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One-Pot Aqueous Synthesis of Nucleoside-Templated Fluorescent Copper Nanoclusters and Their Application for Discrimination of Nucleosides Yong Wang, Tianxia Chen, Qianfen Zhuang, and Yongnian Ni ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09768 • Publication Date (Web): 30 Aug 2017 Downloaded from http://pubs.acs.org on September 2, 2017

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ACS Applied Materials & Interfaces

One-Pot Aqueous Synthesis of NucleosideTemplated Fluorescent Copper Nanoclusters and Their Application for Discrimination of Nucleosides Yong Wang,1,* Tianxia Chen,1 Qianfen Zhuang,1 and Yongnian Ni1,2,* 1

2

College of Chemistry, Nanchang University, Nanchang, Jiangxi 330031, China

State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047,China

* Corresponding author. Telephone +86 791 83969500. Fax: +86 791 83969500. E-mail address: [email protected] and [email protected]

1 Environment ACS Paragon Plus


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ABSTRACT: A facile, one-pot synthetic method has been proposed to prepare water-soluble fluorescent copper nanoclusters (CuNCs) templated by nucleosides. The nucleoside-templated fluorescent CuNCs were further characterized by using various analytical techniques, such as transmission electron microscopy, X–ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and fluorescence spectroscopy. The role of various reactants such as ascorbic acid, nucleoside and citrate buffer in the synthesis process of fluorescent CuNCs was explored. The results showed that nucleoside and ascorbic acid were very likey to be respectively acted as a stabilizer and a reductant to form nanoclusters, and citrate buffer acted as both pH regulator solution and a reducing agent. The fluorescence spectra of various nucleoside-templated CuNCs were finally combined with multivariate chemometrics analysis for discrimination of different nucleosides. KEYWORDS: copper nanoclusters, nucleosides, fluorescence, chemometrics, pattern recognition


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1. INTRODUCTION In the past few years, the synthesis of noble metal nanoclusters (NMNCs) have become a hot topic in the scientific research field due to their extraordinary physical, chemical, electric and catalytic characteristics.1-3 Particularly, the NMNCs usually possess very small sizes comparable to the Fermi wavelength of electrons, resulting in their molecular-like fluorescence property.4-6 The fluorescent noble metal nanoclusters possess a series of virtues over those currently available fluorescent materials like organic dyes or quantum dots in terms of photoblinking, biocompatibility, non-toxicity, and so on.4-6 Therefore, great effort has been devoted to the preparation of fluorescent noble nanoclusters (gold or silver, for example) in recent times. Compared with gold and silver, copper is earth abundant and significantly cheaper nonprecious metal, but it has many similar properties to gold (Au) and silver (Ag). Theoretical and experimental have shown that fluorescent copper nanoclusters (CuNCs) can be formed.7-10 However, at present, reports on the preparation of fluorescent CuNCs are far more scarce than other fluorescent AuNCs and AgNCs, primarily because of their susceptibility to oxidation on exposure to air and the synthetic difficulty in controlling subnanometer size.7-10 To this end, various template ligands have been currently employed to synthesize fluorescent CuNCs, including dendrimers,11 synthetic polymers,12,13 proteins,14,15 and peptides.16,17 These template molecules usually contain thiol/mercaptan functional groups because these groups display good reducibility to copper salts and affinity to CuNCs. Lately, researchers started to pay more attention to the use of environmentally friendly non-thiol DNA biomacromolecules as template for preparation of water-soluble fluorescent CuNCs on the basis of the well-known interactions of DNA with copper ions,18-22 and found that the nucleoside sequences in DNA oligonucleotide had a huge influence on the fluorescent property of CuNCs.18-20 In addition, these high-cost



