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Infrared attenuated total reflection spectroscopy for the characterization of gold nanoparticles in solution Angela Inmaculada López-Lorente, Markus Sieger, Miguel Valcarcel, and Boris Mizaikoff Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 09 Dec 2013 Downloaded from http://pubs.acs.org on December 13, 2013
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Analytical Chemistry
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Infrared attenuated total reflection spectroscopy for the
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characterization of gold nanoparticles in solution
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Ángela Inmaculada López-Lorente1, Markus Sieger2, Miguel Valcárcel1*,
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Boris Mizaikoff2*
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1
Department of Analytical Chemistry, University of Córdoba, E-14071 Córdoba, Spain
6 7
Phone/Fax +34 957 218616; E-mail: [email protected] 2
Institute of Analytical and Bioanalytical Chemistry, University of Ulm, Ulm, Germany
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In-situ synthesis of bare gold nanoparticles mediated by stainless steel as reducing agent was
9
monitored via infrared attenuated total reflection (IR-ATR) spectroscopy. Gold nanoparticles
10
were directly synthesized within the liquid cell of the ATR unit taking immediate advantage of
11
the stainless steel walls of the ATR cell. As nanoparticles were formed, a layer of particles was
12
deposited at the SiO2 ATR waveguide surface. Incidentally, the absorption bands of water
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increased resulting from surface enhanced infrared absorption (SEIRA) effects arising from the
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presence of the gold nanoparticles within the evanescent field. Next to the influence of the
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Au(III) precursor concentration and the temperature, the suitability of IR-ATR spectroscopy as
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an innovative tool for investigating changes of nanoparticles in solution including their
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aggregation promoted by an increase of the ionic strength or via a pH decrease, and for detailing
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the sedimentation process of gold nanoparticles was confirmed.
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Keywords: attenuated total reflection, ATR, infrared spectroscopy, surface-enhanced infrared
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absorption, SEIRA, gold nanoparticles, nanoparticle synthesis, aggregation.
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*Corresponding
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[email protected];
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[email protected]
authors.
Boris Miguel
Mizaikoff. Valcárcel.
Tel.:
+49
Tel./Fax:
731 +34
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22750;
E-mail:
957
218616;
E-mail:
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1. INTRODUCTION
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Metallic nanoparticles and especially gold nanoparticles (AuNPs) provide exceptional
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properties, which have promoted their usage in a diverse range of areas including biomedical
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imaging and diagnostic assays1, biological applications2, catalysis3, and for applications based
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on the enhanced optical properties of nanoparticles such as enhanced Rayleigh scattering or
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surface-enhanced Raman scattering (SERS) of adsorbed molecules4.
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Furthermore, a diversity of synthetic procedures for the generation of AuNPs has emerged in
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recent literature5. Consequently, advanced characterization tools for nanoparticles synthesized at
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a wide variety of conditions are increasingly demanded. Microscopic techniques are most
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commonly utilized, although they suffer from several drawbacks, i.e., they are time-consuming
35
in order to ensure representative sampling, and imaging problems may arise from the
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aggregation of nanoparticles when studied in solution. Optical analysis techniques have proved
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to be useful for the characterization of nanoparticles, in particular when taking advantage of the
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pronounced surface plasmon resonances attributed to gold nanoparticles. Dynamic light
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scattering (DLS) is a common method for the size characterization of nanoparticles6. In
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addition, Raman spectroscopy has been applied for the characterization and quantification of
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gold nanoparticles previously extracted by ionic liquids7. In addition, separation techniques
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including electrophoresis8 have extensively been used for the characterization of synthesized
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gold nanoparticles. Finally, inductively coupled plasma mass spectrometry9 (ICP-MS) has
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emerged as a method of choice for quantitatively determining the presence of gold
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nanoparticles.
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In general, gold nanoparticles do not show pronounced mid-infrared (2.5-25 µm; MIR)
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absorptions, unless they are functionalized with organic ligands attached to the particle surface,
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which
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cetyltrimethylammonium bromide (CTAB) molecules adsorbed in ionic liquid media10 have
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been characterized by Fourier transformation infrared (FT-IR) spectroscopy. Furthermore, IR
gives
rise
to
characteristic
IR
spectra.
For
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example,
AuNPs
with
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spectra of 2-(12-mercaptododecyloxy)methyl-15-crown-5-ether modified gold nanoparticles11,
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4-mercaptobenzoic acid modified AuNPs12, and AuNPs-metallocarbonyl conjugates13 have been
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published. Infrared attenuated total reflectance (IR-ATR) spectroscopy has been used to
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quantitatively determine competitive molecular adsorption of thiolated poly(ethylene glycol) at
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gold nanoparticle surfaces14. However, to date infrared spectroscopy has not been applied for
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the characterization of gold nanoparticles without any surface modification.
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Hartstein et al.15 were the first to report on so-called surface enhanced infrared absorption
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(SEIRA) effects occurring at corrugated noble metal surfaces. In general, two enhancement
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mechanisms – electromagnetic and chemical enhancement - are considered providing the main
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contributions to the enhancement of molecular absorption features in SEIRA spectroscopy16.
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The electromagnetic mechanism increases the local electric field at the surface, while chemical
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enhancement gives rise to an increase of the absorption due to chemical interactions between
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molecules and gold nanoparticles17. Among others, the SEIRA effect has been used for the
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determination of molecules such as p-nitrobenzoic acid18, carbon monoxide19, octadecanethiol20-
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22
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In the present study, the adsorption of water molecules at gold nanoparticles has been utilized
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for in-situ monitoring of the synthesis of bare gold nanoparticles via surface-enhanced infrared
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attenuated total reflection (SEIRA-ATR) spectroscopy. Gold nanoparticles were directly grown
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within the liquid cell of the ATR unit from an aqueous tetrachloroauric acid solution mediated
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by the stainless steel housing of the ATR cell following a mechanism previously discussed by
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the authors25,26. As nanoparticles were formed and deposited at the surface of the ATR
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waveguide, an enhancement of the water absorption features was observed, which correlated
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with the amount of AuNPs present at the surface. A similar effect has been observed in the case
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of gold nanoparticles synthesized in D2O with the absorption bands of deuterium oxide
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revealing similar enhancement during the formation of AuNPs. Moreover, the generic suitability
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of SEIRA-ATR spectroscopy for the investigation of gold nanoparticle solutions has been
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studied via the evaluation of effects such as the addition of salts or variation of the pH on the
, 4-pyridinethiol23, and p-mercaptoaniline24.
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aggregation state of gold nanoparticles in solution, as well as monitoring the involved
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sedimentation processes. Thus, in the present study IR-ATR spectroscopy is confirmed as an
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innovative analytical tool applicable for the characterization of bare AuNPs providing a
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straightforward and non-destructive characterization strategy.
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2. METHODS
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2.1. Materials and reagents
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HAuCl4 solution (200 mgdL-1 in deionized water)
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Steinheim, Germany) were used to synthesize the gold nanoparticles. Prior to synthesis, the
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material was washed with a mixture of nitric acid and hydrochloric acid (HNO3:HCl = 1:3
87
mixture, aqua regia) (Panreac, Barcelona, Spain). A flat piece of 304-stainless steel was used as
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solid reducing agent of gold precursor in order to form gold nanoparticles. Sodium citrate
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dehydrate 99.5% (Sigma Aldrich, St. Louis, MO, USA) was used to synthesize citrate-coated
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gold nanoparticles. Silver nitrate, potassium nitrate, and sodium chloride purchased from
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Sigma-Aldrich (St. Louis, MO, USA) were used to investigate the aggregation of the
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nanoparticles. Cetyltrimethylamonium chloride (CTAC) was obtained from Fluka (Buchs,
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Switzerland). Deuterium oxide (D2O, 99.8%) was provided by Merck (Darmstadt, Germany).
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The stock solutions used herein were invariably prepared in Milli-Q water.
