Research article Received: 16 July 2013,
Revised: 11 October 2013,
Accepted: 26 October 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2613
Bovine serum albumin-confined silver nanoclusters as fluorometric probe for detection of biothiols Zhen Chen, Dongtao Lu, Zongwei Cai, Chuan Dong and Shaomin Shuang* ABSTRACT: Fluorescent bovine serum albumin-confined silver nanoclusters (BSA–AgNCs) were demonstrated to be a novel and environmentally friendly probe for the rapid detection of biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH). The sensing was ascribed to the strong affinity between the mercapto group of the biothiols and the silver nanoclusters. The fluorescence intensity of BSA–AgNCs was quenched efficiently on increasing the concentration of biothiol, corresponding with a red-shift in emission wavelength. However, the fluorescence of the silver nanoclusters was almost unchanged in the presence of other α-amino acids at 10-fold higher concentrations. By virtue of this specific response, a new, simple and rapid fluorescent method for detecting biothiols has been developed. The linear ranges for Cys, Hcy and GSH were 2.0 × 10-6 to 9.0 × 10-5 M (R2 = 0.994), 2.0 × 10-6 to 1.2 × 10-4 M (R2 = 0.996) and 1.0 × 10-5 to 8.0 × 10-5 M (R2 = 0.980), respectively. The detection limits were 8.1 × 10-7 M for Cys, 1.0 × 10-6 M for Hcy and 1.1 × 10-6 M for GSH. Our proposed method was successfully applied to the determination of thiols in human plasma and the recovery was 94.83–105.24%. It is potentially applicable to protein-stabilized silver nanoclusters in a chemical or biochemical sensing system. Copyright © John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: bovine serum albumin; silver nanoclusters; biothiols; fluorescence
Introduction
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* Correspondence to: Shaomin Shuang, Department of Chemistry and Chemical Engineering, Institute of Environmental Science, Shanxi University, Taiyuan 030006, People’s Republic of China. E-mail: smshuang@sxu. edu.cn Department of Chemistry and Chemical Engineering, Institute of Environmental Science, Shanxi University, Taiyuan 030006, People’s Republic of China
Copyright © 2014 John Wiley & Sons, Ltd.
1
Noble metal nanoclusters (NCs) with distinct, unique physical, electrical and optical properties have attracted a great deal of attention in recent years for their promising applications in optical sensing, catalysis, biological labeling and imaging (1–3). Noble metal NCs consisting of a few to tens of metal atoms are of ultra-small size (approximately the Fermi wavelength of the conduction electrons), which leads to discrete energy levels and strong quantum confinement. Therefore, they show remarkable photoluminescence (1). Silver nanoclusters are more reactive than their gold analogs, and the study of AgNCs is an active issue (2–4). Because AgNCs are prone to aggregation and grow to form larger clusters or nanoparticles (NPs), a variety of stabilizers such as dendrimers (5), polyelectrolytes (6), thiolates (7), small organic molecules (8), biomacromolecules, DNA oligonucleotides (9,10) and peptides/proteins (11–13) have been used for the modification of AgNCs. Among these modifications, a recent breakthrough in the synthesis of fluorescence AgNCs, with protein as scaffolds, has received special attention (12,13). Protein-protected AgNCs have outstanding spectral and photophysical properties, combined with low toxicity and good biocamptibility, and so they hold great promise as optical probes, in biological labeling and as imaging agents (14). However, most of the recent studies focus on using a facile method to synthesize stable and highly luminescent AgNCs (13,15), few studies have involved the analytical application of protein-stabilized AgNCs. Zhou et al. (16) assemblied lysozyme-stabilized AgNCs to determine Hg2+. Yang et al. (17) proposed a spectrophotometric and colorimetric method for ascorbic acid based on the catalysis of bovine serum
albumin (BSA)-protected AgNCs. Therefore, a practical use for protein-stabilized AgNCs is desirable. Biological thiols (biothiols), such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), participate in reversible redox reactions and have important cellular functions, including detoxification and metabolism (18). A normal level of biothiols in the human body is essential and plays a vital role in maintaining healt. A Cys deficiency correlates with slow growth, hair depigmentation, edema, liver damage, skin lesions and weakness (19). An elevated Hcy level may be a sign of disorders such as cardiovascular disease and Alzheimer’s disease (20). GSH is one of the most abundant cellular biothiols, and is typically associated with beneficial antioxidant effects and might quench reactive free-radical species that can damage genetic material (21). Thus, sensitive and selective detection of these biothiols is very important in the early diagnosis of disease. To date, various methods to determine biothiols have involved highperformance liquid chromatography (HPLC) (22), mass spectrography (23), and capillary electrophoresis coupling optical and electrochemical detectors (24–26). However, these methods have several disadvantages: they require expensive and sophisticated instrumentation, complicated sample preparation and they are time-consuming. Fluorimetry is a favorable method
Z. Chen et al. with high sensitivity and selectivity compared with other methods. Several attempts have been made to use fluorescent AgNCs as the optical probe for detecting biothiols. However, limited ligand-capped fluorescent silver nanoclusters including polymer-stabilized AgNCs (27,28), DNA oligonucleotide-stabilized AgNCs (25,26) and glutathione-protected AgNCs (4) have been developed as a biothiol fluorometric probe. We try to expand the use of protein as a scaffold for AgNCs to determine biothiols using a fluorometric method. We synthesized AgNCs using BSA as the stabilizer and further developed the detection method for biothiols including Cys, Hcy and GSH. The fluorescence intensity of BSA–AgNCs was found to be quenched effectively in the presence of biothiols. However, 18 other α-amino acids (without a thiol group) might lead to negligible changes in the fluorescence intensity of the nanoclusters. This method was successfully used to detect thiols in human plasma.
Experimental
month. In addition, the cluster solution could be freeze-dried into a powder form for further storage.
Fluorescence measurements Cys, Hcy and GSH aqueous solutions at different concentrations were freshly prepared before use. A typical detection procedure was conducted as follows: 150 μL BSA–AgNCs solution was diluted to 2 mL with 0.2 mol/L PBS solutions (pH 9.00 for Cys, 8.02 for Hcy and 8.55 for GSH). Owing to the lack of quality information for BSA–AgNCs, we present the concentration of BSA–AgNCs using the quality of BSA. An appropriate volume of a 0.01 M biothiol stock solution was mixed with the BSA–AgNCs solution (1.875 mg/mL) at room temperature (22 ± 1°C) and equilibrated for 1 min. The fluorescence emission spectrum of AgNCs was recorded in the wavelength range 485–750 nm upon excitation at 465 nm. To investigate the selectivity of this assay, the influence of the other 18 α-amino acids (at a 10-fold higher concentration than Cys, Hcy and GSH) on the BSA–AgNCs was investigated. All experiments were performed in triplicate.
Chemicals Silver nitrate (AgNO3, 99%) and sodium borohydride (NaBH4, powder, 98%) were from Sigma Aldrich Chemical Co. (ST. Louis, USA). BSA (> 96%) was purchased from Shanghai Sangon Biotechnology Co. Ltd (Shanghai, China). Cys and other α-amino acids were purchased from Bei-jing Chemical Co. (Beijing, China). GSH (88%, for analysis, reduced) was obtained from ACROS ORGANICS (NJ, USA). Homocysteine (> 95%, titration) was from Sigma Aldrich Chemical Co. (ST. Louis, USA). All other reagents were of analytical reagent grade, and used as received. -1 Double-distilled water (> 18.2 MΩ cm ) from the Millipore Milli Q system (Massachusetts, USA) was used throughout.