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macromolecular DNA ligands lead to the formation of CuNCs with large hydrodynamic radius, which limited the scope of potential applications. Therefore, it is highly valuable to carry on research on the synthesis of fluorescent CuNCs templated by small molecule nucleosides.23,24 However, to the best of our knowledge, there has been no report on the preparation of nucleoside-templated fluorescent CuNCs to date. On the other hand, nucleosides are the basic unit of DNA. Different nucleosides usually played an extremely different role in biological activities such as anti-platelet aggregation, anticonvulsant activity, anti-arrhythmic and anti-seizure effect, stimulating axon growth in vitro and in the central nervous system (CNS), maintaining the immune response, and affecting the growth and differentiation of the gastro intestinal tract.25-29 And they have the potential to act as biochemical biomarkers for many diseases, particularly neoplastic diseases.25-29 Hence, the development of a simple and inexpensive method for discrimination of the nucleosides is always desirable. However, the small chemical differences between the nucleosides make their differentiation challenging.25-30 In this work, for the first time, we tried to exploit nucleosides as templates to develop a facile and rapid method for the preparation of water-soluble fluorescent CuNCs. Then, the fluorescence spectra of the CuNCs synthesized with different nucleosides together with advanced multivariate chemometrics analysis allowed to differentiate the nucleosides. This brief protocol is schematically described in Scheme 1.

2. EXPERIMENTAL SECTION 2.1. Reagents and Chemicals. Adenosine (A), cytidine (C), guanosine (G), thymidine (T), ascorbic acid and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Cupric nitrate trihydrate was bought from Tianjin Chemical Reagent 4th


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Factory Kaida Chemical Plant (Tianjin, China). Citric acid and sodium hydroxide were bought from Xilong Chemical Reagent Co., Ltd. (Shantou, China). Tris(hydroxymethyl)aminomethane (Tris) was obtained from Lanji Science and Technology Development Co., Ltd. (Shanghai, China). All chemicals were used as received without further purification. Deionized distilled water was used throughout. 2.2. Apparatus. All fluorescence (FL) spectra were recorded on a PerkinElmer LS−55 fluorescence spectrophotometer. Transmission electron microscopy (TEM) images were obtained on a JEOL JEM−2100 transmission electron microscope. X−ray photoelectron spectra (XPS) were carried out on an ESCALab 250Xi using 200 W monochromated Al Kα radiation. Fourier transform infrared spectra (FTIR) were measured in a Nicolet 380 spectrometer equipped with a DTGS KBr detector and a KBr beam splitter in the transmission mode. All of UV-vis spectra were collected by an Agilent 8453 UV-visible spectrometer (Agilent Technologies, USA). 2.3. Synthesis of Nucleoside-Templated Fluorescent CuNCs. Copper (II) nitrate solution (final concentration: 40 µM) was mixed with various nucleoside solution (final concentration: 1.8 mM), followed by citrate buffer (final concentration: 3.0 mM, pH=7). Then, ascorbic acid solution (finally concentration: 1.2 mM) was added into the resulting solution. After that, the mixture was heated under 80 °C water bath for 3 hours to form fluorescent CuNCs solution. 2.4. Measurement of Fluorescence Quantum Yields. The quantum yield (QY) of the nucleoside-templated fluorescent CuNCs were calculated by comparing the integrated fluorescence intensity at 300 nm excitation and the absorbance at 300 nm of the CuNCs samples with those of the reference (quinine sulfate dissolved in 0.1 M H2SO4 solution, literature QYref is



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0.546).31 Absorbance values of samples (CuNCs or quinine sulfate) were kept under 0.1 at 300 nm.32 The QY was estimated using the following equation:32 QY = QYref × (η2/η2ref ) × (K/ Kref)

(1)

where the subscript ref denotes quinine sulfate, the absence of the subscript implies an unknown CuNCs sample, η is the refractive index (1.33 for water and a 0.1 M H2SO4), K represents the slope of the curve from the plot of the integrated fluorescence intensity against absorbance. 2.5. Discrimination of Adenosine, Cytidine, Guanosine and Statistical Analysis. Adenosine, cytidine, and guanosine were employed as the template molecules for the synthesis of CuNCs. More importantly, we used the fluorescence spectra of CuNCs obtained in the aforementioned synthesis process to differentiate the three nucleosides. 80 µL of the CuNCs solution prepared in the aforementioned synthesis process was diluted with water to 2.0 mL, and then mixed thoroughly. After that, the resultant solution were transferred into a 3.5 mL quartz cell, and the fluorescence spectrum of such the solution was scanned every 0.5 nm in the wavelength range from 325.5 nm to 555.5 nm at the excitation wavelength of 300 nm. The excitation and emission monochromator slit widths were respectively 10 nm and 20 nm. All the measurements were performed at room temperature. At a given concentration, seven independent measurements were performed for each individual nucleoside. The intensities over the wavelength range between 325.5 nm and 555.5 nm were selected as the input variables. These responses of the three nucleosides could be represented as a data set matrix [(7 replicates × 3 nucleosides) × 461 wavelengths]. To remove the effect of the fluctuating signal, the data matrix was first normalized along the wavelength direction as following:33-35


