M. S.
Bootharaju
a,
Kamalesh
Chaudhari
ab and
T.
Pradeep
*a
aDST Unit of Nanoscience and Thematic Unit of Excellence, Department of Chemistry, Indian Institute of Technology, Madras, Chennai - 600 036, India. E-mail: pradeep@iitm.ac.in
bDepartment of Biotechnology, Indian Institute of Technology, Madras, Chennai - 600 036, India
First published on 28th August 2012
In this work, we use dark-field optical microscopy (DFM) and hyper spectral imaging (HSI) to study the interactions of single Ag and Au nanoparticles (NPs) with Hg2+ in real time, at room temperature (25 °C). NPs were immobilized on glass substrates using 3-aminopropyltrimethoxysilane (APTMS) as the anchoring agent. Red, green and blue colors were assigned to the particles in hyper spectral images on the basis of their relative scattering intensities at 640, 550 and 460 nm, where the particles showed maxima in their scattering spectra. While Ag NPs showed all the colors, Au NPs were mostly red and rarely green in DFM images. The scattering spectra of Ag NPs were more blue shifted (with an average shift of 46 nm in the case of red particles) in a given time compared to Au NPs, after passing over Hg2+ solution and these shifts increased with time. Depending on the extent of blue shift, the colors of the particles got modified. Red particles appeared more reactive than green and blue, as revealed from the larger extent of shifts and their time dependence. The greater reactivity of red particles is attributed to their anisotropic nature possessing reactive tips, edges and more surface area due to their large size. The effect of quality of water on the scattering spectrum was checked by passing over deionized (DI) and tap waters separately, which showed that the effect is minimal compared to the presence of Hg2+, when data at a given time, flow rate and temperature were compared. Solution phase interactions of NPs with Hg2+ were also performed for comparison. These were characterized by UV-vis absorption spectroscopy, transmission electron microscopy (TEM) and energy dispersive analysis of X-rays (EDAX). Solution phase experiments showed citrate-induced aggregation of Ag NPs and partial reduction of Hg2+ to Hg0 upon exposure to Hg2+. Immobilized particles cannot aggregate and they show only reduction.
The scattering properties of NPs have been used for sensing applications as extinction-based methods require relatively smaller quantities of NPs in comparison to other colorimetric reagents. Scattering intensity is highly advantageous as a single 80 nm Au particle exhibits a light scattering power equivalent to the emission of ∼106 fluorescein molecules.12 Recently, a technique called single particle plasmon spectroscopy18 has evolved which measures the scattering spectrum of isolated plasmonic NPs. This spectroscopy has several advantages including greater sensitivity and smaller sample volumes than traditional methods.19 The NPs can be imaged by collecting scattered light from the particles and the image is called a hyper spectral image (HSI). This technique has been utilized for the real time study of chemical reactions,20 bio-molecule binding,18 detection of conformational changes of protein,21 sensing of small organic molecules22 and receptors23 as well as for DNA hybridization24. The application of such a technique is necessary to understand the real time interactions of NPs with inorganic contaminants such as heavy metal ions (Hg2+, Cd2+, Pb2+, etc.) and organic contaminants such as phenols, nitro aromatics, etc. present in water. Mercury is known to cause the minamita25 disease and its permissible limit in drinking water is 2 ppb set by the US environmental protection agency (EPA).26 Contamination of mercury in the environment occurs due to natural and anthropogenic processes.26 Various sensors such as monolayer and protein protected noble metal quantum clusters,26,27 NPs,28etc. have been reported for mercury sensing where the interactions are ensemble averaged. Recently, functionalized single plasmonic gold NPs have been utilized for Hg2+ sensing.29 In this paper, the mechanistic aspects of sensing are not discussed. There is no study so far on the utilization of scattering spectra of Ag NPs for understanding the real time interactions with Hg2+.
In this work, we studied the interaction of immobilized Ag and Au@citrate NPs with Hg2+ in real time using DFM and X-ray photoelectron spectroscopy (XPS). Interactions of Hg2+ with mobile (solution phase) NPs were also done for comparison, which were characterized with UV-vis spectroscopy, TEM and EDAX. After interaction of immobilized NPs with Hg2+, representative colors of NPs were changed with a blue shift and decrease in the intensity of the scattering. The color change and blue shifts were attributed to the redox reaction of Ag NPs and reduction of Hg2+ leading to the formation of Hg0. It may be pointed out that the particles are immobile which prevents consequent aggregation and red shift. The effect of water quality on the scattering spectrum of NPs was also studied. The decrease in silver content due to the reaction was confirmed by XPS quantification data. A visual detection limit of Hg2+ on the basis of color change of NPs in HSI is 1 ppm in our experimental conditions. This can be brought down further to ppb level by optimizing the experimental conditions such as decreasing the flow rate of Hg2+ and increasing the incubation time. Here, our objective was to understand the real time interactions of Ag NPs with Hg2+ spectroscopically and microscopically. Enhancing the detection limit was not an aspect of the investigation.