Apparatus Fluorescence measurements were made on a F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) with a quartz cell (1 cm × 1 cm). Excitation and emission slit widths were set at 10 nm and the photomultiplier tube voltage was set at 700 V. The emission spectra were recorded in the wavelength range 485–750 nm upon excitation at 465 nm. UV/vis absorption spectra were recorded in the wavelength range 350–800 nm on a UV-3010 spectrophotometer (Hitachi). Transmission electron microscopy (TEM) images were obtained using a FEI TECNAL 20 at an accelerating voltage of 200 kV. The hydrodynamic diameter was measured by dynamic light scattering (DLS) using a Malvern Autosizer ZS90 spectrometer.
Preparation of the BSA-confined Ag nanoclusters
2
BSA–AgNCs were prepared using a modification of a simple wet route modified (13). In a typical synthesis, 0.5 mL of 0.1 M AgNO3 solution was added to 10 mL water containing 250 mg BSA powder with vigorous stirring at room temperature. The mixture was left to incubate for 5 min under vigorous stirring before the dropwise addition of 0.3 mL NaOH (1 M). After stirring for 1 h at room temperature, NaBH4 solution (10 mM, freshly prepared) was added dropwised until the solution turned from colorless to reddish-brown, indicating the formation of various amounts of clusters. To collect clean and stable BSA–AgNCs, the obtained solution was then dialyzed using a 8–14 kDa dialysis bag in double-distilled water for 24 h. The BSA–AgNCs solution was kept at 4°C in the dark and the luminescence remained almost unchanged for least one
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Measurement of biothiols in human plasma samples Fresh human blood samples were obtained from the local hospital. After centrifugation at 4000rpm (2680g) for 10 min, the supernatant solution containing the proteins and amino acids was collected and used as the source of the plasma. The collected supernatant was reduced by addition of triphenylphosphine (PPh3) as a catalyst and then mixed with acetonitrile to precipitate proteins (22–27). The supernatant containing amino thiols in plasma was used for further analysis, and the amount of biothiols was estimated using a standard addition method. The plasma sample was diluted with 0.2 M PBS solution (pH 9.00) before measurement to be within the dynamic range of the proposed method. For recovery studies, known concentrations of Cys solution were added to plasma samples and the total thiol concentrations were then determined at the same condition.
Results and discussion Characteristics of BSA–AgNCs The as-prepared BSA–AgNCs were characterized by TEM, DLS, UV/vis absorption and fluorescence spectroscopy as indicated in Figs 1 and 2. As shown in Fig. 1, the UV/vis absorption spectrum of BSA–AgCNs exhibited a small broad peak around 450 nm due to quantum confinement effects, but no characteristic surface plasmon resonance peak in the range of 400–500 nm was observed, indicating the formation of nanoclusters rather than lager nanoparticles (29,30). The TEM image indicates that the BSA–AgNCs were spherical and well dispersed, with an average diameter of 2 nm, whereas the hydrodynamic diameter measured using DLS was ~ 1.75 nm (Fig. 2). The maximum fluorescence emission of BSA–AgCNs could be observed at 598 nm when excitation wavelength was 465 nm (Fig. 1, curves b and c). The inset to Fig. 1 shows that the BSA–AgNCs solution was reddish-brown in color under visible light, although it exhibited a bright red fluorescence under UV light at 365 nm. The fluorescence quantum yield (QY) of the BSA–AgNCs was calculated to be 2.6% by comparison with rhodamine 6G in ethanol (with a standard QY of 95%).
Copyright © 2014 John Wiley & Sons, Ltd.
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Detection of biothiols based on BSA–AgNCs
Figure 1. UV/vis absorption spectrum (a) and fluorescence spectra (b, c) of the asprepared BSA–AgNCs. (Inset) BSA–Ag NCs under visible and UV light at 365 nm.
Optical response of BSA–AgNCs to Cys The fluorescence response of the BSA–AgNCs to Cys was investigated and the results are shown in the Fig. 3. Upon addition of 100 μM Cys, the absorption spectra had an apparent red-shift from 465 nm to ~ 495 nm; the absorption intensity showed virtually no change (Fig. 3a). The fluorescence intensity of the nanoclusters solution at 598 nm decreased significantly, corresponding with a red-shift from 598 to 617 nm (Fig. 3b). The inset in Fig. 3(b) showed that the color of the BSA–AgNCs solution changed from reddish-brown to colorless in the presence of Cys
under visible light (Fig. 3b, upper), while a weak blue fluorescence was observed under 365 nm UV light (Fig. 3b, lower). This phenomenon indicated that BSA–AgNCs as the probe could achieve naked-eye recognition. In order to study the interaction between BSA–AgNCs and biothiols, absorption spectra of BSA–AgNCs in the presence of varying concentrations of Cys were examined (Fig. S1). It was found that the interaction process could be distinguished into two parts from the absorption spectra. When the concentration of Cys varied in the range of 1–100 μM, the absorption peak of BSA–AgNCs had an obvious red-shift and the intensity was almost unchanged (Fig. S1a). This may be because Cys could penetrate the BSA protective layer and combine with the AgNCs core based on the high affinity of sulfhydryl to Ag (27). This combination influenced the optical properties of the BSA–AgNCs. When the concentration of Cys was increased to > 100 μM, the absorption peak no longer had a red-shift and gradually disappeared (Fig. S1b). This may result from the destruction of the BSA–AgNCs, which might be ascribed to oxidative etching from both the oxygen and the mercapto group (31). The sulfide in the mercapto group could donate an electron pair to the Ag atoms on the surface, which were coordinately unsaturated and therefore an unoccupied orbital existed (32). On this occasion, the added biothiols could be readily absorbed on the surface of Ag clusters by partially removing the residues from BSA. In the meantime, Fig. S2 shows that the lifetime of BSA–AgNCs did not change appreciably in the presence of Cys, which indicated that there was a change in ground state of the fluorescence BSA–AgNCs. Both the UV spectra and lifetime
Figure 2. (a) TEM image of BSA–AgNCs. (b) Hydrodynamic diameter of the as-prepared BSA–AgNCs measured with DLS.
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Figure 3. The absorption (A) and fluorescence (B) spectra of BSA–AgNCs (1.875 mg/mL) in the absence (a) and presence (b) of 100 μM Cys. (B, inset) AgNCs in the absence (1) and presence (2) of Cys (100 μM) under visible and UV light at 365 nm.
