A visual physiological temperature sensor developed with gelatin-stabilized luminescent silver nanoclusters

Author’s Accepted Manuscript A visual physiological temperature sensor developed with gelatin-stabilized luminescent silver nanoclusters† Jing Lan, Hong Yan Zou, Ze Xi Liu, Ming Xuan Gao, Bin Bin Chen, Yuan Fang Li, Cheng Zhi Huang www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(15)00406-3 http://dx.doi.org/10.1016/j.talanta.2015.05.042 TAL15634

To appear in: Talanta Received date: 13 March 2015 Revised date: 15 May 2015 Accepted date: 18 May 2015 Cite this article as: Jing Lan, Hong Yan Zou, Ze Xi Liu, Ming Xuan Gao, Bin Bin Chen, Yuan Fang Li and Cheng Zhi Huang, A visual physiological temperature sensor developed with gelatin-stabilized luminescent silver nanoclusters†, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.05.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A visual physiological temperature sensor developed with gelatin-stabilized luminescent silver nanoclusters†

Jing Lana, Hong Yan Zoua, Ze Xi Liua, Ming Xuan Gaob, Bin Bin Chenb, Yuan Fang Lib, Cheng Zhi Huang a, b, * a

Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest

University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China b

College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China

Abstract A visual physiological temperature sensor was successfully developed with newly hydrothermally prepared fluorescent silver nanoclusters (AgNCs) at room temperature using gelatin as the protective and reducing agent. The as-prepared gelatin-stabilized AgNCs was water-soluble, uniform and exhibited a narrow distribution with an average size of 1.16 nm, showing a maximum emission band at 552 nm (2.45 eV) when excited at 445 nm (2.79 eV). The large Stokes shift of 110 nm of the gelatin-stabilized AgNCs makes it actually applicable with very low background and light scattering interferences. It was found that the as-prepared gelatin-stabilized AgNCs is temperature-sensitive over the range from 5 oC to 45 oC, and thus a visual physiological temperature sensor could be developed with the gelatin-AgNCs as under the irradiation of visible light. Key words: Temperature sensor; Fluorescence; Gelatin; Ag nanoclusters

* Corresponding author. Tel.: +86-23-68254659; Fax: +86-23-68367257, E-mail address: [email protected] (C. Z. Huang)

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1. Introduction

Temperature

is

one

of

the

most

important

physical

factors

for

the

temperature-dependent systems, such as environment and organism. In life system, the temperature is closely related to the physiological state, and any change of temperature could prompt our bodies to suffer from some diseases. Up to now, numerous temperature sensors have been developed and widely applied in environmental temperature measurement, biomedical sensing, and thermal monitoring of microprocessors. [1] Of these sensors, optical temperature sensing based on the luminescence properties (e.g., excited state lifetime or emission intensity) is a versatile technique to monitor the local temperature. [2] Quantum dots (QDs), [3] inorganic phosphors, [2] and organic dyes [4] have been employed for the temperature sensing and indicating. However, the further applications of these luminescent materials in environment or organism are limited because the fluorescence emissions at these cases need stable illuminated and non-toxic fluorescence probes.

Fluorescent noble metal nanoclusters (NCs, i.e., AuNCs, AgNCs, and PtNCs), consisting of several to tens of metal atoms and possessing size comparable to the Fermi wavelength of electrons, have been the subject of intense research for their widely potential applications in bioimaging, [5，6] biosensing, [7-9] and catalysts [10, 11]. Among these clusters, low-nuclearity AgNCs are particularly attractive candidates for their low toxicity and potential to combine small size and potentially ultrabright emission in a variety of scaffolds. [12] Up to now, a lot of efforts have

2

been made to direct the synthesis of fluorescent and water-stable AgNCs with the help of various stabilizers, including polyelectrolytes, [13-15] thiolates, [16, 17] and biomolecules. [18-21] Especially, proteins or peptides have attracted increasing attention in the synthesis of fluorescent AgNCs due to their excellent biocompatibility and abundant functional groups. [20,21]

