- Email: [email protected]
ionization mass spectrometry of low molecular weight compounds
Chemical Physics Letters 576 (2013) 61–64
Contents lists available at SciVerse ScienceDirect
Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett
Silver nanoparticles on zeolite surface for laser desorption/ionization mass spectrometry of low molecular weight compounds Mengrui Yang, Tatsuya Fujino ⇑ Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji-shi, Tokyo 192-0397, Japan
a r t i c l e
i n f o
Article history: Received 20 April 2013 In final form 15 May 2013 Available online 23 May 2013
a b s t r a c t Silver nanoparticles loaded on NHþ 4 -type zeolite, AgNPs–NH4ZSM5, was developed as an inorganic matrix for laser desorption/ionization mass spectrometry of low molecular weight compounds. It was found that AgNPs–NH4ZSM5 could work as an efficient Ag+ donor to ionize analytes and that zeolite worked as a heat bath to prevent the destruction of AgNPs after the photoexcitation. The AgNPs–NH4ZSM5 was applied to laser desorption/ionization mass spectrometry of biologically active substances with low molecular weights including acetylsalicylic acid, L-histidine, glucose, urea, cholesterol, and those in human serum. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Metal nanoparticles (NPs) are one of the promising materials for analytical chemistry because of their unique physicochemical properties, such as large surface areas, electrical and thermal conductivities and size-dependent light absorption. Metal NPs were also applicable to mass spectrometry, and studies using metal NPs such as Au, Ag, Pt, Cu, and Fe oxides have seen a significant thrust [1–5]. Among the several kinds of metal NPs, silver nanoparticles (AgNPs) were often used in laser desorption/ionization mass spectrometry because of their effective absorption in the UV region [6]. AgNPs enabled selective ionization of olefins such as phytoene and b-carotene [7]. Although the application of AgNPs in mass spectrometry showed great potential, direct mixing of AgNPs and an analyte still poses several problems, such as aggregation of AgNPs and low ionization efficiency. In order to improve the functionality of AgNPs, chemical modification on AgNPs was also reported. Several kinds of fatty acids in mouse liver and retinal tissues were detected using AgNPs modified with alkylcarboxylate and alkylamine [8]. The AgNPs-enhanced target was used for high-sensitive detection of low molecular weight compounds [9], and the feasibility of AgNPs on porous silicon was demonstrated [10]. Zeolites, which are widely used as catalysts and sorbents, are porous aluminosilicates whose structures consist of a threedimensional framework of the SiO4 and AlO4 tetrahedron linked by oxygen bridges. As a consequence of the isomorphous replacement of Si4+ by Al3+ in the crystal structure, negative charges on the Si–O–Al bridge sites are balanced by cations. On the surface of proton type (H+-type) zeolites, for example, there exist hydroxyl ⇑ Corresponding author. Fax: +81 42 677 2525. E-mail address: [email protected] (T. Fujino). 0009-2614/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cplett.2013.05.030
groups with strong Brönsted acidity, and they are known to be responsible for various catalytic reactions on zeolite surface. Recently, zeolites were found to be applicable to mass spectrometry of small molecules, and enhancement of the peak intensity of protonated analyte and suppression of alkali-cation adducted peaks were observed by using H+-type zeolite [11,12]. As many compounds are difficult to ionize by protons, alkali-cation-substituted zeolites were also developed for the ionization of low molecular weight compounds [13,14]. Zeolites have also been used in the present letter, but as an effective heat bath for excess vibrational energy after the photoexcitation, instead of the proton (cation) source. For this purpose, zeolitic protons on the surface was exchanged to ammonium ions to prohibit the proton (cation) supply from zeolites. Then, AgNPs were loaded on the NHþ 4 -substituted zeolite surface using infiltration methods, and thus-obtained AgNPs–NH4ZSM5 was used as an inorganic matrix for laser desorption/ionization mass spectrometry of low molecular-weight compounds. Compounds such as acetylsalicylic acid, L-histidine, glucose, urea, cholesterol, and those in human serum were clearly observed as Ag+-adducted ions on the mass spectra. In addition, the peak intensities of Ag+-adducted analytes were moderately enhanced, and peaks assignable to Na+, K+-adducted species, usually observed in conventional laser desorption/ionization mass spectrometry, were suppressed by the advantage of zeolites. 2. Experimental Silver nanoparticles (water suspension, 0.1 wt%), with sizes between 20 nm and 35 nm, were purchased from HAMAMATSU Nano Technology (Japan). Zeolites (ZSM5) were supplied by the Catalysis Society of Japan. Loading of AgNPs on zeolite surface was carried out by infiltration.
