Organic Gold: A study of the effects of the cysteine content of protein-bound gold nanoparticles in novel biochemical sensing.THIRD YEAR PROJECT PRESENTATIONCHRISTOPHER HUCKLE

Gold nanoparticles Nanoparticle research remains a dynamic area of research with many potential uses across different scientific fields.

Gold nanoparticles (AuNPs) give rapid detection of miniscule concentrations of material diagnosis of disease, forensics, quality control for food and water, etc.

Capped with citrate, AuNPs repel one-another via their negative charges, giving a deep red colour in solution.

The introduction of positively-charged ions displaces this negative charge, causing the AuNPs to aggregate, giving a distinct colour change to blue.

Introduction

Detection of heavy metals Small concentrations of heavy metals are needed in the body for various metabolic processes.

Can be very harmful in high concentrations (ingestion, inhalation, skin absorption).

Accumulation of heavy metals in water or soil has detrimental effects on entire food chains.

The testing of water or soil quality via heavy metal detection ensures safe food and water distribution and increased crop yield.

AuNPs have suitable properties for detection:◦ Low detection limits◦ Small concentration needed (cost-effective)◦ Easy to synthesise◦ Intense and immediate detection◦ Good signal/noise ratio (small sensor element with much larger transduction event)

Introduction

Question Guo et al. (2011) have suggested that binding protein (specifically papain) to the surfaces of AuNPs increases their detection ability – both the S/N ratio and the range of metals that can be detected.

Papain apparently increases detection ability due to the presence of seven cysteine residues.

Does the more costly and time-consuming addition of proteins to AuNPs increase their detection ability of heavy metal ions? What are the implications either way?

Introduction

Outline Majority of procedures are identical to those found in Guo et al. (2011).

Synthesis of AuNPs via the Turkevich method (Kimling et al., 2006).

AuNP properties largely depend on their size, determined by their synthesis.

Sodium citrate is added to aqueous, boiling, pale yellow chloroauric acid and stirred.

Yellow dark blue: citrate reduces Au3+ to Au+.

Dark blue deep red: 3Au+ Au+ + 2Au.

2 gold atoms induce reduction of additional Au+ ions, creating an atomic gold nucleus.

Citrate caps the nanoparticles to prevent aggregation and further growth.

Materials and Methods

Chemicals Hydrochloric and nitric acid mixed 3:1 aqua regia.

Chloroauric acid and sodium citrate to synthesise AuNPs.

Heavy metal solutions dissolved in ultrapure water:◦ Copper(II) sulphate pentahydrate◦ Mercury(II) nitrate

Proteins of varying cysteine content, purchased from Sigma Aldrich:◦ Papain (fewer)◦ BSA (greater)

Materials and Methods

Adsorption of proteins to AuNPs Solid papain and BSA dissolved in ultrapure water to give 10-5 M solutions.

Excess amounts of each protein added to 1ml AuNPs.

Shaken for 30 minutes, left to stand for 24 hours.

Proteins bind non-covalently via electrostatic interactions (Brewer et al., 2005).

Centrifuged at 10,000x speed for 20 minutes.

Supernatant removed and pellet resuspended in ultrapure water. Repeat twice more.

Similarly to Guo et al. (2011), protein-bound AuNPs were purple rather than red.

Materials and Methods

Sensing heavy metals In each experiment, 500μl AuNPs + 500μl metal solution.

Visible changes, absorption spectra, maximum absorbance and peak wavelength recorded.

Comparisons were drawn between different combinations of proteins adsorbed onto AuNPs (or lack thereof) and different metal ions detected.

This allowed comparisons to be made concerning different colour change intensities and S/N ratios.

Repeated to gain mean values and exclude anomalies.

Measured relative intensities, S/N ratios, limits of detection and stability, as well as controls.

Materials and Methods

Sensing heavy metals – intensity and S/N

In each experiment, 500μl AuNPs + 500μl metal solution.

Visible changes, absorption spectra, maximum absorbance and peak wavelength recorded.

Comparisons were drawn between different combinations of proteins adsorbed onto AuNPs (or lack thereof) and different metal ions detected.

This allowed comparisons to be made concerning different colour change intensities and S/N ratios.

Repeated to gain mean values and exclude anomalies.

Materials and Methods

Sensing heavy metals – limit of detection

As before, but with serial dilutions of heavy metal ions.

Procedure used to detect the minimum concentration required for AuNPs to provide a measureable signalling event in response to detection.

Since equal volumes of AuNPs and metal solutions were mixed together, any metal solution dilutions would be doubled to give the final concentration

◦ E.g. 500μl 0.1M metal solution added to 500μl AuNPs = 0.05M final concentration

Materials and Methods

Sensing heavy metals – stability Stocks of AuNPs and protein-bound AuNPs were kept in the fridge for one month.

Then used in detection experiments identical to those seen in intensity and S/N ratio experiments.

Results compared with original intensity experiments carried out on newly-synthesised AuNPs and protein-bound AuNPs to determine if they have degraded at all.