Herein, we firstly report a novel, rapid one-step synthesis of fluorescent AgNCs starting from Ag+ using gelatin as protective and reducing agent, the as-prepared gelatin-stabilized AgNCs have a Stokes shift of 110 nm, which is very large and makes the gelatin-stabilized AgNCs makes it actually applicable with very low background and light scattering interferences. In principle, Ag+ is reduced to Ag0 by the phenolic group of tyrosine under alkaline conditions and the formed AgNCs are captured by the -SH group of cysteine. [21] Gelatin significantly restricts the size of AgNCs during the nucleation and growth processes, [22] and the application of gelatin for AgNCs preparation prompts an new easy one-step synthesis route without any additional reagents compared with previous reports, [23] wherein green-emitting AgNCs have successfully prepared with dihydrolipoic acid (DHLA) as an etching ligand to remove the Ag atom on the surface of gelatin-protected Ag nanoparticles (AgNPs).

Noteworthily,

the

as-prepared

gelatin-AgNCs

could

respond

to

physiological temperatures with reversibly and sensitively.

2. Experimental

2.1. Materials. 3

Gelatin from bovine skin (Type B) was purchased from Dingguo Changsheng Biotechnology Co., Ltd (Beijing, China) and AgNO3 was obtained from Shanghai Shenbo Chemical Co., Ltd. (Shanghai, China). Other reagents were of analytical reagent grade. Mili-Q purified water (18.2 M cm) was used to prepare solutions throughout the experiment.

2.2. Apparatus The fluorescence spectroscopy and absorption spectroscopy were performed with an F-2500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) and a UV-3600 spectrophotometer (Hitachi, Tokyo, Japan), respectively. A high resolution transmission electron microscope (Tecnai G2 F20 S-TWIN, FEI Company, USA) with an accelerating voltage of 200 kV was used to record the high-resolution TEM (HRTEM) images. Atomic force microscopy (AFM) images were recorded on a Dimension Icon Scan Asyst atomic force microscope (Bruker Co.) Elemental and functional group analysis were carried out using an ESCALAB 250 X-ray photoelectron spectrometer (XPS) and a FTIR-8400S Fourier transform infrared spectrometer (FTIR, Toyota, Japan), respectively. LabRAM HR800 Laser confocal Raman spectrometer was used to record the Raman spectrum of nature gelatin and gelatin-AgNCs. An FL-TCSPC fluorescence spectrophotometer (Horiba Jobin Yvon Inc., France) measured the fluorescence life time of gelatin-AgNCs. Dynamic laser light scattering (ZEN3600, Malvern) was used to characterize the zeta potential on the surface of AgNCs.

4

2.3 The synthesis of luminescent gelatin-AgNCs

Gelatin-AgNCs were efficiently synthesized by one-pot. Briefly, 20 mg/mL of gelatin was mixed with 30 mM of freshly prepared AgNO3 and the aqueous solution was under vigorous magnetic stirring in the dark at 25 oC for 5 min. After that, 1 M of NaOH was added to adjust the pH of solution approximately to 12 and the mixture was kept stirring at 25 oC for 4 h, which was further purified through centrifugation at 10000 rpm for 20 min and stored at 4 oC for further use. The solid AgNCs was prepared by freezing at -80 oC and dried under vacuum for cytotoxicity investigation and characterization by Fourier transform infrared spectroscopy.

2.4 Cytotoxicity investigation of gelatin-AgNCs

Human epidermoid cancer cells (Hep-2) were used to investigate the cell viability of the as-prepared AgNCs through CCK-8 method. Briefly, 100 L of Hep-2 cells, approximately 2×106 cells per mL in Roswell Park Memorial Institute 1640 medium (RPMI 1640), were plated into a 96-well plate and cultured at 37 oC, 5% CO2 for 24 h. Then, 100 L of RPMI 1640 containing 10 L of AgNCs at different concentrations (0.01mg/mL, 0.05mg/mL and 0.1 mg/mL) replaced the culture medium for culturing Hep-2 cells. After 24 h incubation, the culture medium was removed from every cell well, which was washed with PBS buffer twice and contained 90 L RPMI 1640. Finally, the cells were further incubated for 1 h and the optical density (OD) of the mixture was measured at 450 nm with a Microplate Reader Model. The following equation was used to estimate the cell viability (Vcell): 5

VCell (%) 

ODtreated  ODblank ODcontrol  ODblank

(1)

Wherein, the ODtreated and ODcontrol were obtained in the presence and absence of AgNCs, respectively, and ODblank could be obtained only in the presence of PBS buffer.