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For the preparation of AgNPs–NH4ZSM5, the NH4+-type ZSM5 zeolite (16 mg, SiO2/Al2O3 ratio = 30) and AgNPs (0.48 mg; 3 wt%) were shaken for 3 h in 4 mL of acetonitrile and water (v/v, 1:1). The mixture was then dried at 75 °C. Thus-obtained powder, AgNPs–NH4ZSM5, was suspended in a solution of acetonitrile and water (v/v, 1:1) with the assistance of a supersonic bath. All samples were dissolved in their original solutions and each sample concentration was 1 mg/mL except for cholesterol; a 0.1 mg cholesterol sample was dissolved in 1 mL of methanol. Proteins in human serum were precipitated by the addition of acetonitrile. The volume ratio of acetonitrile:human serum sample was 2:1 for the sufficient precipitation of protein. Once acetonitrile was added, the sample was shaken vigorously for 5 min. The solution was centrifuged at 3000 rpm for 10 min and the supernatant was filtered and diluted for mass analysis. For mass analysis, 0.5 lL of the sample solution was deposited on a stainless steel sample plate. This was followed by dropping 0.5 lL of AgNPs–NH4ZSM5 suspension on the same spot and mixing the mixture gently. This step was repeated again on the same spot. Then, the solvent was evaporated in air. Mass spectrometry was performed with a commercial instrument for matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS; Waters) in the positive ion and reflecting mode. The excitation laser energy was adjusted to 5 lJ at 337 nm. Mass spectra were obtained from 100 averaging in the same spot. 3. Results and discussion 3.1. Characterization of AgNPs–NH4ZSM5 Figure 1a shows the diffuse reflectance spectrum of AgNPs– NH4ZSM5. For comparison, the spectrum of NH4+-type ZSM5 (NH4ZSM5) is also displayed with dotted lines. By loading AgNPs on the zeolite surface, the absorption bands around 400–500 nm newly appeared. In contrast, NH4ZSM5 had no absorption in the visible region. The bands around 400–500 nm are considered to be surface plasmon absorption by AgNPs [15–17]. In addition, an increase in
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the absorption was observed around 200 nm increased by loading AgNPs. This observation is ascribable to the Ag+ cations on the zeolite surface [15,18]. Although AgNPs were loaded on the zeolite surface, generation of some Ag+ cations was expected because of the high catalytic property of zeolite: Brönsted acid sites remained unchanged by NH4+ substitution might be the reason for the generation of Ag+ cation. In this letter, excitation at 337 nm by N2 laser was adopted. Therefore, AgNPs are mainly photoexcited rather than Ag+ cations. The mass spectrum of AgNPs–NH4ZSM5 is shown in Figure 1b. For comparison, the mass spectrum measured only with AgNPs (without zeolite) was shown in Figure 1c. In Figure 1b, the strong peaks of [Ag]+ as well as cluster ions such as [2Ag]+ and [3Ag]+, were observed, whereas the peak intensities of nAg+ (n > 3) cluster ions were too low to be detected. These peaks, [Ag]+, [2Ag]+, and [3Ag]+ were characterized by several sets of peaks, and they were ascribed to the isotopes of Ag (107Ag and 109Ag). For example, [2Ag]+ consisted of three peaks: 107Ag + 107Ag, 107Ag + 109Ag, and 109 Ag + 109Ag. By comparison of Figure 1b with c, one of the merits, denoted as (1), of using zeolite surface (AgNPs–NH4ZSM5) was recognized as follows: The peak intensities of [Ag]+ and clusters are much higher, which indicates AgNPs–NH4ZSM5 can be a rich ion source rather than AgNPs only. After a certain delay of photoexcitation, Ag-related ions as well as charge–neutral species would be evaporated from AgNPs by vibrational excitation of the Ag lattice. In the presence of zeolite, however, it is assumed that excess evaporation, melting, and explosion of AgNPs are likely to be prevented since zeolite lattice vibrations can work as an effective heat bath [12,19]. This argument has been confirmed by a measuring diffuse reflectance spectrum of AgNPs–NH4ZSM5 before and after the laser irradiation (Figure 2a). Laser light was focused and irradiated onto AgNPs–NH4ZSM5 for 3 min with the laser power of 5 lJ. In Figure 2a, both spectra were slightly different especially around 600– 800 nm. However, the absorption due to AgNPs around 400– 500 nm was found to be unchanged, indicating the nano-sized structures of AgNPs were preserved even after the laser irradiation. In the case of AgNPs only, however, nano-sized structures were not kept by the photoexcitation, indicating AgNPs were destroyed, melted, or evaporated, and this morphological change of AgNPs
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m/z Figure 1. Diffuse reflectance spectra of (a) AgNPs–NH4ZSM5 and NH4ZSM5. Mass spectra of (b) AgNPs–NH4ZSM5 and (c) AgNPs.
Figure 2. Diffuse reflectance spectra of (a) AgNPs–NH4ZSM5 before and after the laser irradiation (337 nm, 3 min, 5 lJ). The spectrum observed before irradiation is the same as that in Figure 1(a). Images of scanning electron microscopy of AgNPs (b) before and (c) after the laser irradiation (337 nm, 3 min, 5 lJ). In (b), dotted circles indicate one of the Ag nano particles.
