final draft

School of Biological Sciences

BS3010INDIVIDUAL 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: [email protected]

School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX

PROJECT SUPERVISOR: Dr James McEvoyWet Project Dry Project

TURNITIN RECEIPT NUMBER:

X

2014-15

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

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.

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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

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

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).

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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

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

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.

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2.5.4 Control experiments

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.

10

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

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).11

4. Discussion

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

12

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

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

13

causes this difference. Further investigations into AuNP interactions with different

elements are required.

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 Hg 2+

(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.

14

4.3 Evaluation

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

15

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

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.

16

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

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.

17

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

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.

18

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]

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]

19

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]

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]

20

8. Figures and tables

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

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

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.

23

AuNPs + copper AuNPs + copper after several

minutes

AuNPs + mercury

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.

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.

24

A

50mM 5mM 2.5mM 2mM 1.5mM 1mM 500μM

B 50mM 5mM 2.5mM 2mM 1.5mM 1mM 500μM

Una

vaila

ble

Figure 5 – A visual comparison of the colours observed in control experiments.

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

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

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.

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

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.

Mixture Dilution of metal solution

Final concentration of metal ions

Colour change?

AuNPs + copper

0.1M (100mM) 0.05M (50mM) Yes0.01M (10mM) 0.005M (5mM) Yes0.005M (5mM) 0.0025M (2.5mM) Yes0.004M (4mM) 0.002M (2mM) Yes0.003M (3mM) 0.0015M (1.5mM) Yes0.002M (2mM) 0.001M (1mM) No0.001M (1mM) 0.0005 (500μM) No

AuNPs + mercury

0.01M (10mM) 0.005M (5mM) Yes0.005M (5mM) 0.0025M (2.5mM) Yes0.004M (4mM) 0.002M (2mM) Yes0.003M (3mM) 0.0015M (1.5mM) No0.002M (2mM) 0.001M (1mM) No0.001M (1mM) 0.0005 (500μM) No

P-AuNPs + copper

0.1M (100mM) 0.05M (50mM) Yes0.01M (10mM) 0.005M (5mM) Yes0.005M (5mM) 0.0025M (2.5mM) Yes0.004M (4mM) 0.002M (2mM) No0.003M (3mM) 0.0015M (1.5mM) No0.002M (2mM) 0.001M (1mM) No0.001M (1mM) 0.0005 (500μM) No

P-AuNPs + mercury

0.01M (10mM) 0.005M (5mM) Yes0.005M (5mM) 0.0025M (2.5mM) Yes0.004M (4mM) 0.002M (2mM) No0.003M (3mM) 0.0015M (1.5mM) No0.002M (2mM) 0.001M (1mM) No0.001M (1mM) 0.0005 (500μM) No

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.

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9. Appendix I – Project diary

DateTime 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.

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 1Installed and experimented with protein visualisation

software (Discovery Studio)

09/10/14 14:30-14:45 0.25Final 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 2Initial mixing and shaking of P-AuNP and BSA-AuNP

solutions. Left to stand for 24 hours.

24/10/14 15:30-16:30 1Second (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)

29

nitrate solution.

05/11/14 12:00-16:00 4Intensity and S/N ratio experiments with all types of

AuNPs using copper.

06/11/14 13:30-16:30 3Continuation 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 3Control experiments and finished intensity and S/N

ratio 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 6Edited materials and methods. Finished introduction

and began write-up of results.

04/01/15 16:00-20:00 4Investigation into purple colour of protein-bound

AuNPs for discussion section

13/01/15 12:00-17:00 5Intensity and S/N ratio experiments with all types of

AuNPs using mercury.

14/01/15 13:30-15:30 2Began limits of detection experiments with AuNPs +

copper/mercury.

15/01/15 13:00-16:00 3Finished 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.30

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.



22/02/15 13:00-16:00 3Further research into therapeutic uses for AuNPs for

discussion section and conclusions.

24/02/15 13:00-16:00 2Further research into therapeutic uses for AuNPs for

discussion section and conclusions.

26/02/15 12:00-17:00 5 Additional experimental work

27/02/15 12:00-17:00 5 Additional experimental work


02/03/15 19:30-22:00 2.5 Continuation of write-up.

04/03/15 17:00-20:00 3In-depth investigation of papain structure relating to

cysteine.











18/03/15 18:00-22:00 4 Finished conclusion and abstract. Formatted figures.

31






24/03/15 15:30-22:00 6.5Tidied laboratory space. Final stages of write-up and

editing.

25/03/15 10:00-14:00 4Signed form D. Final editing and formatting.

Completion of final draft.

Total hours: 181.25

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final draft

Documents