2.5 Temperature sensing using gelatin-AgNCs The temperature sensing based on gelatin-AgNCs was as follows. Typically, 400 L of as-purified AgNCs was put into a 1.5 mL of Eppendorf (Ep) tube. After incubated in a series of temperature, increasing from 5 oC to 45 oC, for 10 min, the fluorescence spectra emitted at 552 nm and absorption spectra at 425 nm of AgNCs were recorded on an F-2500 fluorescence spectrophotometer and UV-3600 spectrophotometer, respectively.

3. Results and Discussion

3.1 Synthesis and characterizations of as-prepared gelatin-AgNCs

As depicted in Scheme 1, gelatin, AgNO3, and NaOH were added into one pot and continuous stirred at 25 oC for 4 h. Ag+ could be reduced to Ag0 by the phenolic group of tyrosine under alkaline conditions and the AgNCs were captured by the -SH group, released under alkaline conditions, of Cysteine. [21] No fluorescent AgNCs could be obtained without gelatin, or NaOH (Fig. S1, ESI†). The as-prepared AgNCs were yellowish-brown under daylight and yellow under 365 nm UV lamp, respectively

6

(Inset of Fig. 1A). As we could see, the AgNCs showed an absorption band over 260 nm to 700 nm with an obvious peak at 425 nm (Fig. 1A). It was interesting that the emission showed significantly red shift when excited with the light beam of wavelength lower than 440 nm, and the red shift greatly reduced when the excitation wavelength was higher than 440 nm (Fig 1B), indicating that the distribution of as-prepared AgNCs was very uniform. [24] When excited with 445 nm, the gelatin-AgNCs showed a strong fluorescence peak at 552 nm with a nearly 110 nm Stokes shift, which implied that they could be used as excellent fluorescent nanoprobes for chemo/biosensing and imaging. [25] The average fluorescence life time of AgNCs was calculated as 0.38 ns (Fig.S2, Table S1, ESI†), indicating the luminescence mechanism was most likely the radioactive recombination nature of excitations. [26, 27] The uniform distribution of the gelatin-AgNCs could be further identified by measuring the morphology of AgNCs. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) as shown in Fig. 2A and 2B implied that the AgNCs were mono-dispersed and nanospherical with a narrow size and height distribution. The average size of gelatin-AgNCs was 1.16 nm with a lattice spacing of 0.35 nm (Upper left inset of Fig. 2A). The height distribution (Fig. S3, ESI†) of gelatin-AgNCs was uniform and approximately 0.5-1.6 nm, which was close to the diameter of AgNCs. What’s more, the zeta potential, measured with dynamic laser light scattering (DLS), was approximately -25.0 mV (Fig. S4, ESI†), revealing that the gelatin-AgNCs were negatively charged on their surface because of the capping

7

agent gelatin. The X-ray photoelectron spectrometer (XPS) survey spectra of gelatin-AgNCs implied the presence of the expected elements, including Na, O, N, Ag, C, and S, which were derived from NaOH and gelatin (Fig. S5A, ESI†). As shown in Fig.2C, the binding energy of 367.72 eV and 373.77 eV were assigned to the Ag 3d5/2 and Ag 3d3/2 of AgNCs, respectively, while the S 2p peak (Fig. S5B, ESI†) at 162.28 eV and 160.88 eV indicated the interaction of AgNCs and gelatin through the covalent binding of Ag-S. The weakened of the gelatin S-S stretching frequency located at 527 cm-1 in the Raman spectra also demonstrated the interaction of Ag and S (Fig. S6, ESI†). [25] In addition, the Fourier transform infrared (FTIR) spectra of denatured gelatin under pH 12 in Fig. 2D displayed the characteristic band of -SH at 2457 cm-1, while the FTIR spectra of natural gelatin and gelatin-AgNCs did not present, indicating that the AgNCs were stabilized by gelatin via Ag-S interaction. [28]

