Fabrication of Stable and Luminescent Copper Nanocluster-Based

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Fabrication of Stable and Luminescent Copper Nanocluster-Based AIE Particles and Their Application in #-Galactosidase Activity Assay Meizhi Zhao, Zhaosheng Qian, Mengting Zhong, Zhentian Chen, Hang Ao, and Hui Feng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09659 • Publication Date (Web): 01 Sep 2017 Downloaded from http://pubs.acs.org on September 3, 2017

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ACS Applied Materials & Interfaces

Fabrication of Stable and Luminescent Copper Nanocluster-Based

AIE

Particles

and

Their

Application in β-Galactosidase Activity Assay Meizhi Zhao,† Zhaosheng Qian,† Mengting Zhong, Zhentian Chen, Hang Ao and Hui Feng* College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, People’s Public of China * Corresponding author. E-mail: [email protected]; Tel. & Fax. +86-579-82282269. †

These authors contributed to this work equally.

College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, China

ACS Paragon Plus Environment

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ABSTRACT: Thiolated copper nanoclusters with aggregation-induced emission characteristic have becoming as a novel luminescent material, but it is still a challenging task to retain its bright luminescence in neutral solution. In this work, we report a new copper nanocluster with aggregation-induced emission (AIE) enhancement property using a hydrophobic molecule as the protecting ligand, and brightly luminescent AIE particles of copper nanocluster were prepared via hydrophobic interaction. These CuNCs AIE particles possess uniform rod-like shapes with sizes in hundreds of nanometer and intense luminescence, and more importantly its luminescence remains stable in neutral and alkaline solutions. It is found that 4-nitrophenol is able to effectively quench the luminescence of CuNC AIE particles through strong hydrophobic interaction and electron transfer between them. This strong quenching effect was adopted to develop a luminescent assay for β-galactosidase at physiological condition. This work presents a demonstration of preparing CuNC AIE particles with bright luminescence at neutral condition, and gives an example of the use of AIE particles in monitoring enzyme activity.


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INTRODUCTION Aggregation-induced emission luminogens (AIEgens) have been becoming a promising luminescent material with great potential applications in bioimaging and chemo/biosensing areas because they overcome the notorious aggregation-caused quenching (ACQ) effect of conventional luminogens.1-3 Organic aggregation-induced-emission dots (AIE dots) have also been proposed to attain high emission efficiency, and great advance in hexaphenylsilole or tetraphenylethene-containing AIE dots with fluorescence emission has been made in both preparation and utilization in biosensing and bioimaging.4,5 Apart from these organic AIEgens, the unique AIE behavior has also been found for thiolated copper nanoclusters (CuNCs) in recent studies,6,7 and these thiolated CuNCs frequently possess long lifetimes in microseconds and red/NIR luminescence, which are much different from fluorescent CuNCs protected by polymers or macromolecules.8,9 These merits of thiolated CuNCs over organic AIEgens enable them to facilitate the imaging tissues and avoid the autofluorescence of biosystems. Up to date various thiolated CuNCs with different capping ligands have been prepared and investigated in-depth in mechanochromic property, and their use in sensing pH, temperature, water or hydrogen sulfide was examined.10-15 However, these CuNCs only possess bright luminescence in acidic solution but cannot work in neutral or alkaline solution, and their luminescence is very sensitive to the change of environmental temperature and presence of oxidizing substances like hydrogen peroxide. Glutathione-protected CuNCs reported in our previous paper also showed pHdependent luminescence behavior and can only intensely luminesce under acidic condition, which makes it only suited for assaying enzymes that can remain catalysis activity in acidic solution such as acid phosphatase.16 This drawback of dispersed CuNCs greatly hinders their


