Comparison of ZnS Semiconductor Nanoparticles Capped with

munological assays,24 capillary electrophoresis,25 and liquid chro- matography tandem mass spectrometry (LC-MS/MS).26 Ubiq- uitin is a small protein t...
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Anal. Chem. 2008, 80, 9681–9688

Comparison of ZnS Semiconductor Nanoparticles Capped with Various Functional Groups as the Matrix and Affinity Probes for Rapid Analysis of Cyclodextrins and Proteins in Surface-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Suresh Kumar Kailasa, Kamatam Kiran, and Hui-Fen Wu* Department of Chemistry, National Sun Yat-Sen University, Kaohsiung 80424, Taiwan, and Center for Nanoscience and Nanotechnology, National Sun Yat-Sen University, Kaohsiung, 80424, Taiwan Zinc sulfide (ZnS) semiconductor nanoparticles (NPs) capped with a variety of functional groups including bare ZnS NPs, 3-mercaptopropanoic acid (ZnS-3-MPA), sodium citrate (ZnS-citrate), cysteamine (ZnS-Cys), and 2-mercaptoethane sulfonate (ZnS-2-MES) have been investigated as the matrix and affinity probes for analysis of r-, β-, and γ-cyclodextrins (CDs), ubiquitin, and insulin in biological samples by using surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF-MS). Various parameters that would influence the ionization efficiency and sensitivity of these ZnS NPs in SALDI-TOF-MS were examined including the effect of capping agents, sample pH, ion abundance, and concentration of ZnS NPs. Among these ZnS NPs, our results have demonstrated that ZnS-3-MPA exhibited the highest efficiency toward CDs, ubiquitin, and insulin for high-sensitivity detection in SALDI-TOF-MS. The detection limits were 20-55 nM for CDs, 91 nM for ubiquitin, and 85 nM for insulin. The applicability of the present method is demonstrated by detection of ubiquitin-like proteins in oyster mushroom and also in the analysis of analytes in biological samples such as human urine and plasma. To our best knowledge, this is the first time semiconductor NPs were used as the matrix and affinity probes for highsensitivity detection of organic and biomolecules in SALDITOF-MS. This approach exhibits the advantages of being simple, rapid, efficient, and straightforward for direct analysis of organic and biological samples in SALDI-TOFMS without the need for time-consuming separation processes, tedious washing steps, or further laborious purification. In addition, it also can provide a sensitive and reliable quantitative assay for small- and largemolecule analysis with the detectable mass up to 8500 Da. We believe that this novel ZnS nanoprobe is simple, efficient, lower cost (compared with Au, Ag, and Pt NPs), fast, and with the potential for high-throughput analysis in SALDI-TOF-MS. * Corresponding author. Phone: 886-7-5252000-3955. Fax: 886-7-525-3908. E-mail: [email protected]. 10.1021/ac8015664 CCC: $40.75  2008 American Chemical Society Published on Web 11/08/2008

Nanotechnology provides an opportunity to establish novel, sensitive, and selective methods by means of intensive research and the effective transfer of research findings into innovative products. Recent years, scientists have paid much attention on the development of novel and dynamic nanoparticles and their applications in interdisciplinary science.1-4 The bond formation of specific molecules onto the surfaces of the nanoparticles can cause a change in photophysical, photochemical, photoelectronic, and photocatalytic properties. These changes can provide strong interactions and increase the binding efficiency of nanoparticles with the target molecules. The absorption of biomolecules onto the surfaces of the nanoparticles can produce significant data, and these efforts have also contributed to the contact angle of liquids on solids increasing more interests in the field of nanobiotechnology and bioanalytical chemistry.4-8 Recently, more researchers have focused their attention on applications of semiconductor nanoparticles for biological application.9 The use of water-soluble zinc sulfide (ZnS) nanoparticles (NPs) coated with different capping agents9 can permit greater affinity to bind or interact with biotarget molecules, and it also can increase selectivity and sensitivity for the detection of biomolecules.10 Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is an important and powerful tool for the analysis of sample compositions, particularly complex of biological materials. It is a rapid, selective, and sensitive analytical (1) Yang, H.; Santra, S.; Holloway, P. H. J. Nanosci. Nanotechnol. 2005, 5, 1364–1375. (2) Stroh, M.; Zimmer, J. P.; Duda, D. G.; Levchenko, T. S.; Cohen, K. S.; Brown, E. B.; Scadden, D. T.; Torchilin, V. P.; Bawendi, M. G.; Fukumura, D.; Jain, K. R. Nat. Med. 2005, 11, 678–682. (3) Schuerenberg, M.; Luebbert, C.; Eickhoff, H.; Kalkum, M.; Lehrach, H.; Nordhoff, E. Anal. Chem. 2000, 72, 3436–3442. (4) Han, M. Y.; Gao, X. H.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631– 635. (5) Horbett, T. A. Adv. Chem. Ser. 1982, 199, 233–244. (6) Andrade, J. D.; Hlady, V. Adv. Polym. Sci. 1986, 79, 1–63. (7) Andrade, J. D. Surface and Interfacial Aspects of Biomedical Polymers; Plenum Press: New York, 1985; Vol. 1, pp 470-485. (8) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267–340. (9) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016–2018. (10) Josephson, L.; Tung, C. H.; Moore, A.; Weissleder, T. Bioconjugate Chem. 1999, 10, 186–191.

