Glutathione-stabilized Cu nanoclusters as fluorescent probes for sensing pH and vitamin B1

Author’s Accepted Manuscript Glutathione-stabilized Cu nanoclusters as fluorescent probes for sensing pH and vitamin B1 Yawen Luo, Hong Miao, Xiaoming Yang

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S0039-9140(15)30129-6 http://dx.doi.org/10.1016/j.talanta.2015.07.001 TAL15766

To appear in: Talanta Received date: 17 April 2015 Revised date: 26 June 2015 Accepted date: 2 July 2015 Cite this article as: Yawen Luo, Hong Miao and Xiaoming Yang, Glutathionestabilized Cu nanoclusters as fluorescent probes for sensing pH and vitamin B1 Talanta, http://dx.doi.org/10.1016/j.talanta.2015.07.001 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.

Glutathione-stabilized Cu nanoclusters as fluorescent probes for sensing pH and vitamin B1 Yawen Luo, Hong Miao, Xiaoming Yang* College of Pharmaceutical Sciences, Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, Southwest University, Chongqing 400715, China Abstract Glutathione (GSH), playing roles as both a reducing reagent and protecting ligand, has been successfully employed for synthesizing Cu nanoclusters (CuNCs@GSH) on the basis of a simple and facile approach. The as-prepared CuNCs exhibited a fluorescence emission at 600 nm with a quantum yield (QY) of approximately 3.6%. Subsequently, the CuNCs described here was employed as a broad-range pH sensor by virtue of the fluorescence intensity of CuNCs responding sensitively to pH fluctuating in a linear range of 4.0-12.0. Meanwhile, these prepared CuNCs were applied for detections of vitamin B1 (VB1) on the basis of positively charged VB1 neutralizing the negative surface charge of CuNCs, thus leading to the instability and aggregations of CuNCs, and further facilitating to quench their fluorescence. In addition, the proposed analytical method permitted detecting VB1 with a linear range of 2.0×10-8-1.0×10-4 mol L-1 as well as a detection limit of 4.6×10-9 mol L-1. Eventually, the practicability of this sensing approach was validated by assaying VB1 in human urine samples and pharmaceutical tablets, confirming its potential to *

To whom correspondence should be addressed. Tel: 86-23-68251225; Fax: 86-23-68251225; E-mail: [email protected] 1

broaden avenues for assaying VB1. Key word: Glutathione; Cu nanoclusters; pH sensing; Vitamin B1. 1. Introduction Metal nanoclusters (NCs), usually consist of several to tens of atoms with properties regulated by their subnanometer dimensions [1-4]. As one type of fluorescent material, metal NCs exist in ultra-small size with low toxicity compared with quantum dots. Besides, unique characteristics of metal NCs have attracted numerous interestings, potentiating it as a satisfactory candidate for biosensing, catalysis, and imaging [1, 4-6]. Over the past decade, extensive studies have been performed on luminescent Au and Ag nanocluster due to their attractive features such as chemical stability, ultra-small hydrodynamic diameters, and tailorable surface properties [3, 6]. Besides, non-noble metal Cu is earth-abundant, much lower cost and widely used in industry. Thereby, development of CuNCs has been attracting more and more attentions. Recently, several studies have been successfully proposed for the synthesis and application of CuNCs [7, 8]. For example, a polyethyleneimine (PEI)-coated CuNCs was prepared in water solution and further employed for the quantification of Sudan dyes [9]. Moreover, tannic acid capped CuNCs and their application for Hg2+ sensing was reported [10]. Similarly, L-cysteine protected CuNCs was employed for assaying Fe3+ [11]. Again, D-penicillamine (DPA) stabilized CuNCs with an aggregation induced emission feature has been explored for pH sensing and catalysis applications [12]. However, compared to the exciting progress in the development of Au and Ag 2