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2.2. Synthesis of bare gold nanoparticles
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Gold nanoparticles were synthesized following a procedure previously described by the authors
97
using stainless steel is used as solid reducing agent25,26. All glassware was cleaned with freshly
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prepared aqua regia (HNO3:HCl = 1:3 mixture) and then thoroughly rinsed with distilled H2O
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prior to use. A piece of 304-stainless steel (12.9 mm2 of total surface) was introduced into 100
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µL of a 0.2 mg mL-1 aqueous solution of HAuCl4. The reaction was carried out at room
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temperature; the stainless steel substrate was simultaneously used as stirrer during the reaction.
and gold(III) chloride (Sigma Aldrich,
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2.3. Synthesis of bare gold nanoparticles inside the ATR liquid cell
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Gold nanoparticles were also directly synthesized inside the liquid cell of the ATR unit by
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taking direct advantage of the stainless steel, which forms the walls of the ATR liquid cell. In
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this case, no stainless steel piece was added to the solution of tetrachloroauric acid, which was
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again used as a gold(III) precursor matrix. Once the tetrachloroauric acid solution was located
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within the ATR unit, the immediate onset of the formation of gold nanoparticles was observed.
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Thus, obtained AuNPs sedimented at the ATR crystal surface, thereby enabling direct
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monitoring via SEIRA-ATR spectroscopy. The reaction was performed at a variety of
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temperatures: room temperature (i.e., 22ºC), 40ºC, 50ºC, and 60ºC, as controlled by a thermostat
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unit.
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2.4. Synthesis of citrate-coated gold nanoparticles
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Gold nanoparticles obtained via HAuCl4 reduction mediated by sodium citrate were also
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prepared according to the method described by Turkevich et al.27 applying modifications
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described elsewhere7. Firstly, all glassware were cleaned with freshly prepared aqua regia
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(HNO3:HCl 1:3 mixture), and then thoroughly rinsed with distilled H2O prior to usage.
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Tetrachloroauric acid and sodium citrate solutions were filtered through a 0.45 µm nylon
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membrane prior to use. Once a 0.01% solution of HAuCl4 was boiling, 0.254 mL of a 1%
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sodium citrate solution were added. The system was then left to react while stirring for a period
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of 15 min. Afterwards, 5mL of 0.01% HAuCl4 solution were added to the system followed by
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0.254 mL of 1% sodium citrate solution, and again stirred while heating for 15 min. Then, the
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heater was switched off, and the solution was stirred until cooling to room temperature. Thus
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obtained AuNPs were stored at 4ºC in an amber bottle.
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2.5. Infrared spectroscopic analysis
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Infrared spectroscopic studies were performed using a Vertex 70 FT-IR spectrometer (Bruker
127
Optics, Ettlingen, Germany) equipped with a BioATR-II unit, and a liquid nitrogen cooled
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mercury-cadmium-telluride (MCT) detector (Bruker Optics, Ettlingen, Germany). Figure 1 5 ACS Paragon Plus Environment
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shows a schematic of the experimental setup. As evident although not to scale, the top ATR
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crystal interfacing with the sample is made from a circular section of a silicon wafer providing
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8-10 internal reflections. IR radiation is coupled into this silicon ATR plate via a secondary zinc
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selenide (ZnSe) ATR waveguide (which is not in contact with the sample) providing a
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hemispherical shape for efficient coupling of photons from the FT-IR spectrometer into the Si-
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ATR. The entire unit was connected to a circulating water bath ensuring a defined and constant
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temperature during the IR-ATR analysis. Data acquisition and processing was performed using
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the OPUS 6.5 software package (Bruker Optics, Ettlingen, Germany). Spectra were acquired by
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averaging 300 spectra in the wavelength window of 4000 to 400 cm−1 at a spectral resolution of
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4 cm−1. Prior to analyzing each set of samples, a background spectrum of the tetrachloroauric
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acid solution was collected when monitoring the subsequent in-situ synthesis of AuNPs. If
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AuNPs were synthesized ex-situ, prior to a sample measurement a background spectrum of
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deposited AuNPs at time zero was recorded. Finally, measurements of the synthesis of gold
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nanoparticles in D2O media were acquired averaging 500 spectra within 4000 to 400 cm-1 at a
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spectral resolution of 4 cm-1.
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3. RESULTS AND DISCUSSION
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3.1. In-situ synthesis of gold nanoparticles within the IR-ATR liquid cell
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Gold nanoparticles were directly synthesized inside the ATR unit taking advantage of the
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stainless steel ring establishing the confining walls of the liquid cell, as previously demonstrated
148
by the authors25,26. While AuNPs are formed, they sediment at the ATR waveguide surface until
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Coulombic repulsion from already adsorbed AuNPs leads to an equilibrium.
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Gold nanoparticles are characterized by a strong plasmon resonance in the UV/Vis region as a
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consequence of the collective oscillation of electrons within the conduction band. However,
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bare gold nanoparticles do not inherently show a pronounced absorption in the MIR. Despite the
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IR-inactivity of AuNPs, it was found that their formation could still be monitored via IR
154
spectroscopy by analyzing the increase of the water absorption features during the synthesis 6 ACS Paragon Plus Environment
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Analytical Chemistry
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process, which arises from a SEIRA effect, as discussed below. Figure 2 shows a series of in-
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situ IR-ATR spectra recorded during gold nanoparticles being synthesized within the ATR unit.
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According to literature28, the vibrational modes of water in the infrared region are the H2O
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bending mode at 1644 cm-1, the combination of H2O bending and libration mode at 2128 cm-1,
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and a pronounced vibration around 3404 cm-1, which is composed of the overtone of the
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bending mode at 3250 cm-1, the symmetric OH stretch at 3450 cm-1, and the antisymmetric OH
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stretch at 3600 cm-1 22. As gold nanoparticles are formed and deposited at the ATR waveguide
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surface, an increase of the H2O bending and the OH stretching bands is observed despite the
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effective decrease of water molecules present within the evanescent field being displaced by
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gold nanoparticles. This increase is attributed to an enhancement of the water absorptions within
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the evanescent field resulting from the presence of AuNPs. While the magnitude of the
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enhancement certainly decreases with increasing the distance from the waveguide surface (i.e.,
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the nanoparticulate Au film), it compensates for the decreasing number of water molecules
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steadily displaced by AuNPs. The hypothesis of enhancing the IR signature of water via the
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formation of AuNPs is further corroborated by the observed increase of the water absorption
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signal vs. the background spectrum.
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For monitoring the kinetics of the synthesis and deposition of AuNPs at the ATR waveguide
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surface, the peak height of the water bending mode (δ(H2O)) was analyzed in detail. Figure 2a
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shows the peak height of the H2O bending mode vs. time with t=0 indicating the initiation of the
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in-situ synthesis of gold nanoparticles from tetrachloroauric acid solution. The obtained data
175
readily fit to a Langmuir model, which describes a random adsorption process within the first
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monolayer (Figure 2c). The obtained goodness-of-the-fit was r2=0.997.
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Likewise, the absorption band corresponding to the OH stretching vibration revealed an increase
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during the synthesis of gold nanoparticles (figure 2b). Plotting the peak height vs. time again fits
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a Langmuir isotherm with a goodness-of-the-fit of r2=0.999. Table 1 summarizes the main
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parameters obtained when fitting both vibrational water bands to a Langmuir isotherm.
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In order to further study the in-situ formation of gold nanoparticles within the ATR cell, a
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stainless steel O-ring of similar dimensions was glued onto a piece of silicon wafer, in order to
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simulate the scenario inside the ATR liquid cell (i.e., mock-up ATR cell; MU-ATR) enabling
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access to subsequent scanning electron microscopy (SEM) studies of the obtained AuNPs
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without additional manipulation. Similar to the in-situ synthesis monitored by IR-ATR
186
spectroscopy, a small amount of HAuCl4 (200 mg dL-1) was added to the MU-ATR in order to
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synthesize AuNPs. The reaction was stopped after 20 min and 100 min, respectively, by
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removing the aqueous solution followed by rinsing with dried air.
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After 20 min, a sub-monolayer coverage with few agglomerates of AuNPs was determined at
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the Si wafer surface (Figure 3 left), as expected from the in-situ IR-ATR studies. After 100 min,
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the AuNPs in solution show significant agglomeration with already adsorbed gold
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nanoparticles, as evident in Figure 3 (right). During the process of deposition of AuNPs at the
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ATR crystal surface, the distribution, size, and shape of the NPs as well as their interaction
194
change. This morphological development results in changes of the electromagnetic field
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properties near the ATR waveguide surface. Firstly, a monolayer of AuNPs is formed at the
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surface (Figure 3 left), which leads to a strong water bending and OH stretching signal
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enhancement in the first few minutes. The SEIRA activity results from an enhanced near-field
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around the NPs, which decays with increasing distance from the particle surface. As previously
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explained, the near-field enhancement around AuNPs overcompensates the reduced number of
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water molecules present within the evanescent field, as they are increasingly replaced by AuNPs
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during their adsorption on the waveguide surface. Once gold nanoparticles are deposited on the
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surface, aggregates or islands of nanoparticles are observed, as evident within the SEM images
203
(Figure 3, right), along with no further increase of the water absorption features. At such
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islands, the electromagnetic near-field properties change, as accordingly the absorption of IR
205
radiation21.