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Z. Chen et al. results were consistent with the related literature and confirmed that the biothiols were chemisorbed on the surface of AgNCs (25,32). It is well known that the redox potential of silver decreases upon absorption of nucleophiles on a particle surface (33). Therefore, it might be expected that the
redox potential of silver in the few-atom Ag clusters would show a large negative shift upon the further adsorption of biothiols. Thus, the oxidation of Ag by oxygen could be greatly facilitated and the gradual oxidation of AgNCs occurred in the presence of biothiols. Similar phenomena were observed in the absorption spectra for Hcy and GSH, as shown in Figs S3 and S4. Optimization of factors influencing the detection of biothiols Prior to the application of our fluorescence probe for the detection of biothiols, several factors that may affect the interaction of BSA–AgNCs with biothiols were optimized, such as the concentration of BSA–AgNCs, pH, incubation time and temperature. As shown in Fig. S5, the relative fluorescence intensity of the system increased gradually as the concentration of BSA–AgNCs changed from 0.5 to 1.875 mg/mL, but decreased at higher concentrations. Considering the fluorescence background and the sensitivity of the detection, 1.875 mg/mL was chosen as the optimum concentration of BSA–AgNCs. The effect of pH on the detection of biothiols by BSA–AgNCs was investigated and optimized. The fluorescence intensity of BSA–AgNCs in the absence and presence of 50 μM Cys in the pH range 3.00–12.00 is shown in Fig. S6. It is evident that the fluorescence of BSA–AgNCs had a tendency toward gradual decrease as pH increases from 3.00 to 9.00. The quenching effect is closely related to the patterns of mercapto. Mercapto of biothiol was mainly present in a protonated pattern in strong acidic media and resulted in a weak quenching effect (34). In neutral and weak basic media the deprotonated mercapto had strong affinity to the AgNCs and readily caused the fluorescence quenching of BSA–AgNCs. The maximum quenching action occurred at pH 9.00. However, quenching decreased dramatically at a pH > 9.00 because the mercapto group in Cys was oxidized to generate the disulfide bond. Therefore, an optimum pH of 9.00 was chosen for detection. A similar influence of pH could be observed for BSA–AgNCs in the presence of Hcy and GSH. In order to find optimum values for incubation time and temperature, their efects on the BSA–AgNCs-based detection
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Figure 4. Fluorescence spectra (λex = 465 nm) of BSA–AgNCs (1.875 mg/mL) in the presence of biothiol solutions at different concentrations. (a) Cys: 0, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140 μM. (b) Hcy: 0, 2, 6, 10, 15, 20, 30, 40, 60, 80, 100, 120, 140 μM. (c) GSH: 0, 2, 4, 6, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 160, 200 μM. (Insets) Relationship between (F0 – F) and concentration of biothiols.
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Figure 5. Relative fluorescence intensity of BSA–AgNCs (1.875 mg/mL) in the presence of 100 μM Cys, GSH and Hcy, and 1000 μM of various other amino acids.
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Detection of biothiols based on BSA–AgNCs Table 1. Determination of biothiols in human plasma samples based on BSA–AgNCs Plasma sample
Determined biothiols (10-4 M)
1
2.38
2
2.74
3
2.62
Added Cys (10-6 M) 2.00 4.00 2.00 4.00 2.00 4.00
of biothiols were tested. As shown in Fig. S7, the relative fluorescence intensity F0 – F increased rapidly within 1 min and no obvious change was found over time, which indicated that the interaction between BSA–AgNCs and biothiols could be carried out within 1 min. Figure S8 shows that the higher the incubation temperature, the lower the quenching efficiency. Considering the stability of the system and the simplicity of operation, all the titration experiments were carried out at room temperature. Analytical merits of BSA–AgNCs to biothiols Figure 4 shows the fluorescence spectra of BSA–AgNCs in the presence of varying amounts of Cys, Hcy and GSH. The emission at 598 nm of BSA–AgNCs was seen to be strongly quenched by biothiols, whereas an obvious red-shift in the emission wavelength was observed. As shown in Fig. 4(insets), the calibration curves for biothiols exhibited linear relationships in the range from 2.0 × 10-6 to 9.0 × 10-5 M for Cys, 2.0 × 10-6 to 1.2 × 10-4 M for Hcy and 1.0 × 10-5 to 8.0 × 10-5 M for GSH. The corresponding detection limits were 8.1 × 10-7 M for Cys, 1.0 × 10-6 M for Hcy and 1.1 × 10-6 M for GSH. Cys : F 0 –F ¼ 7:849 þ 1:554C R2 ¼ 0:994 Hcy : F 0 –F ¼ 0:009 þ 1:207C R2 ¼ 0:996 GSH : F 0 –F ¼ 10:946 þ 1:198C R2 ¼ 0:980 where F0 and F are the fluorescence intensity of BSA–AgNCs in the absence and presence of biothiols, and C presents the concentration of biothiols. We were able to confirm that the quenching ability of the three biothiols follow the order Cys ≈ Hcy > GSH. GSH exhibited relatively lower reactivity than Cys and Hcy, which might be due to steric hindrance (4,35). Here, biothiols acted as nucleophile reagents that could be chemisorbed on the surface of AgNCs (28,32). Upon addition, Cys could penetrate the BSA protective layer and be adsorbed onto the surface of AgNCs, because silver atoms have low-lying vacant d orbitals and electron pairs from the mercapto of Cys can enter vacant electron orbitals on the silver atoms (36). The interaction would partially break the AgNCs–BSA bond, which weakens the interaction between the AgNCs and BSA (37). The mercapto group of the biothiols can not only stabilize, but also destabilize silver nanoclusters (31). Because the AgNCs were only poorly stabilized with BSA, thiol-induced fluorescence quenching of BSA–AgNCs occurred (32,37).
To test the selectivity of the quenching behavior of biothiols (Cys, Hcy and GSH), the fluorescence responses of BSA–AgNCs against 18 other α-amino acids at 10-fold higher concentrations than
1.90 4.21 2.02 3.98 2.06 3.99
Recovery (%) 94.83 105.24 100.77 99.57 103.23 99.86
RSD (n = 3, %) 2.90 2.88 3.31 3.16 3.39 1.57
Cys, Hcy and GSH were investigated under the same conditions. Figure 5 shows the changes in the relative fluorescence [(F0 – F)/F0] of BSA–AgNCs that occurred after the separate addition of Cys, Hcy and GSH (100 μM) and 18 other α-amino acids (1000 μM). It is clear that the variations in fluorescence intensity of BSA–AgNCs in the presence of Cys, Hcy and GSH were strikingly larger than that of other common α-amino acids. In addition, there was no red-shift as in Cys, Hcy and GSH in the other amino acids. These results indicated that the proposed assay had high selectivity for biothiols and has potential in the practical detection of biothiols in biological samples.
Determination of biothiols in human plasma In order to investigate the feasibility of our proposed method for practical applications, we performed recovery experiments. Cys was chosen as the reference substance to test the recovery of this method because it is the main component of biothiols in human plasma and other thiols reacted with BSA–AgNCs in a similar manner. Pretreated human plasma samples were diluted with an appropriate phosphate buffer to ensure the concentration of biothiols was in the linear range and to obtain quantitative recovery of the spiked thiols. Owing to variations in the chemical composition of biological samples, a standard addition method was employed. The obtained concentrations of biothiols in three human plasmas are listed in Table 1, and were in agreement with the results in the literature (25,28). Recoveries of 94.83–105.24% of the known amount Cys in the plasma samples were obatined, thus demonstrating the reliability of BSA–AgNCs for detecting biothiols in human plasma.
Conclusions BSA–AgNC was explored as a novel signal-off fluorescent probe for simple, selective, sensitive and cost-effective detection of biothiols, such as Cys, Hcy and GSH. The fluorescence of BSA–AgNCs was quenched by biothiols because of the strong affinity between mercapto and Ag atoms. This method, with good interference immunity, was successfully used for the detection of thiols in human plasma samples. As far as we awared, protein-protected AgNCs are used first for the detection of biothiols with fast, simply, sensitive and selective features. In addition, our method has great potential for the quantitative analysis of thiol levels in biological fluids. More importantly, the proposed method is a novel application of protein-stabilized AgNCs, and provides a better understanding of the interaction between noble metal nanoclusters and protein.
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Selectivity of BSA–AgNCs for the detection of biothiols
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Measured (10-6 M)
Z. Chen et al. Acknowledgments Financial supports from the National Natural Science Foundation of China (Nos. 21175086 and 21175087), Shanxi Province Hundred Talent Project Support.