NCs, studies on CuNCs are still in a preliminary stage. Therefore, exploring new strategies for synthesizing stable and high fluorescent CuNCs, and further broadening their application fields are still desired. Among thiolate-protected nanoclusters, GSH capped Ag/Au NCs have been widely studied for several years due to its relatively high QY, water solubility and near-IR emission [13, 14]. Additionally, these GSH protected Au/Ag NCs were extensively applied for biosensing [15, 16], bioimaging [17], and novel catalysts [18, 19] benefit from their superior optical, electrical and catalytic properties. Significantly, copper as a member of transition metals shows similar properties as gold or silver, providing the possibility for synthesising CuNCs. The pH sensing has received more and more concerns owing to pH plays a critical role in fields of biomedicine and pharmaceutics. For instance, pH is a fundamental parameter for biological systems towards synthesizing adenosine triphosphate (ATP), which is driven by a proton gradient [20]. In addition, diseases like cancer [21] and Alzheimers [22] are associated with abnormal cellular pH. Thus, building up new simple and effective strategies for pH sensing is highly valuable. To date, kinds of nano-scale materials have been reported for pH sensing by taking advantage of their unique structural and photophysical features, such as quantum dots (QDs) [23-25], modified nanoparticles [26-28] and metal NCs [29,30]. During recent years, developing metal NCs have been becoming increasingly widespread for pH sensing due to their attractive features such as low toxicity, ultra-small hydrodynamic diameters, and good biocompatibility [29, 30]. 3

Thiamine (vitamin B1), a member of water-soluble vitamins, exists in many types of foods. As an important biological compound, it participates during the metabolic processes of sugars, proteins, and lipids [31]. Additionally, it is mainly used to cure beriberi and varies of polyneuritis in clinical. Various methods have been developed to assay thiamine, especially traditional techniques such as thin-layer chromatography [32],

capillary

electrophoresis

(CE)

[33],

and

high

performance

liquid

chromatography (HPLC) [34,35]. However, sophisticated instrumentation and/or complicated sample pretreatment usually limited the application of these methods. Additionally, several nano-scale materials have been successfully introduced into detections of VB1. For example, Wang et al. reported a resonance rayleigh scattering (RRS) technique for the VB1 sensing via copper nanoparticles [36]. Again, Sun et al. reported a VB1 assaying method based on quenching the photoluminescence (PL) of CdSe QDs by VB1[37]. Nevertheless, the previous methods exhibited either the limitation of the narrow linear range or interference from coexisted. Consequently, simple, economical, sensitive and selective methods for detecting VB1 are still meaningful. In this study, a modified method for preparing water-soluble CuNCs has been well built up while GSH served as both a reducing reagent and a protecting ligand (CuNCs@GSH). Compared to the previously reported protocol [38], the current synthesis method showed obvious superiority. First, the synthesis procedure was performed at the physiological temperature (37 đ ), and the reaction has been completed within 1h. Second, an enhanced fluorescent QY of 3.6% was obtained after 4

the optimization of synthesis conditions. Meanwhile, a series of characterization data were employed to investigate the properties of the current CuNCs. Significantly, the CuNCs described here was employed for sensing pH and VB1. To be specific, the CuNCs showed obvious sensitivity as well as pH value varying from acidic to alkaline. This phenomenon was originated from the protonation and deprotonation of their surface carboxyl groups, which may cause electrostatic doping/charging to the CuNCs and shift the Fermi level, thus resulting in the fluorescence variation [39, 40]. For the mechanism of VB1-induced fluorescence quenching of CuNCs, the reasonable explanation was that positively charged VB1 neutralized negative surface charge of CuNCs, thereby leading to the instability and aggregations of CuNCs, and further facilitating to quench their fluorescence [41]. Additionally, the practicability of the proposed strategy has been validated by applying this probe to assay VB1 in human urine samples and pharmaceutical tablets. Overall, the as-prepared CuNCs@GSH implied potential to broaden avenues for biosensing and fantastic applications by virtue of various advantages. 2. Experimental 2.1. Materials and reagents Copper sulfate (CuSO4), glutathione, vitamin B1, vitamin B3, vitamin B6, vitamin B12, vitamin C and all the metal ions were obtained from Shanghai Sangon Biotechnonlogy Co. Ltd. (Shanghai, China). Sodium hydroxide (NaOH), phosphoric acid (H3PO4), glacial acetic acid (CH3COOH) and boric acid (H3BO3) were purchased