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tool for the characterization of biomolecules.11 It also can provide mass and structural information of compounds in multidisciplinary science. However, in the MALDI-MS analysis, matrix selection plays an important role for the analysis of target molecules because it serves both as laser energy absorbent and energy transport medium. To date, a number of matrixes have been introduced, and these matrixes possess their own characteristics with regard to mass of the analyte molecules and improvement of the signal intensity. However, small-molecule analysis is limited in MALDIMS due to the low-mass interferences from the organic matrix. Thus, inorganic materials were introduced as the matrix in order to reduce this problem in the MALDI-MS analysis. Sunner et al. applied graphite particles (2-150 µm) mixed with glycerol as the matrix for the analysis of proteins and peptides. This technique is named surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS).12 In recent years, the applications of nanoparticles to bind or interact with biomolecules and their analysis by surface-assisted laser desorption/ionization time-offlight mass spectrometry (SALDI-TOF-MS) have been intensively investigated.12-17 These approaches provide better understanding of the affinity and capability of binding efficiency of NPs toward target biomolecules. Since 2005, we have been focused on developing novel microextraction methods in applying various NPs such as Au,18 SiO2,19 and Ag20,21 as affinity probes for rapid analysis of peptides and proteins by using AP-MALDI ion trap MS18-21 and MALDI-TOF-MS.19,20 The detection principle was based on the electrostatic or hydrophobic interactions of NPs with the target molecules. In the present work, the feasibility of the ZnS NPs for the analysis of cyclodextrins (CDs), ubiquitin, and insulin have been successfully demonstrated using SALDI-TOFMS. CDs comprise a family of cyclic oligosaccharides, and these are known as R-, β-, and γ-cyclodextrin according to anhydroglucose units (6, 7, and 8) with molecular weights 972, 1135, and 1297, respectively (Supporting Information Figure S1). The structures of CDs exhibit a truncated cone shape of tubular configuration containing a hydrophobic cavity interiors and hydrophilic cavity exteriors. These characteristics are responsible for their solubility and excellent ability to form inclusion complexes in aqueous media, and these features account for their significant applications in pharmaceutical and food materials.22,23 Insulin is a peptide hormone consisting of 51 amino acid residues with a molecular mass of 5807 Da. It is secreted by the pancreas that controls the level of the sugar glucose in the blood. It prevents (11) Pan, C.; Xu, S.; Zhou, H.; Fu, Y.; Ye, M.; Zou, H. Anal. Bioanal. Chem. 2007, 387, 193–204. (12) Sunner, J.; Dratz, E.; Chen, Y. C. Anal. Chem. 1995, 67, 4335–4341. (13) Teng, C. H.; Ho, K. C.; Lin, Y. S.; Chen, Y. C. Anal. Chem. 2004, 76, 4337– 4342. (14) Huang, Y. F.; Chang, H. T. Anal. Chem. 2006, 78, 1485–1493. (15) Su, C. L.; Tseng, W. L. Anal. Chem. 2007, 79, 1626–1633. (16) Wen, X.; Dagan, S.; Wysocki, V. H. Anal. Chem. 2007, 79, 434–444. (17) Vanderpuije, B. N. Y.; Han, G.; Rotello, V. M.; Vachet, R. W. Anal. Chem. 2006, 78, 5491–5496. (18) Sudhir, P.-R.; Wu, H. F.; Zhou, Z. C. Anal. Chem. 2005, 77, 7380–7385. (19) Agrawal, K.; Wu, H. F. Rapid Commun. Mass Spectrom. 2008, 22, 283– 290. (20) Shrivas, K.; Wu, H. F. Anal. Chem. 2008, 80, 2583–2589. (21) Shrivas, K.; Wu, H. F. Rapid Commun. Mass Spectrom. 2008, 22, 2863– 2872. (22) Liftsson, T.; Duchene, D. Int. J. Pharm. 2007, 329, 1–11. (23) Shaw, P. J. Food Sci. 1983, 48, 646–647.