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3.2. Influence of the gold(III) precursor concentration
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The influence of the concentration of the gold(III) precursor (i.e., tetrachloroauric acid) was
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studied. While the kinetics of the reaction follow a different rate depending on the
210
concentration, in all cases the obtained rates may be fitted with a Langmuir isotherm.
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If the concentration of gold precursor within the ATR unit is high (i.e., 200 mgdL-1), the
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increase of the absorption features related to the water spectrum is rapidly observed (i.e., within
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1 min). At lower concentrations (i.e., 66 mgdL-1), the formation of gold nanoparticles is
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correspondingly slower, which is reflected in the IR spectrum (Figure S1, Supporting
215
Information). Figure S2 (Supporting Information) shows the H2O bending region at different
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times of reaction. Initially, AuNPs deposited at the surface appear isolated with only few
217
particles agglomerated. With increasing reaction time, packed island of connected nanoparticles
218
are evident. This behavior agrees with findings previously reported in literature22 for the water
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OH stretching band during the adsorption of a submonolayer of AuNPs at a substrate initially
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treated with (aminopropyl)triethoxysilane (APTES).
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3.3. Influence of the temperature
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If AuNPs are synthesized outside the ATR unit by inserting a piece of stainless steel piece into
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the solution25, an increase of the rate of production of gold nanoparticles is observed with
224
increasing temperature. The in-situ synthesis was performed at different temperatures (23, 40,
225
50, and 60ºC) for spectroscopically analyzing this behavior. With increasing temperature, the
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enhancement of water bands more rapidly appears, which in turn confirms that AuNPs are more
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rapidly formed.
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3.4. Reproducibility
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A study of the reproducibility of the in-situ AuNPs synthesis and the observed SEIRA-ATR
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water signal was performed by repeating the analysis four times during different days for the
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synthesis procedure based on a 200 mg dL-1 HAuCl4 solution at 60ºC. As reported in Table 1,
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the relative standard deviation calculated from the H2O bending peak height was 10.55%, while
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the relative standard deviation obtained from the OH stretching band was 8.16%.
234
3.5. In-situ synthesis of gold nanoparticles in D2O
235
The synthesis of the gold nanoparticles mediated by stainless steel inside the ATR unit was
236
finally also performed in D2O, using AuCl3. The aim of this study was to potentially observe the
237
SEIRA effect on the associated D2O bands in a different spectral regime for confirmation of the
238
observations initially made for H2O. No significant difference was noted concerning the
239
formation of AuNPs during the chemical reaction compared to the studies in H2O. Similar to the
240
observations in water, as gold nanoparticles were emerging, the absorption signals of the υ(OD)
241
and the δ(D2O) band appear with enhanced intensity. Again, thus obtained data were fitted using
242
a Langmuir model with a goodness-of-the-fit of r2=0.994 for the δ(D2O) band (see Figure S3,
243
Supporting Information), and of r2=0.998 when evaluating the OD stretching band (Table 1).
244
The same behavior was observed when performing the synthesis in a 1:1 mixture of H2O and
245
D2O resulting in the corresponding simultaneous enhancement of water and deuterium oxide
246
infrared absorption features, respectively.
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3.6. Influence of the ionic strength
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As IR spectra are also sensitive to variations of the chemical environmental, next to non-
249
destructively monitoring the synthesis of gold nanoparticles one may furthermore analyze
250
changes in aggregation or stability of the obtained AuNP dispersions. The aggregation of
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AuNPs causes coupling of their plasmonic modes, thereby resulting in a red shift and
252
broadening of the longitudinal plasmon resonance band in the optical spectrum, as evident in the
253
UV/Vis absorption spectra shown in Figure 4 (top). It is known that variation of the ionic
254
strength is a particle-independent strategy to affect the repulsion distance between charged
255
particles. Increasing the ionic strength decreases the Debye length, thus also decreasing the
256
averaged distance between particles29. Two hypotheses have been proposed to explain the
257
observed change in the color of such solutions when increasing the ionic strength: (i) gold 10 ACS Paragon Plus Environment
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nanoparticles physically coalesce to form larger particles, and (ii) the mean interaction distance
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is reduced such that their surface plasmons may interact29.
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Herein, the effect of the addition of silver(I) nitrate and potassium nitrate was investigated. As
261
gold nanoparticles are synthesized and deposited within the evanescent field at the ATR
262
waveguide surface, an enhancement of the water absorption features is observed. If the ionic
263
strength of the solution is altered by addition of salts, a sudden increase of the absorption of the
264
water bands is observed, without any further change with time.
265
While AuNPs synthesized in-situ using the stainless steel assisted method do not contain citrate
266
ligands bound to the nanoparticle surface, chloride ions are present in abundance and may shield
267
the obtained nanoparticles. Such a chloride shell may prevent nanoparticle agglomeration, and
268
may also facilitate the stabilization of the solution due to the achieved spacing between
269
individual NPs. When adding silver nitrate to the solution, which increases the ionic strength,
270
the enhancement of the water bands terminates (see Figure 4, bottom). Apparently, silver ions
271
interact with chloride ions, and thus, AuNPs will be destabilized and rapidly deposit at the
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waveguide surface. Once deposited as islands on the surface, the increase of the water
273
absorption bands terminates in analogy to the observations during the deposition of AuNPs at
274
in-situ synthesis conditions at extended periods of time.
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A similar behavior was observed when adding potassium nitrate; once nanoparticles aggregate,
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an increase in water signal is observed followed by no further changes of the IR absorption
277
band.
278
3.7. Influence of the pH value
279
The influence of a decrease in pH during the synthesis of gold nanoparticles within the ATR
280
unit was investigated by adding HNO3 during the synthesis. 10 µL of a 10% (v/v) HNO3
281
solution were added to 40 µL of 200 mg dL-1 HAuCl4 solution previously placed into the ATR
282
liquid cell. It has previously been described that low pH values induce the aggregation of gold
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nanoparticles30. Again, the suitability of IR-ATR spectroscopy to determine changes in 11 ACS Paragon Plus Environment
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aggregation state of the synthesizing gold nanoparticles was demonstrated observing a behavior
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similar to that when increasing the ionic strength.
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3.8. Investigating the sedimentation behavior of ex-situ synthesized unmodified and citrate
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coated gold nanoparticles via IR-ATR spectroscopy
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The sedimentation behavior of gold nanoparticles has been studied via UV/Vis spectroscopy
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under normal gravitational force31. The optical detection during sedimentation of particles in
290
solution enables the accurate determination of the particle size distribution in a polydisperse
291
nanoparticle sample31. Herein, IR-ATR spectroscopy is proposed as an alternative strategy for
292
monitoring gravitational sedimentation of gold nanoparticles. Hence, the sedimentation rate of
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both unmodified gold nanoparticles obtained via stainless steel assisted reduction outside the
294
ATR liquid cell, and citrate-capped gold nanoparticles was compared. In order to achieve
295
acceptable sedimentation rates, CTAC was added, as previously reported in the literature32.
296
Cationic surfactant causes aggregation by neutralizing the negative charge at the nanoparticle
297
surface.
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Experiments were carried out by placing a mixture of 20 µL of stainless steel assisted bare gold
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nanoparticles synthesized outside the cell with 20 µL of 0.1 mol L-1 CTAC solution into the
300
ATR unit. An increase of water absorption features is again observed with time with an
301
accelerated enhancement in the presence of CTAC in the solution. A similar experiment was
302
conducted by placing a mixture of 20 µL of citrate-coated gold nanoparticles with 20 µL of 0.1
303
mol L-1 CTAC solution into the ATR unit. Figure 5 shows a comparison of the behavior of both
304
types of nanoparticles during sedimentation at the ATR surface.