5

from Dingguo Changsheng Biotechnology Co. Ltd. (Beijing, China). Ultrapure water, 18.25 MΩ cm, produced by an Aquapro AWL-0520-P ultrapure water system (Chongqing, china), was employed for all the following experiments. 2.2. Apparatus All fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) with excitation slit set at 10 nm band pass and emission at 10 nm band pass in 1 cm×1 cm quartz cells. In addition, UV-vis spectra were recorded by a Shimadzu UV-1750 spectrophotometer (Tokyo, Japan). High resolution transmission electron microscopy (HR-TEM) images were obtained by using a TECNAI G2 F20 microscope (Hillsboro, America) at 200 KV. Images were taken with an Olympus E-510 digital camera (Tokyo, Japan). Fourier transform infrared (FTIR) spectra were recorded on a Shimadzu IR prestige-21 spectrometer (Tokyo, Japan). Elemental analysis was obtained by ESCALAB 250 X-ray photoelectron spectrometer (XPS) (Massachusetts, America). The quantum yields were obtained by using Absolute PL quantum yield spectrometer C11347 (Hamamatsu, Japan). A Fangzhong pHS-3C digital pH meter (Chengdu, China) was used to measure pH values of the aqueous solutions and a vortex mixer QL-901 (Haimen, China) was applied to blend the solution. The thermostatic water bath (DF-101s) was purchased from Gongyi Experimental Instruments Factory (Gongyi, China). 2.3. Preparation and purification of CuNCs. As a typical experiment, 5.0 mL of GSH solution (50 mg mL-1) was added into 5.0 6

mL of CuSO4 solution (10 mM). The transparent solution changed to white suspension liquid. Then, a NaOH solution (1M) was added dropwise until the turbid liquid turned to transparent light yellow, and the corresponding pH value was 4-5. Subsequently, the solution was stirred vigorously at 37ć for 1 h (Fig. 1A), and the products were subjected to 500 MWCO of dialysis membrane for purification before further characterization and applications. (Fig. 1 is inset here) 2.4. Response for pH Sensing pH was accomplished by the following steps: 200μL of CuNCs, 160 μL of water, and 40 μL of various pH of Britton-Robinson (BR) buffers were mixed together with reaction time of 10 min, and then the fluorescence intensity of these mixtures were measured upon being excited at 380 nm. In addition, to evaluate the reversibility of the pH sensitive change of fluorescence, the as-prepared CuNCs were subjected to pH cycling between pH 4.0 and 12.0 using acid and base as modulators. 2.5. Detection of vitamin B1 Towards the purpose of detecting VB1, stock solutions of VB1 were prepared respectively. Then, 40 μL of BR buffer solutions (pH 4.0) was mixed with 200 μL of CuNCs initially, and 160 μL of various concentrations of VB1 were added. After incubation for 10 min at 40ć, the fluorescence intensity of these mixtures were measured upon being excited at 380 nm. 2.6. Interference and selectivity studies As been well known, human urine usually contains large amounts of amino acids, 7

urea, uric, and creatinin. Additionally, several common metal ions and excipients also exist in pharmaceutical tables. To evaluate the interference from coexisting component in pharmaceutical tables and urine, a variety of common metal ions (Ag+, Na+, K+, Mg2+, Mn2+, Cd2+, Ni2+, Zn2+, Ba2+, Fe2+, Co2+), excipients (glucose, sucrose, mannitol, and lactose), amino acids (valine, glutamic, serine, proline), urea, uric, and creatinin were introduced with the identical concentration of 100 μM. Furthermore, effects originated from 10 μM of other water-soluble vitamins (vitamin B3, vitamin B6, vitamin B12, and vitamin C) were further assessed towards the selectivity of current assay. 2.7. Preparation of actual samples The morning urine samples by five healthy volunteers were collected from Southwest University Hospital (Chongqing, China). All the raw samples were centrifuged (1000rpm, 10min) and filtered through a 0.25-μm membrane to remove the impurities. Then, a series of samples were prepared by spiking standard VB1 solutions (5 μM) into the purified urine which contain 10% BR buffer (pH=4.0). Pharmaceutical VB1 tablets produced by Baijingyu pharmaceutical factory (Nanjing, China) were purchased from Tongjunge Pharmaceutical Store (Chongqing, China). The analysis of VB1 in pharmaceutical preparations was carried out by following procedure. Firstly, VB1 tablets were grinded into powder. Next, a certain amount of this powder was dissolved in hydrochloric acid solution (0.01 M) and filtered through a 0.25 μm membrane. 3. Results and discussions 8