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application scope. As a result, it is critical to develop CuNC AIE particles with bright luminescence and good stability under various conditions. β-Galactosidase (β-Gal, EC, 3.2.1.23) is an exoglycosidase that catalyzes the hydrolysis of βgalactosides of a variety of substrates including ganglioside GM1, lactose and various glycoproteins.17,18 LacZ β-galactosidase is commonly used in molecular biology as a marker to monitor gene expression.19 It is also found that β-galactosidase is closely associated with senescence of cells,20 and a recent study showed that it can serve as a molecular target for visualizing peritoneal metastases from ovarian cancers.21 A variety of fluorescent substrates have been synthesized and applied in detection of β-galactosidase activity, and the fluorophore in substrates varies from coumarins to rhodamines.22-25 Despite of good performance of these molecular probes in tracking β-galactosidase in living cells, luminescent nanomaterials have been used to prevent the interference from the adsorption of big macromolecules in biosystems.26-28 Zeng et al.26 utilized enzyme-triggered aggregation of gold nanoparticles to develop a colorimetric assay of β-galactosidase, and another colorimetric assay based on gold nanorods was established for further detection of Escherichia coli.27 Our recent study demonstrated that β-cyclodextrin modified carbon quantum dot nanoprobe is capable to quantify β-galactosidase in vitro and imaging its level in primary overran cancer cells.28 However, the fluorescence emission and quantum yield of this nanoprobe need to be improved for practical application. In this work, a facile synthesis of 4-methylthiophenol protected copper nanoclusters with aggregation-induced emission enhancement characteristic was achieved, and stable and bright luminescent particles were prepared by hydrophobicity-controlled assembly of copper nanoclusters. To overcome the drawbacks of preceding CuNCs capped with hydrophilic ligands


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that the luminescence cannot be retained in neutral and alkaline solutions, we adopted a hydrophobic ligand, 4-methylthiophenol, to synthesize hydrophobic copper nanoclusters with the assistance of a reducing agent NH2NH2. The morphology and optical properties of as-prepared CuNCs were examined as well as its AIE behavior. Hydrophobicity of the protecting ligand of CuNCs makes it possible to self-assemble into brightly luminescent AIE particles. The size, luminescence performance and stability of these AIE particles were investigated in detail. Bright luminescence of AIE particles in neutral and alkaline solutions enables them to be used to assay enzyme activity at physiological conditions. The detection of β-galactosidase in neutral buffer using the AIE particles is a demonstration of its application in biosensing. The schematic illustration for preparation of CuNC particles and its use in monitoring β-galactosidase was shown in Scheme 1. Weakly emissive CuNCs were first assembled into bright luminescent CuNC particles via hydrophobic interaction. 4-Nitrophenyl-β-D-galactopyranoside (NPGal) was selected as the substrate of β-galactosidase. The presence of β-galactosidase triggers the rapid hydrolysis of the substrate into galactose and 4-nitrophenol which could adsorb on CuNC particles and efficiently quench its luminescence. The evaluation of β-galactosidase level can be conducted in the continuous and real-time way.

EXPERIMENTAL SECTION Materials and Reagents. Triple-distilled water was used throughout the experimental process. Copper nitrate (Cu(NO3)2·3H2O), hydrazine hydrate (NH2NH2, 50%), 4-nitrophenol (NP), and 4nitrophenyl-β-D-galactopyranoside (NPGal) and other inorganic salts were obtained from J&K Chemical Company (Shanghai, China). β-Galactosidase (β-Gal, EC 3.2.1.23, 25kU) from aspergillus oryzae and 4-methylthiolphenol were bought from Sigma-Aldrich Company


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(Shanghai, China). Phosphate-buffered saline (PBS, pH 7.0) was prepared by mixing stock solution of NaH2PO4 and Na2HPO4. All reagents were of analytical grade and without any further purification. Synthesis of 4-methylthiophenol-capped CuNCs. In a typical procedure, Cu(NO3)2·3H2O (0.01 g) and 1 mL NH2NH2 (50%) were added into 20 mL of water in a 100-mL round bottom flask. The solution was vigorously stirred at room temperature for 10 min, and then 4methylthiolphenol (0.05 g) was added to the preceding solution. A yellow turbid solution was formed after 30 min of stirring. The appearance of red luminescence under a UV lamp indicated the completion of the synthesis. In order to remove excessive reactants such as 4methylthiolphenol, NH2NH2 and Cu(NO3)2, the resulting solution containing CuNCs was further centrifuged at 10,000 rpm for 10 min, and the precipitate was washed with ethanol three times. The resultant 4-methylthiolphenol-capped CuNCs were collected and then freeze-dried under vacuum, and as-prepared yellow powder was stored in the refrigerator for long-term preservation. Preparation of Stable AIE Particles of CuNCs. A certain amount of 4-methylthiolphenolcapped CuNCs (0.01 g) was dissolved in 20 mL of ethanol under the refluxing. A portion of the resulting solution (2 mL) was dropped into 10 mL of water drop by drop under the ultrasonic treatment. The resultant mixture was further allowed for 30 minutes’ ultrasonic treatment. Stable AIE particles of CuNCs dispersed in water were obtained at the end. To prepare AIE particles dispersion in buffer solution, the preceding procedure was slightly modified by using PBS buffer solution instead of water. Determination of Luminescence Quantum Yield of CuNC AIE Particles. Determination of luminescence quantum yield of CuNC AIE particles was achieved by comparison of the wavelength integrated intensity of CuNC AIE particles to that of rhodamine 6G as the standard