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was confirmed by observing the images of scanning electron microscope (SEM) (Figure 2b and c). It is noted that SEM image of AgNPs–NH4ZSM5 could not be measured because of conductive and non-conductive properties of AgNPs and zeolite, respectively. Consequently, it was understood that zeolite could be a heat bath to prevent the destruction of AgNPs, and this permitted AgNPs– NH4ZSM5 to work as efficient, long-living, and stable ion supply, leading high intensity of [Ag]+, clusters, and Ag+-adducted analytes. As for the merit (2) of using zeolite support, the peaks from [Na]+ and [K]+ can be prevented. Although we do not discuss the peaks appearing in this mass region in more detail, the mass spectrum could be simplified by the reduction of these peaks. Appearance of the peaks of [Na]+ and [K]+ could not be prevented when AgNPs only were used since intermixing of [Na]+ and [K]+ in the sample preparation could not be avoided as in the case of conventional MALDI MS. By using zeolite, however, those peaks were not observed since zeolite could also work as cation reserver [11]. As for the third metri (3), the high polarity of zeolite could be used to stabilize the complex among analyte, Ag+, and zeolite, during the desorption/ionization process as stated in our previous study [11]. This stabilization might be also affected to the intensity enhancement of Ag+-adducted analyte species. 3.2. Application of AgNPs–NH4ZSM5 to low molecular weight compounds Application of AgNPs–NH4ZSM5 was explored for laser desorption/ionization mass spectrometry of a low molecular compound, acetylsalicylic acid (ASA). Detection of ASA is difficult by the conventional MALDI MS using a typical organic matrix molecule such as 2,4,6-trihydroxyacetophenone (THAP). Figure 3a shows the mass spectrum of ASA measured with AgNPs only (without zeolite support). The total amount of ASA on the sample plate was 5.6 nmol. Peaks of Ag+-adducted ASA, [ASA + 107Ag]+ and [ASA + 109Ag]+, were observed at m/z = 287 and 289; however, these intensities were very weak. Strong peaks around m/z = 390, also observed in Figure 3a, are assignable to a complex formed between AgNPs and solvents, such as acetonitrile. By using AgNPs– NH4ZSM5, clear detection of ASA was achieved as the Ag+-adducted species. Figure 3b shows the mass spectrum of ASA obtained with AgNPs–NH4ZSM5. The total amount of ASA on the sample plate was 5.6 nmol, being equal to that in Figure 3a. The intensities of
(a) ASA/AgNPs (only)
the two characteristic peaks of [ASA + 107Ag]+ and [ASA + 109Ag]+ were moderately enhanced in comparison with Figure 3a. In addition, two peaks by [Na]+ and [K]+ were suppressed by using zeolite support. In here, AgNPs were loaded on NH4+-type ZSM5 zeolite to prevent unnecessary proton or cation supply from the zeolite surface in order to use silver ions as an ionization probe. To confirm the function of NH4+ termination of zeolite, AgNPs were loaded onto H+-type ZSM5 zeolite having the equal SiO2/ Al2O3 ratio (30) to produce AgNPs–HZSM5, and then laser desorption/ionization mass spectrometry of ASA was examined. Figure 3c shows the mass spectrum of ASA measured with AgNPs–HZSM5. No analyte-related peaks were observed. In addition, the peak intensities of [Ag]+ and cluster ions were much weaker than the results shown in Figure 3a and b. Therefore, it was confirmed that the NH4+ termination of Brönsted acid sites was important for using the silver ions as an ionization probe of low molecular weight compounds. Generation of acidic protons from Brönsted OH groups after the photoexcitation, which have the same positive charge as Ag+, is prevented in the NH4+-type zeolite. Actually, the NH4+ termination of Brönsted acid sites is sometimes used to prevent destruction of the zeolite framework by adsorbates, such as water molecules in the atmosphere, as the OH groups in H+-type zeolites have high catalytic acidity. In order to evaluate the applicability of AgNPs–NH4ZSM5, laser desorption/ionization mass spectrometry was performed for several compounds. Figure 4a shows the mass spectrum of L-histidine (L-his, 6.5 nmol). Although L-his is difficult to observe by conventional MALDI using organic matrix, the peaks of [L-his + Ag]+ were clearly observed at m/z = 262 and 264. Figure 4b shows the mass spectrum of glucose (Glu, 5.6 nmol), one of the highly polar molecules that are difficult to protonate. Figure 4b shows that Glu was ionized by Ag+ adduction; strong peaks were observed at m/z = 287 and 289, whereas the peaks of Na+- and K+-adducted Glu were not detected due to the advantage of the use of zeolite. Figure 4c and d show the mass spectra of urea (16.7 nmol) and cholesterol (Cho, 2.6 nmol), respectively. The peaks of these analytes were observed as Ag+-adducted species at m/z = 167 and
(a) L-his/AgNPs-NH4ZSM5 [L-his+Ag]+
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0 0
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0
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m/z Figure 4. Mass spectra of (a) L-histidine (L-his), (b) glucose (Glu), (c) urea, and (d) cholesterol (Cho) measured with AgNPs–ZSM5.
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M. Yang, T. Fujino / Chemical Physics Letters 576 (2013) 61–64
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m/z Figure 5. Mass spectrum of human serum sample. Peaks of (a) urea, (b) glucose (Glu), and (c) cholesterol (Cho) are enlarged in the insets.