3.1. synthesis of CuNCs The existence of thiol group in the molecule structure of GSH induced the formation of high-affinity metal ligand clusters. In addition, the unctional groups of carboxyl and amino in GSH molecule provided a protective layer to ensure the stability of metal NCs. More importantly, GSH has been served as a reducing agent for synthesizing nanoparticles due to its amino groups [42]. Therefore, GSH was employed as both a reducing reagent and a protecting ligand for preparing CuNCs in this contribution. To identify the optimized conditions for synthesizing CuNCs, a series of experiments were performed. As revealed in Fig. S1A, S1B and S1C, the fluorescent intensities of CuNCs exhibited variations along with varying reaction time, temperature and concentrations of GSH, demonstrating that synthesis of CuNCs were dependent on these selected conditions. Thus, 60 min, 37ć and 50 mg mL-1 of GSH served as the optimal conditions toward synthesizing CuNCs. 3.2. Characterization of CuNCs In this study, the fluorescent CuNCs was synthesized in aqueous solution by an ease-manipulate method, and its quantum yield was determined to be 3.6%. These CuNCs as described were characterized successively by fluorescence spectroscopy (Fig. 1B), UV-visible absorption (Fig. 1C), FTIR (Fig. 2B), HR-TEM (Fig. 2C) and XPS (Fig. 2D). As Fig. 1B indicated, a standard fluorescence emission peak was centered at 600 nm as well as excited at 380 nm. The CuNCs solution was light yellow under day light (photograph ĉ), meanwhile it emitted an obvious red 9

fluorescence under UV light (photograph Ċ). Subsequently, UV−vis absorption spectra of GSH and CuNCs were displayed respectively (Fig. 1C). Only CuNCs exhibited an obvious absorption peak around 270 nm, while GSH showed no peaks. Additionally, the emission spectra of CuNCs at excitations from 350 to 390 nm were recorded. Interestingly, the emission peak indicated no shift as the excitation wavelength varied (Fig. 2A). Next, the surface groups of these synthesized CuNCs were characterized by FTIR technique. As shown in Fig. 2B, the peaks at 3375 cm-1 can be ascribed to the characteristic absorption of O-H stretching vibration, while a C=O stretching vibration peaks was at 1688 cm-1. Data above revealed that the surface of these obtained CuNCs were equipped with carboxylic groups. Similarly, the peaks at 2893 and 1378 cm-1 were attributed to the C-H stretching vibration and C-H bending vibration, and a peak at 3134 cm-1 was associated with N-H stretching vibration. Consequently, these FTIR data suggested that the CuNCs prepared here were equipped with characteristic functional groups including –COOH, -OH, and –NH2, which were originated from the protecting ligands of GSH. Compared with the FTIR spectra of GSH (Fig. S2), disappearance of -SH stretching vibration peak at 2525 cm-1 in that of CuNCs@GSH (Fig. 2B) demonstrated copper atoms of CuNCs were covered by GSH through Cu-S bonding. To further visualize the shape and measure the diameter of CuNCs, their related HR-TEM images were obtained. As depicted in Fig. 2C, the majority of CuNCs existed within the size range of 1–3 nm together with no distinct aggregations 10

observed, suggesting that the synthetic procedure allowed the sizes of this new type of nanoclusters effectively controlled. Finally, XPS analysis was carried out to confirm the components of the current CuNCs. As revealed in Fig. S3, six major peaks of S(2p), C(1s), N(1s), O(1s), Cu(2p), and Na(1s) obviously emerged in the survey spectrum, indicating that CuNCs prepared here were mainly composed of C, N, O, S, Cu, and Na [11]. To further describe the oxidation state of Cu, the amplified peak was showed in Fig. 2D. Two intense peaks at 932.2 and 952.0 eV were assigned to Cu 2p3/2 and Cu 2p1/2, which were the feature peaks due to Cu0. Moreover, there were no peak displayed around 942 eV (Fig. 2D), demonstrating the absence of Cu2+ for CuNCs. Importantly, the 2p3/2 binding energy of Cu0 was only 0.1 eV different from that of Cu+. Therefore, the presence of Cu0 cannot be ruled out [43, 44]. (Fig. 2 is inset here) 3.3. Sensing pH As shown in Fig. 3A, the fluorescence signal of CuNCs showed obvious response to pH fluctuations. In detail, the fluorescence intensity at 600 nm dramatically decreased with varying the pH value from acidic to alkaline. Significantly, the fluorescence intensity versus the pH value displayed a linear range from 4.0 to 12.0 (Fig. 3B). Meanwhile, the colour of the CuNCs solution transformed from light yellow to yellow brown under daylight (Fig. 3C, top) and their fluorescence changed from red to colorless under UV night (Fig. 3C, bottom). This pH induced fluorescence variation of CuNCs was originated from the protonation and deprotonation of their 11