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or reduces the long-term problems of diabetes, because most cells of the body have insulin receptors which attach the insulin to the cell surface. The attached insulin cell surface activated the other receptors that absorb glucose from the blood stream into the cells such as muscle cells, red blood cells, and fat cells. Hence, insulin performs its most important role for controlling glucose levels in blood. Insulin was quantitatively measured by employing immunological assays,24 capillary electrophoresis,25 and liquid chromatography tandem mass spectrometry (LC-MS/MS).26 Ubiquitin is a small protein that occurs in all eukaryotic cells, and it main function is to mark other proteins for proteolysis. It consists of 76 amino acids and has a molecular mass of about 8.5 kDa. Ubiquitin and ubiquitin-like proteins activities are the most important to control cell-cycle progression, signal transcriptional regulation, receptor down-regulation, immune response, endocytosis, and apoptosis.27,28 Wang and Ng isolated a ubiquitin-like protein with translation-inhibiting, ribonuclease and anti-HIV-1 reverse transcriptase activities from oyster mushroom (Pleurotus ostreatus).29 Hence, it is essential to isolate and detect ubiquitin and ubiquitin-like proteins in mushroom. In this study, the feasibility of the various ZnS NPs including bare ZnS NPs, 3-mercaptopropanoic acid (ZnS-3-MPA), sodium citrate (ZnScitrate), cysteamine (ZnS-Cys), and 2-mercaptoethane sulfonate (ZnS-2-MES) have been examined and compared as the SALDI matrix and affinity probes for the analysis of R-, β-, and γ-CDs, ubiquitin, and insulin in aqueous solutions and biological samples and ubiquitin and ubiquitin-like proteins in oyster mushroom by SALDI-TOF-MS. EXPERIMENTAL SECTION Materials and Reagents. R-, β-, and γ-cyclodextrins, ubiquitin, insulin, ZnCl2, sinapic acid, sodium citrate, cysteamine hydrochloride, Tris-HCl (pH 7.2), and 2-mercaptoethane sulfonate were purchased from Sigma-Aldrich (St. Louis, MO). Sodium sulfide was obtained from Nihon Shiyaku Industries Ltd., Japan. 3-Mercaptopropanoic acid was purchased from Fluka (Steinheime, Germany). Milli-Q ultrapure water was used for all the experiments. All chemicals used were of analytical grade. UV-Vis Detection and the Measurement of Sample pH. A double-beam UV-vis spectrophotometer (Hitachi, Japan) was used to measure the absorbance of the aqueous ZnS NPs. The sample pH was measured by using a digital pH meter (Istek Model 720P, South Korea). Synthesis of Various ZnS NPs. Bare ZnS NPs. ZnS semiconductor colloidal NPs were synthesized according to the reported method.30 The synthesis was carried out in a 250 mL three-necked round-bottom flask. A volume of 30 mL of deionized water was transferred into the round-bottom flask and stirred well to remove excess of oxygen present in the water under N2 (24) Duckworth, W. C.; Bennett, R. G.; Hamel, F. G. Endocr. Rev. 1998, 19, 608–624. (25) Shihabi, Z. K.; Friedberg, M. J. Chromatogr., A 1998, 807, 129–133. (26) Thevis, M.; Thomas, A.; Delahaut, P.; Bosseloir, A.; Scha1nzer, W. Anal. Chem. 2006, 78, 1897–1903. (27) Hershko, A. The ubiquitin system. In Ubiquitin and the Biology of the Cell; Peter, A., Ed.; Plenum Press: New York, 1998; pp 1-17. (28) Ng, T. B.; Lam, S. K.; Chan, S. Y. Peptides 2002, 23, 1361–1365. (29) Wang, H. X.; Ng, T. B. Biochem. Biophys. Res. Commun. 2000, 276, 587– 593. (30) Mu, J.; Gu, D.; Xu, Z. Appl. Phys. A: Mater. Sci. Process. 2005, 80, 1425– 1429.

atmosphere for 15 min. To this, 5 mL of 0.1 M zinc chloride solution was added to the above solution. After that, 5 mL of 0.1 M sodium sulfide was added dropwise slowly into the flask. ZnS semiconductor NPs were collected, and this NPs solution was kept under ultrasonication for 30 min. ZnS NPs Capped with 3-Mercaptopropionic Acid. ZnS NPs capped with 3-mercaptopropionic acid (ZnS-3-MPA) were prepared according to literature.31 Briefly, 30 mL of deionized was taken in a 250 mL three-necked round-bottom flask and through it was passed N2 gas while stirring for 15 min to remove excess oxygen in water. To this, 4 mL of 0.1 M ZnCl2 solution was added, and the mixture was stirred several minutes. Then 4 mL of 0.1 M sodium sulfide solution was added, and the mixture was stirred for 10 min. Finally, 4 mL of 1 M 3-mercaptopropionic acid (3-MPA) solution was added while stirring in a N2 atmosphere for 45 min. After 1 h, a milky white-colored solution was formed. This indicated that the formation of aqueous ZnS NPs capped with 3-MPA was achieved. The above solution was transferred into a 100 mL beaker, ultrasonicated for 30 min, and finally stored the NPs solution at 4 °C. ZnS NPs Capped with Citrate. We removed excess oxygen in a reaction flask by the described procedure. To this flask, 0.1 M ZnCl2 solution was added, and the mixture was stirred for 15 min. A 0.01 M solution of sodium citrate was added, and then a 0.1 M solution of sodium sulfide was added while stirring under N2 atmosphere for 1 h. The freshly prepared ZnS NPs were ultrasonicated for 30 min and then stored at 4 °C. ZnS NPs Capped with Cysteamine. The excess oxygen in the flask solution was removed by the previous procedure. An amount of 5 mL of 0.1 M ZnCl2 solution was added to the flask, and the mixture was stirred for 15 min. After that, 0.1 M sodium sulfide was added dropwise slowly, and then 1 M cysteamine hydrochloride solution was added under N2 atmosphere. The ZnS NPs capped with cysteamine solution were ultrasonicated for 30 min. ZnS NPs Capped with 2-Mercaptoethane Sulfonate. After removal of excess oxygen in the test solution, we added 1 M 2-mercaptoethane sulfonate solution, and then a 0.1 M solution of ZnCl2 was added while stirring in a N2 atmosphere for 30 min. To this, 0.1 M sodium sulfide solution was added slowly, the mixture was stirred for 1 h under N2 atmosphere, and the solution of ZnS-2MES was ultrasonicated for 30 min. Structures of the ZnS nanoparticles capped with different functional groups are shown in Supporting Information Figure S2a. Preparation of Sample Solution. Stock solutions of CDs (0.7-1.0 mM) and insulin (0.17 mM) were prepared by dissolving 1 mg of the above substances in deionized water, and ubiquitin (0.11 mM) was prepared by dissolving of 1 mg in 50 mM NH4HCO3 solution. The above stock solutions were diluted further for working concentrations. An aliquot of 900 µL of the CDs, ubiquitin, and insulin solutions were taken in a 1 mL polyethylene vial. The pH of the sample solution was adjusted by adding 0.1 M HCl or NaOH. An amount of 100 µL of (0.5-2.5 µM) ZnS NPs solution was added, and the mixture was vortexed for 30 min with the speed of 900 rpm. The sample solutions (1 µL) were pipetted onto a stainless steel target plate and allowed to air-dry for 10 min before the SALDI-TOF-MS analysis. Schematic procedures of the NPs interactions with the target analytes are shown in Supporting Information Figure S2b. (31) Li, H.; Shih, W. Y.; Shih, W. H. Ind. Eng. Chem. Res. 2007, 46, 2013–2019.