References 1. Maity P, Xie S, Yamauchi M, Tsukuda T. Stabilized gold clusters: from isolation toward controlled synthesis. Nanoscale 2012;4:4027–37. 2. Shang L, Dong SJ, Nienhaus GU. Ultra-small fluorescent metal nanoclusters: synthesis and biological applications. Nano Today 2011;6:401–18. 3. Lu Y, Chen W. Sub-nanometre sized metal clusters: from synthetic challenges to the unique property discoveries. Chem Soc Rev 2012;41:3594–623. 4. Yuan X, Tay YQ, Dou XY, Luo ZT, Leong DT, Xie JP. Glutathioneprotected silver nanoclusters as cysteine-selective fluorometric and colorimetric probe. Anal Chem 2013;85:1913–19. 5. Xu H, Suslick KS. Sonochemical synthesis of highly fluorescent Ag nanoclusters. ACS Nano 2010;4:3209–14. 6. Díez I, Pusa M, Kulmala S, Jiang H, Walther A, Goldmann AS, et al. Color tunability and electrochemiluminescence of silver nanoclusters. Angew Chem Int Ed 2009;48:2122–5. 7. Rao TUB, Pradeep T. Luminescent Ag7 and Ag8 clusters by interfacial synthesis. Angew Chem Int Ed 2010;49:3925–9. 8. Muhammed MAH, Aldeek F, Palui G, Trapiella-Alfonso L, Mattoussi H. Growth of in situ functionalized luminescent silver nanoclusters by direct reduction and size focusing. ACS Nano 2012;6:8950–61. 9. Petty JT, Fan CY, Story SP, Sengupta B, Iyer AS, Prudowsky Z, et al. DNA encapsulation of 10 silver atoms producing a bright, modulatable, near-infrared-emitting cluster. J. Phys. Chem. Lett. 2010;1:2524–29. 10. Martinez JS, Sharma J, Yeh HC, Yoo H, Werner JH. Silver nanocluster aptamers: in situ generation of intrinsically fluorescent recognition ligands for protein detection. Chem Commun 2011;47:2294–96. 11. Adhikari B, Banerjee A. Short-peptide-based hydrogel: a template for the in situ synthesis of fluorescent silver nanoclusters by using sunlight. Chem Eur J 2010;16:13698–705. 12. Guo CL, Irudayaraj J. Fluorescent Ag clusters via a protein-directed approach as a Hg(II) ion sensor. Anal Chem 2011;83:2883–9. 13. Mathew A, Sajanlal PR, Pradeep T. A fifteen atom silver cluster confined in bovine serum albumin. J Mater Chem 2011;21:11205–12. 14. Xavier PL, Chaudhari K, Baksi A, Pradeep T. Protein-protected luminescent noble metal quantum clusters: an emerging trend in atomic cluster nanoscience. Nano Rev 2012;3:14767. 15. Anand U, Ghosh S,Mukherjee S. Toggling between blue- and redemitting fluorescent silver nanoclusters. J Phys Chem Lett 2012;3:3605–9. 16. Zhou TY, Huang YH, Li WB, Cai ZM, Luo F, Yang CJ, et al. Facile synthesis of red-emitting lysozyme-stabilized Ag nanoclusters. Nanoscale 2012;4:5312–15. 17. Yang XH, Ling J, Peng J, Cao QE, Wang L, Ding ZT, et al. Catalytic formation of silver nanoparticles by bovine serum albumin protected-silver nanoclusters and its application for colorimetric detection of ascorbic acid. Spectrochim Acta A 2013;106:224–30. 18. Chen X, Zhou Y, Peng X, Yoon J. Fluorescent and colorimetric probes for detection of thiols. Chem Soc Rev 2010;39:2120–35. 19. Shahrokhian S Lead phthalocyanine as a selective carrier for preparation of a cysteine-selective electrode. Anal Chem 2001;73:5972–8.
20. Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, D’Agostino RB, et al. Plasma homocysteine as a risk factor for dementia and Alzheimer’s disease. N Engl J Med 2002;346:476–83. 21. Hong R, Han G, Fernández JM, Kim BJ, Forbes NS, Rotello VM. Glutathione-mediated delivery and release using monolayer protected nanoparticle carriers. J Am Chem Soc 2006;128:1078–9. 22. Janes L, Lisjak K, Vanzo A. Determination of glutathione content in grape juice and wine by high-performance liquid chromatography with fluorescence detection. Anal Chim Acta 2010;674:239–42. 23. Satterfield MB, Sniegoski LT, Welch MJ, Nelson BC, Pfeiffer CM. Comparison of isotope dilution mass spectrometry methods for the determination of total homocysteine in plasma and serum. Anal Chem 2003;75:4631–8. 24. Chen G, Zhang L, Wang J. Miniaturized capillary electrophoresis system with a carbon nanotube microelectrode for rapid separation and detection of thiols. Talanta 2004;64:1018–23. 25. Han BY, Wang EK. Oligonucleotide-stabilized fluorescent silver nanoclusters for sensitive detection of biothiols in biological fluids. Biosens Bioelectron 2011;26:2585–9. 26. Thiagarajan S, Umasankar Y, Chen SM. Functionalized multi walled carbon nanotubes nano biocomposite film for the amperometric detection of L-cysteine. Nanosci Nanotechnol 2010;10:702–10. 27. Shang L, Yin JY, Li J, Jin LH, Dong SJ. Gold nanoparticle-based nearinfrared fluorescent detection of biological thiols in human plasma. Biosens Bioelectron 2009;25:269–74. 28. Zhang N, Qu F, Luo HQ, Li NB. Sensitive and selective detection of biothiols based on target-induced agglomeration of silver nanoclusters. Biosens Bioelectron 2013;42:214–18. 29. Liu GL, Feng DQ, Mu XY, Zheng WJ, Chen TF, Qi L, et al. DNAfunctionalized silver nanoclusters as a chemopalette: tunable fluorescence for turn-on detection of cysteine. J Mater Chem B 2013;1:2128–31. 30. Wen T, Qu F, Li NB, Luo HQ. Polyethyleneimine-capped silver nanoclusters as a fluorescence probe for sensitive detection of hydrogen peroxide and glucose. Anal Chim Acta 2012;749:56–62. 31. Choi S, Dickson RM, Yu JH. Developing luminescent silver nanodots for biological applications. Chem Soc Rev 2012;41:1867–91. 32. Shang L, Dong SJ. Sensitive detection of cysteine based on fluorescent silver clusters. Biosens Bioelectron 2009;24:1569–73. 33. Henglein A Small-particle research: physicochemical properties of extremely small colloidal metal and semiconductor particles. Chem Rev 1989;89:1861–73. 34. Bian W, Ma J, Liu QL, Wei YL, Li YF, Dong C, et al. A novel 2+ phosphorescence sensor for Co ion based on Mn-doped ZnS quantum dots. J Luminesc 2013;DOI 10.1002/bio.2520 35. Lee KH, Chen SJ, Jeng JY, Cheng YC, Shiea JT, Chang HT. Fluorescence and interactions with thiol compounds of Nile Redadsorbed gold nanoparticles. J Colloid Interf Sci 2007; 307:340–8. 36. Mulvaney P, Linnert T, Henglein A. Surface chemistry of colloidal silver in aqueous solution: observations on chemisorption and reactivity. J Phys Chem 1991;95:7843–6. 37. Su YT, Lan GY, Chen WY, Chang HT. Detection of copper ions through recovery of the fluorescence of DNA-templated copper/ silver nanoclusters in the presence of mercaptopropionic acid. Anal Chem 2010;82:8566–72.
Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site.
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Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014
Revised: 11 October 2013,
Accepted: 26 October 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2613
Bovine serum albumin-confined silver nanoclusters as fluorometric probe for detection of biothiols Zhen Chen, Dongtao Lu, Zongwei Cai, Chuan Dong and Shaomin Shuang* ABSTRACT: Fluorescent bovine serum albumin-confined silver nanoclusters (BSA–AgNCs) were demonstrated to be a novel and environmentally friendly probe for the rapid detection of biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH). The sensing was ascribed to the strong affinity between the mercapto group of the biothiols and the silver nanoclusters. The fluorescence intensity of BSA–AgNCs was quenched efficiently on increasing the concentration of biothiol, corresponding with a red-shift in emission wavelength. However, the fluorescence of the silver nanoclusters was almost unchanged in the presence of other α-amino acids at 10-fold higher concentrations. By virtue of this specific response, a new, simple and rapid fluorescent method for detecting biothiols has been developed. The linear ranges for Cys, Hcy and GSH were 2.0 × 10-6 to 9.0 × 10-5 M (R2 = 0.994), 2.0 × 10-6 to 1.2 × 10-4 M (R2 = 0.996) and 1.0 × 10-5 to 8.0 × 10-5 M (R2 = 0.980), respectively. The detection limits were 8.1 × 10-7 M for Cys, 1.0 × 10-6 M for Hcy and 1.1 × 10-6 M for GSH. Our proposed method was successfully applied to the determination of thiols in human plasma and the recovery was 94.83–105.24%. It is potentially applicable to protein-stabilized silver nanoclusters in a chemical or biochemical sensing system. Copyright © John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: bovine serum albumin; silver nanoclusters; biothiols; fluorescence
Introduction
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* Correspondence to: Shaomin Shuang, Department of Chemistry and Chemical Engineering, Institute of Environmental Science, Shanxi University, Taiyuan 030006, People’s Republic of China. E-mail: smshuang@sxu. edu.cn Department of Chemistry and Chemical Engineering, Institute of Environmental Science, Shanxi University, Taiyuan 030006, People’s Republic of China
Copyright © 2014 John Wiley & Sons, Ltd.
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Noble metal nanoclusters (NCs) with distinct, unique physical, electrical and optical properties have attracted a great deal of attention in recent years for their promising applications in optical sensing, catalysis, biological labeling and imaging (1–3). Noble metal NCs consisting of a few to tens of metal atoms are of ultra-small size (approximately the Fermi wavelength of the conduction electrons), which leads to discrete energy levels and strong quantum confinement. Therefore, they show remarkable photoluminescence (1). Silver nanoclusters are more reactive than their gold analogs, and the study of AgNCs is an active issue (2–4). Because AgNCs are prone to aggregation and grow to form larger clusters or nanoparticles (NPs), a variety of stabilizers such as dendrimers (5), polyelectrolytes (6), thiolates (7), small organic molecules (8), biomacromolecules, DNA oligonucleotides (9,10) and peptides/proteins (11–13) have been used for the modification of AgNCs. Among these modifications, a recent breakthrough in the synthesis of fluorescence AgNCs, with protein as scaffolds, has received special attention (12,13). Protein-protected AgNCs have outstanding spectral and photophysical properties, combined with low toxicity and good biocamptibility, and so they hold great promise as optical probes, in biological labeling and as imaging agents (14). However, most of the recent studies focus on using a facile method to synthesize stable and highly luminescent AgNCs (13,15), few studies have involved the analytical application of protein-stabilized AgNCs. Zhou et al. (16) assemblied lysozyme-stabilized AgNCs to determine Hg2+. Yang et al. (17) proposed a spectrophotometric and colorimetric method for ascorbic acid based on the catalysis of bovine serum
albumin (BSA)-protected AgNCs. Therefore, a practical use for protein-stabilized AgNCs is desirable. Biological thiols (biothiols), such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), participate in reversible redox reactions and have important cellular functions, including detoxification and metabolism (18). A normal level of biothiols in the human body is essential and plays a vital role in maintaining healt. A Cys deficiency correlates with slow growth, hair depigmentation, edema, liver damage, skin lesions and weakness (19). An elevated Hcy level may be a sign of disorders such as cardiovascular disease and Alzheimer’s disease (20). GSH is one of the most abundant cellular biothiols, and is typically associated with beneficial antioxidant effects and might quench reactive free-radical species that can damage genetic material (21). Thus, sensitive and selective detection of these biothiols is very important in the early diagnosis of disease. To date, various methods to determine biothiols have involved highperformance liquid chromatography (HPLC) (22), mass spectrography (23), and capillary electrophoresis coupling optical and electrochemical detectors (24–26). However, these methods have several disadvantages: they require expensive and sophisticated instrumentation, complicated sample preparation and they are time-consuming. Fluorimetry is a favorable method
Z. Chen et al. with high sensitivity and selectivity compared with other methods. Several attempts have been made to use fluorescent AgNCs as the optical probe for detecting biothiols. However, limited ligand-capped fluorescent silver nanoclusters including polymer-stabilized AgNCs (27,28), DNA oligonucleotide-stabilized AgNCs (25,26) and glutathione-protected AgNCs (4) have been developed as a biothiol fluorometric probe. We try to expand the use of protein as a scaffold for AgNCs to determine biothiols using a fluorometric method. We synthesized AgNCs using BSA as the stabilizer and further developed the detection method for biothiols including Cys, Hcy and GSH. The fluorescence intensity of BSA–AgNCs was found to be quenched effectively in the presence of biothiols. However, 18 other α-amino acids (without a thiol group) might lead to negligible changes in the fluorescence intensity of the nanoclusters. This method was successfully used to detect thiols in human plasma.
Experimental
month. In addition, the cluster solution could be freeze-dried into a powder form for further storage.
Fluorescence measurements Cys, Hcy and GSH aqueous solutions at different concentrations were freshly prepared before use. A typical detection procedure was conducted as follows: 150 μL BSA–AgNCs solution was diluted to 2 mL with 0.2 mol/L PBS solutions (pH 9.00 for Cys, 8.02 for Hcy and 8.55 for GSH). Owing to the lack of quality information for BSA–AgNCs, we present the concentration of BSA–AgNCs using the quality of BSA. An appropriate volume of a 0.01 M biothiol stock solution was mixed with the BSA–AgNCs solution (1.875 mg/mL) at room temperature (22 ± 1°C) and equilibrated for 1 min. The fluorescence emission spectrum of AgNCs was recorded in the wavelength range 485–750 nm upon excitation at 465 nm. To investigate the selectivity of this assay, the influence of the other 18 α-amino acids (at a 10-fold higher concentration than Cys, Hcy and GSH) on the BSA–AgNCs was investigated. All experiments were performed in triplicate.
Chemicals Silver nitrate (AgNO3, 99%) and sodium borohydride (NaBH4, powder, 98%) were from Sigma Aldrich Chemical Co. (ST. Louis, USA). BSA (> 96%) was purchased from Shanghai Sangon Biotechnology Co. Ltd (Shanghai, China). Cys and other α-amino acids were purchased from Bei-jing Chemical Co. (Beijing, China). GSH (88%, for analysis, reduced) was obtained from ACROS ORGANICS (NJ, USA). Homocysteine (> 95%, titration) was from Sigma Aldrich Chemical Co. (ST. Louis, USA). All other reagents were of analytical reagent grade, and used as received. -1 Double-distilled water (> 18.2 MΩ cm ) from the Millipore Milli Q system (Massachusetts, USA) was used throughout.