3.2 Stability of the as-prepared gelatin-AgNCs

It was worth mentioning that the gelatin-AgNCs showed good properties of water solubility, photostability, and ion strengths resistance because of abundant functionalities such as -OH, -NH2, -COOH, and Ag-S (Fig. S7, ESI†). Meanwhile, except Hg2+ could weakly quench the fluorescence of AgNCs via the d10-d10 metallophilic interaction (5d10(Hg2+)-4d10(Ag+), [20] other common metal ions such as Ca2+, Cr3+, Mn2+, Ni+, Cd2+, Co2+, K+, Na+, Pb2+, Zn2+ scarcely exerted influence on the fluorescence of gelatin-AgNCs even the concentration was up to 125 mM (Fig. S8,

8

ESI†). What’s more, the as-prepared gelatin-AgNCs had a low toxicity even the concentration was 0.1 mg/mL (Fig. S9, ESI†), indicating that the AgNCs hold a potential to be applied to in vivo.

3.3 The temperature sensing by using gelatin-AgNCs.

The fluorescence of gelatin-AgNCs was temperature-sensitive in a wide region. It was found that the fluorescence intensity, located at 552 nm, exhibited a monotonous decrease with the increase of temperature (Fig. 3A), indicating that the gelatin-AgNCs could be used as a fluorometric sensor to monitor and indicate temperature. The decreased fluorescence emission showed a linear correlation with temperature over the range from 5 oC to 45 oC. The linearity could be fitted with I=-22.36 T (oC) + 2064.55 with a related coefficient r of 0.999, wherein I was the fluorescence intensity of gelatin-AgNCs at 552 nm with different temperature (Fig. 3B). This decreased emission with the increase of temperature could be recovered if the temperature reduced, and the reversible process could experience 4 cycles between 45 oC and 5 oC with a little decrease of fluorescence intensity, which was resulted from the oxidation of AgNCs when the temperature increased (Fig. S10, ESI†). Meanwhile, the fluorescence of gelatin-AgNCs under 365 nm UV lamp decreased with the increase of temperature (Insert of Fig. 3B). What’s more, we also observed that the color of solution darkened with the increase of temperature (Fig. S11A, ESI†) and the absorbance at 425 nm showed a linear relationship with temperature over the range from 7 oC to 45 oC (Fig. S11B, ESI†), indicating that the colorimetric monitoring of

9

temperature also could be achieved. In order to clearly understand the temperature-dependent fluorescence emission of the as-prepared AgNCs, the Arrhenius plot of fluorescence intensity of gelatin-AgNCs was drawn. As shown in Fig. 4A, we could deduce an equation fitted as 

0 . 0 0 5 3 T 0 . 0 1 5

I  1 9 1 9 .e8 5

(2)

Where I was the fluorescence intensity and T (oC) was the absolute temperature. With the empirical equation expressed as, [29] I0  R  K BaT  1  ( )e I 0 E

(3)

wherein Rand were the fluorescence life times of gelatin-AgNCs at T (K) and T0 (K), respectively. Accordingly, we could calculate the activation energy, which was 418.9 meV and larger than that of Au10@histidine, [29] suggesting that decreased surface/defect states occurred due to protection and stabilization of gelatin as well as reasonable high fluorescence quantum efficiency at low temperature. [29] Similar to other temperature-sensitive metal nanoclusters, [30, 31] the lowering of fluorescence emission intensity of gelatin-AgNCs with the increase of temperature could be due to enhanced internal conversion. [32] The thermal energy aided in internal conversion, where electronic energy was converted to the vibrational energy of the clusters shifting the equilibrium towards nonradiative decay process, [33] which was verified by the decrease of fluorescence life time from 0.96 ns at 5 oC to 0.38 ns at 25 oC (Fig. 4B).