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x

raw ij

∑ (x n

j =1

norm x = ij

− xi

raw ij

−x)

2

+1

i

（n − 1） 2

(2)

where xijraw and xijnorm denote respectively the raw data and the normalized data at specified sample i and wavelength j, n represents the number of emission wavelength, and xi is the average value of the specified sample along the wavelength direction. Then, the treated data were subjected to advanced multivariate chemometrics analysis like principal component analysis (PCA) and hierarchical cluster analysis (HCA).33-35 After that, the score plot and dendrogram of the three nucleosides from different samples could be respectively acquired from PCA and HCA.33-35 The resultant plots made it easier to discriminate the three nucleosides. Both the data treatment and multivariate chemometrics analysis were performed with Matlab software version 7.0 (MathWorks Co., USA) under Windows XP.

3. RESULTS AND DISCUSSION 3.1. Characterization of Nucleoside-Templated Fluorescent CuNCs. The fluorescence characterics of CuNCs in the presence or absence of four different nucleosides, i.e., adenosine (A), cytidine (C), guanosine (G), or thymidine (T) were shown in Figure 1. It was easily observed by naked eye that under 365 nm UV lamp, A, C, or G as template could produce blue fluorescence, but T or absence of nucleoside (blank) failed. These suggested that all nucleosides except T played a crucial role in the preparation process of fluorescent CuNCs. The fluorescent spectrum of the A-templated CuNCs (A-CuNCs) displayed an emission and excitation maximum wavelength at 380 nm and 285 nm, respectively. The C-templated CuNCs (C-CuNCs) and G-templated CuNCs (G-CuNCs)



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possessed a fluorescence maxima at 300 nm/380 nm (Ex/Em) and 300 nm/380 nm (Ex/Em), respectively. The fluorescence quantum yield (QY) of A-CuNCs, C-CuNCs and G-CuNCs at 300 nm excitation was respectively measured to be 1.34%, 0.44% and 0.27% by using the comparative approach with quinine sulfate in 0.1 mol L-1 H2SO4 aqueous solution (QY = 0.546).31 Therefore, the order of the fluorescence QY of CuNCs templated by the four nucleosides was A>C>G>>T. On the basis of earlier literature,20,21,36,37 we suppose that this result can be caused by the affinity of nucleoside for Cu2+ and the donating capability of nucleoside, as well as the nucleoside quenching efficiency. Moreover, it was worth noting that thymidine as a template failed to generate fluorescent CuNCs in this work. On the other hand, recent literature reported the dependence of the formation of T-templated fluorescent AgNCs on the pH value.38 Therefore, we herein tried to test the influence of pH value on the fluorescence intensity of the T-templated CuNCs. As can be seen from Figure S1, it seemed that the T-templated fluorescent CuNCs had nothing to do with the pH level of the solution, and exhibited the fluorescence spectra similar to that in the absence of nucleoside. Moreover, several researchers has lately observed that single-stranded poly-(thymine) (polyT) DNA could be used as a highly efficient template to produce fluorescent CuNCs.39-41 Simultaneously, it was pointed out that only the relatively long polyT could template fluorescent CuNCs formation, while the fluorescence induced by polyT of less than 15 bases was almost negligible.39-41 The result implied that the formation of fluorescent CuNCs is highly dependent on the polymerization degree and the length of polyT DNA. Hence, the failure to synthesize Ttemplated fluorescent CuNCs was very likely because thymidine was a short-length monomer.