169 for urea and m/z = 493 and 495 for Cho. The Cho, an olefin compound, was particularly difficult to measure by conventional MALDI MS. However, AgNPs–NH4ZSM5 has the capability of measuring the mass spectra of such compounds that are hardly ionized by conventional MALDI, as Ag+-adducted ions. In addition, the mass spectra were simplified since peaks of alkali metal related ions were suppressed. Finally, AgNPs–NH4ZSM5 was applied to human serum samples. The human serum sample from which high molecular-weight proteins were precipitated with acetonitrile was used for laser desorption/ionization mass spectrometry. The filtered and diluted human serum sample was mixed with AgNPs–NH4ZSM5 suspension on the sample plate. Note that human serum sample is very complicated and contains a large amount of compounds that markedly lower the sensitivity of measurements. Figure 5 shows the mass spectra of the treated human serum sample. In inset figures a–c, the mass regions of Ag+-adducted urea, Glu, and Cho were enlarged. The intensities of those peaks were weak; they were observed at m/z = 167 and 169 (urea), m/z = 287 and 289 (Glu), and m/z = 493, 495 (Cho), respectively. For Glu and Cho, the intensities of the two peaks are nearly equal, reflecting the abundance of the two isotopes of Ag. For urea, however, the peak at m/z = 167 was observed with higher intensity than the other peak at m/z = 169. It is probable that the peak at m/z = 167 is overlapped with the peaks of other species from unidentified compounds in the human serum sample having an equal mass number. Those compounds in human serum can hardly be observed by using AgNPs only, AgNPs– NH4ZSM5 has allowed us to analyze compounds with low molecular weights in the human serum clearly with the help of the zeolite surface. 4. Conclusion Silver nanoparticles (AgNPs) loaded on NH4-type ZSM5 zeolite (AgNPs–NH4ZSM5) was developed as an inorganic matrix for laser desorption/ionization mass spectrometry. It was understood that AgNPs–NH4ZSM5 could be a rich ion source rather than AgNPs only. In addition, it was also understood that zeolite could work as an efficient heat bath to prevent the destruction of AgNPs after
the photoexcitation. The AgNPs–NH4ZSM5 was applied to some biological active substances including acetylsalicylic acid, L-histidine, glucose, urea, cholesterol, and those in human serum. Acknowledgements M.Y. acknowledges an Asian Human Resources Fund (International Student Special Selection at Tokyo Metropolitan University) by the Tokyo Metropolitan Government. T.F. acknowledges a Grant-in-Aid for Scientific Research (C) (No. 24550030) from JSPS, and a Grant-in-Aid for Scientific Research on Priority Area (477) from MEXT. References [1] N. Goto-Inoue, T. Hayasaka, N. Zaima, Y. Kashiwagi, M. Yamamoto, M. Nakamoto, M. Setou, J. Am. Soc. Mass Spectrom. 21 (2010) 1940. [2] K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshida, Rapid Commun. Mass Spectrom. 2 (1988) 151. [3] M.-C. Tseng et al., J. Am. Soc. Mass Spectrom. 21 (2010) 1930. [4] T. Yao, H. Kawasaki, T. Watanabe, R. Arakawa, Int. J. Mass Spectrom. 291 (2010) 145. [5] T. Yonezawa, H. Kawasaki, A. Tarui, T. Watanabe, R. Arakawa, T. Shimada, F. Mafune, Anal. Sci. 25 (2009) 339. [6] T. Chiu, L. Chang, C. Chiang, H. Chang, J. Am. Soc. Mass Spectrom. 19 (2008) 1343. [7] S.D. Sherrod, A.J. Diaz, W.K. Russell, P.S. Cremer, D.H. Russell, Anal. Chem. 80 (2008) 6796. [8] T. Hayasaka et al., J. Am. Soc. Mass Spectrom. 21 (2010) 1446. [9] J. Niziol, W. Rode, Z. Zielin´ski, T. Ruman, Int. J. Mass Spectrom. 335 (2013) 22. [10] H. Yan, N. Xu, W.-Y. Huang, H.-M. Han, S.-J. Xiao, Int. J. Mass Spectrom. 281 (2009) 1. [11] Y. Komori, H. Shima, T. Fujino, J.N. Kondo, K. Hashimoto, T. Korenaga, J. Phys. Chem. C 114 (2010) 1593. [12] R. Yamamoto, T. Fujino, Chem. Phys. Lett. 543 (2012) 76. [13] J. Suzuki et al., Chem. Phys. Lett. 546 (2012) 159. [14] J. Suzuki, T. Fujino, Anal. Sci. 28 (2012) 901. [15] N.S. Flores-López, J. Castro-Rosas, R. Ramírez-Bon, A. Mendoza-Córdova, E. Larios-Rodríguez, M. Flores-Acosta, J. Mol. Struct. 1028 (2012) 110. [16] K.-C. Lee, S.-J. Lin, C.-H. Lin, C.-S. Tsai, Y.-J. Lu, Surf. Coat. Technol. 202 (2008) 5339. [17] W.S. Szulbinski, Inorg. Chim. Acta 266 (1998) 253. [18] L.B. Gulina, G. Korotcenkov, B.K. Cho, S.H. Han, V.P. Tolstoy, J. Mater. Sci. 46 (2011) 4555. [19] T. Fujino et al., J. Chem. Phys. 105 (1996) 279.