SALDI-TOF-MS Analysis. All mass spectra were obtained in positive ion mode using a Microflex (Bruker Daltonics, Bremen, Germany) MALDI-TOF-MS, and a 337 nm nitrogen laser was used for irradiation of the sample. Ions produced by laser desorption were stabilized during a delayed extraction period of 200 ns before entering the mass analyzer and then accelerated through the TOF analyzer. The accelerating voltages existed in the range from +20 to -20 kV. All experiments were performed in a linear (for m/z > 6000) and reflectron (for m/z < 6000) mode of TOF-MS. RESULTS AND DISCUSSION Identification of the ZnS NPs by Scanning Electron Microscopy. ZnS nanoparticles capped with different functional groups (3-MPA, citrate, cysteamine, 2-MES) and bare ZnS were synthesized and then further investigated as the matrix and affinity probes in SALDI-TOF-MS to determine selected molecules including CDs, ubiquitin, and insulin from aqueous solution and urine samples. Figure 1 displays the scanning electron microscopy (SEM) images of the ZnS NPs capped with different functional groups. It can be seen that the different SEM images of the ZnS nanoparticles with 3-MPA, citrate, cysteamine, 2-MES, and bare ZnS including nanocrystals, nanosheets, nanoflowers, or nanowires are shown in Figure 1a-e. This indicates that the capping agents exhibit great impact on the shape and size of the nanoparticles. These results were in good agreement from those reported in the literature.32 Since, it has been proposed that the capping agents have demonstrated with significant influence on the morphology of ZnS NPs.32,33 Moreover, by adding capping agents to the cubic ZnS NPs it can gradually grow into bigger nanocrystals with different morphologies accompanying a partial phase transformation from the cubic to hexagonal ZnS, and this leads to the formation of multimorphological ZnS nanoparticles.32 In addition, images of ZnS NPs have proven that the solublization and cross-linking steps ZnS NPs did not result in aggregation. The ZnS NPs characterized by transmission electron microscopy (TEM) images were reported in the literature with the size of 200 nm.30 UV-Vis Absorption Spectra of Various ZnS NPs. The UV-vis absorption spectra of the above five different types of ZnS NPs are shown in Supporting Information Figure S3. These absorption spectra clearly indicate that the ZnS NPs capped with different functional groups exhibited the maximum absorbance from 290 to 310 nm, which can confirm the formation of these five types of different ZnS NPs. Effects of Capping Agents. Aqueous ZnS colloidal semiconductor NPs were capped with different functional groups such as 3-MPA, citrate, cysteamine, and 2-MES. They can disperse the nanoparticles in the suspension and also control the particle sizes of NPs. In the cases of ZnS-3-MPA, ZnS-cys, and ZnS-2-MES, the thiol groups directly bind to the surfaces of the ZnS NPs via a ZnS-S bond; the other end of these NPs are carboxyl and sulfonate groups containing negative charges, and the amino group does not carry charges (Supporting Information Figure S2a). In the ZnS-citrate NPs, although the citrate functional group does not contain thiol groups on the surfaces of ZnS NPs, it acts (32) Tong, H.; Zhu, Y. J.; Yang, L. X.; Li, L.; Zhang, L.; Chang, J.; An, L. Q.; Wang, S. W. J. Phys. Chem. C 2007, 111, 3893–3900. (33) Huang, F.; Banfield, J. F. J. Am. Chem. Soc. 2005, 127, 4523–4529.