Materials and Methods

Control experiments Two sets of experiments carried out:

1. Copper/mercury + papain/BSA◦ Used to confirm that the AuNPs and not the proteins alone were responsible for any colour changes

2. Ultrapure water + AuNPs◦ Used to confirm that the AuNPs selectively detected metal ions

Materials and Methods

Intensity and S/N ratio All experiments:

◦ Immediate red blue colour change◦ Shift in max. absorbance and peak wavelength◦ Band broadening

AuNPs alone:◦ Most intense colour changes and greater differences between max.

absorbances and peak wavelengths (>10nm)

P-AuNPs:◦ Moderate colour changes with smaller differences between max.

absorbances and peak wavelengths (<10nm)

BSA-AuNPs:◦ Least intense colour change with mercury, no colour change at all with

copper◦ No further experiments carried outTherefore, AuNPs alone have better signalling events and lower S/N ratios than protein-bound AuNPs.

Results

Copper and mercury Both metals were visibly detected but with different signalling events.

Copper:◦ Red blue colour change◦ Solution remained transparent

Mercury:◦ Red purple colour change◦ Mercury-AuNP complexes precipitated out of solution

Results

Limits of detection Serial dilutions of copper and mercury allowed for an ‘intensity scale’, showing the ‘cut-off point’.

Limits of detection for copper and mercury were similar but not identical.

AuNPs + metal:◦ Copper: 1.5mM◦ Mercury: 2.0mM

P-AuNPs + metal:◦ Copper: 2.5mM◦ Mercury: 2.5mM

Therefore, AuNPs alone have lower limits of detection than P-AuNPs.

Results 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

Stability 1 month-old AuNPs + metal:

◦ No significant differences in results

1 month-old P-AuNPs + metal:◦ No visible colour changes observed at all

Therefore, AuNPs remain stable for longer than P-AuNPs.

Results

Control experiments Copper + papain:

◦ No significant changes in colour, absorbance or wavelength

Mercury + papain:◦ No significant changes in colour, absorbance or wavelength

Water + AuNPs:◦ Paler red colour due to dilution. No significant changes in colour, absorbance or wavelength.

Water + P-AuNPs:◦ Paler red colour due to dilution. No significant changes in colour, absorbance or wavelength.

Therefore, the signalling event is entirely due to AuNP/P-AuNP aggregation alone.

Results

Protein coronas All protein-bound AuNPs have lower colour change intensities and S/N ratios, higher limits of detection and less stability. Hypothesis refuted.

BSA-AuNPs: highest cysteine content, poorest detectors.

Possibly due to cysteines and other positively-charged amino acids already inducing aggregation.

Indicated by Jonjinakool et al. (2014) where isolated cysteine induced AuNP aggregation.

Further additions of cations induced minimal further colour changes.

Alternatively, protein coronas provide greater stability at higher ionic strengths, making aggregation and detection more difficult.

Discussion

Alternative implications Protein-bound AuNPs could instead become specifically tailored towards biological environments.

Match certain tissues or subcellular locations improve cellular uptake, prevent degradation, reduce harmful side effects (Saptarshi et al., 2013).

Far-reaching implications, e.g. protein-capped AuNPs as novel vehicles for drug delivery in nanomedicine.

AuNP protein corona ‘fingerprint’ library currently undergoing construction (Walkey et al., 2014).

Countless other materials are being investigated as AuNP ligands to target specific subcellular locations (Zeng et al., 2011).

Discussion

Mercury selectivity Different signalling events.

Mercury able to induce greater AuNP aggregation.

Possibly due to mercury being more thiophilic. Confirmed in experiments with dithioerythritol (Kim et al., 2010).

Appears to be main basis of Guo et al. (2011) hypothesis. However, greater aggregation was still seen in AuNPs alone.

Likely that papain and BSA tertiary structures shielded cations from cysteines.

Dithioerythritol is a far smaller molecule with exposed thiol groups.

Protein corona preventing aggregation at high ionic strengths is already a poor detector, but ensuring that this protein is tailored towards a different cation reinforces this further.

Discussion

Evaluation Any significant differences between data obtained from the same stock of AuNPs or P-AuNPs were due to differences in heavy metal or protein content.

No anomalies noted.

Any alterations in materials and methods compared to Guo et al. (2011) were due to lack of identical resources. Alternatives were used. None of these resulted in less reliable data.

AuNPs in this investigated were slightly larger than the originals. Did not affect results.

All copper salts have a light blue colour in solution. Affects S/N ratio. However, control experiments allowed for comparison.

Discussion

Comparison to original paper Through near-identical materials and methods, the results could not be replicated.

AuNPs alone are better detectors in all criteria.

More cost-effective and require less time to synthesise.

Remain stable for longer.

Unique thiol-mercury interactions were inappropriately applied to P-AuNPs.

Likely induced early aggregation before addition of metal cations, negating detection ability.

Nonsensical to functionalise AuNPs with proteins which induce an identical effect to that of the analyte.

Conclusions

Next steps More thorough experimentation using more advanced techniques are required.

Better understanding of sulphur-metal ion interactions.

Protein coronas appear to hinder AuNP detection ability, so perhaps this should not be pursued.

Instead, may be applied to other circumstances:◦ Enzyme coronas to tailor AuNPs to metabolic substrates

In vivo research concerning therapeutic uses (drug delivery, tumour detection, combatting pathogens, etc).

Requires safe and successful delivery to specific locations in the body.

Surpass biological barriers, pH extremes, enzymatic degradation, etc.

Conclusions

Questions

Thank you for your attention