Contents lists available at SciVerse ScienceDirect
Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett
Silver nanoparticles on zeolite surface for laser desorption/ionization mass spectrometry of low molecular weight compounds Mengrui Yang, Tatsuya Fujino ⇑ Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji-shi, Tokyo 192-0397, Japan
a r t i c l e
i n f o
Article history: Received 20 April 2013 In final form 15 May 2013 Available online 23 May 2013
a b s t r a c t Silver nanoparticles loaded on NHþ 4 -type zeolite, AgNPs–NH4ZSM5, was developed as an inorganic matrix for laser desorption/ionization mass spectrometry of low molecular weight compounds. It was found that AgNPs–NH4ZSM5 could work as an efficient Ag+ donor to ionize analytes and that zeolite worked as a heat bath to prevent the destruction of AgNPs after the photoexcitation. The AgNPs–NH4ZSM5 was applied to laser desorption/ionization mass spectrometry of biologically active substances with low molecular weights including acetylsalicylic acid, L-histidine, glucose, urea, cholesterol, and those in human serum. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Metal nanoparticles (NPs) are one of the promising materials for analytical chemistry because of their unique physicochemical properties, such as large surface areas, electrical and thermal conductivities and size-dependent light absorption. Metal NPs were also applicable to mass spectrometry, and studies using metal NPs such as Au, Ag, Pt, Cu, and Fe oxides have seen a significant thrust [1–5]. Among the several kinds of metal NPs, silver nanoparticles (AgNPs) were often used in laser desorption/ionization mass spectrometry because of their effective absorption in the UV region [6]. AgNPs enabled selective ionization of olefins such as phytoene and b-carotene [7]. Although the application of AgNPs in mass spectrometry showed great potential, direct mixing of AgNPs and an analyte still poses several problems, such as aggregation of AgNPs and low ionization efficiency. In order to improve the functionality of AgNPs, chemical modification on AgNPs was also reported. Several kinds of fatty acids in mouse liver and retinal tissues were detected using AgNPs modified with alkylcarboxylate and alkylamine [8]. The AgNPs-enhanced target was used for high-sensitive detection of low molecular weight compounds [9], and the feasibility of AgNPs on porous silicon was demonstrated [10]. Zeolites, which are widely used as catalysts and sorbents, are porous aluminosilicates whose structures consist of a threedimensional framework of the SiO4 and AlO4 tetrahedron linked by oxygen bridges. As a consequence of the isomorphous replacement of Si4+ by Al3+ in the crystal structure, negative charges on the Si–O–Al bridge sites are balanced by cations. On the surface of proton type (H+-type) zeolites, for example, there exist hydroxyl ⇑ Corresponding author. Fax: +81 42 677 2525. E-mail address: [email protected] (T. Fujino). 0009-2614/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cplett.2013.05.030
groups with strong Brönsted acidity, and they are known to be responsible for various catalytic reactions on zeolite surface. Recently, zeolites were found to be applicable to mass spectrometry of small molecules, and enhancement of the peak intensity of protonated analyte and suppression of alkali-cation adducted peaks were observed by using H+-type zeolite [11,12]. As many compounds are difficult to ionize by protons, alkali-cation-substituted zeolites were also developed for the ionization of low molecular weight compounds [13,14]. Zeolites have also been used in the present letter, but as an effective heat bath for excess vibrational energy after the photoexcitation, instead of the proton (cation) source. For this purpose, zeolitic protons on the surface was exchanged to ammonium ions to prohibit the proton (cation) supply from zeolites. Then, AgNPs were loaded on the NHþ 4 -substituted zeolite surface using infiltration methods, and thus-obtained AgNPs–NH4ZSM5 was used as an inorganic matrix for laser desorption/ionization mass spectrometry of low molecular-weight compounds. Compounds such as acetylsalicylic acid, L-histidine, glucose, urea, cholesterol, and those in human serum were clearly observed as Ag+-adducted ions on the mass spectra. In addition, the peak intensities of Ag+-adducted analytes were moderately enhanced, and peaks assignable to Na+, K+-adducted species, usually observed in conventional laser desorption/ionization mass spectrometry, were suppressed by the advantage of zeolites. 2. Experimental Silver nanoparticles (water suspension, 0.1 wt%), with sizes between 20 nm and 35 nm, were purchased from HAMAMATSU Nano Technology (Japan). Zeolites (ZSM5) were supplied by the Catalysis Society of Japan. Loading of AgNPs on zeolite surface was carried out by infiltration.