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reference. The optical absorbance was kept below 0.05 to avoid inner filter effects. The luminescence quantum yield of CuNC AIE particles was calculated using the following formula: Φ =Φs [I·AS·n2]/[ IS·A·nS2], where Φ is the quantum yield of the substance, I is the integrated intensity, A is the optical density and n is the refractive index of the solvent. The subscript S refers to the standard reference of known quantum yield. Rhodamine 6G was chosen as the standard reference, and its quantum yield in ethanol is 0.95. Luminescence Quenching of AIE particles of CuNCs by 4-Nitrophenol. For optimization of incubation time for luminescence quenching by 4-nitrophenol, 30 µL of 4-nitrophenol solution (10.0 mM) was added into 3.0 mL of AIE particles of CuNCs (0.08 mg/mL) buffer solution (PBS, pH 7.0). Then the luminescence of mixed solution was recorded after different incubation times of 1, 2, 3, 4, 5, 6, and 7 min, respectively. Under the optimum incubation time (3 min), the luminescence spectra of AIE particles (0.08 mg/mL) in the presence of different concentrations of 4-nitrophenol (0.0 - 100.0 µM) were recorded at the optimal excitation wavelength of 345 nm. All experiments were conducted at 37 °C. Luminescent Assay for β-Galactosidase Activity Based on AIE Particles.

The β-

galactosidase activity was first assessed as extending incubation time with a certain amount of βgalactosidase level. The sensing system containing AIE particles (0.08 mg/mL), NPGal (100.0 µM), and β-galactosidase (260.0 U/L) in PBS solution (pH 7.0) was monitored by luminescence spectra every minute in a period of 10 min. Under the optimal incubation time (6 min), different levels of β-Gal ranging from 0.0 to 260.0 U/L was, respectively, added into 3.0 mL of assay solution containing AIE particles (0.08 mg/mL) and NPGal (100.0 µM). After an incubation time of 6 min at 37 °C, the luminescence spectra of the assay solution with different amounts of


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β-galactosidase were recorded at an excitation wavelength of 345 nm, respectively. Another three assays were performed using 50.0, 150.0 and 200.0 µM of NPGal in the same way, respectively. Characterization Methods. The UV-vis spectra were recorded on a Perkin-Elmer Lambda 950 spectrometer. The luminescence spectra and time-resolved luminescence decay tests were performed using Edinburgh Instruments Model FLS980 fluorescence spectrophotometer. Transmission electron microscopy (TEM) was conducted on a JEOL-Model 2100F instrument with an accelerating voltage of 200 kV. A Kratos Axis ULTRA X-ray photoelectron spectroscopy was used for the X-ray photoelectron spectroscopy analyses. The size distribution of CuNC AIE particles in aqueous solution was determined using Zetasizer Nano ZS90 (Malvern, UK).

Scheme 1. Schematic Illustration of Self-Assembly of AIE Particles of CuNCs Mediated by Hydrophobic Interaction and Application in Detection of β-Galactosidase Activity Using 4-Nitrophenol-Releasing Substrate.


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RESULTS AND DISCUSSION Synthesis and Characterization of 4-Methylthiophenol Capped CuNCs. In the previous reports, some hydrophilic thiol-containing molecules like glutathione, penicillamine, cysteine and 4-thiolbenzoic acid, were used to synthesize CuNCs. Their hydrophilicity makes as-prepared CuNCs readily to disperse in neutral and alkaline solutions, and for this reason these CuNCs show very weak luminescence in such solutions. To overcome this fault of CuNCs, we chosen 4methylthiophenol as the protecting ligand and used hydrazine hydrate as the reducing agent. After 30 minutes’ reaction, bright red luminescent CuNCs precipitate can be obtained in the solution. As-prepared CuNCs are easily dispersed in DMF, DMSO and ethanol, but are insoluble in water. CuNCs have significant absorption in the whole UV-visible range and have a maximum absorption at 333 nm, which is easily distinguished from that of 4-methylthiophenol in Figure S1. CuNCs dispersion in ethanol shows faint red emission under the UV light, but CuNCs powder exhibits bright luminescence upon the irradiation as shown in Figure 1A. This intensely emissive behavior of CuNCs in solid state implies that as-prepared CuNCs possess aggregation-induced emission or aggregation-induced emission enhancement property. Their luminescence spectra clearly indicate that CuNCs dispersion and powder have same emission maximum of 625 nm, but their luminescence intensity differs largely and their excitation maxima are also different by 140 nm. The morphology and element composition of CuNCs dispersion in ethanol were further characterized by transmission electron microscope (TEM) and X-ray photoelectron spectroscopy analysis (XPS), respectively. TEM image of CuNCs dispersion in ethanol in Figure 1B clearly illustrates that CuNCs are in well dispersed state in such an ethanol solution, and their sizes are concentrated at 2 – 3 nm in diameter. Similar crystalline structures for CuNCs can be observed from its high-resolution TEM image. XPS wide spectrum in Figure 1C displays the predominant