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4. Conclusions

In summary, we have successfully established an efficient and facile synthesis strategy to prepare fluorescent AgNCs using gelatin as protective and reducing agent for the first time. The whole procedure of preparing AgNCs did not introduce any other extra agents and could be completed within 4 h at room temperature. The gelatin-AgNCs owned excellent properties of water-soluble, stable toward ion strength, common metal ions, and continuous excitation. Furthermore, the gelatin-AgNCs exhibited a rapid response of temperature, indicating that the gelatin-AgNCs could be a candidate for monitoring the temperature of environment.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (NSFC, No. 21035005).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, http://www.sciencedirect.com.

References [1] P.R. N. Childs, J.R. Greenwood and C.A. Long, Rev. Sci. Instrum. 71 (2000) 2959-2978.

11

[2] S.W. Allison and G.T. Gillies, Rev. Sci. Instrum. 68 (1997) 2615-2650. [3] R.Z. Liang, R. Tian, W.Y. Shi, Z.H. Liu, D.P. Yan, M. Wei, D.G. Evans and X. Duan, Chem. Commun. 49 (2013) 969-971. [4] D. Yan, J. Lu, J. Ma, M. Wei, D.G. Evans and X. Duan, Angew. Chem. Int. Ed. 50 (2011) 720-723. [5] J.J. Yin, X.X. He, K.M. Wang, Z.H. Qing, X. Wu, H. Shi and X.H. Yang, Nanoscale. 4 (2012) 110-112. [6] C.P. Liu, T.H. Wu, C.Y. Liu and S.Y. Lin, Anal Chim Acta. 849 (2014) 57-63. [7] X.L. Xiong, Y. Tang, L.L. Zhang, S.L. Zhao, Talanta. 132 (2015) 790-795. [8] P. Zhang, Y. Wang, Y. Chang, Z.H. Xiong and C.Z. Huang, Biosens Bioelectron. 49 (2013) 433-437. [9] X.Y. Wang, R.Y. Lin, Z.H. Xu, H.D. Huang, L.M. Li, F. Liu, N. Li, X.D. Yang, Anal Chim Acta. (2013) 79-85. [10] Y. Tao, Y.H. Lin, J.S. Ren and X.G. Qu, Biosens Bioelectron. 42 (2013) 41-46. [11] Y. Zhu, H. Qian and R. Jin, Chem. Eur. J. 16 (2010) 11455-11462. [12] L. Shang and S.J. Dong, Chem. Commun. (2008) 1088-1090. [13] J. Lan, P. Zhang, T.T. Wang, Y. Chang, S.Q. Lie, Z.L. Wu, Z.D. Liu, Y.F. Li and C.Z. Huang, Analyst. 139 (2014) 3441-3445. [14] E. Falletta, M. Bonini, E. Fratini, A.L. Nostro, G. Pesavento, A. Becheri, P.L. Nostro, P. Canton and P. Baglioni, J. Phys. Chem. C. 112 (2008) 11758-11766. [15] Y.R. Tang, Y. Zhang, Y.Y. Su, Y. Lv, Talanta. 115 (2013) 830-836. [16] T. Udayabhaskararao, M.S. Bootharaju and T. Pradeep, Nanoscale. 5 (2013) 9404-9411. [17] M. Ganguly, J. Jana, C. Mondal, A. Pal and T. Pal, Phys. Chem. Chem. Phys. 16 (2014) 18185-18197. [18] J. Ai, W.W. Guo, B.L. Li, T. Li, D. Li, E.K. Wang, Talanta. 88 (2012) 450-455. [19] D. Schultz and E. Gwinn, Chem. Commun. 47 (2011) 4715-4717. [20] C.L. Guo and J. Irudayaraj, Anal. Chem. 83 (2011) 2883-2889. [21] Y.Y. Cui, Y.L. Wang, R. Liu, Z.P. Sun, Y.T. Wei, Y.L. Zhao and X.Y. Gao, ACS Nano. 5 (2011) 8684-8689. 12