Apparatus Fluorescence measurements were made on a F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) with a quartz cell (1 cm × 1 cm). Excitation and emission slit widths were set at 10 nm and the photomultiplier tube voltage was set at 700 V. The emission spectra were recorded in the wavelength range 485–750 nm upon excitation at 465 nm. UV/vis absorption spectra were recorded in the wavelength range 350–800 nm on a UV-3010 spectrophotometer (Hitachi). Transmission electron microscopy (TEM) images were obtained using a FEI TECNAL 20 at an accelerating voltage of 200 kV. The hydrodynamic diameter was measured by dynamic light scattering (DLS) using a Malvern Autosizer ZS90 spectrometer.
Preparation of the BSA-confined Ag nanoclusters
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BSA–AgNCs were prepared using a modification of a simple wet route modified (13). In a typical synthesis, 0.5 mL of 0.1 M AgNO3 solution was added to 10 mL water containing 250 mg BSA powder with vigorous stirring at room temperature. The mixture was left to incubate for 5 min under vigorous stirring before the dropwise addition of 0.3 mL NaOH (1 M). After stirring for 1 h at room temperature, NaBH4 solution (10 mM, freshly prepared) was added dropwised until the solution turned from colorless to reddish-brown, indicating the formation of various amounts of clusters. To collect clean and stable BSA–AgNCs, the obtained solution was then dialyzed using a 8–14 kDa dialysis bag in double-distilled water for 24 h. The BSA–AgNCs solution was kept at 4°C in the dark and the luminescence remained almost unchanged for least one
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Measurement of biothiols in human plasma samples Fresh human blood samples were obtained from the local hospital. After centrifugation at 4000rpm (2680g) for 10 min, the supernatant solution containing the proteins and amino acids was collected and used as the source of the plasma. The collected supernatant was reduced by addition of triphenylphosphine (PPh3) as a catalyst and then mixed with acetonitrile to precipitate proteins (22–27). The supernatant containing amino thiols in plasma was used for further analysis, and the amount of biothiols was estimated using a standard addition method. The plasma sample was diluted with 0.2 M PBS solution (pH 9.00) before measurement to be within the dynamic range of the proposed method. For recovery studies, known concentrations of Cys solution were added to plasma samples and the total thiol concentrations were then determined at the same condition.
Results and discussion Characteristics of BSA–AgNCs The as-prepared BSA–AgNCs were characterized by TEM, DLS, UV/vis absorption and fluorescence spectroscopy as indicated in Figs 1 and 2. As shown in Fig. 1, the UV/vis absorption spectrum of BSA–AgCNs exhibited a small broad peak around 450 nm due to quantum confinement effects, but no characteristic surface plasmon resonance peak in the range of 400–500 nm was observed, indicating the formation of nanoclusters rather than lager nanoparticles (29,30). The TEM image indicates that the BSA–AgNCs were spherical and well dispersed, with an average diameter of 2 nm, whereas the hydrodynamic diameter measured using DLS was ~ 1.75 nm (Fig. 2). The maximum fluorescence emission of BSA–AgCNs could be observed at 598 nm when excitation wavelength was 465 nm (Fig. 1, curves b and c). The inset to Fig. 1 shows that the BSA–AgNCs solution was reddish-brown in color under visible light, although it exhibited a bright red fluorescence under UV light at 365 nm. The fluorescence quantum yield (QY) of the BSA–AgNCs was calculated to be 2.6% by comparison with rhodamine 6G in ethanol (with a standard QY of 95%).
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Detection of biothiols based on BSA–AgNCs
Figure 1. UV/vis absorption spectrum (a) and fluorescence spectra (b, c) of the asprepared BSA–AgNCs. (Inset) BSA–Ag NCs under visible and UV light at 365 nm.
Optical response of BSA–AgNCs to Cys The fluorescence response of the BSA–AgNCs to Cys was investigated and the results are shown in the Fig. 3. Upon addition of 100 μM Cys, the absorption spectra had an apparent red-shift from 465 nm to ~ 495 nm; the absorption intensity showed virtually no change (Fig. 3a). The fluorescence intensity of the nanoclusters solution at 598 nm decreased significantly, corresponding with a red-shift from 598 to 617 nm (Fig. 3b). The inset in Fig. 3(b) showed that the color of the BSA–AgNCs solution changed from reddish-brown to colorless in the presence of Cys
under visible light (Fig. 3b, upper), while a weak blue fluorescence was observed under 365 nm UV light (Fig. 3b, lower). This phenomenon indicated that BSA–AgNCs as the probe could achieve naked-eye recognition. In order to study the interaction between BSA–AgNCs and biothiols, absorption spectra of BSA–AgNCs in the presence of varying concentrations of Cys were examined (Fig. S1). It was found that the interaction process could be distinguished into two parts from the absorption spectra. When the concentration of Cys varied in the range of 1–100 μM, the absorption peak of BSA–AgNCs had an obvious red-shift and the intensity was almost unchanged (Fig. S1a). This may be because Cys could penetrate the BSA protective layer and combine with the AgNCs core based on the high affinity of sulfhydryl to Ag (27). This combination influenced the optical properties of the BSA–AgNCs. When the concentration of Cys was increased to > 100 μM, the absorption peak no longer had a red-shift and gradually disappeared (Fig. S1b). This may result from the destruction of the BSA–AgNCs, which might be ascribed to oxidative etching from both the oxygen and the mercapto group (31). The sulfide in the mercapto group could donate an electron pair to the Ag atoms on the surface, which were coordinately unsaturated and therefore an unoccupied orbital existed (32). On this occasion, the added biothiols could be readily absorbed on the surface of Ag clusters by partially removing the residues from BSA. In the meantime, Fig. S2 shows that the lifetime of BSA–AgNCs did not change appreciably in the presence of Cys, which indicated that there was a change in ground state of the fluorescence BSA–AgNCs. Both the UV spectra and lifetime
Figure 2. (a) TEM image of BSA–AgNCs. (b) Hydrodynamic diameter of the as-prepared BSA–AgNCs measured with DLS.
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Figure 3. The absorption (A) and fluorescence (B) spectra of BSA–AgNCs (1.875 mg/mL) in the absence (a) and presence (b) of 100 μM Cys. (B, inset) AgNCs in the absence (1) and presence (2) of Cys (100 μM) under visible and UV light at 365 nm.