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Figure 1. SEM images of ZnS nanoparticles: (a) ZnS-3-MPA, (b) ZnS-citrate, (c) ZnS-cysteamine, (d) ZnS-2-MES, and (e) bare ZnS.

as a good capping and stabilizing agent.34 In most of the inorganic colloids formation, sodium citrate acts as a strong stabilizing agent. Thus, for the ZnS-citrate NPs, the citrate group acts as both a stabilizing and capping agent. In this study, the sensitivity and ionization efficiency of analytes were detected by SALDI-TOFMS using the ZnS NPs strongly depends on the light-absorbing properties of the ZnS NPs and the number of adsorbed molecules onto the ZnS NPs. Thus, the optimum parameters including the effects of sample pH, ion abundance, capping agents, and concentration of the ZnS NPs were investigated. In the evaluation of the effect of capping agent, various types of ZnS NPs including bare ZnS, citrate-, Cys-, 2-MES-, and 3-MPA-capped ZnS NPs were examined as the matrix and affinity probes for the analysis of CDs in SALDI-TOF-MS, and the results are shown in Figure 2. Figure 2 displays the results of effect of concentration of NPs and different types of ZnS NPs as the matrix and affinity probes for the analysis of R-CD in SALDI-TOF-MS. Among these ZnS NPs, the ZnS-3MPA NPs exhibited the best efficiency for the signal intensity of R-CD for all concentrations (from 0.01 M to 6 µM) of NPs. In addition, preparation of all these ZnS NPs was carried out in a N2 atmosphere to prevent the formation of disulfide bonds resulting in aggregation of NPs or dissociation of the capping agents from the ZnS NP surfaces.7 We also found that the molar ratio of ZnS NPs versus capping agents was 1:2, in order to observe the best signal intensities with the analytes, and also at this ratio, the obtained NPs can be stabilized in the aqueous solution for a long period of time. Effect of Concentration of Various ZnS NPs. Since the concentration of the ZnS NPs also plays an important role on the signal intensities detected in SALDI-TOF-MS, we also examined the optimum concentration of these five types of ZnS NPs ranging from 0.01 M to 6 µM for the analysis of R-CD (as a model compound for the concentration effect of NPs) in SALDI-TOF(34) Henglein, A.; Giersig, M. J. Phys. Chem. B 1999, 103, 9533–9539.

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Figure 2. Effect of concentration of NPs and different capping agents of ZnS NPs as the matrix and affinity probes for the analysis of R-CD in SALDI-TOF-MS: concentration of R-CD, 45 nM; incubation time, 30 min; pH, 7. A total of 100 pulsed laser shots were applied under a laser fluence of 60.7 µJ.

MS, and the results are also demonstrated in Figure 2. Figure 2 indicates that the best ZnS NPs concentration is 1 µM for all five different types of ZnS NPs with regard to the signal intensity. In addition, at this concentration, the quality of SALDI-TOF mass spectra is clean and also with lower interference signals (data not shown). Therefore, 1 µM ZnS NPs were applied as the matrix and affinity probes for enrichment of R-, β-, and γ-cyclodextrins, ubiquitin, and insulin molecules for further studies. Effect of Sample pH. Since the most efficient ZnS NPs in SALDI-TOF-MS is the ZnS-3-MPA NPs and the surfaces of these

Figure 3. Effect of sample pH for the analysis of CDs using ZnS NPs capped with 3-MPA: concentration of R-, β-, and γ-cyclodextrins, 45, 56, and 60 nM; incubation time, 30 min; concentration of ZnS3-MPA NPs, 1 µM. A total of 100 pulsed laser shots were applied under a laser fluence of 60.7 µJ.

NPs are carboxyl groups containing negative charges, the effect of sample pH was very important because the electrostatic interactions between these NPs with the target analytes would greatly influence the signal intensity in SALDI-TOF-MS.18,19,21 Thus, we have investigated the effect of sample pH for the analysis of CDs, ubiquitin, and insulin in the pH range from 3.0 to 10.0. For the analysis of CDs using the ZnS-3-MPA NPs as the matrix and the affinity probes, the maximum signal intensity was shown at pH 7.0 for all three CDs (Figure 3). The reason is because CDs are neutral molecules. In the analysis of proteins such as ubiquitin and insulin, we obtained excellent signal intensities at pH 6.0 for ubiquitin and pH 5.0 for insulin. This clearly indicates that the pH effect strongly depends on the pI values of proteins as already discussed in the literatures.18,19,21 Because the isoelectric points (pI) of ubiquitin and insulin are 6.79 and 5.50, respectively, they exhibit net positive charges at pH values below 6.79 for ubiquitin and 5.50 for insulin, respectively. Thus, they can bind with ZnS-3-MPA NPs via electrostatic interactions because ZnS-3-MPA NPs exhibited negative charges at their surfaces. Trapping Capability of ZnS NPs. The trapping capability of ZnS NPs for CDs, ubiquitin, and insulin has been studied by UV-vis absorption spectroscopy. Trapping capability of the nanoparticles were calculated according to the reported methods in literature.12,35 UV-vis absorption spectra were recorded at wavelengths of 270-320 nm. The absorbance measurements of the analytes (0.01-1.0 µM) were recorded before adding the NPs (0.5-5 µM). The calibration curve was constructed based on the absorption values of analytes and concentration of NPs. The trapping capabilities of colloidal nanoparticles for the analytes were found to be 0.025-0.3 µM/mg. This parameter was satisfied with the ability of nanoparticles to interact with the analytes. Hence, these observations could be useful for the manipulation of small quantities of analytes using ZnS NPs. Ion Abundance of r-CD with Five ZnS NPs at Different Laser Fluences. In order to give a better insight into the laser desorption/ionization process, we performed a study on the (35) Kirk, J. S.; Bohn, P. W. J. Am. Chem. Soc. 2004, 126, 5920–5926.