305
4. CONCLUSIONS
306
In the present study, we confirm he utility of IR-ATR spectroscopy as an analytical strategy for
307
studying gold nanoparticles in solution. IR-ATR was used to monitor the in-situ synthesis of
308
gold nanoparticles within the ATR unit mediated by the stainless steel enclosure of the ATR
309
liquid cell. As gold nanoparticles were synthesized, they sedimented within the evanescent field 12 ACS Paragon Plus Environment
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at the ATR waveguide surface, thereby displacing the water molecules. Despite the fact that
311
bare gold nanoparticles are inherently not IR-active, the deposition of gold nanoparticles at the
312
ATR waveguide surface may indirectly be observed via the surface enhanced infrared
313
absorption (SEIRA) features of water molecules simultaneously present within the evanescent
314
field. The observed SEIRA effect overcompensates the steadily reduced number of water
315
molecules present within the evanescent field as they are increasingly replaced by AuNPs, and
316
therefore facilitates the determination of the presence of gold nanoparticles at the waveguide
317
surface. A particular benefit of the described procedure is the fact that the obtained gold
318
nanoparticles are not covalently attached to the ATR waveguide surface, thereby providing the
319
potential for future SEIRA-assay applications base upon the in-situ generation and subsequent
320
removal/renewal of the SEIRA-active layer at the waveguide surface. Moreover, IR-ATR not
321
only enables monitoring the particle sedimentation/density at the ATR waveguide surface, but
322
also allows studying chemical changes in solution that may affect AuNPs. Finally, it has been
323
confirmed that IR-ATR spectroscopy is a useful strategy for the physico-chemical
324
characterization of the sedimentation process of gold nanoparticles at a waveguide surface.
325
Consequently, future studies will further investigate the properties of such in-situ synthesized
326
bare gold nanoparticles deposited at the ATR waveguide surface for SEIRA studies of
327
additional (bio)molecules present in solution, thereby evaluating the potential of SEIRA-
328
bioassay applications.
329
ACKNOWLEDGMENTS
330
A.I. López-Lorente and M. Valcárcel wish to thank Spain’s Ministry of Innovation and Science
331
for funding Project CTQ2011-23790 and Junta de Andalucia for Project FQM4801. A.I. López-
332
Lorente also wishes to thank the Ministry for the award of a Research Training Fellowship
333
(Grant AP2008-02939), and gratefully acknowledges funding of her stay in Germany at the
334
University of Ulm to conduct the research reported in the present study. M. Sieger and B.
335
Mizaikoff gratefully acknowledge support of this study by the Kompetenznetz Funktionelle
336
Nanostrukturen Baden Wuerttemberg, Germany. Finally, the authors acknowledge the Focused 13 ACS Paragon Plus Environment
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Ion Beam Center UUlm supported by FEI Company (Eindhoven, Netherlands), the German
338
Science Foundation (INST40/385-F1UG), and the Struktur- und Innovationsfonds Baden-
339
Württemberg for support during nanoparticle characterization.
340
The authors declare no competing financial interest.
341
REFERENCES
342
(1) Cai, W.; Gao, T.; Hong, H.; Sun, J. Nanotechnol. Sci. Appl. 2008, 1, 17-32.
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(4) López-Lorente, A.I.; Simonet, B.M.; Valcárcel, M. Comprehensive Analytical Chemistry
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Takahashi, K.; Nakamura, A.; Kinugasa, S. Toxicol. In Vitro 2009, 23, 927-934.
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(7) López-Lorente, A.I.; Simonet, B.M.; Valcárcel, M. Analyst 2012, 137, 3528-3534.
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(8) López-Lorente, A.I.; Simonet, B.M.; Valcárcel, M. TrAC, Trends Anal. Chem. 2011, 30, 58-
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(11) Lin, S.Y.; Liu, S.W.; Lin, C.M.; Chen, C.H. Anal. Chem. 2002, 74, 330-335.
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(12) Park, J.H.; Park, J.; Dembereldorj, U.; Cho, K.; Lee, K.; Yang, S.I.; Lee, S.Y.; Joo, S.-W.
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Anal. Bioanal. Chem. 2011, 401, 1631–1639.
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(14) Tsai, D.-H.; Davila-Morris, M.; DelRio, F.W.; Guha, S.; Zachariah, M.R.; Hackley, V.A.
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Langmuir 2011, 27, 9302–9313.
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(15) Harstein, A.; Kirtley, J.R.; Tsang, J.C. Phys. Rev.. Lett. 1980, 45, 201-204.
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(16) Baia, M.; Toderas, F.; Baia, L.; Maniu, D.; Astilean, S. Chem. Phys. Chem. 2009, 10, 1106-
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(19) Miyake, H.; Ye, S.; Osawa, M. Electrochem. Commun. 2002, 4, 973-977.
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(20) Pucci, A.; Neubrech, F.; Weber, D.; Hong, S.; Toury, T.; Lamy de la Chapelle, M. Phys.
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Status Solidi B 2010, 247, 2071-2074.
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(21) Enders, D.; Nagao, T.; Pucci, A.; Nakayama, T.; Aono, M. Phys. Chem. Chem. Phys. 2011,
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(22) Enders, D.; Nagao, T.; Nakayama, T.; Aono, M. Surf. Interface Anal. 2008, 40, 1681-1683.
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(23) Zhang, Z.; Imac, T. J. Colloid Interface Sci. 2001, 233, 107-111.
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(24) Kundu, J.; Le, F.; Nordlander, P.; Halas, N.J. Chem. Phys. Lett. 2008, 452, 115-119.
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(25) López-Lorente, A.I.; Simonet, B.M.; Valcárcel, M.; Mizaikoff, B. Anal. Chim. Acta 2013,
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788, 122-128.
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(26) López-Lorente, A.I.; Simonet, B.M.; Valcárcel, M.; Eppler, S.; Schindl, S.; Kranz, C.;
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Mizaikoff, M. Talanta 2014, 118, 321-327.
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(27) Turkevich, J.; Stevenson, P.C.; Hillier, J. Discuss. Faraday Soc. 1951, 11, 55-75.
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(28) Venyaminov, S.Y.; Pendegast, F.G. Anal. Biochem. 1997, 248, 234-245.
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(29) Burns, C.; Spendel, W.U.; Puckett, S.; Pacey, G.E. Talanta 2006, 69, 873-876.
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(31) Alexander, C.M.; Dabrowiak, J.C.; Goodisman, J. J. Colloid Interf. Sci. 2013, 396, 53-62.
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(32) Norman Jr., T.J.; Grant, C.D.; Magana, D.; Zhang, Z.; Liu, J.; Cao, D.; Bridges, F.; Van
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FIGURES
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Figure 1
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(a)
1,6
(b) 0,09
1,4
0,08
1 0,8 AuNPs 0,6
AuNPs+AgNO3
0,4
υ(H2 O) band height
1,2
absorbance (a.u.)
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0,07 0,06 0,05 0,04 0,03 0,02 0,01
0,2
0
0 400
500
600
700
0
800
20
Wavelength (nm)
425 426
40 Time (min)
Figure 4
427 428 429 430 431 432 433 434
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60
80
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0.0025 δ(H2O) band height
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0.002 0.0015 stainless steel
0.001
citrate 0.0005 0 0
20
40
60
80
100
Time (min)
435 436
Figure 5
437 438 439 440 441 442 443 444 445 446 447 448
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FIGURE CAPTIONS
450
Figure 1. Schematic of the ATR configuration used during the present studies (not to scale; for
451
details see Bruker BioATR-II unit, Ettlingen, Germany). As evident, the walls of the liquid cell
452
are composed of stainless steel, which acts as reducing agent leading to the in-situ formation of
453
gold nanoparticles from tetrachloroauric acid solution.
454
Figure 2. In-situ SEIRA-ATR spectra obtained during the synthesis of gold nanoparticles inside
455
the ATR unit in an aqueous environment (from a 200 mg dL-1 HAuCl4 solution). (a) H2O
456
bending mode, (b) OH stretching mode. In order to evaluate the SEIRA effect of gold
457
nanoparticles enhancing the water absorption bands, a spectrum of tetrachloroauric acid solution
458
in water was recorded prior to initiating the synthesis, and was used as the background
459
(reference) spectrum. Thus, the water features shown in the spectra herein reflect the observed
460
enhancement as gold nanoparticles sediment at the surface vs. the absorption of pure water in
461
absence of AuNPs. (c) Height of the δ(H2O) bending vibration band at 1644 cm-1 with fitted
462
Langmuir isotherm describing the evolvement of band height vs. time (r2 =0.997). At t=0, the
463
HAuCl4 solution was injected into the ATR liquid cell.