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Because thymidine cannot be used as a template to synthesize fluorescent CuNCs, we only characterized A-CuNCs, C-CuNCs and G-CuNCs in this study. The three nucleoside-templated CuNCs were characterized by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and Fourier-transform infrared spectroscopy (FTIR). It was clearly noticed from TEM images that the three CuNCs were quasi-spherical and nearly monodisperse (Figure 2A-C). Based on the size statistics of more than 60 nanoclusters, the size of A-CuNCs, C-CuNCs and G-CuNCs were respectively estimated to 3.2±0.7 nm, 1.5±0.5 nm, and 1.5±0.4 nm. XPS analysis was performed to measure the oxidation state of copper in the CuNCs. As displayed in Figure 2D-F, the two intense peaks appeared at ca. 933.5 eV and 953.5 eV, which were assigned to the binding energy of 2p3/2 and 2p1/2 of Cu or Cu+.42 The 2p3/2 satellite peak of Cu2+ at ca. 942.0 eV was not observed.42,43 All these results showed that the valence state of the Cu in ACuNCs, C-CuNCs and G-CuNCs were possibly 0 and/or +1, and the Cu2+ precursor was completely reduced. The FTIR spectra of the three CuNCs showed two strong, prominent, stakeshaped bands positioned about 1400 cm-1 and 1595 cm-1 (Figure 2G-I), which are respectively ascribed to the symmetric stretching and asymmetric stretching of COO-.44,45 Moreover, it appears as a strong, broad band covering a wide range of around 3000-3700 cm-1, which likely corresponds to the O-H stretch.46 All these FTIR results suggested that the three CuNCs possibly contained COO- and O-H functional groups. 3.2. Role of Each Reactant. To further disclose the role of each reactant like ascorbic acid, nucleoside and citrate buffer, in the synthesis process of CuNCs, we collected the fluorescence spectra of various nucleoside-templated CuNCs under different experimental conditions (Figure 3A-C). As can be seen from curves a, b and c in Figure 3A-C, all the three nucleoside-templated fluorescent CuNCs cannot be formed without ascorbic acid, regardless of incubation time. This



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result implied that ascorbic acid played an essential role as a reductant in the synthesis process of nucleoside-templated CuNCs. Looking at curves a and d in Figure 3A-C, it was found that the absence of nucleoside almost led to a failure in the preparation of fluorescent CuNCs, indicating that nucleoside very possibly acted as a stabilizer to form nanoclusters. Careful analysis of the data from curves a and e in Figure 3A-C showed that the pH value of citrate buffer had a significant influence on the band shape and relative intensity of the fluorescence spectra of nucleoside-templated CuNCs. The observation illustrated that citrate buffer acted as pH regulator solution. It was clearly noted from curves a and f in Figure 3A-C that the fluorescence intensity of CuNCs enhanced significantly as buffer concentration increased in citrate buffer (pH=7.0). This result suggested that citrate buffer maybe also serve as a reducing agent, but its reducing ability only took effect in the presence of ascorbic acid. When Tris buffer with the same concentration and pH level was made to replace citrate buffer, the fluorescence intensity of CuNCs diminished remarkably (curves a and g in Figure 3A-C). The observation was very likely because the reducing ability of Tris was inferior to that of citrate. Furthermore, we further examined the likelihood of the formation of fluorescent CuNCs from the mixture of citrate and Cu2+ under different buffers and temperatures. It was clearly noted from Figure S2 that almost no fluorescence for the mixture of citrate and Cu2+ appeared under different buffers and temperatures, suggesting that no fluorescent CuNCs were produced. The results confirmed the importance of nucleosides as templates in the synthesis of fluorescent CuNCs. At the same time, it was found from Figure S2 that the fluorescence intensity of all the nucleoside-templated fluorescent CuNCs increased with the increasing temperature up to approximately 80 °C. Looking at curve h in Figure 3A-C, we could observe that almost no fluorescence appeared at room temperature. All these results indicated that high temperature facilitated the formation of


