed form D. Final editing and formatting.
Completion of final draft.
Total
hours: 181.25
31

32. 32

33. 33

34. 34

35. 35

36. 36

37. 37

38. 38

39. 39

40. 40

41. 41