An anti-galvanic reduction single-molecule fluorescent probe for detection of Cu(II)

Abstract

An anti-galvanic reduction method using [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ nanoclusters as the metal ion probe for detecting Cu²⁺ is reported here. We found [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ can reduce more reactive Cu²⁺ with a quantitative relationship. The single molecule fluorescence imaging shows that Ag₆₂ NCs could be used as a single molecule probe for detecting Cu²⁺.

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Fluorescent nanomaterials have recently attracted increasing attention in optical sensing¹ and biolabeling.² Among these materials, much attention has been paid to noble metal clusters (Ag, Au) consisting of a few to dozens of metal atoms.³ Several ultra-small fluorescent nanoparticles (NPs) of noble metal such as gold and silver have been synthesized by using different ligands like DHLA,⁴ dendrimers,⁵ polymers,⁶ DNA,⁷ peptides and proteins.⁸ They are particularly stable and can exhibit fluorescence ranging from the visible to the near infrared region depending on the number of metal atoms.⁹ Meanwhile, Single-nanoparticle imaging has been widely used to elucidate the optical properties of nanoparticles by correlating the optical responses with their well-defined structures and morphologies.¹⁰ However, up to now, the applications of these NPs in analyzing and sensing are mainly based on the fluorescence resonance energy transfer (FRET) mechanism with NPs used as the reporting groups, which means an organic fluorophores would be connected as a receptor group.¹¹ Now, researchers pay more attention to the direct detection of the target ions,¹² but a fundamental understanding of its process is still far from complete.

Recently, Wu reported an anti-galvanic reduction (AGR) in which the reducing activity of the ultra-small silver nanoparticles (less than 3 nm) is drastically enhanced and they can even reduce some more reactive metal ions (e.g. reducing Cu²⁺ to Cu⁰).¹³ It is very interesting to answer questions such as: (i) how does the fluorescence change during the AGR process? (ii) Could this changing process meet the requirements of ion recognition, such as a fast, accurate and quantitative determination?

Herein, we synthesized [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ NCs referring to the reported method with the definite structure in the proper size,¹⁴ and the ligand species of [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ also meet the requirements of the AGR process. Meanwhile, we found that the Cu²⁺ ions can be reduced by [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ NCs with a quantitative relationship. Both the optical properties and the single-molecule fluorescence of [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ NCs are studied. This discovery will help us design nanocluster sensors for detecting metal ions.

Ion selectivity study was performed in MeCN : H₂O = 1 : 1 solution (as shown in Fig. 1). The data also illustrate that Co²⁺, Cd²⁺, Mn²⁺, Hg²⁺, Fe²⁺, Ni²⁺, and Cu²⁺ cause negligible interference to the fluorescence of [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ while Hg²⁺ had a slight effect. The aqueous solutions of [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ with the addition of different ions clearly demonstrated that Cu²⁺ changed the color of the solution dramatically, while other ions caused no perceivable changes. This result suggests that AGR process is too limited to reduce metal ions which have much higher reducing activity (e.g. Pb²⁺ and Fe³⁺).

Fig. 1 Fluorescence responses of [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ upon addition of various metal ions. Experimental conditions: 500 nM [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ in MeCN : H₂O = 1 : 1. The light bars represent the fluorescence emission of the solution of [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ and 1 equiv. of the cation of interest. The dark bars show the fluorescence change that occurs upon addition of 1 equiv. of Cu(II) to the solution containing [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺and the cation (λ_ex = 497 nm. λ_em = 598 nm).

It is imperative to confirm whether the reaction of [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ and Cu²⁺ ions is quantitative, which is a prerequisite for the quantitative measurement. To illustrate this, absorption (Fig. 2a) and fluorescence (Fig. 2b) spectra were collected in MeCN : H₂O = 1 : 1 solution. As shown in Fig. 2a, the absorption spectrum of [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ exhibits two shoulders in the UV region and a distinct absorption peak in the visible region (543 nm), which is consistent with the reported results.¹⁴ Upon addition of Cu²⁺, the absorption peak at 543 nm shifted to 534 nm gradually, with a color change of the solution from red to yellow. After addition of Cu²⁺, the emission at 598 nm was quenched (Fig. 2b). The change of fluorescence is not the same as Au NCs. Li reported that the fluorescence of Au₁₆@BSA had a blue shift and was enhanced after the addition of Ag⁺.^12d The mechanism of the fluorescence quenching by Cu²⁺ ions still needs to be studied in future work. It should be noted that the minimal fluorescence intensity emerges when the [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺/Cu²⁺ molar ratio reaches 1 with a detection limit of 0.5 nM at a signal-to-noise ratio (S/N) of 3. But with the increase of the concentration of [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ NCs (e.g. 50 μM), more copper ions will be required to render the fluorescence completely quenched. We deduced that one [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ molecular could react with more than one copper ions, thereby complete quenching fluorescence of [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ needs a large amount of copper ions.