ionization process of R-CD with five types of ZnS NPs at various laser energies, and a graph was constructed with the obtained ion abundances versus laser fluence (Figure S4 of the Supporting Information). In the comparison of the capability of five types of ZnS NPs for ionization of R-CD by SALDI, the R-CD has significantly demonstrated higher ion abundance efficiency with ZnS-3-MPA than the other ZnS NPs. Moreover, at the higher laser fluence (above saturation), the ion abundance of R-CD is slightly reduced due to visible depletion of the sample on the surface.36 On the basis of the above results, we noticed that the ZnS-3-MPA NPs shows the best quality of spectra, and it has good ability to be ionized by SALDI than other types of ZnS NPs. Comparison of the Capability of Various ZnS NPs as the Matrix and Affinity Probes in SALDI-TOF-MS for Analysis of Cyclodextrins and Proteins. Cyclodextrins can interact or bind with a number of molecules in aqueous solutions.37 They may form inclusion complexes via host-guest interactions, hydrophobic interactions, hydrogen-bonding interactions, or binding through chemisorption with nanoparticles based on the functional groups or charges of the NPs38-40 because they possess many hydroxyl groups (18 for R-CD, 21 for β-CD, and 24 for γ-CD, respectively).41 Figure 2 already proved that these five types of ZnS NPs capped with different functional groups can be used as the matrix and affinity probes to detect cyclodextrins in SALDITOF-MS. In order to further understand the different types of ZnS NPs on the ionization efficiency and detection sensitivity for SALDI-TOF-MS, we have carried out a series of experiments on these ZnS NPs including ZnS-3-MPA, ZnS-citrate, ZnS-Cys, ZnS2-MES, and bare ZnS in order to study their ability to interact with the CDs, ubiquitin, and insulin, then detected with SALDITOF-MS (Supporting Information Figures S5-S9). Supporting Information Figures S5-S7 and S10 show the detailed results of R-, β-, and γ-cylcodextrins by using these five types of ZnS NPs as the matrix and affinity probes detected in SALDI-TOF-MS. The SALDI-TOF mass spectra were obtained from the concentrations of 45, 56, and 60 nM for R-CD, β-CD, and γ-CD. The main ions observed for R-CD, β-CD, and γ-CD were shown at m/z 995.04, m/z 1156.90, and m/z 1318.70, respectively, which were assigned as sodium adduct ions ([CD + Na]+). All these CDs mainly produced sodium adduct ions ([CD + Na]+) by using these NPs as the matrix, while sometimes small intensity of the potassium adduct ions ([CD + K]+ were observed too. No protonated molecule ions were ever observed in these CDs because alkali ions are easily attached to the CDs ions due to high affinity of the uncharged carbohydrate molecules toward alkali ions.37 All the results in Supporting Information Figures S5-S7 and S10 indicated that the ZnS-3-MPA NPs exhibit the best sensitivity for all CDs analyses. In addition, the affinity order for the ZnS NPs to interact with the CDs is ZnS-3-MPA > ZnS-citrate > ZnS-Cys > ZnS-2-MES > bare ZnS. Because the carboxylic groups exhibit (36) Fournier, I.; Marinach, C.; Tabet, J. C.; Bolbach, G. J. Am. Soc. Mass Spectrom. 2003, 14, 893–899. (37) Mohr, M. D.; Bornsen, K. O.; Widmer, H. M. Rapid Commun. Mass Spectrom. 1995, 9, 809–814. (38) Nepogodiev, A. S.; Stoddart, J. F. Chem. Rev. 1998, 98, 1959–1979. (39) Zhao, B.; Chen, H. Mater. Lett. 2007, 61, 4890–4893. (40) Liu, Y.; Male, B. K.; Bouvrette, P.; Luong, J. H. T. Chem. Mater. 2003, 15, 4172–4180. (41) Szejtli, J. In Comprehensive Supramolecular Chemistry; Permogaon-Elsevier: New York, 1996; Vol. 3, pp 5-40.

