Mercury is one of the most toxic elements and is a well-known environmental pollutants released from several human activities. It is important to have a sensitive and selective detection method for mercury. In this project, mercury(II) was detected by modified gold nanoparticles. The gold nanoparticles (~13 nm) are synthesized by reducing chloroauric acid (HAuCl4) in hot aqueous solution by sodium citrate. The gold nanoparticles are characterized by UV-visible spectrum and scanning electron microscope (SEM). The detection of mercury(II) ions is done by using two kinds of gold nanoparticles (AuNPs): 3-mercaptopropionic acid(MPA)-coated gold nanoparticles and DNA-AuNPs hybrid. For both AuNPs, red-shift is intensified as higher concentration of Hg2+ is added. This is due to aggregation of AuNPs induced by mercury(II) ions. Upon the addition of Hg2+, the coordination between MPA (or citrate) and Hg2+ results in the aggregation of AuNPs via the bridging of neighbouring AuNPs. As a result, the solution colour is changed from red to purple. For DNA-AuNPs, Hg2+ interacts with thymine forming Hg2+-bis-thymine complexes which detach nucleic acid from the AuNPs. This leads to the aggregation of AuNPs and as a result the solution changes from red to blue. For the two functionalized gold nanoparticles, DNA-AuNPs is more sensitive since its colour changes can be visible to naked eyes down to 0.5M Hg2+ whereas, for MPA-coated AuNPs, is only 0.1 mM.
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The guideline value from WHO is 0.006 mg/L (6 ppb) for inorganic mercury in drinking water [i] . For the United States Food and Drug Administration (FDA) and Environmental Protection Agency (EPA), the maximum contaminant level for inorganic mercury is 0.002 mg/L (2 ppb) in water. [ii] In Hong Kong, the maximum permitted concentration of mercury for all food in liquid and solid form is 0.5 ppm. [iii]
1.3 Conventional Detection Methods for Mercuric Ions
1.4 Aims of the Project
In this project, the main focus was the detection of inorganic mercuric ion (Hg2+). Mercuric ion is soluble in water, thus human may ingest it through drinking water. Due to its toxicity, it is important to develop an effective method for detecting mercuric ions. As previously mentioned, there are several detection methods that are commonly used for the analysis. They all have very low detection limit. However, these methods need cumbersome instrumentation and are very sophisticated. Therefore, a simpler system was used in this project which was using functionalized gold nanoparticles.
Chapter 2 Experimental
HAuCl4, mercury(II) perchlorate (Hg(ClO4)2), 3-mercaptopropionic acid (MPA), sodium citrate, sodium perchlorate (NaClO4) and 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) were purchased from Aldrich and used as received. DNA was purchased from
All solutions were prepared with filtered MilliQ water except MPA solution was prepared with methanol and DNA solution was prepared with autoclaved water.
2.2 Synthesis of Functionalized Gold Nanoparticles
2.2.1 Synthesis of Gold Nanoparticles
All glassware and magnetic stir bars used in this synthesis were thoroughly cleaned in aqua regia (HCl/HNO3 3:1), v/v), rinsed in distilled water, and then oven-dried prior to use.
AuNPs were synthesized by reduction of HAuCl4 with sodium citrate. [iv] Briefly, 5 mL of sodium citrate solution (38.8mM) was added rapidly to a 50mL boiled aqueous solution of HAuCl4 (1 mM) that was heated under reflux with stirring. The mixture was heated for 10 more minutes, during the time the colour of solution changed from light yellow to wine red. Then, the obtained gold nanoparticles solution was cooled to room temperature with stirring.
2.2.2 Modification of Gold Nanoparticles by 3-Mercaptoprionic acid (MPA)
MPA solution was prepared by dissolving purchased stock MPA powders in methanol. MPA solution was added in 3000:1 ratio to gold nanoparticles (i.e. concentration of MPA:AuNPs = 3000:1). The reaction mixture was stirred for 2 hours.
2.2.3 Modification of Gold Nanoparticles by DNA
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All the apparatus and solutions (except mercury(II) solution) were autoclaved before use.
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) was used as buffer. 50 mM HEPES buffer was prepared by dissolving HEPES powder in MilliQ water and the pH was tuned to 7.4 by adding sodium hydroxide solution. The buffer was filtered then autoclaved.
Poly-thymine nucleic acid, 5'-TTCTTTCTTCCCTTGTTTGTT-3', was used. 750 nM stock DNA solution was prepared in autoclaved water. DNA (100 nM), HEPES (10 mM) was mixed with AuNPs (5 nM) and stood for 15 minutes.
2.3 Detection of Mercuric Ions
2.3.1 Detection of Mercuric Ions by MPA-capped Gold Nanoparticles
Mercury(II) stock solution (2 mM) was prepared by dissolving Hg(ClO4)2 in filtered MilliQ water. Hg2+ ions in different concentrations were added to a solution of MPA-capped AuNPs (3.0 nM, 500 Î¼L). The concentration of MPA-capped AuNPs was assumed to be the same as before addition of MPA to citrate-capped AuNPs. The resulting solutions were allowed to stand for 30 minutes. The UV-vis absorption spectra of the solutions were recorded.