Fig. 2 (a) The UV-vis spectra of [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ (500 nM) in the presence of Cu²⁺. (b) Emission spectra of [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ (500 nM) with the excitation at 497 nm upon titration with Cu²⁺ in the MeCN : H₂O = 1 : 1. The inset digital photo was taken at a much higher concentration (0.25 mM).

XPS is employed to determine whether the Cu²⁺ is reduced to Cu⁰ or not. After an equivalent of Cu²⁺ was added, the [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ NCs were washed with deionized water and treated by vacuum drying before XPS measurement. The Ag 3d peak (Fig. 3a) in [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺–Cu²⁺ (367.32 eV) was observed on the oxidation side comparing to that of [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ NCs (367.45 eV), indicating that the Ag atoms in Ag₆₂–Cu²⁺ are positively charged. In contrast, the Cu 2p peak (Fig. 3b) in Ag₆₂–Cu²⁺ is 932.7 eV, which is assigned to Cu 2p_3/2, indicating that the incorporated copper is neutral (932.6 eV for pure copper Cu 2p_3/2). The observation implies that copper ions can be reduced by more inert Ag NCs.

Fig. 3 (a) Binding energies of Ag 3d in [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ NCs and Ag₆₂–Cu²⁺, respectively. (b) Binding energies of Cu 2p in Ag₆₂–Cu²⁺.

TEM can effectively determine whether the size of clusters has changed after reaction, in particular whether the incorporation of Cu²⁺ decomposes the clusters. As shown in Fig. S3a,† the TEM images reveal that the synthesized [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ NCs are in high monodispersity. After an equimolar amount of Cu²⁺ was added (Fig. S3b†), no significant size change was observed, which indicates that the fluorescence quenching is not induced by the decomposition or size growth of the [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ NCs.

MALDI-TOF-MS analysis is further carried out to measure the molecular weight changes of the [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ NCs. Unlike the most thiolate-protected Au NCs (e.g. Au₂₅, Au₃₈), thiolate-protected [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ NCs didn't display a single peak at 9958 Da (Fig. S1†), but a broad peak around 9500 Da, instead. After adding one equivalent of Cu²⁺, the broad peak slightly shifted to the higher mass, suggesting that Cu is not replacing the Ag atoms in the NCs, but binding to the silver core. Further increasing copper ions until reaching Ag₆₂ : Cu²⁺ = 1 : 4, the clusters had decomposed, which also proved from the side, thus a high concentration of [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ requires a greater amount of Cu²⁺ ions to make the fluorescence completely quenched.

The performance of [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ NCs in single-molecule applications was assessed using the previously described immobilization and imaging methods¹⁵ (Fig. 4). The single-molecule experiments were carried out with a confocal fluorescence microscope, using a 720 nm laser as the two-photon excitation source. The confocal fluorescence images of [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ exhibit many bright spots (Fig. 4a), whereas the [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ treated with 1 equiv. of Cu²⁺ shows no bright spots (Fig. 4b). The extent of surface immobilization for both NCs are indeed similar, thus the difference can only be attributed to the addition of Cu²⁺ ions which quench the fluorescence of the [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ NCs. This result enlightens us that Ag NCs hold potential in single molecule detection.

Fig. 4 Fluorescence images of immobilized [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ NCs (a) and after addition of 1 equiv. Cu²⁺ (b) with pseudo-colors.

In summary, we synthesized [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ NCs to reduce more reactive Cu²⁺ metal ions. The UV-vis and fluorescence spectra suggested that one copper(II) metal ion can quench the fluorescence of one [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ nanocluster. The response mechanism was revealed in detail by means of MALDI-TOF mass spectra, XPS and TEM. In addition, single molecule fluorescence imaging illustrated that [Ag₆₂S₁₃(SBu^t)₃₂]⁴⁺ NCs can be used as a single molecule probe for detecting Cu²⁺. More broadly, this finding gives exciting results for the possible development of a series of new-generation probes for detection of metal ions with the AGR process. A set of single-molecule fluorescence probes with such properties are expected to find widespread applicability as tools in biological studies that utilize fluorescence.

We acknowledges financial support by NSFC (20871112, 21072001, 21372006), Chang Jiang Scholars Program and the Scientific Research Foundation for Returning Overseas Chinese Scholars, State Education Ministry and Ministry of Human Resources and Social Security, Anhui Province International Scientific and Technological Cooperation Project, 211 Project of Anhui University.

