B American Society for Mass Spectrometry, 2013

J. Am. Soc. Mass Spectrom. (2013) 24:305Y308 DOI: 10.1007/s13361-012-0541-5

APPLICATION NOTE

Analysis of the Formation Process of Gold Nanoparticles by Surface-Assisted Laser Desorption/Ionization Mass Spectrometry Iva Tomalová,1 Chia-Hsin Lee,2 Wen-Tsen Chen,2 Cheng-Kang Chiang,2 Huan-Tsung Chang,2 Jan Preisler1 1 2

CEITEC MU and Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno( Czech Republic Department of Chemistry, National Taiwan University, 1, Section 4, Roosevelt Road, Taipei 106, Taiwan Abstract. Chemical reactions of reducing agents in the gold nanoparticle (AuNP) formation process were characterized using surface-assisted laser desorption/ ionization mass spectrometry (SALDI-MS). As the reaction of the AuNPs progresses, the produced AuNPs can serve as an efficient SALDI substrate. SALDI-MS revealed that the reducing agents and their oxidation products can be determined in the mass spectra. With respect to the transmission electron microscopic and UV-Vis spectroscopic examination of AuNPs, SALDI-MS results confirm not only the tendency toward AuNPs formation, but also reflect the information of the redox reaction process. Our results provide useful information for developing SALDI-MS methods to explore the chemical information regarding the surface behavior between adsorbates and nanomaterials. Key words: Nanomaterials, Gold nanoparticle, Surface-assisted laser desorption/ionization mass spectrometry, SALDI, Nanoparticle formation process Received: 30 August 2012/Revised: 9 November 2012/Accepted: 14 November 2012/Published online: 11 January 2013

D

ue to their stability, size-related electric, magnetic and optical properties, as well as the ease of the surface modification with various ligands, gold nanoparticles (AuNPs) have been the subject of extensive interest in many fields including nanotechnology, electronics, and material science [1, 2]. AuNPs have been synthesized by various methods that are based usually on the reduction of chloroauric acid in the presence of a stabilizing (capping) agent. The most common method is the reduction of chloroauric acid by trisodium citrate [3] or NaBH4 [4]. However, with the increasing attention paid to nanomaterials, less traditional reducing agents including amino acids [5–7], proteins [8, 9] or even lemongrass extract [10] have been employed to prepare AuNPs in recent years. Up to now, numerous methodologies have been employed to explore the surface morphologies and lattice structures of AuNPs, including Auger electron spectrometry,

Electronic supplementary material The online version of this article (doi:10.1007/s13361-012-0541-5) contains supplementary material, which is available to authorized users. Correspondence to: Huan-Tsung Chang; e-mail: [email protected], Jan Preisler; e-mail: [email protected]

energy dispersive spectrometry, secondary ion mass spectrometry [11], X-ray absorption near-edge spectroscopy, and X-ray photoelectron spectroscopy [12]. Although several optical and electrochemical approaches have been extensively applied to study the formation mechanism of AuNPs and the role of reducing agents [13, 14], it has rarely been examined by mass spectrometric approaches. Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) is an approach based on NPs or other substrate materials mediating the desorption/ionization process [15]. A range of nanomaterials of various sizes, shapes and compositions has been adopted as SALDI matrices in analytical and bio-analytical applications [16]. Recently, SALDI-MS has also provided strong evidence for the chemical reactions of functionalized AuNPs with analytes and in the analysis of adsorbates on gold nanorods [17]. In this study, we monitored the formation process of AuNPs in the presence of various reducing agents using SALDI-MS [18]. We examined the general aspects of the chemical reactions of reducing agents in the AuNP formation process. As the formation of AuNPs progresses, the resulting AuNPs act as the SALDI substrate to explore the oxidation products of reducing agents adsorbed on the particle surfaces. Thus, the progress of the chemical redox reaction can be monitored.

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Experimental

Results and Discussion

Chemicals

Analysis of Dopamine-Reduced AuNPs by SALDI-MS

The reducing agents including dopamine, ascorbic acid, and catechin were purchased from Aldrich (Milwaukee, WI, USA). Trisodium citrate and sodium phosphate dibasic were obtained from Sigma (St. Louis, MO, USA), hydrogen tetrachloroaurate (III) trihydrate (HAuCl4) from Acros (Geel, Belgium). All solvents and chemicals were of the highest purity grade commercially available and were used without further purification. Sodium phosphate buffer (10 mM, pH 7.2) was used in the experiment.