Scheme 1 Schematic representation of HSI set-up. A is a representation of immobilized Ag@citrate NPs using APTMS on glass substrate. B and C are the scattering spectrum of a silver particle and HSI of Ag@citrate NPs, respectively. |
Immobilized particles were subjected to HSI analysis and a large area hyper spectral image is shown in Fig. 1A. It shows different representative particles in red, green and blue. The scattering spectra from single red, green and blue (traces a, b and c, respectively) particles are shown in Fig. 1B. The corresponding particles are shown in the inset of B in which particles are marked with dotted circles. In our specific case, the red, green and blue particles show maxima at 659, 540 and 470 nm, respectively. It is reported that when Au NP monomer is converted to its dimer, a color change from green to yellow is observed with red shift in the scattering spectrum.32 Intensities of scattering spectra of blue particles are less than green and green is lesser than red. It is known that particles of high aspect ratio and larger size scatter more light than the smaller objects.20 To verify the existence of isolated nanoparticles on the glass substrates, FESEM measurements were carried out on the nanoparticle-immobilized surfaces (inset of Fig. 1A). The large area FESEM image of the same sample is shown in Fig. S4A. The EDAX spectrum clearly shows the presence of silver in a single NP (Fig. S4B). FESEM images reveal that each colored particle in HSI is a single particle with polydispersity in size. The scattering position is therefore related to the size of the particle. To validate the nature of a single particle in HSI, we have compared the number of particles in HSI and FESEM in a given area. For an area of 7.2 × 6.2 μm2, the number of particles in HSI and FESEM images are 33 ± 3 and 35 ± 3, respectively (Fig. S5). However, as the particles appear larger in HSI images, they seem to present a larger number density.
Fig. 1 (A) Large area HSI of immobilized Ag@citrate NPs. (B) Scattering spectra of Ag@citrate particles which are shown in the inset. Particles from which spectra are collected are labelled in the inset. The particles and the spectral traces have the same colors. Scale bars: 3 μm in (A) and 1 μm in the inset of (B), showing the typical distances between the particles. The sizes of the particles are exaggerated as they are imaged under the optical diffraction limit. Inset of A is an FESEM image of immobilized Ag@citrate NPs on a glass substrate. |
Fig. 2 HSI (A) of immobilized Ag@citrate NPs after passing 5 ppm Hg2+ for 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0 and 6.0 h (a–i, respectively). Scattering spectra (B) of a red particle with time (a–i). Scale bars of images in A are 500 nm. The particle from which spectra are collected is marked. |
Similarly, conversion of a green particle to blue is shown in Fig. 3. The λmax of the particle at 0.0, 0.5 and 6.0 h of passing Hg2+ are 528, 518 and 501 nm, respectively. The particle is almost completely converted to blue at 3.0 h (Fig. 3Ag) itself and a further blue shift of the scattering position is less than 5 nm. This indicates that similar to the red particle, there could be adsorption of Hg2+ followed by reduction on NPs which prevents subsequent interaction with Hg2+. The HSI of the blue particle with time is shown in Fig. S8. The maxima were observed at 495, 491, 484 and 478 nm at 0.0, 0.5, 1.0 and 6.0 h of treatment of Hg2+. Seemingly interaction of the blue particle with Hg2+ is less compared to red and green particles, under the given experimental conditions.
Fig. 3 HSI (A) of immobilized Ag@citrate NPs after passing 5 ppm Hg2+ for 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0 and 6.0 h (a–i, respectively). Scattering spectra (B) of a green particle (marked) with time (a–i). Scale bars of images in A are 400 nm. |
Interestingly, some particles (red and green) show a blue shift with decrease in scattering intensity after reacting with Hg2+ for 1.5 h. These effects were attributed to a redox reaction.33,34 Thereafter, they show a small red shift and increase in scattering intensity up to 4.0 h (Fig. S9). These were due to the deposition of reduced mercury on partially reacted silver particles leading to a small increase in size. After 4.0 h, a small blue shift with diminishing scattering intensity was noticed which may be due to the continuous reactivity of Ag particle. The dominant blue shift (due to oxidation of Ag0) compared to red shift (due to deposition of reduced mercury) of scattering wavelength may be due to the requirement of oxidation of two Ag0 for reduction of one Hg2+. Uptake of mercury by the particle was manifested in the EDAX spectrum of Ag@citrate particles immobilized on the functionalized glass substrate. The FESEM image of the particle from which the EDAX was taken is shown in Fig. S10 as an inset. The presence of Hg and Ag was confirmed by the EDAX spectrum collected from one of the particles (Fig. S10).