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signals from Cu2p1/2 (952.2 eV), Cu2p3/2 (932.2 eV), Cu3s (122.1 eV), Cu3p (75.2 eV), C1s (284.2), S2s (226.2 eV), S2p (162.2 eV) and O1s (532.1), respectively. XPS signals from Cu, C and S are originated from the corresponding starting materials, but the additional signal from O may be from the molecular oxygen in air during the determination. The XPS peak of 932.2 eV for Cu2p3/2 electron of CuNCs is substantially different from that of Cu(II), indicating that copper atoms in CuNCs exist mainly in lower valence states Cu(I) or Cu0. This is also consistent to the finding that specific satellite peaks at around 943 eV of Cu(II) does not appear in the highresolution XPS spectrum for Cu2p in Figure S2. Figure 1D shows aggregation-induced emission enhancement behavior of CuNCs. Only very weak emission can be recorded for CuNCs dispersion in ethanol; however, as the water content in the water-ethanol mixture is gradually increased from 0% to 100%, an apparent luminescence enhancement was observed in both luminescence spectra and by naked eyes. This observation further verified the AIE characteristic of the CuNCs capped by 4-methylthiophenol. It is generally accepted that intramolecular vibration and rotation predominantly contribute to aggregation-induced emission behavior of organic AIEgens.1 In a recent review, Xie’s group proposed metallophilic interaction as the major factor for AIE property of thiolated metal nanoclusters.29 By considering the two aspects, it is reasonable to deduce that both vibration and rotation of surface ligands of CuNCs and metallophilic interaction play significant roles in the aggregation-induced emission process. However, we also found that these CuNCs with AIE property also respond well to the change of temperature, and their luminescence is gradually quenched as increasing the solvent temperature. This leads us to speculate that significant inhibition of dynamic quenching to this long-lived luminescence may also greatly contribute the AIE phenomenon of CuNCs. This underlying


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nature of AIE phenomenon of thiolated metal nanoclusters is still under investigation for its complexity. Self-assembly of CuNCs into AIE Particles Mediated by Hydrophobic Interaction. As revealed by the preceding section, 4-methylthiophenol capped CuNCs possess aggregationinduced emission enhancement characteristic. To attain high luminescence efficiency, CuNCs

Figure 1. (A) Luminescence excitation and emission spectra of CuNCs dispersion in ethanol and CuNCs powder. Inset: images of CuNCs dispersion (a) and CuNCs powder (b) under UV lamp. (B) TEM image and high-resolution TEM image of CuNCs dispersion in ethanol. (C) XPS wide survey spectrum of CuNCs. (D) Luminescence spectra of CuNCs (0.05 mg/mL) in different ratios of ethanol-water solutions. Inset: photographs of CuNCs in different solutions upon the irradiation of UV light.


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Figure 2. (A) TEM image of CuNC AIE particles (0.08 mg/mL) in water. (B) Excitation and emission spectra of CuNCs dispersion in ethanol and CuNC AIE particles in water. Inset: photographs of CuNCs dispersion in ethanol (a) and CuNC AIE particles in water (b) upon the irradiation of UV light.

were assembled into AIE particles through hydrophobic interaction. When CuNCs dispersion in ethanol were added into water under the ultrasonic treatment, apparent aggregation induced by hydrophobic nature of the ligand occurred, and aggregated CuNCs in water exhibit very intense red luminescence under the irradiation of UV light. The morphology of aggregated CuNCs in water was examined by TEM as shown in Figure 2A. A great number of nanoparticles consisting of CuNCs were observed, and they exist in rod-like shape and with the size of around (189±28) ×(72±6) nm. These nanoparticles composed of CuNCs are obviously different with bare organic AIE dots prepared from tetraphenylethene derivatives in morphology and size distribution.30,31 Furthermore, size distribution of these CuNC AIE particles in aqueous solution determined by dynamic light scattering technique was shown in Figure S3, and one can note that their sizes are concentrated at 242.5 nm in average, which is consistent with the observation from TEM image. In comparison with those organic AIE dots, bright red luminescence is easily