surface carboxyl groups, which may cause electrostatic doping/charging to the CuNCs and shift the Fermi level [39, 40]. To further evaluate the reversibility of the pH sensitive change of fluorescence, these prepared CuNCs were subjected to pH cycling between pH 4.0 and 12.0. As revealed in Fig. 3D, the CuNCs displayed an excellent reversibility towards pH variations. Additionally, sensing pH range by our approach has been compared with previously reported methods. As listed in Table S1, this CuNCs@GSH for pH showed a relatively broad detection range. (Fig. 3 is inset here) 3.4. Fluorescence quenching of CuNCs by vitamin B1 As shown in Fig. 4A, the fluorescence intensity of CuNCs at 600 nm dramatically decreased upon addition of 10 μM VB1. Meanwhile, their fluorescence of the CuNCs solution changed from red (photograph ĉ) to disappeared (photograph Ċ) under UV night. To explore the quenching mechanism, zeta potential measurements were performed. In Fig. 4B, the initial zeta potential of CuNCs was -19.2 mV, indicated that a large number of negative charge were exposed on the surfaces of the CuNCs. Nevertheless, this value changed to -16.8 mV and -14.0 mV by introducing 30 and 50 μM VB1. Thus, the reasonable explanation for this VB1-induced fluorescence quenching was that the negative surface charges of CuNCs were neutralized by the positively charged VB1, which induced the instability and aggregations of CuNCs, thus leading to quenching their fluorescence [41]. These results indicated that the CuNCs prepared here could potentially be applied as fluorescence probes for detecting VB1. 12

To optimize detection conditions for VB1, effects by the incubation temperature and time were studied and optimized. As shown in Fig. 4C, the fluorescence intensity of CuNCs gradually decreased as the temperature varying from 35ć to 55ć, suggesting that the CuNCs are instable for the temperature rising. Nevertheless, the VB1-induced fluorescent intensity decrease of CuNCs (F0-F) reached the maximum at 40ć. Likewise, effect from incubation time (1 min, 5 min, 10 min, 15min, 20 min, 25 min) were investigated with 10 μM VB1. As Fig. 4D indicated, the VB1-induced fluorescence quenching process of CuNCs has completed within 10 min. Therefore, the incubation temperature of 40ć and incubation time of 10 min served as the optimal conditions during the following experiments. In addition, the whole procedure for testing VB1 was carried out at pH 4.0, since VB1 were instable in alkaline environment, and spoiled easily when pH value exceed 5. (Fig. 4 is inset here) 3.5. Detection of vitamin B1 As shown in Fig. 5A, the fluorescence intensity of CuNCs decreased accompanied with different amounts of VB1 added (0.02, 0.08, 0.2, 0.4, 0.8, 1, 4, 6, 10, 20, 60, 100 μM). In particular, the fluorescence intensity decrease (F0-F) versus the logarithmic plots of VB1 various concentrations displayed a linear range from 2.0×10-8 mol L-1 to 1.0×10-4 mol L-1 (Fig. 5B). Meanwhile, the detection limit of VB1 was obtained as 4.6×10-9 mol L-1 at a signal-to-ratio of 3. Moreover, the sensitivities of various previous reports were compared with the proposed method here (Table S2). From the table, the VB1 assay based on CuNCs@GSH not only 13