To understand the interactions better, HSI and scattering spectra of 5 particles of each color (red, green and blue) were collected. The average of shifts (Δλmax) of each colored particle with time of treatment with Hg2+ are plotted in Fig. 4. Note that the bars (I) represented in the graph are not error bars. They show the range of Δλmax in which all the five particles are present. Shifts are larger for red particles and smaller for green and very small for blue particles, at a given time of exposure. From this, it appears that red particles are most reactive towards Hg2+ in comparison to blue particles. There are two issues being discussed here. One is the larger shift of the red particles and the second is the nature of the shift itself. While the plasmon resonances are normally found to red shift upon interaction with analytes, a blue shift is observed here.
Fig. 4 A plot of average blue shift (Δλmax) of 5 particles of each color (red, blue and green) with time. A large shift is observed in the case of red particles and it decreases for green and blue analogues. |
The apparent larger reactivity of red particles may be due to the following reasons. 1. As in the Ag@citrate particle system, there is a possibility of non-spherical particles to exist which possess reactive sharp edges and tips.35,36 Anisotropic particles are expected to scatter light of higher wavelengths due to large aspect ratios (red colored particles). Maybe due to the greater reactivity of these anisotropic particles, more of a blue shift was seen in the case of red particles. Although we looked for anisotropic particles and their shape dependent Hg2+ reaction leading to inhomogeneous mercury distributions on them in TEM, these effects were not detected. It could be that mercury diffusion in such nanoscale particles was fast and imaging time scale was slow to detect inhomogeneity in the distribution. 2. From the FESEM and TEM images, we know that the particles are polydispersed in size and shape. The red particles in HSI are bigger in size. Due to the greater surface area of bigger particles, a greater extent of reactivity may be expected in such red particles.
Various research groups reported the red shift of the scattering spectrum of NPs when molecules/ions interact/bind with NPs.18,20,37 They attributed the red shift to an increase in the local refractive index.38 In our experiment, we see a blue shift which may be due to Hg2+ interaction with the NP core. There is the feasibility of a redox reaction which may be dominating here as particles were immobilized whereas in the mobilized case, aggregation is dominant (discussed later). The possibility of reduction of Hg2+ by citrate was avoided as there was a continuous flow which flushes excess citrate. The reduced Hg2+ may interact with the remaining silver core and change its composition leading to the formation of an amalgam, shifting the plasmon resonance. This leads to a blue shift.39 The deposited Ag+, likely to be in the form of poorly soluble salts reduces further interaction of the core with Hg2+ and as a result, the shift reduces with time. The Ag+ ions formed are partly detectable in the solution. Another reason for the blue shift is the reduction in the size of the silver particles.33 Further support for this suggestion comes from XPS (see below). The results presented suggest that all of these events occur and the observed process is predominantly due to redox chemistry in the case of silver. However, such reduction slows down with time as Ag+ is deposited on the surface of the particle.
Similarly immobilized Au@citrate NPs were also treated with 5 ppm Hg2+. HSI images with time are shown in Fig. S11. Initially, Au particles were in green and red colors (with maxima ∼540 and ∼640 nm, respectively). Some of the red particles turned red–yellow after treating with Hg2+. Here again the scattering spectra were blue shifted due to reduction of Hg2+ leading formation of an amalgam.34 The magnitude of the shifts was less compared to Ag particles for a given time which indicates more reactivity of silver compared to gold particles in our experimental conditions. This suggests that nano silver reduces Hg2+ more efficiently than nano gold. Ag particles of red color show an average blue shift of 12 nm within 30 min whereas Au particles of red color show a shift of 15 nm after 2.0 h (Fig. S12). It could be due to the strong metallophilic interactions of Au1+–Hg2+.27,40 Since large shifts were seen in the case of silver, more analyses were done in this case compared to gold NPs.