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achieved using CuNC AIE particles due to its simple preparation process. The luminescence of CuNC AIE particles in water is nearly identical to that of CuNCs dispersion in ethanol with the maximum emission of 625 nm. However, its luminescence is much brighter than that of CuNCs dispersion in ethanol as shown in Figure 2B. The quantum yield of CuNC AIE particles in water was determined to be 9%, which is much higher than that in ethanol (2.4%), and rhodamine 6G (QY 0.95 in ethanol) was chosen as standard reference in the determination of quantum yields. The lifetime of CuNC AIE particle in water from its time-resolved decay curve in Figure S4 was estimated to be 1.96 µs, and its solid powder has a longer lifetime of 7.65 µs. Their luminescence can be classified as phosphorescence in terms of emission duration of the microsecond magnitude because it is generally accepted that the phosphorescence frequently possesses such a long lifetime in microseconds or longer. The longer lifetime of its solid state indicates that physical aggregation in solid greatly restricts the vibration and rotation of the capping ligands in CuNCs, and further leads to the inhibition of non-radiative de-excitation pathways which results in the prolongation of the lifetime and sharp increase of the quantum yield. Their relatively long microsecond lifetimes are helpful to avoid the interference from autofluorescence with short lifetime in nanosecond domain.32-35 The effect of variable pHs on the luminescence of CuNC AIE particles was examined as shown in Figure 3A. It is found that almost no impact was exerted by the change of pHs in the range of pH 2 – pH 12, suggesting that the CuNC AIE particles can work well in different solutions at various pHs. The stability of CuNC AIE particles in three solutions at pH 2.0, pH 7.0 and pH 12.0 was also investigated. Figure 3B indicates that the luminescence of CuNC AIE particles at three pHs has only changed less than 5% within 1 h, and after 15 h the majority of their luminescence (over 90%) is still retained, indicating that these CuNC AIE particles are very stable in aqueous solution. The excellent stability of CuNC AIE


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particles is attributed to the hydrophobic nature of 4-methylthiophenol as the surface-capping ligand of CuNCs. Its high hydrophobicity of the protecting ligand makes CuNCs to aggregate in a compact way, and the existence of hydroxide ions exerts no impact on its aggregation. In addition, salt effect and the influence of different buffer solutions including Britton-Robinson,

Figure 3. (A) Luminescence intensity of CuNC AIE particles (0.08 mg/mL) versus variable pHs from 2.0 to 12.0. (B) Luminescence intensity of CuNC AIE particles (0.08 mg/mL) versus standing time from 0 to 15 h in pH 2, pH 7 and pH 12 solutions respectively. (C) Luminescence spectra of AIE particles (0.08 mg/mL) as a function of temperature from 0 to 70°C. (D) Luminescence spectra of AIE particles (0.08 mg/mL) in the presence of different amounts of H2O2 in the range of 0.0 to 18.0 mM.


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HAc-NaAC, PBS and Tris-HCl buffer solutions on the luminescence of AIE particles were carried out to verify its good stability. Figure S5 shows that only slight variation in luminescence can be observed for the four buffer solutions, and the presence of up to 1.1 mM NaCl solution hardly exert any impact on the luminescence. In addition, the resistance to photobleaching of CuNC AIE particles was also tested. Figure S6 displays good photostability of CuNC AIE particles from its only slight decrease in luminescence after ten hours’ irradiation of UV light centered at 365 nm. Moreover, it has been found that CuNCs reported previously are subjected to temperature and the presence of hydrogen peroxide. When the temperature is raised to 70 °C most luminescence was quenched for GSH-capped CuNCs;11 however, the luminescence of AIE particles in this work only was decreased by 30% as shown in Figure 3C. This apparent difference in response to high temperature between dispersed CuNCs and CuNC AIE particles strongly indicates that AIE particles are more stable than CuNCs at variable temperatures. Figure 3D shows that a high amount of hydrogen peroxide (18.0 mM) only results in 40% luminescence quenching for CuNC AIE particles, which shows much resistance to oxidation in comparison with that of penicillamine-protected CuNC aggregates because only 5 mM H2O2 leads to complete quenching of its luminescence.7 Luminescence Quenching of CuNC AIE Particles by 4-Nitrophenol via Hydrophobic Interaction. 4-Nitrophenol as a good electron acceptor and an efficient quencher has been used to quench the fluorescence of CdSe/ZnS quantum dots,36 carbon quantum dots37 and copper nanocluster aggregates13 through electron transfer. As a result, quenching effect of 4-nitrophenol on CuNC AIE particles was also examined. Three minutes’ incubation was required to attain the equilibrium for the luminescence quenching experiment by 4-nitrophenol according to Figure S7. Figure 4A shows the variation in quenching efficiency of CuNC AIE particles by 100.0 µM