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Table 1. Linear Range, LOD, and Correlation Coefficient of Cyclodextrins Using ZnS NPs in SALDI-TOF-MS parameters ZnS NPs

LR (µM)a

3-MPA

0.01-1.8

citrate

0.02-2.0

cysteamine

0.03-2.5

2-MES

0.04-3.0

bare ZnS

0.05-3.0

a

R2

LOD (nM)b

0.9978 0.9925 0.9872 0.9881 0.9921 0.9899 0.9983 0.9832 0.9814 0.9901 0.9822 0.9845 0.9798 0.9905 0.9852

R-CD 20 β-CD 25 γ-CD 28 R-CD 26 β-CD 32 γ-CD 34 R-CD 31 β-CD 39 γ-CD 41 R-CD 37 β-CD 43 γ-CD 49 R-CD 40 β-CD 48 γ-CD 55

Linear range. b Limit of detection.

much higher affinity to form hydrogen bonding with CDs, ZnS3-MPA and ZnS-citrate NPs show superior results than the other three NPs. But unlike the ZnS-3-MPA, the ZnS-citrate NP does not have the citrate group bind onto the surfaces of ZnS NPs. Although it acts as a good capping and stabilizing agent,34 ZnScitrate NPs possess multiple charges in aqueous solutions and these charges can decrease the affinity to CDs.42Meanwhile, some of the hypotheses were explored that the sizes of the nanoparticles were significantly decreased by capping with MPA than citrate and other molecules.43,44 Thus, its affinity is lower than that of ZnS-3-MPA NP toward CDs. As to the ZnS-Cys NP, which does not exhibit charges, it thus possesses lower affinity than both ZnS3-MPA and ZnS-citrate NP because CDs have high affinity to interact with charged nanoparticles containing higher electronegative atoms and also to form stronger hydrogen bonds.39 However, it still can form hydrogen-bonding interactions via the amino group with the CDs. 2-MES NPs exhibited much lower affinity than ZnS-3-MPA, ZnS-citrate, and ZnS-Cys because it does not contain either carboxylic groups or amino groups. Thus, it cannot form any hydrogen-bonding interactions with CDs. Bare ZnS NPs exhibited the lowest affinity toward CDs, and the reason is because it only can interact with CDs via weak van der Waals forces. In addition, all these five types of ZnS NPs can be successfully used to detect proteins like ubiquitin and insulin too. However, similar to the results of CDs, the ZnS-3-MPA NPs exhibited the best sensitivity and the best quality of mass spectra (lower noise) for these two proteins. Quantitative Analysis of Cyclodextrins and Proteins by Using ZnS-3-MPA NPs as the Matrix and Affinity Probes in SALDI-TOF-MS. In order to evaluate the applicability of using the current approach for quantitative analysis, we have performed an array of experiments for quantitative analysis of CDs, ubiquitin, and insulin by SALDI-TOF-MS using ZnS-3-MPA NPs as the matrix and affinity probes. Table 1 shows that the limits of detection (LODs) of the CDs using the five types of ZnS NPs are (42) Gabelica, V.; Galic, N.; Pauw De, E. J. Am. Soc. Mass Spectrom. 2002, 13, 946–953. (43) Neiman, B.; Grushka, E.; Lev, O. Anal. Chem. 2001, 73, 5220–5227. (44) Li, H.; Shih, W. Y.; Shih, W. H. Nanotechnology 2007, 18, 205604–205609.

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Figure 4. SALDI-TOF mass spectra of ubiquitin (0.28 µM) using ZnS-3-MPA NPs as the matrix and affinity probes for (a) direct analysis from aqueous solution and (b) a spiked urine sample at pH 5.0. Incubation time is 30 min; concentration of ZnS-3-MPA NPs is 1 µM. A total of 200 pulsed laser shots were applied under a laser fluence of 68.5 µJ.

20-55 nM with R2 > 0.9798. The results of the proposed methods are significantly well with regard to LODs, and good linearity was observed with ZnS-3-MPA NPs than the other ZnS NPs. In addition, the ZnS-3-MPA NP has been successfully applied to the quantitative analysis of proteins including ubiquitin and insulin by SALDI-TOF-MS due to its significant affinity toward these proteins (Figures 4 and 5). In these experiments, the pH of the ubiquitin and insulin solutions must be controlled lower than their pI values. Thus, they carry net positive charges in order to interact with the ZnS-3-MPA NPs via electrostatic interactions. The LODs were found to be 0.091 µM for ubiquitin with R2 0.9789 and 0.085 µM for insulin with R2 0.9923 by using the ZnS-3-MPA NPs as the matrix and affinity probes in SALDI-TOF-MS. Moreover, ubiquitin and insulin ions can be obtained by using sinapic acid (SA) as a matrix (a model matrix for the representative of conventional matrix), and the results are shown in Figures S8 and S9 of the Supporting Information. Therefore, comparing with the traditional organic matrix, we know that the ZnS-3-MPA NPs (Figures 4a and 5a) were superior than the organic matrix for sensitivity. Applications of ZnS-3-MPA NPs as the Matrix and Affinity Probes in SALDI-TOF-MS for Analysis of Cyclodextrins and Proteins in Biological Samples and Mushroom. Ubiquitin-like proteins were isolated from oyster mushroom according to the reported literature.28 Briefly, oyster mushroom (Pleurotus ostreatus) was purchased from local market, cut into small pieces, homogenized with 10 mM Tris-HCl (pH 7.2), and kept at 0-4 °C for 3 h. The samples were centrifuged, and the supernatant solution was diluted (2-fold) with water and analyzed for ubiquitin-