464
Figure 3. Scanning electron microscopy (SEM) images of in-situ synthesized gold
465
nanoparticles: (left) after 20 min, (right) after 100 min of sedimentation.
466
Figure4. (a) UV-Vis spectra of the gold nanoparticle solution prior and after the addition of
467
NaCl for increasing the ionic strength, which leads to the aggregation of AuNPs. (b) Plot of the
468
height of the water OH stretching band vs. time. At minute 11 (red arrow), 10 µL of 1M AgNO3
469
solution were added.
470
Figure 5. Plot of the height of the H2O bending mode vs. time during the sedimentation of
471
citrate-coated and bare gold nanoparticles in CTAC containing aqueous solution. The SEIRA
472
effect is evidenced by recording the spectrum of the gold nanoparticle solution at t=0 as
473
background (reference) spectrum. Thus, the water absorption features shown in the spectra
474
correspond to the obtained SEIRA-effect as gold nanoparticles deposit at the waveguide surface. 21 ACS Paragon Plus Environment
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Table 1. Parameters obtained for fitting a Langmuir isotherm to the water and deuterium oxide
476
absorption features vs. time in aqueous media.
477
Langmuir fit (r2)
Reproducibility* (%)
H2O bending
0.997
10.55
OH stretching
0.999
8.16
D2O bending
0.994
-
OD stretching
0.998
-
*Study performed in aqueous media
478 479 480 481 482 483 484 485 486 487 488 489
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Article
Infrared attenuated total reflection spectroscopy for the characterization of gold nanoparticles in solution Angela Inmaculada López-Lorente, Markus Sieger, Miguel Valcarcel, and Boris Mizaikoff Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 09 Dec 2013 Downloaded from http://pubs.acs.org on December 13, 2013
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Analytical Chemistry
1
Infrared attenuated total reflection spectroscopy for the
2
characterization of gold nanoparticles in solution
3
Ángela Inmaculada López-Lorente1, Markus Sieger2, Miguel Valcárcel1*,
4
Boris Mizaikoff2*
5
1
Department of Analytical Chemistry, University of Córdoba, E-14071 Córdoba, Spain
6 7
Phone/Fax +34 957 218616; E-mail: [email protected] 2
Institute of Analytical and Bioanalytical Chemistry, University of Ulm, Ulm, Germany
8
In-situ synthesis of bare gold nanoparticles mediated by stainless steel as reducing agent was
9
monitored via infrared attenuated total reflection (IR-ATR) spectroscopy. Gold nanoparticles
10
were directly synthesized within the liquid cell of the ATR unit taking immediate advantage of
11
the stainless steel walls of the ATR cell. As nanoparticles were formed, a layer of particles was
12
deposited at the SiO2 ATR waveguide surface. Incidentally, the absorption bands of water
13
increased resulting from surface enhanced infrared absorption (SEIRA) effects arising from the
14
presence of the gold nanoparticles within the evanescent field. Next to the influence of the
15
Au(III) precursor concentration and the temperature, the suitability of IR-ATR spectroscopy as
16
an innovative tool for investigating changes of nanoparticles in solution including their
17
aggregation promoted by an increase of the ionic strength or via a pH decrease, and for detailing
18
the sedimentation process of gold nanoparticles was confirmed.
19 20
Keywords: attenuated total reflection, ATR, infrared spectroscopy, surface-enhanced infrared
21
absorption, SEIRA, gold nanoparticles, nanoparticle synthesis, aggregation.
22
*Corresponding
23
[email protected];
24
[email protected]
authors.
Boris Miguel
Mizaikoff. Valcárcel.
Tel.:
+49
Tel./Fax:
731 +34
1 ACS Paragon Plus Environment
50
22750;
E-mail:
957
218616;
E-mail:
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25
1. INTRODUCTION
26
Metallic nanoparticles and especially gold nanoparticles (AuNPs) provide exceptional
27
properties, which have promoted their usage in a diverse range of areas including biomedical
28
imaging and diagnostic assays1, biological applications2, catalysis3, and for applications based
29
on the enhanced optical properties of nanoparticles such as enhanced Rayleigh scattering or
30
surface-enhanced Raman scattering (SERS) of adsorbed molecules4.
31
Furthermore, a diversity of synthetic procedures for the generation of AuNPs has emerged in
32
recent literature5. Consequently, advanced characterization tools for nanoparticles synthesized at
33
a wide variety of conditions are increasingly demanded. Microscopic techniques are most
34
commonly utilized, although they suffer from several drawbacks, i.e., they are time-consuming
35
in order to ensure representative sampling, and imaging problems may arise from the
36
aggregation of nanoparticles when studied in solution. Optical analysis techniques have proved
37
to be useful for the characterization of nanoparticles, in particular when taking advantage of the
38
pronounced surface plasmon resonances attributed to gold nanoparticles. Dynamic light
39
scattering (DLS) is a common method for the size characterization of nanoparticles6. In
40
addition, Raman spectroscopy has been applied for the characterization and quantification of
41
gold nanoparticles previously extracted by ionic liquids7. In addition, separation techniques
42
including electrophoresis8 have extensively been used for the characterization of synthesized
43
gold nanoparticles. Finally, inductively coupled plasma mass spectrometry9 (ICP-MS) has
44
emerged as a method of choice for quantitatively determining the presence of gold
45
nanoparticles.
46
In general, gold nanoparticles do not show pronounced mid-infrared (2.5-25 µm; MIR)
47
absorptions, unless they are functionalized with organic ligands attached to the particle surface,
48
which
49
cetyltrimethylammonium bromide (CTAB) molecules adsorbed in ionic liquid media10 have
50
been characterized by Fourier transformation infrared (FT-IR) spectroscopy. Furthermore, IR
gives
rise
to
characteristic
IR
spectra.
For
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AuNPs
with
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spectra of 2-(12-mercaptododecyloxy)methyl-15-crown-5-ether modified gold nanoparticles11,
52
4-mercaptobenzoic acid modified AuNPs12, and AuNPs-metallocarbonyl conjugates13 have been
53
published. Infrared attenuated total reflectance (IR-ATR) spectroscopy has been used to
54
quantitatively determine competitive molecular adsorption of thiolated poly(ethylene glycol) at
55
gold nanoparticle surfaces14. However, to date infrared spectroscopy has not been applied for
56
the characterization of gold nanoparticles without any surface modification.
57
Hartstein et al.15 were the first to report on so-called surface enhanced infrared absorption
58
(SEIRA) effects occurring at corrugated noble metal surfaces. In general, two enhancement
59
mechanisms – electromagnetic and chemical enhancement - are considered providing the main
60
contributions to the enhancement of molecular absorption features in SEIRA spectroscopy16.
61
The electromagnetic mechanism increases the local electric field at the surface, while chemical
62
enhancement gives rise to an increase of the absorption due to chemical interactions between
63
molecules and gold nanoparticles17. Among others, the SEIRA effect has been used for the
64
determination of molecules such as p-nitrobenzoic acid18, carbon monoxide19, octadecanethiol20-
65
22
66
In the present study, the adsorption of water molecules at gold nanoparticles has been utilized
67
for in-situ monitoring of the synthesis of bare gold nanoparticles via surface-enhanced infrared
68
attenuated total reflection (SEIRA-ATR) spectroscopy. Gold nanoparticles were directly grown
69
within the liquid cell of the ATR unit from an aqueous tetrachloroauric acid solution mediated
70
by the stainless steel housing of the ATR cell following a mechanism previously discussed by
71
the authors25,26. As nanoparticles were formed and deposited at the surface of the ATR
72
waveguide, an enhancement of the water absorption features was observed, which correlated
73
with the amount of AuNPs present at the surface. A similar effect has been observed in the case
74
of gold nanoparticles synthesized in D2O with the absorption bands of deuterium oxide
75
revealing similar enhancement during the formation of AuNPs. Moreover, the generic suitability
76
of SEIRA-ATR spectroscopy for the investigation of gold nanoparticle solutions has been
77
studied via the evaluation of effects such as the addition of salts or variation of the pH on the
, 4-pyridinethiol23, and p-mercaptoaniline24.