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fluorescent CuNCs. This was possibly because the adjustment of copper coordination is more easily to take place in high temperature. 3.3. Discrimination of Adenosine, Cytidine, Guanosine Using Advanced Multivariate Analysis. The other aim of the work is to assess whether nucleoside-templated fluorescence CuNCs could be applied to differentiate adenosine (A), cytidine (C), and guanosine (G). On the basis of the above-mentioned fluorescence characteristics of nucleoside-templated CuNCs, we can easily find that the fluorescence emission spectra of the three nucleoside-templated CuNCs overlapped seriously, but the spectral features like peak intensity and band shape have differences. Although the entire spectral overlapping hindered us from using the univariate spectral data to identify of the three nucleosides, we can exploit the changes in peak intensity and band shape together with advanced multivariate chemometrics approaches to realize their identification. The fluorescence spectra of each CuNCs were collected and plotted for each nucleoside at each concentration (Figure 4A, 5A and 6A). At a given concentration, each of the three nucleosides generates a different fluorescence pattern. And each individual nucleoside was examined in seven replicate assays. These responses could be compiled into a data set matrix (7 replicates × 3 nucleosides × 461 wavelengths). The row of the data set matrix was first preprocessed by normalization procedure, and then submitted to advanced multivariate chemometrics analysis like principal component analysis (PCA) and hierarchical cluster analysis (HCA).34,35,47,48 PCA used an orthogonal transformation to transform the recorded fluorescence spectra into a set of values of linearly uncorrelated variables (PCs).34,35,47,48 In the present work, we retained the first two PC scores in the system to create a two-dimensional visualization map (i.e. the PCA score plot). When the concentration of each nucleoside to be differentiated was 1.8 mM, the first two PCs account for 99.2% of all the data information available (Figure 4B). The



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resulting PCA score plot clearly showed three unique clustering of the data, and sample replicates from each nucleoside were grouped close together in well-separated clusters. The result suggested that the PCA multivariate analysis was a useful tool for discrimination of the three nucleosides. The discriminatory power of the system was further substantiated by HCA. HCA took account of complete dimensionality of the data set, and classified the samples based on the relative distances between all samples in the N-dimensional space.35,48 In HCA, the relative distance corresponded to the similarity in behaviour of the original samples. The most similar samples accumulated into a cluster at the first level, which was then paired with other most similar samples or clusters at the second level and so on. Euclidean distance was herein used to measure the similarity between samples, and Ward's (minimum variance) method was applied to define a cluster. HCA generated a graphic diagram in the form of a dendrogram (Figure 4C). It was noted from Figure 4C that three distinct clusters could be produced, corresponding to the different types of nucleotides to some degree. This confirmed that HCA was also applied to differentiate the three nucleosides. The next step was to extend the scope of our system to identify the three nucleosides at a low concentration. When the concentration of each individual nucleoside was lowered to 0.5 mM or 20 µM, three distinct clusters corresponding to the three nucleosides could be still clearly observed in the PCA or HCA plots, suggesting the successful identification of the three nucleosides (Figure 5B-C and 6B-C). Therefore, fluorescence spectra of nucleoside-templated CuNCs coupled with the advanced multivariate chemometrics method could be used to discriminate different nucleosides at a concentration as low as 20 µM.


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4. CONCLUSIONS In summary, we successfully exploited small molecule nucleosides as templates to synthesize fluorescent CuNCs in the presence of ascorbic acid and citrate buffer. The role of various reactants such as ascorbic acid, nucleoside and citrate buffer in the synthesis process was explored. Through application of advanced multivariate chemometrics methods such as PCA and HCA, we can use these fluorescence changes of nucleoside-templated CuNCs to discriminate adenosine (A), cytidine (C), and guanosine (G) in a simple and efficient manner. It is anticipated that the fluorescence of the as-prepared CuNCs can be further modulated and extended to the field of optical data storage, biological labeling, and (bio) sensing applications. Much work toward this direction is under study. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Extra figures of the Fluorescence spectra of T-templated CuNCs under different buffers and the mixture of citrate and Cu2+ at different pH

AUTHOR INFORMATION Corresponding Authors *E−mail: [email protected] *E−mail: [email protected]. Tel.: +86 791 83969500 ORCID Yongnian Ni: 0000-0002-7281-7437 Notes The authors declare no competing financial interest.



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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (NSFC−21305061), the Natural Science Foundation of Jiangxi Province (20171BAB203018 and 20151BAB203021), the Jiangxi Provincial Department of Education (GJJ160006 and GJJ160204), and the State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University (SKLCBC−2013010).