displayed the satisfactory linear range and detection limit, but also seldom required complicated probe preparation and sophisticated instrumentation. Taken together, the results obtained here described a simple and sensitive method for detecting VB1. (Fig. 5 is inset here) 3.6. Interference and selectivity As been well known, there usually co-existing metal ions and excipients in pharmaceutical tables. Additionally, large amounts of amino acids, urea, uric, and creatinine also exist in urine. To address the question whether foreign coexisting substance imply effect on the measurement of VB1 or not, further interference experiments were performed in the presence of VB1 (10 μM) together with various metal ions (Ag+, Na+, K+, Mg2+, Mn2+, Cd2+, Ni2+, Zn2+, Ba2+, Fe2+, Co2+), excipients (glucose, sucrose, mannitol, and lactose), amino acids (valine, glutamic, serine, proline), urea, uric, and creatinine. As described in Fig. 6A and Fig. 6B, there was no obvious variation for fluorescence signals, indicating that foreign coexisting substances showed little effect on the detection of VB1. To identify the selectivity of the current method, experiments were manipulated separately in the presence of several water-soluble vitamins (vitamin B3, vitamin B6, vitamin B12, and vitamin C). As Fig. 6C revealed, dramatic fluorescence decrease was observed for CuNCs upon addition of VB1. In striking contrast, no obvious decrease was founded by introducing other water-soluble vitamins into CuNCs, indicating the satisfactory selectivity of CuNCs for assaying VB1. (Fig. 6 is inset here) 14

3.7. Detecting vitamin B1 in urine and pharmaceutical tablets To validate its applicability, our approach was employed to analyze VB1 in five human urine samples, and three batches of pharmaceutical VB1 tablets. In particular, the urine samples were analyzed by both direct calibration and standard additions. Meanwhile, the results were compared with that obtained by UV spectrophotometry. Additionally, pharmaceutical tablets were all directly determined by this fluorescent strategy. The VB1 contents in all samples were derived from standard curves and regression equations. Importantly, as listed in Table 1 the recovery of the supplemented VB1 was above 90% in all cases, suggesting excellent average recoveries in the urine sample, and the RSD were generally acceptable. Additionally, the results obtained by the current approach and UV spectrophotometry were in a satisfactory agreement. Again, the results of pharmaceutical tablets by the described method were essentially consistent with the labeled amount reported by the manufacturing laboratory (Table 2). Therefore, this fluorescent method has potential for broadening ways of detecting VB1 in actual samples. (Table 1and Table 2 is inset here) 4. Conclusions In summary, we have successfully proposed a modified method for preparing water-soluble CuNCs according to GSH-mediated reduction of CuSO4 under physiological temperature (37ć) within 1 h. Moreover, these prepared CuNCs were applied for sensing pH and VB1. In principle, the sensing of pH was based on the protonation and deprotonation of the surface carboxyl groups of CuNCs, which may 15

cause electrostatic doping/charging to the CuNCs and shift the Fermi level, thus resulting in their fluorescence change. Meanwhile, the mechanism of VB1-induced fluorescence quenching was that positively charged VB1 neutralized the negative surface charge of CuNCs, thereby leading to the instability and aggregations of CuNCs, and further facilitating to quench their fluorescence. Compared with the previously reported, our current protocol for sensing pH and VB1 displayed the advantages of being simple, rapid, selective and sensitive with relatively broad detection range and avoiding complicated probe preparation as well as sophisticated instrumentation. Additionally, this sensing approach was employed to analyze VB1 in human urine samples and pharmaceutical tablets, confirming its potential for actual samples analysis. Acknowledgements We gratefully acknowledge financial support by Research Fund for the Doctoral Program of Higher Education of China (20110182120014), Natural Science Foundation Project of CQ CSTC (cstc2013jcyjA10117), Fundamental Research Funds for the Central Universities (XDJK2015A005, 2362014xk07), and Innovative Research Project for Postgraduate Students of Chongqing (CYS14049).