[22] P. Zhang, X.X. Yang, Y. Wang, N.W. Zhao, Z.H. Xiong and C.Z. Huang, Nanoscale. 6 (2014) 2261-2269. [23] C.X. Zhang, X.Y. Sun, J. Li and Y.N. Liu, Nanoscale. 5 (2013) 6261-6264. [24] S.H. Liu, F.Lu and J.J. Zhu, Chem. Commun. 47 (2011) 2661-2663. [25] Z.Q. Yuan, N. Cai, Y. Du, Y. He and E.S. Yeung, Anal. Chem. 86 (2014) 419-426. [26] H. Zhu, X. Wang, Y. Li, Z. Wang, F. Yang and X. Yang, Chem. Commun. (2009) 5118-5120. [27] Y. Yang, J. Cui, M. Zheng, C. Hu, S. Tan, Y. Xiao, Q. Yang and Y. Liu, Chem. Commun. 48 (2012) 380-382. [28] S. Kuramitsu and K. Hamaguchi, J. Biochem. 85 (1979) 443-456. [29] P. Yu, X.M. Wen, Y.R. Toh and J. Tang, J. Phys. Chem. C. 116 (2012) 6567-6571. [30] H. Huang, H. Li, A.J. Wang, S.X. Zhong, K.M. Fang and J.J. Feng, Analyst. 139 (2014) 6536-6541. [31] X. Chen, J.B. Essner, G.A. Baker, Nanoscale. 6 (2014) 9594-9598. [32] S.K. Sailapu, A.K. Sahoo, S.S. Ghosh, A. Chattopadhyay, Small. 10 (2014) 4067-4071. [33] D. Cauzzi, R. Pattacini, M. Delferro, F. Dini, C.D. Natale, R. Paolesse, S. Bonacchi, M. Montalti, N. Zaccheroni, M. Calvaresi, F. Zerbetto, L. Prodi, Angew. Chem. Int. Ed. 51 (2012) 9662-9665.

Captions for Scheme and Figures Scheme 1. Illustration of the preparation and application of gelatin-AgNCs. Fig. 1 The optical properties of the as-prepared gelatin-AgNCs. (A) UV-vis absorption and fluorescence spectra of gelatin-AgNCs, respectively. Inset: the photographs of gelatin-AgNCs under the irradiation of day light (left) and 365 nm UV lamp (right). (B) The emission spectra of AgNCs excited at different wavelength from 420 nm to 490 nm. Fig. 2 The morphology and composition characterization of gelatin-AgNCs. (A) TEM

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image and (B) AFM image of gelatin-AgNCs. Inset: the HRTEM image (upper left) and size distribution (lower right) of gelatin-AgNCs. (C) XPS spectrum of Ag 3d. (D) FTIR spectra of gelatin, gelatin-AgNCs, and gelatin with NaOH, respectively. Fig. 3 The temperature sensing by using gelatin-AgNCs. (A) The fluorescence spectra of gelatin-AgNCs with different temperatures from 5 oC to 45 oC. (B) The linear relationship between fluorescence intensity of gelatin-AgNCs and temperature. Inset: the photographs under 365 nm UV light of gelatin-AgNCs with different temperatures at 7, 18, 25, 35, and 45 oC. Fig. 4 The mechanism of temperature sensing. (A) Arrhenius plot of fluorescence intensity of gelatin-AgNCs. Inset: the fluorescence spectra of gelatin-AgNCs with temperatures from 5

o

C to 60

o

C. (B) The fluorescence decay curves of

gelatin-AgNCs at 5 oC and 25 oC, respectively.

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Highlights 

Gelatin was firstly used as protective and reducing agent to prepare Ag nanoclusters.



The preparation of Ag nanoclusters could be completed within 4h at room temperature.



A temperature sensor was developed with gelatin-stabilized Ag nanoclusters.

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Graphical Abstract for

A visual physiological temperature sensor developed with gelatin-stabilized luminescent silver nanoclusters † Jing Lan a, Hong Yan Zou a, Ze Xi Liu a, Ming Xuan Gao b, Bin Bin Chen b, Yuan Fang Li b and Cheng Zhi Huang a, b, *

A direct synthesis strategy of water-soluble and fluorescent Ag nanoclusters (AgNCs), using gelatin as protective and reducing agent, has been established in this report. The AgNCs were successfully prepared within 4h at room temperature. Notably, the gelatin-AgNCs were further applied to be a reversible and sensitive temperature sensor.

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