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Figure captions Scheme 1. Schematic of aqueous synthesis of nucleoside-templated fluorescent CuCNs and their application for discrimination of nucleosides.

Figure 1. (A) Fluorescence emission spectra of the CuNCs synthesized with and without nucleoside. Inset: the corresponding photographs CuNCs under 365 nm UV light. Reaction conditions: cCu2+ = 40 µM, cnucleoside = 1.8 mM, cascorbic acid = 1.2 mM, ccitrate buffer = 3.0 mM, pH = 7.0, T = 80 °C, t = 3 h. (B) Fluorescence excitation spectra of the synthesized CuNCs with and without nucleoside. Experimental conditions are as in Figure 1(A).

Figure 2. TEM images of the A-CuNCs (A), C-CuNCs (B), and G-CuNCs (C). XPS spectra of Cu 2p in the A-CuNCs (D), C-CuNCs (E), and G-CuNCs (F). FTIR spectra of the A-CuNCs (G), C-CuNCs (H), and G-CuNCs (I).

Figure 3. Fluorescence emission spectra of A-CuNCs (A), C-CuNCs (B), and G-CuNCs(C), prepared under different experimental conditions: (a) cCu2+ = 40 µM, cnucleotide = 1.8 mM, cascorbic acid =

1.2 mM, ccitrate buffer = 3.0 mM, pH = 7.0, T = 80 °C, t = 3 h; (b) cCu2+ = 40 µM, cnucleotide =

1.8 mM, ccitrate buffer = 3.0 mM, pH = 7.0, T = 80 °C, t = 3 h; (c) cCu2+ = 40 µM, cnucleotide = 1.8 mM, ccitrate buffer = 3.0 mM, pH = 7.0, T = 80 °C, t = 10 h; (d) cCu2+ = 40 µM, cascorbic acid = 1.2 mM, ccitrate buffer = 3.0 mM, pH = 7.0, T = 80 °C, t = 3 h; (e) cCu2+ = 40 µM, cnucleotide = 1.8 mM, cascorbic acid

= 1.2 mM, ccitrate buffer = 3.0 mM, pH = 3.0, T = 80 °C, t = 3 h; (f) cCu2+ = 40 µM, cnucleotide = 1.8

mM, cascorbic acid = 1.2 mM, ccitrate buffer = 0.8 mM, pH = 7.0, T = 80 °C, t = 3 h; (g) cCu2+ = 40 µM,



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cnucleotide = 1.8 mM, cascorbic acid = 1.2 mM, cTris buffer = 3.0 mM, pH = 7.0, T = 80 °C, t = 3 h; (h) cCu2+ = 40 µM, cnucleotide = 1.8 mM, cascorbic acid = 1.2 mM, ccitrate buffer = 3.0 mM, pH = 7.0, T = 27 °C, t = 3 h.

Figure 4. (A) Fluorescence emission spectra of the CuNCs synthesized in the presence of 1.8 mM A, C or G. (B) PCA score plots of three nucleosides measured seven times. (C) HCA dendrogram for 21 samples obtained from three nucleosides; adenosine: sample No. 1-7; cytidine: sample No. 8-14; guanosine: sample No. 15-21.

Figure 5.(A) Fluorescence emission spectra of the CuNCs synthesized in the presence of 0.5 mM A, C and G. (B) PCA score plots of three nucleosides measured seven times. (C) HCA dendrogram for 21 samples obtained from three nucleosides; adenosine: sample No. 1-7; cytidine: sample No. 8-14; guanosine: sample No. 15-21.

Figure 6. (A) Fluorescence emission spectra of the CuNCs synthesized in the presence of 20 µM A, C and G. (B) PCA score plots of three nucleosides measured seven times. (C) HCA dendrogram for 21 samples obtained from three nucleosides; adenosine: sample No. 1-7; cytidine: sample No. 8-14; guanosine: sample No. 15-21.


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Scheme 1



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Figure 1

A

B


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Figure 2

D

E

F

G

H

I



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Figure 3

B

A

C


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Figure 4



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Figure 5

A

B

C


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Figure 6

B

A

C



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For TOC only


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One-Pot Aqueous Synthesis of Nucleoside ... - ACS Publications

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