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aggregation state of gold nanoparticles in solution, as well as monitoring the involved
79
sedimentation processes. Thus, in the present study IR-ATR spectroscopy is confirmed as an
80
innovative analytical tool applicable for the characterization of bare AuNPs providing a
81
straightforward and non-destructive characterization strategy.
82
2. METHODS
83
2.1. Materials and reagents
84
HAuCl4 solution (200 mgdL-1 in deionized water)
85
Steinheim, Germany) were used to synthesize the gold nanoparticles. Prior to synthesis, the
86
material was washed with a mixture of nitric acid and hydrochloric acid (HNO3:HCl = 1:3
87
mixture, aqua regia) (Panreac, Barcelona, Spain). A flat piece of 304-stainless steel was used as
88
solid reducing agent of gold precursor in order to form gold nanoparticles. Sodium citrate
89
dehydrate 99.5% (Sigma Aldrich, St. Louis, MO, USA) was used to synthesize citrate-coated
90
gold nanoparticles. Silver nitrate, potassium nitrate, and sodium chloride purchased from
91
Sigma-Aldrich (St. Louis, MO, USA) were used to investigate the aggregation of the
92
nanoparticles. Cetyltrimethylamonium chloride (CTAC) was obtained from Fluka (Buchs,
93
Switzerland). Deuterium oxide (D2O, 99.8%) was provided by Merck (Darmstadt, Germany).
94
The stock solutions used herein were invariably prepared in Milli-Q water.
95
2.2. Synthesis of bare gold nanoparticles
96
Gold nanoparticles were synthesized following a procedure previously described by the authors
97
using stainless steel is used as solid reducing agent25,26. All glassware was cleaned with freshly
98
prepared aqua regia (HNO3:HCl = 1:3 mixture) and then thoroughly rinsed with distilled H2O
99
prior to use. A piece of 304-stainless steel (12.9 mm2 of total surface) was introduced into 100
100
µL of a 0.2 mg mL-1 aqueous solution of HAuCl4. The reaction was carried out at room
101
temperature; the stainless steel substrate was simultaneously used as stirrer during the reaction.
and gold(III) chloride (Sigma Aldrich,
102
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2.3. Synthesis of bare gold nanoparticles inside the ATR liquid cell
104
Gold nanoparticles were also directly synthesized inside the liquid cell of the ATR unit by
105
taking direct advantage of the stainless steel, which forms the walls of the ATR liquid cell. In
106
this case, no stainless steel piece was added to the solution of tetrachloroauric acid, which was
107
again used as a gold(III) precursor matrix. Once the tetrachloroauric acid solution was located
108
within the ATR unit, the immediate onset of the formation of gold nanoparticles was observed.
109
Thus, obtained AuNPs sedimented at the ATR crystal surface, thereby enabling direct
110
monitoring via SEIRA-ATR spectroscopy. The reaction was performed at a variety of
111
temperatures: room temperature (i.e., 22ºC), 40ºC, 50ºC, and 60ºC, as controlled by a thermostat
112
unit.
113
2.4. Synthesis of citrate-coated gold nanoparticles
114
Gold nanoparticles obtained via HAuCl4 reduction mediated by sodium citrate were also
115
prepared according to the method described by Turkevich et al.27 applying modifications
116
described elsewhere7. Firstly, all glassware were cleaned with freshly prepared aqua regia
117
(HNO3:HCl 1:3 mixture), and then thoroughly rinsed with distilled H2O prior to usage.
118
Tetrachloroauric acid and sodium citrate solutions were filtered through a 0.45 µm nylon
119
membrane prior to use. Once a 0.01% solution of HAuCl4 was boiling, 0.254 mL of a 1%
120
sodium citrate solution were added. The system was then left to react while stirring for a period
121
of 15 min. Afterwards, 5mL of 0.01% HAuCl4 solution were added to the system followed by
122
0.254 mL of 1% sodium citrate solution, and again stirred while heating for 15 min. Then, the
123
heater was switched off, and the solution was stirred until cooling to room temperature. Thus
124
obtained AuNPs were stored at 4ºC in an amber bottle.
125
2.5. Infrared spectroscopic analysis
126
Infrared spectroscopic studies were performed using a Vertex 70 FT-IR spectrometer (Bruker
127
Optics, Ettlingen, Germany) equipped with a BioATR-II unit, and a liquid nitrogen cooled
128
mercury-cadmium-telluride (MCT) detector (Bruker Optics, Ettlingen, Germany). Figure 1 5 ACS Paragon Plus Environment
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shows a schematic of the experimental setup. As evident although not to scale, the top ATR
130
crystal interfacing with the sample is made from a circular section of a silicon wafer providing
131
8-10 internal reflections. IR radiation is coupled into this silicon ATR plate via a secondary zinc
132
selenide (ZnSe) ATR waveguide (which is not in contact with the sample) providing a
133
hemispherical shape for efficient coupling of photons from the FT-IR spectrometer into the Si-
134
ATR. The entire unit was connected to a circulating water bath ensuring a defined and constant
135
temperature during the IR-ATR analysis. Data acquisition and processing was performed using
136
the OPUS 6.5 software package (Bruker Optics, Ettlingen, Germany). Spectra were acquired by
137
averaging 300 spectra in the wavelength window of 4000 to 400 cm−1 at a spectral resolution of
138
4 cm−1. Prior to analyzing each set of samples, a background spectrum of the tetrachloroauric
139
acid solution was collected when monitoring the subsequent in-situ synthesis of AuNPs. If
140
AuNPs were synthesized ex-situ, prior to a sample measurement a background spectrum of
141
deposited AuNPs at time zero was recorded. Finally, measurements of the synthesis of gold
142
nanoparticles in D2O media were acquired averaging 500 spectra within 4000 to 400 cm-1 at a
143
spectral resolution of 4 cm-1.
144
3. RESULTS AND DISCUSSION
145
3.1. In-situ synthesis of gold nanoparticles within the IR-ATR liquid cell
146
Gold nanoparticles were directly synthesized inside the ATR unit taking advantage of the
147
stainless steel ring establishing the confining walls of the liquid cell, as previously demonstrated
148
by the authors25,26. While AuNPs are formed, they sediment at the ATR waveguide surface until
149
Coulombic repulsion from already adsorbed AuNPs leads to an equilibrium.
150
Gold nanoparticles are characterized by a strong plasmon resonance in the UV/Vis region as a
151
consequence of the collective oscillation of electrons within the conduction band. However,
152
bare gold nanoparticles do not inherently show a pronounced absorption in the MIR. Despite the
153
IR-inactivity of AuNPs, it was found that their formation could still be monitored via IR
154
spectroscopy by analyzing the increase of the water absorption features during the synthesis 6 ACS Paragon Plus Environment
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process, which arises from a SEIRA effect, as discussed below. Figure 2 shows a series of in-
156
situ IR-ATR spectra recorded during gold nanoparticles being synthesized within the ATR unit.
157
According to literature28, the vibrational modes of water in the infrared region are the H2O
158
bending mode at 1644 cm-1, the combination of H2O bending and libration mode at 2128 cm-1,
159
and a pronounced vibration around 3404 cm-1, which is composed of the overtone of the
160
bending mode at 3250 cm-1, the symmetric OH stretch at 3450 cm-1, and the antisymmetric OH
161
stretch at 3600 cm-1 22. As gold nanoparticles are formed and deposited at the ATR waveguide
162
surface, an increase of the H2O bending and the OH stretching bands is observed despite the
163
effective decrease of water molecules present within the evanescent field being displaced by
164
gold nanoparticles. This increase is attributed to an enhancement of the water absorptions within
165
the evanescent field resulting from the presence of AuNPs. While the magnitude of the
166
enhancement certainly decreases with increasing the distance from the waveguide surface (i.e.,
167
the nanoparticulate Au film), it compensates for the decreasing number of water molecules
168
steadily displaced by AuNPs. The hypothesis of enhancing the IR signature of water via the
169
formation of AuNPs is further corroborated by the observed increase of the water absorption
170
signal vs. the background spectrum.
171
For monitoring the kinetics of the synthesis and deposition of AuNPs at the ATR waveguide
172
surface, the peak height of the water bending mode (δ(H2O)) was analyzed in detail. Figure 2a
173
shows the peak height of the H2O bending mode vs. time with t=0 indicating the initiation of the
174
in-situ synthesis of gold nanoparticles from tetrachloroauric acid solution. The obtained data
175
readily fit to a Langmuir model, which describes a random adsorption process within the first
176
monolayer (Figure 2c). The obtained goodness-of-the-fit was r2=0.997.