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For the preparation of AgNPs–NH4ZSM5, the NH4+-type ZSM5 zeolite (16 mg, SiO2/Al2O3 ratio = 30) and AgNPs (0.48 mg; 3 wt%) were shaken for 3 h in 4 mL of acetonitrile and water (v/v, 1:1). The mixture was then dried at 75 °C. Thus-obtained powder, AgNPs–NH4ZSM5, was suspended in a solution of acetonitrile and water (v/v, 1:1) with the assistance of a supersonic bath. All samples were dissolved in their original solutions and each sample concentration was 1 mg/mL except for cholesterol; a 0.1 mg cholesterol sample was dissolved in 1 mL of methanol. Proteins in human serum were precipitated by the addition of acetonitrile. The volume ratio of acetonitrile:human serum sample was 2:1 for the sufficient precipitation of protein. Once acetonitrile was added, the sample was shaken vigorously for 5 min. The solution was centrifuged at 3000 rpm for 10 min and the supernatant was filtered and diluted for mass analysis. For mass analysis, 0.5 lL of the sample solution was deposited on a stainless steel sample plate. This was followed by dropping 0.5 lL of AgNPs–NH4ZSM5 suspension on the same spot and mixing the mixture gently. This step was repeated again on the same spot. Then, the solvent was evaporated in air. Mass spectrometry was performed with a commercial instrument for matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS; Waters) in the positive ion and reflecting mode. The excitation laser energy was adjusted to 5 lJ at 337 nm. Mass spectra were obtained from 100 averaging in the same spot. 3. Results and discussion 3.1. Characterization of AgNPs–NH4ZSM5 Figure 1a shows the diffuse reflectance spectrum of AgNPs– NH4ZSM5. For comparison, the spectrum of NH4+-type ZSM5 (NH4ZSM5) is also displayed with dotted lines. By loading AgNPs on the zeolite surface, the absorption bands around 400–500 nm newly appeared. In contrast, NH4ZSM5 had no absorption in the visible region. The bands around 400–500 nm are considered to be surface plasmon absorption by AgNPs [15–17]. In addition, an increase in
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NH4ZSM5 200
300
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500
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reflectance (KM)
1.0
the absorption was observed around 200 nm increased by loading AgNPs. This observation is ascribable to the Ag+ cations on the zeolite surface [15,18]. Although AgNPs were loaded on the zeolite surface, generation of some Ag+ cations was expected because of the high catalytic property of zeolite: Brönsted acid sites remained unchanged by NH4+ substitution might be the reason for the generation of Ag+ cation. In this letter, excitation at 337 nm by N2 laser was adopted. Therefore, AgNPs are mainly photoexcited rather than Ag+ cations. The mass spectrum of AgNPs–NH4ZSM5 is shown in Figure 1b. For comparison, the mass spectrum measured only with AgNPs (without zeolite) was shown in Figure 1c. In Figure 1b, the strong peaks of [Ag]+ as well as cluster ions such as [2Ag]+ and [3Ag]+, were observed, whereas the peak intensities of nAg+ (n > 3) cluster ions were too low to be detected. These peaks, [Ag]+, [2Ag]+, and [3Ag]+ were characterized by several sets of peaks, and they were ascribed to the isotopes of Ag (107Ag and 109Ag). For example, [2Ag]+ consisted of three peaks: 107Ag + 107Ag, 107Ag + 109Ag, and 109 Ag + 109Ag. By comparison of Figure 1b with c, one of the merits, denoted as (1), of using zeolite surface (AgNPs–NH4ZSM5) was recognized as follows: The peak intensities of [Ag]+ and clusters are much higher, which indicates AgNPs–NH4ZSM5 can be a rich ion source rather than AgNPs only. After a certain delay of photoexcitation, Ag-related ions as well as charge–neutral species would be evaporated from AgNPs by vibrational excitation of the Ag lattice. In the presence of zeolite, however, it is assumed that excess evaporation, melting, and explosion of AgNPs are likely to be prevented since zeolite lattice vibrations can work as an effective heat bath [12,19]. This argument has been confirmed by a measuring diffuse reflectance spectrum of AgNPs–NH4ZSM5 before and after the laser irradiation (Figure 2a). Laser light was focused and irradiated onto AgNPs–NH4ZSM5 for 3 min with the laser power of 5 lJ. In Figure 2a, both spectra were slightly different especially around 600– 800 nm. However, the absorption due to AgNPs around 400– 500 nm was found to be unchanged, indicating the nano-sized structures of AgNPs were preserved even after the laser irradiation. In the case of AgNPs only, however, nano-sized structures were not kept by the photoexcitation, indicating AgNPs were destroyed, melted, or evaporated, and this morphological change of AgNPs
0.06
AgNPs-NH4ZSM5 after irradiation
0.04
wavelength (nm)
before irradiation 0.02 300
(b) AgNPs-NH4ZSM5 [Ag]
[2Ag]+
30
10
500
600
700
800
wavelength (nm) (b) AgNPs before irradiation
40
20
400
+
250 nm
[3Ag]+
(c) AgNPs after irradiation 250 nm
(c) AgNPs (only) [Na]+
intensity (arb. Unit)
50
[K]+
0 100
200
300
400
m/z Figure 1. Diffuse reflectance spectra of (a) AgNPs–NH4ZSM5 and NH4ZSM5. Mass spectra of (b) AgNPs–NH4ZSM5 and (c) AgNPs.
Figure 2. Diffuse reflectance spectra of (a) AgNPs–NH4ZSM5 before and after the laser irradiation (337 nm, 3 min, 5 lJ). The spectrum observed before irradiation is the same as that in Figure 1(a). Images of scanning electron microscopy of AgNPs (b) before and (c) after the laser irradiation (337 nm, 3 min, 5 lJ). In (b), dotted circles indicate one of the Ag nano particles.