2.3.2 Detection of Mercuric Ions by DNA-Gold Nanoparticles
Different concentrations of mercuric ions solutionHg2+ ions were added to solutions of DNA-capped AuNPs (5.0 nM) and were allowed to react for 20 minutes. Then NaClO4 (100 mM) was added.
Chapter 3 Results and Discussion
3.1 Characterization of Gold Nanoparticles
3.1.1 Scanning Electron Microscopy
Scanning electron microscopy can be used to image the size and shape of gold nanoparticles. Fig. 1a is the SEM image of gold nanoparticles synthesized using the citrate reduction method. It showed that the gold nanoparticles were monodispersed. They were sphere in shape and had similar size (~13 nm)
Fig. a SEM image of gold nanoparticles
Fig. b showed the SEM image of MPA-capped gold nanoparticles. When compare with the bare gold nanoparticles (Fig. a), there were no significant differences in size and shape. This can be interpreted as only a monolayer-MPA was coated on the gold nanoparticles. Since the layer was so thin that was not observed in SEM.
Fig. b SEM image of MPA-capped gold nanoparticles
3.1.2 UV-visible Spectroscopy
Fig. UV-visible spectrum of gold nanoparticles
For gold nanoparticles with their size about 13 nm, there is a characteristic peak at the wavelength of 520 nm. It corresponds to the wine-red colour of the gold nanoparticles solution.
3.1.3 Calculation of Size and Concentration of Gold Nanoparticles
The size is crucial to gold nanoparticles since the optical functions of gold are dependent on the particle size for particle sizes smaller than the mean free path in bulk gold. Thus, a precise calculation of gold nanoparticle size is important. Moreover, known concentration of AuNPs is necessary for calculating the amount of MPA and DNA needed.
In the experiments, the sizes and concentrations of gold nanoparticles were calculated with reference to Haiss. [v] According to Haiss, when the size of AuNPs with diameters smaller than 35 nm, the ratio of the absorbance at the surface plasma resonance peak to the absorbance at 450 nm can be used to determine the particle size without knowing the concentration.
Aspr/A450 is the ratio of the absorbance at the surface plasma resonance peak (Aspr) to the absorbance at 450 nm (A450). B1 = 3.00 and B2 = 2.20 and d is the diameter. The calculated sizes of AuNPs synthesized using the above method is normally 11 - 14 nm.
With the size of AuNPs determined, the number density of the particles (N) can be determined.
A450 is the absorbance at Î»= 450 nm, and d is the particle diameter in nanometer. The concentration (in nM) can then be calculated by multiplying N by 1.67Ã-10-6.
Fig 1. UV-visible spectrum of AuNPs.
With the equations (1) and (2), the size and concentration of gold nanoparticles can be calculated by substituting in the values of A450 and Aspr obtained from UV-visible spectrum. Fig. 1 showed the UV-visible spectrum of gold nanoparticles. The peak appeared at 520 nm with the absorbance of 0.515299 (Aspr) and the absorbance at 450 nm was 0.322606 (A450). The size and concentration of AuNPs calculated were 13.35513 nm and 10.7458968 nM respectively. The size found by SEM was ~14 nm which was in a quite good agreement with the calculated value.
3.2 Detection of Mercuric Ions
3.2.1 Detection of Mercuric Ions by 3-Mercaptoproprionic acid-capped Gold Nanoparticles
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Before deciding the amount of MPA added, different concentrations of MPA to gold nanoparticles were tested. With the coating of MPA, changes of UV-visible spectra were expected. Fig. 2a showed the UV-visible spectra of gold nanoparticles in different concentrations of MPA. There was no significant observable colour change of solution. There were changes in the peak absorbance but there was no significant trend.
Fig. 2a UV-visible spectra changes of AuNPs capped with different concentration of MPA. (i.e. 1:50 = [AuNPs] : [MPA])
On the other hand, it was observed that at ~600 nm region, the absorbance increased as the MPA concentration increased. For MPA concentration 1 to 100 times of AuNPs, the trend was not obvious. This may because the concentration of MPA was too low that it did not exert significant effect on absorbance of AuNPs. For 500 - 3000 times, the width between each spectrum increased indicating that the AuNPs had not yet reach saturation with MPA.
Fig. 2b Part of UV-visible spectra (599 - 602 nm) from Fig. 2a. (bottom to top: 1:50, 1:100, 1:0, 1:1, 1:500, 1:1000, 1:2000, 1:3000)
Since the ratio 1:3000 was already quite large, the gold nanoparticles modified with 3000 times more concentrated MPA were used to detect mercuric ions. A positive result was obtained.
Fig. 3 showed the SEM images of MPA-capped gold nanoparticles in the (a) absence and (b) presence of mercury(II). Obviously, before addition of mercury(II) ions, individual gold colloids can be seen. After the addition of mercury(II) ions, the gold nanoparticles aggregated and formed lumps. This was due to the interaction between mercury(II) and MPA leading to bridging of neighbouring AuNPs.
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3.2.2 Detection of Mercuric Ions by DNA-Gold Nanoparticles