As mentioned above, we applied SALDI-MS to investigate the oxidation products of the reductants used for the AuNP preparation. Dopamine was chosen as the first model reductant: Figure 1 shows the SALDI spectra of solutions of 200 μM Au(III) ions in a phosphate buffer (pH 7.2) after the addition of dopamine (0-1 mM). The signals at m/z 170.02, 172.04, 174.05, and 176.07 were assigned to the monoisotopic ions [indolequinone + Na]+, [5,6-dihydroxyindole + Na]+, [leucodopaminechrome + Na]+, and [dopamine + Na]+, respectively. As indicated

Generation of AuNPs Using Various Reducing Agents

a

0.4 0.2

1.0

0 316 318 320 322 324

0.5

0.4 0.2

176.07

174.05

316 318 320 322 324 0.5

172.04

0 170.02

1.0

b

319.07

1.5

317.05

0.0

176.07 174.05

316 318 320 322 324 1

172.04

0 170.02

2

321.08

0.2

c

323.10

0.4 3

317.05

0.0 319.07

Signal intensity (a.u.; x104)

0 1.5

d

0.4 176.07

Briefly, the growth solution for AuNPs consisted of 0.2 mM HAuCl4 and various concentrations (0–1 mM) of the reducing agent (dopamine, catechin or ascorbic acid). In the catechin and dopamine experiments, reactions were performed in the sodium phosphate buffer (10 mM, pH 7.2). The solutions were equilibrated for 1 h at room temperature. A Cintra 10e double-beam ultraviolet-visible (UV-Vis) spectrophotometer (GBC, Victoria, Australia) was used to measure absorption spectra of the AuNP solution. The size of the AuNPs was determined using an H7100 transmission electron microscope (TEM; Hitachi HighTechnologies Corporation, Tokyo, Japan). For the SALDIMS measurement, aliquots (1.0 μL) of the as-prepared AuNP solutions were pipetted onto a MALDI target and dried at room temperature for 30 min prior to SALDI-MS analysis. For control experiments, 14 nm spherical AuNPs (15 nM) were prepared from a reduction of 1.0 mM HAuCl 4 by 4.0 mM citrate [19]. Control solutions consisting of these AuNPs (7.5 nM), phosphate buffer (10 mM, pH 7.2), and the above-mentioned reducing agents (0–1 mM) were also loaded onto the MALDI target and analyzed by SALDI-MS.

1.5

0.2 1.0

SALDI-MS Measurement

0 316 318 320 322 324

Mass spectrometry experiments were performed in the reflector positive (dopamine experiment) or negative ion mode (catechin and ascorbic acid experiments) using a Microflex MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) with a nitrogen laser (337 nm) operating at 10 Hz. The accelerating voltages on the MALDI target and on the first grid were 19 kV, and 16.25 kV, respectively; the reflector voltage was 20 kV. Voltages of the same magnitude but opposite polarity were applied when measuring in the negative ion mode. The delayed extraction period was 200 ns. A total number of 300 pulsed laser shots were collected from a target position and spectra from 10 MALDI target positions were averaged. The instrument was calibrated with Au clusters ([Aux]+, x01−3).

0.5

0.0 160 162

164 166 168 170

172 174 176 178 180

m/z Figure 1. SALDI mass spectra of solutions containing 200 μM HAuCl4, and phosphate (10 mM, pH 7.2) in the (a) absence and presence of (b) 100, (c) 400, and (d) 1000 μM dopamine. The peaks at m/z 170.02, 172.04, 174.05 and 176.07 were assigned to monoisotopic [indolequinone + Na]+, [5,6dihydroxyindole + Na]+, [leucodopaminechrome + Na]+ and [dopamine + Na]+ ions, respectively. The peaks at m/z 317.05, 319.07, 321.08, and 323.10 (insets) corresponds to various oxidation states of dopamine dimer

I. Tomalová et al.: SALDI MS of Au Nanoparticle Formation Process

in Figure 1b, only peaks corresponding to the oxidized forms of dopamine dominate in the spectrum while adding 100 μM dopamine. Moreover, a series of peaks at m/z 317.05, 319.07, 321.09, and 323.10 belonging to the further oxidation states of dopamine (dimeric forms) were detected in the mass spectra (Figure 1b, c, insets). Upon increasing the concentration of dopamine up to 400 μM, a peak representing the [dopamine + Na]+ ion appears (Figure 1c). Only [dopamine + Na]+ ions were found to dominate in the spectrum and the oxidized forms were hardly detected while adding dopamine in larger concentration (Figure 1d) as a result of competition among the dopamine and other products for the limited surface of the NPs. The background peak at m/z 164.93, which was present in all spectra, including the spectra of the solution with no dopamine added (Figure 1a), was assigned to the monoisotopic [Na3HPO4]+ ion originating from the sodium phosphate buffer. As a control, no oxidized forms of dopamine were observed when using 14 nm AuNPs as SALDI matrixes (Supplemental Figure S1), which confirms that the redox reaction between dopamine and Au (III) could be monitored by SALDI-MS measurement. These observations confirm and broaden the previously suggested mechanism of dopamine oxidation [6, 20]. While the Au(III) ions were reduced to Au, the oxidation of dopamine involved the initial two-electron oxidation of dopamine to yield dopamine-quinone that was subjected to Michael-type cyclization to give leucoaminochrome and subsequent oxidation to aminochrome. Aminochrome rearranged to form 5,6-dihydroxyindole which was further oxidized to form indol-5,6-quinone and then polydopamine (Figure 2). To further understand the SALDI mass spectra features of the generated AuNPs, the UV-Vis spectroscopy and transmission electron microscopy (TEM) were performed in order to characterize the resulting AuNPs. In the UVVis spectra, a broad band at 575 nm was observed after addition of 100 μM dopamine (Figure S2a). While the concentration of dopamine increased, the absorbance characteristic for the surface plasmon of the AuNPs flattened with blue-shifts in the absorbance maxima. The TEM analysis revealed that the higher the concentration of dopamine that was used, the smaller the particles were formed (Figure S2b–d). Besides these small particles, formation of larger AuNP aggregates was observed, presumably due to the inefficient stabilizing properties of dopamine. The size distribution of AuNPs became broad upon increasing dopamine concentration.