XPS analysis was performed to deduce the elemental composition of immobilized Ag NPs before and after passing Hg2+ for 6.0 h. The XPS survey spectra and Ag 3d regions of immobilized Ag@citrate NPs are shown in Fig. 5A and B, respectively. Fig. 5A shows the presence of all possible elements C, O (from APTMS or TSC), N (from APTMS), Si (from glass substrate and APTMS) and Ag (from Ag@citrate NPs) before as well as after treatment with Hg2+ (traces a and b, respectively). The presence of N confirms the immobilization of NPs on APTMS. The presence of Ag 3d5/2 before and after treatment of Hg2+ at 368.2 eV suggests silver in the zero-valent state17 (Fig. 5B). But we were unable to detect the presence of mercury which may be due to its ultra low concentrations. The full width at half maximum (FWHM) of Ag 3d5/2 before and after passing Hg2+ over are 1.3 and 1.7 eV, respectively under the same conditions of XPS measurements. The increase of FWHM may be due to presence of mixed silver oxidation states (Ag+ and Ag0). The elemental quantification data suggest that atomic Ag% decreases with respect to Si% after treating with Hg2+. The atomic ratios of Ag to Si before and after Hg2+ treatment were 0.09 and 0.05 as shown in Table 1. This may support a decrease in the particle size and blue shift of λmax after passing Hg2+ over. This could be due to the dissolution of Ag NPs as Ag0 gives electrons for the reduction of Hg2+ and the ions may dissolve partially in water. Si was taken as an internal reference element because it is largely due to the glass substrate. To check the presence of Ag in the outlet of the flow cell, ICP-OES analysis was performed. An output solution of 20 mL was concentrated using a rotavapor to 2 mL in which 50 ± 7 ppb silver was detected.
Fig. 5 XPS survey spectra and Ag 3d regions (A and B, respectively) of immobilized Ag@citrate NPs before and after passing Hg2+ (traces a and b, respectively). The spectra have been normalized with respect to the Si 2p feature in A and Ag 3d5/2 in B, but shifted vertically for clarity. |
Si(At.%) | Ag(At.%) | Atomic ratio of Ag to Si | |
---|---|---|---|
Before | 91.55 | 8.44 | 0.09 |
After | 95.24 | 4.76 | 0.05 |
Fig. 6 A) UV-vis absorption spectra of Ag@citrate NPs before and after treatment with 5 ppm Hg2+ for 6.0 h (traces a and b, respectively). B–D are XPS survey spectrum, Hg 4f and Ag 3d regions, respectively of Ag@citrate NPs treated with 5 ppm Hg2+ for 6.0 h. Inset: UV-vis absorption spectra of Au@citrate NPs and 5 ppm Hg2+ after interaction for 5 min, 3.0 and 6.0 h (a, b and c, respectively). |
Time-dependent UV-vis absorption spectra of Au@citrate particles and 5 ppm Hg2+ solution are shown in the inset of Fig. 6A. Unlike in the case of Ag particles, no shift of plasmon peak is seen but a small decrease in the absorbance value is noticed after 6.0 h. This supports the poor interaction of Hg2+ with gold particles compared to silver in a given time (6.0 h) and Hg2+ concentration (5 ppm). The reaction mixture of Au NPs and 5 ppm Hg2+ (after 6.0 h) was centrifuged at 5000 rpm. The residue obtained was analyzed with XPS and data are compared with parent Au NPs (Fig. 7). Survey spectra of NPs before and after treatment of Hg2+ (traces a and b, respectively in Fig. 7A) show the presence of expected elements Au, C, Na and O. Gold is present in the zero-valent state (Au 4f7/2 at 84.1 eV) before and after treatment of Hg2+ (traces a and b, respectively in Fig. 7B). The Hg 4f region from Hg2+ treated NPs is shown as an inset in Fig. 7A. Its absence indicates that no or poor interaction of Hg2+ occurs with Au NPs. However, Au@citrate NPs were found to interact with mercuric ion41 at higher concentrations (∼60 ppm, data not shown).
Fig. 7 XPS survey spectra and Au 4f regions (A and B, respectively) of Au@citrate NPs before and after treatment with 5 ppm Hg2+ for 6.0 h (traces a and b, respectively). Inset of A is the Hg 4f region of trace b. |
The reduction of Hg2+ to Hg0 is also supported by redox potentials of Ag+/Ag, Hg2+/Hg+ and Hg+/Hg0 systems. The standard reduction potentials of Ag+/Ag, Hg2+/Hg+ and Hg+/Hg0 are 0.8, 0.91 and 0.82 V, respectively. The cell emf (electromotive force) for the reactions, Ag0 + Hg2+ → Ag+ + Hg+ and Ag0 + Hg+ → Ag+ + Hg0 are therefore +0.11 and +0.02 V, respectively. But at the nanoscale, the reaction may be more feasible. The lower reactivity of gold particles with Hg2+ may be due to the negative emf (−0.5 V) for the reduction (2Au0 + 3Hg2+ → 2Au3+ + 3Hg0) of Hg2+. Although Au+ could be formed, this reaction is also not feasible (Au0 + Hg2+ → Au+ + Hg+, emf: −0.77 V).
Footnote |
† Electronic Supplementary Information (ESI) available: UV-vis absorption spectra and TEM images of Ag and Au@citrate NPs. EDAX spectrum, scattering spectra, FESEM and HSI images of Hg2+ treated NPs. See DOI: 10.1039/c2ra21384b |
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