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Z. Chen et al. results were consistent with the related literature and confirmed that the biothiols were chemisorbed on the surface of AgNCs (25,32). It is well known that the redox potential of silver decreases upon absorption of nucleophiles on a particle surface (33). Therefore, it might be expected that the
redox potential of silver in the few-atom Ag clusters would show a large negative shift upon the further adsorption of biothiols. Thus, the oxidation of Ag by oxygen could be greatly facilitated and the gradual oxidation of AgNCs occurred in the presence of biothiols. Similar phenomena were observed in the absorption spectra for Hcy and GSH, as shown in Figs S3 and S4. Optimization of factors influencing the detection of biothiols Prior to the application of our fluorescence probe for the detection of biothiols, several factors that may affect the interaction of BSA–AgNCs with biothiols were optimized, such as the concentration of BSA–AgNCs, pH, incubation time and temperature. As shown in Fig. S5, the relative fluorescence intensity of the system increased gradually as the concentration of BSA–AgNCs changed from 0.5 to 1.875 mg/mL, but decreased at higher concentrations. Considering the fluorescence background and the sensitivity of the detection, 1.875 mg/mL was chosen as the optimum concentration of BSA–AgNCs. The effect of pH on the detection of biothiols by BSA–AgNCs was investigated and optimized. The fluorescence intensity of BSA–AgNCs in the absence and presence of 50 μM Cys in the pH range 3.00–12.00 is shown in Fig. S6. It is evident that the fluorescence of BSA–AgNCs had a tendency toward gradual decrease as pH increases from 3.00 to 9.00. The quenching effect is closely related to the patterns of mercapto. Mercapto of biothiol was mainly present in a protonated pattern in strong acidic media and resulted in a weak quenching effect (34). In neutral and weak basic media the deprotonated mercapto had strong affinity to the AgNCs and readily caused the fluorescence quenching of BSA–AgNCs. The maximum quenching action occurred at pH 9.00. However, quenching decreased dramatically at a pH > 9.00 because the mercapto group in Cys was oxidized to generate the disulfide bond. Therefore, an optimum pH of 9.00 was chosen for detection. A similar influence of pH could be observed for BSA–AgNCs in the presence of Hcy and GSH. In order to find optimum values for incubation time and temperature, their efects on the BSA–AgNCs-based detection
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Figure 4. Fluorescence spectra (λex = 465 nm) of BSA–AgNCs (1.875 mg/mL) in the presence of biothiol solutions at different concentrations. (a) Cys: 0, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140 μM. (b) Hcy: 0, 2, 6, 10, 15, 20, 30, 40, 60, 80, 100, 120, 140 μM. (c) GSH: 0, 2, 4, 6, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 160, 200 μM. (Insets) Relationship between (F0 – F) and concentration of biothiols.
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Figure 5. Relative fluorescence intensity of BSA–AgNCs (1.875 mg/mL) in the presence of 100 μM Cys, GSH and Hcy, and 1000 μM of various other amino acids.
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Detection of biothiols based on BSA–AgNCs Table 1. Determination of biothiols in human plasma samples based on BSA–AgNCs Plasma sample
Determined biothiols (10-4 M)
1
2.38
2
2.74
3
2.62
Added Cys (10-6 M) 2.00 4.00 2.00 4.00 2.00 4.00
of biothiols were tested. As shown in Fig. S7, the relative fluorescence intensity F0 – F increased rapidly within 1 min and no obvious change was found over time, which indicated that the interaction between BSA–AgNCs and biothiols could be carried out within 1 min. Figure S8 shows that the higher the incubation temperature, the lower the quenching efficiency. Considering the stability of the system and the simplicity of operation, all the titration experiments were carried out at room temperature. Analytical merits of BSA–AgNCs to biothiols Figure 4 shows the fluorescence spectra of BSA–AgNCs in the presence of varying amounts of Cys, Hcy and GSH. The emission at 598 nm of BSA–AgNCs was seen to be strongly quenched by biothiols, whereas an obvious red-shift in the emission wavelength was observed. As shown in Fig. 4(insets), the calibration curves for biothiols exhibited linear relationships in the range from 2.0 × 10-6 to 9.0 × 10-5 M for Cys, 2.0 × 10-6 to 1.2 × 10-4 M for Hcy and 1.0 × 10-5 to 8.0 × 10-5 M for GSH. The corresponding detection limits were 8.1 × 10-7 M for Cys, 1.0 × 10-6 M for Hcy and 1.1 × 10-6 M for GSH. Cys : F 0 –F ¼ 7:849 þ 1:554C R2 ¼ 0:994 Hcy : F 0 –F ¼ 0:009 þ 1:207C R2 ¼ 0:996 GSH : F 0 –F ¼ 10:946 þ 1:198C R2 ¼ 0:980 where F0 and F are the fluorescence intensity of BSA–AgNCs in the absence and presence of biothiols, and C presents the concentration of biothiols. We were able to confirm that the quenching ability of the three biothiols follow the order Cys ≈ Hcy > GSH. GSH exhibited relatively lower reactivity than Cys and Hcy, which might be due to steric hindrance (4,35). Here, biothiols acted as nucleophile reagents that could be chemisorbed on the surface of AgNCs (28,32). Upon addition, Cys could penetrate the BSA protective layer and be adsorbed onto the surface of AgNCs, because silver atoms have low-lying vacant d orbitals and electron pairs from the mercapto of Cys can enter vacant electron orbitals on the silver atoms (36). The interaction would partially break the AgNCs–BSA bond, which weakens the interaction between the AgNCs and BSA (37). The mercapto group of the biothiols can not only stabilize, but also destabilize silver nanoclusters (31). Because the AgNCs were only poorly stabilized with BSA, thiol-induced fluorescence quenching of BSA–AgNCs occurred (32,37).
To test the selectivity of the quenching behavior of biothiols (Cys, Hcy and GSH), the fluorescence responses of BSA–AgNCs against 18 other α-amino acids at 10-fold higher concentrations than
1.90 4.21 2.02 3.98 2.06 3.99
Recovery (%) 94.83 105.24 100.77 99.57 103.23 99.86
RSD (n = 3, %) 2.90 2.88 3.31 3.16 3.39 1.57
Cys, Hcy and GSH were investigated under the same conditions. Figure 5 shows the changes in the relative fluorescence [(F0 – F)/F0] of BSA–AgNCs that occurred after the separate addition of Cys, Hcy and GSH (100 μM) and 18 other α-amino acids (1000 μM). It is clear that the variations in fluorescence intensity of BSA–AgNCs in the presence of Cys, Hcy and GSH were strikingly larger than that of other common α-amino acids. In addition, there was no red-shift as in Cys, Hcy and GSH in the other amino acids. These results indicated that the proposed assay had high selectivity for biothiols and has potential in the practical detection of biothiols in biological samples.
Determination of biothiols in human plasma In order to investigate the feasibility of our proposed method for practical applications, we performed recovery experiments. Cys was chosen as the reference substance to test the recovery of this method because it is the main component of biothiols in human plasma and other thiols reacted with BSA–AgNCs in a similar manner. Pretreated human plasma samples were diluted with an appropriate phosphate buffer to ensure the concentration of biothiols was in the linear range and to obtain quantitative recovery of the spiked thiols. Owing to variations in the chemical composition of biological samples, a standard addition method was employed. The obtained concentrations of biothiols in three human plasmas are listed in Table 1, and were in agreement with the results in the literature (25,28). Recoveries of 94.83–105.24% of the known amount Cys in the plasma samples were obatined, thus demonstrating the reliability of BSA–AgNCs for detecting biothiols in human plasma.
Conclusions BSA–AgNC was explored as a novel signal-off fluorescent probe for simple, selective, sensitive and cost-effective detection of biothiols, such as Cys, Hcy and GSH. The fluorescence of BSA–AgNCs was quenched by biothiols because of the strong affinity between mercapto and Ag atoms. This method, with good interference immunity, was successfully used for the detection of thiols in human plasma samples. As far as we awared, protein-protected AgNCs are used first for the detection of biothiols with fast, simply, sensitive and selective features. In addition, our method has great potential for the quantitative analysis of thiol levels in biological fluids. More importantly, the proposed method is a novel application of protein-stabilized AgNCs, and provides a better understanding of the interaction between noble metal nanoclusters and protein.
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Selectivity of BSA–AgNCs for the detection of biothiols
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Measured (10-6 M)
Z. Chen et al. Acknowledgments Financial supports from the National Natural Science Foundation of China (Nos. 21175086 and 21175087), Shanxi Province Hundred Talent Project Support.