Figure 5. SALDI-TOF mass spectra of insulin solution (0.22 µM) using ZnS-3-MPA NPs as the matrix and affinity probes for (a) direct analysis from aqueous solution and (b) a spiked urine sample at pH 6.0. Incubation time is 30 min; concentration of ZnS-3-MPA NPs is 1 µM. A total of 200 pulsed laser shots were applied under a laser fluence of 68.5 µJ.

like proteins according to the aforesaid procedure. The obtained mass peaks of ubiquitin-like proteins from oyster mushrooms are shown in Figure 6. Four peaks were well-separated with good mass resolution by using ZnS-3-MPA NPs as the matrix and affinity probes (Figure 6a) and resolved mass peaks at m/z 8063, 8384, 8593, and 10 660 which are corresponding to ubiquitin and ubiquitin-like proteins (8.0, 8.3, 8.5, and 10.5 kDa). However, when SA was used as the matrix, the generated peaks containing 1-3 compounds were not resolved and there appeared only one peak (Figure 6b); thus, it was obvious that the ubiquitin-like proteins could not be successfully separated by using SA as the matrix. The present method has proven to be quick, simple, and effective for analysis of ubiquitin-like proteins in real samples than the reported HPLC method.28 To our best knowledge, to date, no reports were made on the analysis of ubiquitin and ubiquitin-like proteins in oyster mushroom by using MALDI-TOF-MS. Additionally, the applicability of ZnS-3-MPA NPs has been also demonstrated for the analysis of CDs, ubiquitin, and insulin in spiked urine and plasma samples. The 80 nM CDs were spiked into urine (Supporting Information Figure S10a) and plasma (Supporting Information Figure S10b) by 4-fold of dilution for urine and 10fold of dilution for plasma, then analyzed by SALDI-TOF-MS. Moreover, 0.30 µM ubiquitin was spiked into the urine samples and analyzed by the described procedure, and mass spectra were shown in Figure 4b. In addition, the potential applicability of the present method has been applied for the analysis of insulin in urine samples for diabetic patents by using ZnS-3-MPA NPs as the matrix and affinity probes, and mass spectra were shown in

Figure 6. SALDI-TOF mass spectra of ubiquitin-like proteins in oyster mushroom by (a) ZnS-3-MPA NPs as the matrix and affinity probes and (b) SA as the matrix. Peaks 1, 2, 3, and 4 at m/z 8063, 8384, 8593, and 10 666 are attributed to the ubiquitin-like protein (8.0 kDa), ubiquitin-like protein (8.3 kDa), ubiquitin (8.5 kDa), and ubiquitinlike protein (10.5 kDa), respectively. Incubation time is 30 min; concentration of ZnS-3-MPA NPs is 1 µM. A total of 200 pulsed laser shots were applied under a laser fluence of 68.5 µJ.

Figure 5b. These experiments were performed using urine samples obtained from diabetic patients; collected samples were stored at 4 °C until analysis. Samples were diluted and analyzed by the described procedures in the literatures.25,45 Insulin (0.25 µM) spiked in the urine samples of diabetic patients has been analyzed, and the mass peak of insulin was obtained using the ZnS-3-MPA NPs by SALDI-TOF-MS (Figure 5b). Because, generally in diabetic patents, blood and urine contains only 19-33 pM insulin,45 and it failed to be detected by this approach, we spiked 0.025-0.45 µM insulin into urine samples of diabetic patents. A calibration graph was constructed between concentration of insulin and signal intensities (Supporting Information Figure S11). The results indicate good linearity of insulin in urine samples using ZnS-3-MPA NPs in SALDI-TOF-MS. CONCLUSIONS A comparison for applications of ZnS NPs capped with different functional groups have been made for the analysis of cyclodextrins, ubiquitin, and insulin in aqueous phase, oyster mushroom, and biological samples by SALDI-TOF-MS. UV-vis spectroscopy and SEM have been used to identify the images, morphology, and trapping capability of the ZnS NPs. This novel technique has proved to be selective with high affinity to the target analytes and good detection limits. In comparison among these ZnS NPs, the ZnS-3-MPA NPs were superior than any other type of ZnS NPs with regard to signal intensity and detection limit. Furthermore, ubiquitin-like proteins in the biological samples can be directly analyzed by this approach without the need of further elutions, washing procedures, or tedious separation processes. (45) Terenteva, S. V.; Matolygina, E. M.; Aptekar, V. D.; Gusakova, A. M.; Ivanovskaya, E. A.; Teplyakov, A. T. Pharm. Chem. J. 2004, 38, 401–403.

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These results demonstrate that the ZnS NPs can be used for simultaneous isolation, concentration, and characterization of lowlevel samples. The advantages of ZnS NPs for MALDI-MS are multifold including being simple, rapid, and sensitive via interaction of ZnS NPs with biomolecules. The ZnS NPs can be applied as an effective probe for small organic compounds and large biomolecule analysis from biological samples.

National Sun Yat-Sen University, and we also thank the Center for Nanoscience and Nanotechnology, National Sun Yat-Sen University for financial support.

ACKNOWLEDGMENT We thank the National Science Council for financial support in the form of project NSC 95-2113-M110-019-MY3 issued to

Received for review July 29, 2008. Accepted October 6, 2008.

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SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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