177
Likewise, the absorption band corresponding to the OH stretching vibration revealed an increase
178
during the synthesis of gold nanoparticles (figure 2b). Plotting the peak height vs. time again fits
179
a Langmuir isotherm with a goodness-of-the-fit of r2=0.999. Table 1 summarizes the main
180
parameters obtained when fitting both vibrational water bands to a Langmuir isotherm.
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In order to further study the in-situ formation of gold nanoparticles within the ATR cell, a
182
stainless steel O-ring of similar dimensions was glued onto a piece of silicon wafer, in order to
183
simulate the scenario inside the ATR liquid cell (i.e., mock-up ATR cell; MU-ATR) enabling
184
access to subsequent scanning electron microscopy (SEM) studies of the obtained AuNPs
185
without additional manipulation. Similar to the in-situ synthesis monitored by IR-ATR
186
spectroscopy, a small amount of HAuCl4 (200 mg dL-1) was added to the MU-ATR in order to
187
synthesize AuNPs. The reaction was stopped after 20 min and 100 min, respectively, by
188
removing the aqueous solution followed by rinsing with dried air.
189
After 20 min, a sub-monolayer coverage with few agglomerates of AuNPs was determined at
190
the Si wafer surface (Figure 3 left), as expected from the in-situ IR-ATR studies. After 100 min,
191
the AuNPs in solution show significant agglomeration with already adsorbed gold
192
nanoparticles, as evident in Figure 3 (right). During the process of deposition of AuNPs at the
193
ATR crystal surface, the distribution, size, and shape of the NPs as well as their interaction
194
change. This morphological development results in changes of the electromagnetic field
195
properties near the ATR waveguide surface. Firstly, a monolayer of AuNPs is formed at the
196
surface (Figure 3 left), which leads to a strong water bending and OH stretching signal
197
enhancement in the first few minutes. The SEIRA activity results from an enhanced near-field
198
around the NPs, which decays with increasing distance from the particle surface. As previously
199
explained, the near-field enhancement around AuNPs overcompensates the reduced number of
200
water molecules present within the evanescent field, as they are increasingly replaced by AuNPs
201
during their adsorption on the waveguide surface. Once gold nanoparticles are deposited on the
202
surface, aggregates or islands of nanoparticles are observed, as evident within the SEM images
203
(Figure 3, right), along with no further increase of the water absorption features. At such
204
islands, the electromagnetic near-field properties change, as accordingly the absorption of IR
205
radiation21.
206
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3.2. Influence of the gold(III) precursor concentration
208
The influence of the concentration of the gold(III) precursor (i.e., tetrachloroauric acid) was
209
studied. While the kinetics of the reaction follow a different rate depending on the
210
concentration, in all cases the obtained rates may be fitted with a Langmuir isotherm.
211
If the concentration of gold precursor within the ATR unit is high (i.e., 200 mgdL-1), the
212
increase of the absorption features related to the water spectrum is rapidly observed (i.e., within
213
1 min). At lower concentrations (i.e., 66 mgdL-1), the formation of gold nanoparticles is
214
correspondingly slower, which is reflected in the IR spectrum (Figure S1, Supporting
215
Information). Figure S2 (Supporting Information) shows the H2O bending region at different
216
times of reaction. Initially, AuNPs deposited at the surface appear isolated with only few
217
particles agglomerated. With increasing reaction time, packed island of connected nanoparticles
218
are evident. This behavior agrees with findings previously reported in literature22 for the water
219
OH stretching band during the adsorption of a submonolayer of AuNPs at a substrate initially
220
treated with (aminopropyl)triethoxysilane (APTES).
221
3.3. Influence of the temperature
222
If AuNPs are synthesized outside the ATR unit by inserting a piece of stainless steel piece into
223
the solution25, an increase of the rate of production of gold nanoparticles is observed with
224
increasing temperature. The in-situ synthesis was performed at different temperatures (23, 40,
225
50, and 60ºC) for spectroscopically analyzing this behavior. With increasing temperature, the
226
enhancement of water bands more rapidly appears, which in turn confirms that AuNPs are more
227
rapidly formed.
228
3.4. Reproducibility
229
A study of the reproducibility of the in-situ AuNPs synthesis and the observed SEIRA-ATR
230
water signal was performed by repeating the analysis four times during different days for the
231
synthesis procedure based on a 200 mg dL-1 HAuCl4 solution at 60ºC. As reported in Table 1,
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the relative standard deviation calculated from the H2O bending peak height was 10.55%, while
233
the relative standard deviation obtained from the OH stretching band was 8.16%.
234
3.5. In-situ synthesis of gold nanoparticles in D2O
235
The synthesis of the gold nanoparticles mediated by stainless steel inside the ATR unit was
236
finally also performed in D2O, using AuCl3. The aim of this study was to potentially observe the
237
SEIRA effect on the associated D2O bands in a different spectral regime for confirmation of the
238
observations initially made for H2O. No significant difference was noted concerning the
239
formation of AuNPs during the chemical reaction compared to the studies in H2O. Similar to the
240
observations in water, as gold nanoparticles were emerging, the absorption signals of the υ(OD)
241
and the δ(D2O) band appear with enhanced intensity. Again, thus obtained data were fitted using
242
a Langmuir model with a goodness-of-the-fit of r2=0.994 for the δ(D2O) band (see Figure S3,
243
Supporting Information), and of r2=0.998 when evaluating the OD stretching band (Table 1).
244
The same behavior was observed when performing the synthesis in a 1:1 mixture of H2O and
245
D2O resulting in the corresponding simultaneous enhancement of water and deuterium oxide
246
infrared absorption features, respectively.
247
3.6. Influence of the ionic strength
248
As IR spectra are also sensitive to variations of the chemical environmental, next to non-
249
destructively monitoring the synthesis of gold nanoparticles one may furthermore analyze
250
changes in aggregation or stability of the obtained AuNP dispersions. The aggregation of
251
AuNPs causes coupling of their plasmonic modes, thereby resulting in a red shift and
252
broadening of the longitudinal plasmon resonance band in the optical spectrum, as evident in the
253
UV/Vis absorption spectra shown in Figure 4 (top). It is known that variation of the ionic
254
strength is a particle-independent strategy to affect the repulsion distance between charged
255
particles. Increasing the ionic strength decreases the Debye length, thus also decreasing the
256
averaged distance between particles29. Two hypotheses have been proposed to explain the
257
observed change in the color of such solutions when increasing the ionic strength: (i) gold 10 ACS Paragon Plus Environment
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nanoparticles physically coalesce to form larger particles, and (ii) the mean interaction distance
259
is reduced such that their surface plasmons may interact29.
260
Herein, the effect of the addition of silver(I) nitrate and potassium nitrate was investigated. As
261
gold nanoparticles are synthesized and deposited within the evanescent field at the ATR
262
waveguide surface, an enhancement of the water absorption features is observed. If the ionic
263
strength of the solution is altered by addition of salts, a sudden increase of the absorption of the
264
water bands is observed, without any further change with time.
265
While AuNPs synthesized in-situ using the stainless steel assisted method do not contain citrate
266
ligands bound to the nanoparticle surface, chloride ions are present in abundance and may shield
267
the obtained nanoparticles. Such a chloride shell may prevent nanoparticle agglomeration, and
268
may also facilitate the stabilization of the solution due to the achieved spacing between
269
individual NPs. When adding silver nitrate to the solution, which increases the ionic strength,
270
the enhancement of the water bands terminates (see Figure 4, bottom). Apparently, silver ions
271
interact with chloride ions, and thus, AuNPs will be destabilized and rapidly deposit at the
272
waveguide surface. Once deposited as islands on the surface, the increase of the water
273
absorption bands terminates in analogy to the observations during the deposition of AuNPs at
274
in-situ synthesis conditions at extended periods of time.
275
A similar behavior was observed when adding potassium nitrate; once nanoparticles aggregate,
276
an increase in water signal is observed followed by no further changes of the IR absorption
277
band.