References 1. Maity P, Xie S, Yamauchi M, Tsukuda T. Stabilized gold clusters: from isolation toward controlled synthesis. Nanoscale 2012;4:4027–37. 2. Shang L, Dong SJ, Nienhaus GU. Ultra-small fluorescent metal nanoclusters: synthesis and biological applications. Nano Today 2011;6:401–18. 3. Lu Y, Chen W. Sub-nanometre sized metal clusters: from synthetic challenges to the unique property discoveries. Chem Soc Rev 2012;41:3594–623. 4. Yuan X, Tay YQ, Dou XY, Luo ZT, Leong DT, Xie JP. Glutathioneprotected silver nanoclusters as cysteine-selective fluorometric and colorimetric probe. Anal Chem 2013;85:1913–19. 5. Xu H, Suslick KS. Sonochemical synthesis of highly fluorescent Ag nanoclusters. ACS Nano 2010;4:3209–14. 6. Díez I, Pusa M, Kulmala S, Jiang H, Walther A, Goldmann AS, et al. Color tunability and electrochemiluminescence of silver nanoclusters. Angew Chem Int Ed 2009;48:2122–5. 7. Rao TUB, Pradeep T. Luminescent Ag7 and Ag8 clusters by interfacial synthesis. Angew Chem Int Ed 2010;49:3925–9. 8. Muhammed MAH, Aldeek F, Palui G, Trapiella-Alfonso L, Mattoussi H. Growth of in situ functionalized luminescent silver nanoclusters by direct reduction and size focusing. ACS Nano 2012;6:8950–61. 9. Petty JT, Fan CY, Story SP, Sengupta B, Iyer AS, Prudowsky Z, et al. DNA encapsulation of 10 silver atoms producing a bright, modulatable, near-infrared-emitting cluster. J. Phys. Chem. Lett. 2010;1:2524–29. 10. Martinez JS, Sharma J, Yeh HC, Yoo H, Werner JH. Silver nanocluster aptamers: in situ generation of intrinsically fluorescent recognition ligands for protein detection. Chem Commun 2011;47:2294–96. 11. Adhikari B, Banerjee A. Short-peptide-based hydrogel: a template for the in situ synthesis of fluorescent silver nanoclusters by using sunlight. Chem Eur J 2010;16:13698–705. 12. Guo CL, Irudayaraj J. Fluorescent Ag clusters via a protein-directed approach as a Hg(II) ion sensor. Anal Chem 2011;83:2883–9. 13. Mathew A, Sajanlal PR, Pradeep T. A fifteen atom silver cluster confined in bovine serum albumin. J Mater Chem 2011;21:11205–12. 14. Xavier PL, Chaudhari K, Baksi A, Pradeep T. Protein-protected luminescent noble metal quantum clusters: an emerging trend in atomic cluster nanoscience. Nano Rev 2012;3:14767. 15. Anand U, Ghosh S,Mukherjee S. Toggling between blue- and redemitting fluorescent silver nanoclusters. J Phys Chem Lett 2012;3:3605–9. 16. Zhou TY, Huang YH, Li WB, Cai ZM, Luo F, Yang CJ, et al. Facile synthesis of red-emitting lysozyme-stabilized Ag nanoclusters. Nanoscale 2012;4:5312–15. 17. Yang XH, Ling J, Peng J, Cao QE, Wang L, Ding ZT, et al. Catalytic formation of silver nanoparticles by bovine serum albumin protected-silver nanoclusters and its application for colorimetric detection of ascorbic acid. Spectrochim Acta A 2013;106:224–30. 18. Chen X, Zhou Y, Peng X, Yoon J. Fluorescent and colorimetric probes for detection of thiols. Chem Soc Rev 2010;39:2120–35. 19. Shahrokhian S Lead phthalocyanine as a selective carrier for preparation of a cysteine-selective electrode. Anal Chem 2001;73:5972–8.
20. Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, D’Agostino RB, et al. Plasma homocysteine as a risk factor for dementia and Alzheimer’s disease. N Engl J Med 2002;346:476–83. 21. Hong R, Han G, Fernández JM, Kim BJ, Forbes NS, Rotello VM. Glutathione-mediated delivery and release using monolayer protected nanoparticle carriers. J Am Chem Soc 2006;128:1078–9. 22. Janes L, Lisjak K, Vanzo A. Determination of glutathione content in grape juice and wine by high-performance liquid chromatography with fluorescence detection. Anal Chim Acta 2010;674:239–42. 23. Satterfield MB, Sniegoski LT, Welch MJ, Nelson BC, Pfeiffer CM. Comparison of isotope dilution mass spectrometry methods for the determination of total homocysteine in plasma and serum. Anal Chem 2003;75:4631–8. 24. Chen G, Zhang L, Wang J. Miniaturized capillary electrophoresis system with a carbon nanotube microelectrode for rapid separation and detection of thiols. Talanta 2004;64:1018–23. 25. Han BY, Wang EK. Oligonucleotide-stabilized fluorescent silver nanoclusters for sensitive detection of biothiols in biological fluids. Biosens Bioelectron 2011;26:2585–9. 26. Thiagarajan S, Umasankar Y, Chen SM. Functionalized multi walled carbon nanotubes nano biocomposite film for the amperometric detection of L-cysteine. Nanosci Nanotechnol 2010;10:702–10. 27. Shang L, Yin JY, Li J, Jin LH, Dong SJ. Gold nanoparticle-based nearinfrared fluorescent detection of biological thiols in human plasma. Biosens Bioelectron 2009;25:269–74. 28. Zhang N, Qu F, Luo HQ, Li NB. Sensitive and selective detection of biothiols based on target-induced agglomeration of silver nanoclusters. Biosens Bioelectron 2013;42:214–18. 29. Liu GL, Feng DQ, Mu XY, Zheng WJ, Chen TF, Qi L, et al. DNAfunctionalized silver nanoclusters as a chemopalette: tunable fluorescence for turn-on detection of cysteine. J Mater Chem B 2013;1:2128–31. 30. Wen T, Qu F, Li NB, Luo HQ. Polyethyleneimine-capped silver nanoclusters as a fluorescence probe for sensitive detection of hydrogen peroxide and glucose. Anal Chim Acta 2012;749:56–62. 31. Choi S, Dickson RM, Yu JH. Developing luminescent silver nanodots for biological applications. Chem Soc Rev 2012;41:1867–91. 32. Shang L, Dong SJ. Sensitive detection of cysteine based on fluorescent silver clusters. Biosens Bioelectron 2009;24:1569–73. 33. Henglein A Small-particle research: physicochemical properties of extremely small colloidal metal and semiconductor particles. Chem Rev 1989;89:1861–73. 34. Bian W, Ma J, Liu QL, Wei YL, Li YF, Dong C, et al. A novel 2+ phosphorescence sensor for Co ion based on Mn-doped ZnS quantum dots. J Luminesc 2013;DOI 10.1002/bio.2520 35. Lee KH, Chen SJ, Jeng JY, Cheng YC, Shiea JT, Chang HT. Fluorescence and interactions with thiol compounds of Nile Redadsorbed gold nanoparticles. J Colloid Interf Sci 2007; 307:340–8. 36. Mulvaney P, Linnert T, Henglein A. Surface chemistry of colloidal silver in aqueous solution: observations on chemisorption and reactivity. J Phys Chem 1991;95:7843–6. 37. Su YT, Lan GY, Chen WY, Chang HT. Detection of copper ions through recovery of the fluorescence of DNA-templated copper/ silver nanoclusters in the presence of mercaptopropionic acid. Anal Chem 2010;82:8566–72.
Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site.
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