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M. Yang, T. Fujino / Chemical Physics Letters 576 (2013) 61–64
was confirmed by observing the images of scanning electron microscope (SEM) (Figure 2b and c). It is noted that SEM image of AgNPs–NH4ZSM5 could not be measured because of conductive and non-conductive properties of AgNPs and zeolite, respectively. Consequently, it was understood that zeolite could be a heat bath to prevent the destruction of AgNPs, and this permitted AgNPs– NH4ZSM5 to work as efficient, long-living, and stable ion supply, leading high intensity of [Ag]+, clusters, and Ag+-adducted analytes. As for the merit (2) of using zeolite support, the peaks from [Na]+ and [K]+ can be prevented. Although we do not discuss the peaks appearing in this mass region in more detail, the mass spectrum could be simplified by the reduction of these peaks. Appearance of the peaks of [Na]+ and [K]+ could not be prevented when AgNPs only were used since intermixing of [Na]+ and [K]+ in the sample preparation could not be avoided as in the case of conventional MALDI MS. By using zeolite, however, those peaks were not observed since zeolite could also work as cation reserver [11]. As for the third metri (3), the high polarity of zeolite could be used to stabilize the complex among analyte, Ag+, and zeolite, during the desorption/ionization process as stated in our previous study [11]. This stabilization might be also affected to the intensity enhancement of Ag+-adducted analyte species. 3.2. Application of AgNPs–NH4ZSM5 to low molecular weight compounds Application of AgNPs–NH4ZSM5 was explored for laser desorption/ionization mass spectrometry of a low molecular compound, acetylsalicylic acid (ASA). Detection of ASA is difficult by the conventional MALDI MS using a typical organic matrix molecule such as 2,4,6-trihydroxyacetophenone (THAP). Figure 3a shows the mass spectrum of ASA measured with AgNPs only (without zeolite support). The total amount of ASA on the sample plate was 5.6 nmol. Peaks of Ag+-adducted ASA, [ASA + 107Ag]+ and [ASA + 109Ag]+, were observed at m/z = 287 and 289; however, these intensities were very weak. Strong peaks around m/z = 390, also observed in Figure 3a, are assignable to a complex formed between AgNPs and solvents, such as acetonitrile. By using AgNPs– NH4ZSM5, clear detection of ASA was achieved as the Ag+-adducted species. Figure 3b shows the mass spectrum of ASA obtained with AgNPs–NH4ZSM5. The total amount of ASA on the sample plate was 5.6 nmol, being equal to that in Figure 3a. The intensities of
(a) ASA/AgNPs (only)
the two characteristic peaks of [ASA + 107Ag]+ and [ASA + 109Ag]+ were moderately enhanced in comparison with Figure 3a. In addition, two peaks by [Na]+ and [K]+ were suppressed by using zeolite support. In here, AgNPs were loaded on NH4+-type ZSM5 zeolite to prevent unnecessary proton or cation supply from the zeolite surface in order to use silver ions as an ionization probe. To confirm the function of NH4+ termination of zeolite, AgNPs were loaded onto H+-type ZSM5 zeolite having the equal SiO2/ Al2O3 ratio (30) to produce AgNPs–HZSM5, and then laser desorption/ionization mass spectrometry of ASA was examined. Figure 3c shows the mass spectrum of ASA measured with AgNPs–HZSM5. No analyte-related peaks were observed. In addition, the peak intensities of [Ag]+ and cluster ions were much weaker than the results shown in Figure 3a and b. Therefore, it was confirmed that the NH4+ termination of Brönsted acid sites was important for using the silver ions as an ionization probe of low molecular weight compounds. Generation of acidic protons from Brönsted OH groups after the photoexcitation, which have the same positive charge as Ag+, is prevented in the NH4+-type zeolite. Actually, the NH4+ termination of Brönsted acid sites is sometimes used to prevent destruction of the zeolite framework by adsorbates, such as water molecules in the atmosphere, as the OH groups in H+-type zeolites have high catalytic acidity. In order to evaluate the applicability of AgNPs–NH4ZSM5, laser desorption/ionization mass spectrometry was performed for several compounds. Figure 4a shows the mass spectrum of L-histidine (L-his, 6.5 nmol). Although L-his is difficult to observe by conventional MALDI using organic matrix, the peaks of [L-his + Ag]+ were clearly observed at m/z = 262 and 264. Figure 4b shows the mass spectrum of glucose (Glu, 5.6 nmol), one of the highly polar molecules that are difficult to protonate. Figure 4b shows that Glu was ionized by Ag+ adduction; strong peaks were observed at m/z = 287 and 289, whereas the peaks of Na+- and K+-adducted Glu were not detected due to the advantage of the use of zeolite. Figure 4c and d show the mass spectra of urea (16.7 nmol) and cholesterol (Cho, 2.6 nmol), respectively. The peaks of these analytes were observed as Ag+-adducted species at m/z = 167 and
(a) L-his/AgNPs-NH4ZSM5 [L-his+Ag]+
60
(b) Glu/AgNPs-NH4ZSM5
50
[solvent+Ag]+
+
intensity (arb. Unit)
[Ag]
30
intensity (arb. Unit)
[2Ag]+
[ASA+Ag]+ [3Ag]+
[Na]+ [K]+
20 (b) ASA/AgNPs-NH4ZSM5
[Glu+Ag]+
40
30 (c) urea/AgNPs-NH4ZSM5 [urea+Ag]+
20
[ASA+Ag]+
10
(d) Cho/AgNPs-NH4ZSM5
10
[Cho+Ag]+
(c) ASA/AgNPs-HZSM5
0
0 0
100
200
300
400
m/z Figure 3. Mass spectra of acetylsalicylic acid (ASA) measured with (a) AgNPs (only), (b) AgNPs–NH4ZSM5 and (c) AgNPs–HZSM5.
0
100
200
300
400
500
m/z Figure 4. Mass spectra of (a) L-histidine (L-his), (b) glucose (Glu), (c) urea, and (d) cholesterol (Cho) measured with AgNPs–ZSM5.
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M. Yang, T. Fujino / Chemical Physics Letters 576 (2013) 61–64
20
(a)
(b)
[urea+107Ag]+ + isobar
40
[Glu+107Ag]+
20
10 20
intensity (arb. Unit)
[Cho+107Ag]+ [Cho+109Ag]+
10
[urea+109Ag]+
0
0 166
200
(c)
[Glu+109Ag]+
167
168
169
170
0 284
286
288
290
292
492
493
494
495
496
497
150 100
[urea+Ag]+
50
[Glu+Ag]+
[Cho+Ag]+
0 0
100
200
300
400
500
m/z Figure 5. Mass spectrum of human serum sample. Peaks of (a) urea, (b) glucose (Glu), and (c) cholesterol (Cho) are enlarged in the insets.