Notes and references

1. (a) J. J. Shi, Y. F. Zhu, X. R. Zhang, W. R. G. Baeyens and A. M. Garcia-Campana, TrAC, Trends Anal. Chem., 2004, 23, 351; (b) G. Aragay, J. Pons and A. Merkoci, Chem. Rev., 2011, 111, 3433; (c) G. Q. Wang, Y. Q. Wang, L. X. Chen and J. Choo, Biosens. Bioelectron., 2010, 25, 1859.
2. (a) T. Jamieson, R. Bakhshi, D. Petrova, R. Pocock, M. Imani and A. M. Seifalian, Biomaterials, 2007, 28, 4717; (b) X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir and S. Weiss, Science, 2005, 307, 538; (c) M. Mahmoudi, V. Serpooshan and S. Laurent, Nanoscale, 2011, 3, 3007.
3. (a) C. J. Lin, C.-H. Lee, J.-T. Hsieh, H.-H. Wang, J. K. Li, J.-L. Shen, W.-H. Chang, H.-I. Yeh and W. H. Chang, J. Med. Biol. Eng., 2009, 29, 276; (b) L. Shang, R. M. Dörlich, V. Trouillet, M. Bruns and G. U. Nienhaus, Nano Res., 2012, 5, 531; (c) M. A. H. Muhammed, S. Ramesh, S. S. Sinha, S. K. Pal and T. Pradeep, Nano. Res., 2008, 1, 333; (d) J. Xie, Y. Zheng and J. Y. Ying, J. Am. Chem. Soc., 2009, 131, 888; (e) X. Yuan, Q. Yao, Y. Yu, Z. Luo, X. Dou and J. Xie, J. Phys. Chem. Lett., 2013, 4, 1811.
4. L. Shang, N. Azadfar, F. Stockmar, W. Send, V. Trouillet, M. Bruns, D. Gerthsen and G. U. Nienhaus, Small, 2011, 7, 2614.
5. (a) J. Zheng and R. M. Dickson, J. Am. Chem. Soc., 2002, 124, 13982; (b) J. Zheng, J. T. Petty and R. M. Dickson, J. Am. Chem. Soc., 2003, 125, 7780.
6. L. Shang and S. J. Dong, Chem. Commun., 2008, 1088.
7. R. Zhou, M. Shi, X. Chen, M. Wang and H. Chen, Chem. – Eur. J., 2009, 15, 4944.
8. (a) W.-Y. Chen, G.-Y. Lan and H.-T. Chang, Anal. Chem., 2011, 83, 9450; (b) H. Kawasaki, K. Hamaguchi, I. Osaka and R. Arakawa, Adv. Funct. Mater., 2011, 21, 3508; (c) T.-H. Cheng and W.-L. Tseng, Small, 2012, 8, 1912.
9. (a) X. Wen, P. Yu, Y.-R. Toh and J. Tang, J. Phys. Chem. C, 2012, 116, 11830; (b) J. Zheng, P. R. Nicovich and R. M. Dickon, Annu. Rev. Phys. Chem., 2007, 57, 409; (c) J. Zheng, C. Zhou, M. Yu and J. Liu, Nanoscale, 2012, 4, 4073.
10. (a) T. Funatsu, Y. Harada, M. Tokunaga, K. Saito and T. Yanagida, Nature, 1995, 374, 555; (b) V. Vukojevic, M. Heidkamp, Y. Ming, B. Johansson, L. Terenius and R. Rigler, Proceedings of the National Academy of Sciences, 2008, 105, 18176; (c) T. Ha and P. Tinnefeld, Annu. Rev. Phys. Chem., 2012, 63, 595; (d) S. K. Yang, X. Shi, S. Park, T. Ha and S. C. Zimmerman, Nat. Chem., 2013, 5, 692.
11. (a) P. C. Ray, G. K. Darbha, A. Ray, J. Walker and W. Hardy, Plasmonics, 2007, 2, 173; (b) F. Liu, J. Y. Choi and T. S. Seo, Biosens. Bioelectron., 2010, 25, 2361; (c) E. Boisselier and D. Astruc, Chem. Soc. Rev., 2009, 38, 1759.
12. (a) J. Xie, Y. Zheng and J. Y. Ying, Chem. Commun., 2010, 46, 961; (b) M. Wang, Z. Wu, J. Yang, G. Wang, H. Wang and W. Cai, Nanoscale, 2012, 4, 4087; (c) Z. Wu, M. Wang, J. Yang, X. Zheng, W. Cai, G. Meng, H. Qian, H. Wang and R. Jin, Small, 2012, 8, 2028; (d) H.-W. Li, Y. Yue, T.-Y. Liu, D. Li and Y. Wu, J. Phys. Chem. C, 2013, 117, 16159.
13. Z. Wu, Angew. Chem., Int. Ed., 2012, 51, 2934.
14. G. Li, Z. Lei and Q.-M. Wang, J. Am. Chem. Soc., 2012, 132, 17678.
15. W. Luo, K. He, T. Xia and X. Fang, Anal. Bioanal. Chem., 2013, 405, 43.

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Footnote

† Electronic supplementary information (ESI) available: Experimental procedures, TEM, XPS and MALDI-TOF. See DOI: 10.1039/c3ra46877a

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