278
3.7. Influence of the pH value
279
The influence of a decrease in pH during the synthesis of gold nanoparticles within the ATR
280
unit was investigated by adding HNO3 during the synthesis. 10 µL of a 10% (v/v) HNO3
281
solution were added to 40 µL of 200 mg dL-1 HAuCl4 solution previously placed into the ATR
282
liquid cell. It has previously been described that low pH values induce the aggregation of gold
283
nanoparticles30. Again, the suitability of IR-ATR spectroscopy to determine changes in 11 ACS Paragon Plus Environment
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284
aggregation state of the synthesizing gold nanoparticles was demonstrated observing a behavior
285
similar to that when increasing the ionic strength.
286
3.8. Investigating the sedimentation behavior of ex-situ synthesized unmodified and citrate
287
coated gold nanoparticles via IR-ATR spectroscopy
288
The sedimentation behavior of gold nanoparticles has been studied via UV/Vis spectroscopy
289
under normal gravitational force31. The optical detection during sedimentation of particles in
290
solution enables the accurate determination of the particle size distribution in a polydisperse
291
nanoparticle sample31. Herein, IR-ATR spectroscopy is proposed as an alternative strategy for
292
monitoring gravitational sedimentation of gold nanoparticles. Hence, the sedimentation rate of
293
both unmodified gold nanoparticles obtained via stainless steel assisted reduction outside the
294
ATR liquid cell, and citrate-capped gold nanoparticles was compared. In order to achieve
295
acceptable sedimentation rates, CTAC was added, as previously reported in the literature32.
296
Cationic surfactant causes aggregation by neutralizing the negative charge at the nanoparticle
297
surface.
298
Experiments were carried out by placing a mixture of 20 µL of stainless steel assisted bare gold
299
nanoparticles synthesized outside the cell with 20 µL of 0.1 mol L-1 CTAC solution into the
300
ATR unit. An increase of water absorption features is again observed with time with an
301
accelerated enhancement in the presence of CTAC in the solution. A similar experiment was
302
conducted by placing a mixture of 20 µL of citrate-coated gold nanoparticles with 20 µL of 0.1
303
mol L-1 CTAC solution into the ATR unit. Figure 5 shows a comparison of the behavior of both
304
types of nanoparticles during sedimentation at the ATR surface.
305
4. CONCLUSIONS
306
In the present study, we confirm he utility of IR-ATR spectroscopy as an analytical strategy for
307
studying gold nanoparticles in solution. IR-ATR was used to monitor the in-situ synthesis of
308
gold nanoparticles within the ATR unit mediated by the stainless steel enclosure of the ATR
309
liquid cell. As gold nanoparticles were synthesized, they sedimented within the evanescent field 12 ACS Paragon Plus Environment
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at the ATR waveguide surface, thereby displacing the water molecules. Despite the fact that
311
bare gold nanoparticles are inherently not IR-active, the deposition of gold nanoparticles at the
312
ATR waveguide surface may indirectly be observed via the surface enhanced infrared
313
absorption (SEIRA) features of water molecules simultaneously present within the evanescent
314
field. The observed SEIRA effect overcompensates the steadily reduced number of water
315
molecules present within the evanescent field as they are increasingly replaced by AuNPs, and
316
therefore facilitates the determination of the presence of gold nanoparticles at the waveguide
317
surface. A particular benefit of the described procedure is the fact that the obtained gold
318
nanoparticles are not covalently attached to the ATR waveguide surface, thereby providing the
319
potential for future SEIRA-assay applications base upon the in-situ generation and subsequent
320
removal/renewal of the SEIRA-active layer at the waveguide surface. Moreover, IR-ATR not
321
only enables monitoring the particle sedimentation/density at the ATR waveguide surface, but
322
also allows studying chemical changes in solution that may affect AuNPs. Finally, it has been
323
confirmed that IR-ATR spectroscopy is a useful strategy for the physico-chemical
324
characterization of the sedimentation process of gold nanoparticles at a waveguide surface.
325
Consequently, future studies will further investigate the properties of such in-situ synthesized
326
bare gold nanoparticles deposited at the ATR waveguide surface for SEIRA studies of
327
additional (bio)molecules present in solution, thereby evaluating the potential of SEIRA-
328
bioassay applications.
329
ACKNOWLEDGMENTS
330
A.I. López-Lorente and M. Valcárcel wish to thank Spain’s Ministry of Innovation and Science
331
for funding Project CTQ2011-23790 and Junta de Andalucia for Project FQM4801. A.I. López-
332
Lorente also wishes to thank the Ministry for the award of a Research Training Fellowship
333
(Grant AP2008-02939), and gratefully acknowledges funding of her stay in Germany at the
334
University of Ulm to conduct the research reported in the present study. M. Sieger and B.
335
Mizaikoff gratefully acknowledge support of this study by the Kompetenznetz Funktionelle
336
Nanostrukturen Baden Wuerttemberg, Germany. Finally, the authors acknowledge the Focused 13 ACS Paragon Plus Environment
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Ion Beam Center UUlm supported by FEI Company (Eindhoven, Netherlands), the German
338
Science Foundation (INST40/385-F1UG), and the Struktur- und Innovationsfonds Baden-
339
Württemberg for support during nanoparticle characterization.
340
The authors declare no competing financial interest.
341
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FIGURES
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Figure 1
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Figure 2
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Figure 3
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(a)
1,6
(b) 0,09
1,4
0,08
1 0,8 AuNPs 0,6
AuNPs+AgNO3
0,4
υ(H2 O) band height
1,2
absorbance (a.u.)
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0,07 0,06 0,05 0,04 0,03 0,02 0,01
0,2
0
0 400
500
600
700
0
800
20
Wavelength (nm)
425 426
40 Time (min)
Figure 4
427 428 429 430 431 432 433 434
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80
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0.0025 δ(H2O) band height
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0.002 0.0015 stainless steel
0.001
citrate 0.0005 0 0
20
40
60
80
100
Time (min)
435 436
Figure 5
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FIGURE CAPTIONS
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Figure 1. Schematic of the ATR configuration used during the present studies (not to scale; for
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details see Bruker BioATR-II unit, Ettlingen, Germany). As evident, the walls of the liquid cell
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are composed of stainless steel, which acts as reducing agent leading to the in-situ formation of
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gold nanoparticles from tetrachloroauric acid solution.
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Figure 2. In-situ SEIRA-ATR spectra obtained during the synthesis of gold nanoparticles inside
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the ATR unit in an aqueous environment (from a 200 mg dL-1 HAuCl4 solution). (a) H2O
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bending mode, (b) OH stretching mode. In order to evaluate the SEIRA effect of gold
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nanoparticles enhancing the water absorption bands, a spectrum of tetrachloroauric acid solution
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in water was recorded prior to initiating the synthesis, and was used as the background
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(reference) spectrum. Thus, the water features shown in the spectra herein reflect the observed
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enhancement as gold nanoparticles sediment at the surface vs. the absorption of pure water in
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absence of AuNPs. (c) Height of the δ(H2O) bending vibration band at 1644 cm-1 with fitted
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Langmuir isotherm describing the evolvement of band height vs. time (r2 =0.997). At t=0, the
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HAuCl4 solution was injected into the ATR liquid cell.
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Figure 3. Scanning electron microscopy (SEM) images of in-situ synthesized gold
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nanoparticles: (left) after 20 min, (right) after 100 min of sedimentation.
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Figure4. (a) UV-Vis spectra of the gold nanoparticle solution prior and after the addition of
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NaCl for increasing the ionic strength, which leads to the aggregation of AuNPs. (b) Plot of the
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height of the water OH stretching band vs. time. At minute 11 (red arrow), 10 µL of 1M AgNO3
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solution were added.
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Figure 5. Plot of the height of the H2O bending mode vs. time during the sedimentation of
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citrate-coated and bare gold nanoparticles in CTAC containing aqueous solution. The SEIRA
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effect is evidenced by recording the spectrum of the gold nanoparticle solution at t=0 as
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background (reference) spectrum. Thus, the water absorption features shown in the spectra
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correspond to the obtained SEIRA-effect as gold nanoparticles deposit at the waveguide surface. 21 ACS Paragon Plus Environment
Analytical Chemistry
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Table 1. Parameters obtained for fitting a Langmuir isotherm to the water and deuterium oxide
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absorption features vs. time in aqueous media.
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Langmuir fit (r2)
Reproducibility* (%)
H2O bending
0.997
10.55
OH stretching
0.999
8.16
D2O bending
0.994
-
OD stretching
0.998
-
*Study performed in aqueous media
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For TOC only:
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