169 for urea and m/z = 493 and 495 for Cho. The Cho, an olefin compound, was particularly difficult to measure by conventional MALDI MS. However, AgNPs–NH4ZSM5 has the capability of measuring the mass spectra of such compounds that are hardly ionized by conventional MALDI, as Ag+-adducted ions. In addition, the mass spectra were simplified since peaks of alkali metal related ions were suppressed. Finally, AgNPs–NH4ZSM5 was applied to human serum samples. The human serum sample from which high molecular-weight proteins were precipitated with acetonitrile was used for laser desorption/ionization mass spectrometry. The filtered and diluted human serum sample was mixed with AgNPs–NH4ZSM5 suspension on the sample plate. Note that human serum sample is very complicated and contains a large amount of compounds that markedly lower the sensitivity of measurements. Figure 5 shows the mass spectra of the treated human serum sample. In inset figures a–c, the mass regions of Ag+-adducted urea, Glu, and Cho were enlarged. The intensities of those peaks were weak; they were observed at m/z = 167 and 169 (urea), m/z = 287 and 289 (Glu), and m/z = 493, 495 (Cho), respectively. For Glu and Cho, the intensities of the two peaks are nearly equal, reflecting the abundance of the two isotopes of Ag. For urea, however, the peak at m/z = 167 was observed with higher intensity than the other peak at m/z = 169. It is probable that the peak at m/z = 167 is overlapped with the peaks of other species from unidentified compounds in the human serum sample having an equal mass number. Those compounds in human serum can hardly be observed by using AgNPs only, AgNPs– NH4ZSM5 has allowed us to analyze compounds with low molecular weights in the human serum clearly with the help of the zeolite surface. 4. Conclusion Silver nanoparticles (AgNPs) loaded on NH4-type ZSM5 zeolite (AgNPs–NH4ZSM5) was developed as an inorganic matrix for laser desorption/ionization mass spectrometry. It was understood that AgNPs–NH4ZSM5 could be a rich ion source rather than AgNPs only. In addition, it was also understood that zeolite could work as an efficient heat bath to prevent the destruction of AgNPs after
the photoexcitation. The AgNPs–NH4ZSM5 was applied to some biological active substances including acetylsalicylic acid, L-histidine, glucose, urea, cholesterol, and those in human serum. Acknowledgements M.Y. acknowledges an Asian Human Resources Fund (International Student Special Selection at Tokyo Metropolitan University) by the Tokyo Metropolitan Government. T.F. acknowledges a Grant-in-Aid for Scientific Research (C) (No. 24550030) from JSPS, and a Grant-in-Aid for Scientific Research on Priority Area (477) from MEXT. References [1] N. Goto-Inoue, T. Hayasaka, N. Zaima, Y. Kashiwagi, M. Yamamoto, M. Nakamoto, M. Setou, J. Am. Soc. Mass Spectrom. 21 (2010) 1940. [2] K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshida, Rapid Commun. Mass Spectrom. 2 (1988) 151. [3] M.-C. Tseng et al., J. Am. Soc. Mass Spectrom. 21 (2010) 1930. [4] T. Yao, H. Kawasaki, T. Watanabe, R. Arakawa, Int. J. Mass Spectrom. 291 (2010) 145. [5] T. Yonezawa, H. Kawasaki, A. Tarui, T. Watanabe, R. Arakawa, T. Shimada, F. Mafune, Anal. Sci. 25 (2009) 339. [6] T. Chiu, L. Chang, C. Chiang, H. Chang, J. Am. Soc. Mass Spectrom. 19 (2008) 1343. [7] S.D. Sherrod, A.J. Diaz, W.K. Russell, P.S. Cremer, D.H. Russell, Anal. Chem. 80 (2008) 6796. [8] T. Hayasaka et al., J. Am. Soc. Mass Spectrom. 21 (2010) 1446. [9] J. Niziol, W. Rode, Z. Zielin´ski, T. Ruman, Int. J. Mass Spectrom. 335 (2013) 22. [10] H. Yan, N. Xu, W.-Y. Huang, H.-M. Han, S.-J. Xiao, Int. J. Mass Spectrom. 281 (2009) 1. [11] Y. Komori, H. Shima, T. Fujino, J.N. Kondo, K. Hashimoto, T. Korenaga, J. Phys. Chem. C 114 (2010) 1593. [12] R. Yamamoto, T. Fujino, Chem. Phys. Lett. 543 (2012) 76. [13] J. Suzuki et al., Chem. Phys. Lett. 546 (2012) 159. [14] J. Suzuki, T. Fujino, Anal. Sci. 28 (2012) 901. [15] N.S. Flores-López, J. Castro-Rosas, R. Ramírez-Bon, A. Mendoza-Córdova, E. Larios-Rodríguez, M. Flores-Acosta, J. Mol. Struct. 1028 (2012) 110. [16] K.-C. Lee, S.-J. Lin, C.-H. Lin, C.-S. Tsai, Y.-J. Lu, Surf. Coat. Technol. 202 (2008) 5339. [17] W.S. Szulbinski, Inorg. Chim. Acta 266 (1998) 253. [18] L.B. Gulina, G. Korotcenkov, B.K. Cho, S.H. Han, V.P. Tolstoy, J. Mater. Sci. 46 (2011) 4555. [19] T. Fujino et al., J. Chem. Phys. 105 (1996) 279.