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media

by

high-performance

liquid

chromatography

with

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nanosensor (AuC@Urease) for the selective detection of blood urea, Small. 9 (2013) 2673-2677. [42] C. Wang, D. Zhang, L. Xu, Y. Jiang, F. Dong, B. Yang, K. Yu, Q. Lin, A simple reducing approach using amine to give dual functional EuSe nanocrystals and morphological tuning, Angew. Chem. Int. Ed. 50 (2011) 7587-7591. [43] N. Goswami, A. Giri, M.S. Bootharaju, P.L. Xavier, T. Pradeep, S.K. Pal, Copper quantum clusters in protein matrix: potential sensor of Pb2+ ion, Anal. Chem. 83 (2011) 9676-9680. [44] R. Ghosh, A.K. Sahoo, S.S. Ghosh, A. Paul, A. Chattopadhyay, Blue-Emitting Copper Nanoclusters Synthesized in the Presence of Lysozyme as Candidates for Cell Labeling, Acs Appl. Mater. Inter. 6 (2014) 3822-3828.

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Figure Captions:

Fig. 1 (A) Schematic illustration of the synthesis of water-soluble CuNCs; (B) Fluorescence excitation and emission spectra of CuNCs. Inset: photographs of CuNCs solution under daylight (ĉ) and UV light (Ċ); (C) UV-vis absorption spectra of GSH and CuNCs.

Fig. 2 (A) Fluorescence emission spectra of CuNCs for varying excitation wavelengths; (B) FT-IR spectrum of CuNCs; (C) HR-TEM image of CuNCs; (D) Amplified XPS of Cu 2p electrons.

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Fig. 3 (A) Fluorescence emission spectra of CuNCs in BR buffers at different pH values; (B) Plot of fluorescence intensity versus the pH value of BR buffers introduced; (C) Photographs of CuNCs solution with the increase of pH value from 4.0 to 12.0 under daylight (top) and UV light (bottom); (D) pH reversibility study of CuNCs between pH 4.0 and 12.0.

Fig. 4 (A) Fluorescence emission spectra of CuNCs in the absence (a) and presence (b) of 10 μM VB1. Inset: photographs of CuNCs solution in the absence (ĉ) and presence (Ċ) of VB1 under UV light; (B) Zeta potential of CuNCs, and on 24

addition of 30 μM and 50 μM of VB1, respectively; (C, D) Influence of incubation temperature and time on the fluorescence intensity of CuNCs in the absence (black) and presence (red) of 10 μM VB1.

Fig. 5 (A) Fluorescence emission spectra of CuNCs in the presence of various concentrations of VB1; (B) Plot of fluorescence intensity decrease (F0-F) versus the logarithm of concentration of VB1 added.

Fig. 6 (A, B) Interference of coexisting metal ions, creatinine, urea, uric, amino acid, and excipients (100 μM) on the fluorescence intensity decrease (F0-F) of CuNCs in the presence of VB1 (10 μM); (C) F0-F of CuNCs in the presence of vitamin B1, vitamin B3, vitamin B6, vitamin B12, and vitamin C (10 μM). 25

Tables: Table 1 Recovery for the determination of VB1 in human urine samples Samples

VB1 in original

VB1 spiked

a

a

Sample fSD

a

current method

UV spectrophotometry VB1 found afSD Recovery

VB1 found fSD

Recovery

(µM)

(%)

(µM)

(%)

(µM)

(µM)

1

0.35f0.28

5.0

5.16f0.35

96.2

5.42f0.37

101.4

2

0.81f0.17

5.0

5.97f0.42

103.2

6.03f0.31

104.4.

3

0.42f0.25

5.0

5.68f0.21

105.2

5.75f0.43

106.6

4

Not detected

5.0

4.85f0.18

97.0

5.08f0.29

101.6

5

Not detected

5.0

4.69f0.32

93.8

4.79f0.36

95.8

Mean of three determinations.

Table 2 Detections of VB1 in pharmaceutical tablets Batch number

a

b

Labelled amount (µM)

TC measured a (µM)

Recovery b (%)

RSD (%)

130203

20.0

21.02

105.1

3.5

131105

20.0

20.54

102.7

2.8

140502

20.0

19.46

97.3

3.2

Mean of three determinations. Recoveries were calculated considering that the pharmaceutical tablets contain the

amount reported by the manufacturing laboratory.

Highlights A facile, mild strategy for synthesizing GSH-protected CuNCs was proposed. CuNCs described here showed obvious sensitivity along with pH varying . A sensitive and selective method for assaying VB1 was well established.

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*Graphical Abstract (for review)