SALDI-MS Assays for Catechin-Reduced AuNPs In our previous work, we developed a method to produce three-dimensional branched Au nanomaterials by the reaction of sodium tetrachloroaurate with an aqueous tea extract (unpublished data). The results indicate that the polyphenols in tea extract played an important role in the reduction of Au

307

dopamine (176.07)

dopaminequinone (174.05)

OH

O OH

O +

-2H -2e

-

oxidation

NH2

NH2

dopaminechrome (172.04)

leucodopaminechrome (174.05)

OH

OH O

OH -2H+-2e oxidation

N

HN

5,6-dihydroxyindole (172.04)

indole-5,6-quinone (170.02)

OH

O OH

O

-2H +-2e-

HN

oxidation

HN

polydopamine Figure 2. The pathway of dopamine oxidation with m/z values observed in mass spectra corresponding to monoisotopic [M + Na]+ ions

(III) to Au and in stabilizing the branched Au nanomaterials. Therefore, we employed different concentrations of catechin, one of the common polyphenols in tea, to synthesize the AuNPs in the aqueous solution and characterized the reaction products by SALDI-MS measurements in the negative ion mode. Two peaks at m/z 287.05 and 289.07 corresponding to the ions [catechin quinone – H]– and [catechin – H]– were observed in mass spectra when adding catechin at concentrations of 200 μM or higher (Figure S3). The ratio of their signal intensities upon increasing catechin concentration follows the similar trend as the ratio of dopamine and its oxidized forms: the signal intensity of [quinone – H]– ion gradually decreases while the [catechin – H]– ion signal becomes dominant in the mass spectra. In order to further characterize the changes in the morphology of the formed Au nanostructures, the TEM images and UVVis spectra of the prepared AuNPs were recorded (for details, see Characterization of catechin-reduced AuNPs and Figure S4 in the Supporting information).

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SALDI-MS Assay with Ascorbic Acid as Reducing Agent To further demonstrate the general applicability of this SALDIMS approach for the characterization of the chemical reaction, we also monitored the oxidized- and reduced-form signals of ascorbic acid from ascorbic acid synthesized AuNPs. In general, the reducing property of ascorbic acid is attributed to the enediol group [21]. Two peaks at m/z 173.01 and 175.02 assigned to the monoisotopic ions [dehydroascorbic acid – H]– and [ascorbic acid – H]– were observed in SALDI spectra (Figure S5). Easy oxidation of ascorbic acid in the neutral or alkaline conditions [22] may distort the signal ratio of the two peaks, but a trend was evident: the signal ratio did not change significantly upon increasing the concentration of ascorbic acid from 100 to 500 μM. The intensities of [ascorbic acid – H]– ions increased slightly when adding 1 mM ascorbic acid presumably because all oxidizing agents were used up. On the contrary, only a peak representing the [ascorbic acid – H]– ions was detected in the spectrum when using 14 nm AuNP SALDI matrix. As in the previous cases, TEM images and UV-Vis spectra were recorded to illustrate the effect of ascorbic acid concentration on the AuNP morphology; see Characterization of ascorbic acid-reduced AuNPs and Figure S6 in the Supporting information for further details.

Conclusion In this study, we have demonstrated the general applicability of SALDI-MS for the investigation of the formation process of AuNPs by various reducing agents in the neutral condition. As the formation of AuNPs proceeds, the generated AuNPs served as a SALDI substrate to explore the chemical properties of reducing agents on the particle surfaces. The relationships between the oxidized- and reduced-forms of reducing agents were examined in detail both in the positive- or negative-ion mode. Moreover, SALDI-MS results also confirmed the tendencies observed in TEM images and UV-Vis spectra of the resulting AuNPs.

Acknowledgment This study was supported by the Czech Science Foundation P206/10/J012 and the Ministry of Education, Youth, and Sports of the Czech Republic CZ.1.05/1.1.00/02.0068, and the National Science Council of Taiwan under contracts NSC 101-2113-M-002-002-MY3 and NSC 99-2923-M-002004-MY3. I.T. is supported by Brno City Municipality Scholarships for Talented Ph.D. Students.

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