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Figure 4. (A) Quenching efficiency of the luminescence of CuNC AIE Particles (0.08 mg/mL) by 4-nitropheol versus variable pHs in the range of pH 6.0 – pH 8.0. (B) Change of luminescence spectra of CuNC AIE particles (0.08 mg/mL) in the presence of different concentrations of 4-nitrophenol from 0.0 to 100.0 µM.

4-nitrophenol at variable pHs in the range of pH 6.0 – pH 8.0. It is found that the quenching efficiency reached almost 80% in the range of pH 6.0 – pH 7.0, but it was markedly decreased as the pH value was raised over pH 7.0. This observation is as a result of the fact that more hydrophobic nature of 4-nitrophenol in neutral or acid solution leads to stronger interaction between 4-nitrophenol and CuNC AIE particles. As a result, pH 7.0 is suited for sensing 4nitrophenol and relating enzyme activity. The gradual PL quenching trend of CuNC AIE particles as the concentration of 4-nitrophenol was shown in Figure 4B. To explore the underlying nature for this quenching, the value I0/I versus the concentration of 4-nitrophenol in the range of 0.0 − 46.0 µM was plotted in Figure S8, which is consistent with Stern-Volmer equation I0/I = 1.00 + 0.0328[Q] where R2 is 0.997. Furthermore, the luminescence lifetime of CuNC AIE particles is nearly unchanged in the presence of varying amount of 4-nitrophenol from time-resolved fluorescence decay curves in Figure S9. As a result, it is concluded that this effective quenching induced by 4-nitrophenol is probably originated from static quenching


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Figure 5. (A) Change of luminescence spectra of CuNC AIE particles in the presence of different components: (a) CuNC AIE particles (0.08 mg/mL); (b) CuNC AIE particles, and 4-nitrophenol (100.0 µM); (c) CuNC AIE particles and NPGal (100.0 µM); (d) CuNC AIE particles, NPGal (100.0 µM) and β-galactosidase (260.0 U/L). (B) Luminescence quenching efficiency vs incubation time at different β-galactosidase levels (150.0, and 260.0 U/L) in the mixture containing CuNC AIE particles (0.08 mg/mL) and NPGal (100.0 µM). (C) Luminescence spectra of the mixture containing CuNC AIE particles and NPGal (150.0 µM) at varied β-

galactosidase levels from 0.0 to 332.0 U/L. (D) Fitting curve between quenched luminescence and βgalactosidase activity in the range from 2.5 to 212.0 U/L. All the data have been repeated three times because no significant difference between data sets from three and seven parallel tests is observed. All experiments were conducted at 37 °C and in PBS buffer solution (pH 7.0), and the optimum incubation time of 6 min was used.


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caused the formation of non-luminescent complex between CuNC AIE particles and 4nitrophenol. Stronger hydrophobic interaction between them mainly contributes to the close proximity for the formation of the nonluminescent complex. Real-Time Luminescent Assay for β-Gal Based on CuNC AIE Particles. The effective quenching ability of 4-nitrophenol to the luminescence of CuNC AIE Particles provides the possibility to establish a real-time assay for β-galactosidase using 4-nitrophenyl-β-Dgalactopyranoside (NPGal) as the substrate. The feasibility was first tested as shown in Figure 5A. The presence of 100.0 of NPGal results in only about 20% of luminescence quenching of CuNC AIE particles; however, the introduction of the same amount of 4-nitrophenol leads to a quenching efficiency of more than 80%. This large difference in quenching efficiency provides the possibility to detect β-Gal level, which is confirmed by the sharp decrease in luminescence after the addition of 260.0 U/L of β-Gal into the system containing CuNC AIE particles and NPGal. Thus it is feasible to measure β-Gal level based this detection strategy in a real-time manner. Figure 5B shows the optimization of incubation time for luminescence quenching in the presence of two levels β-galactosidase (150.0 and 260.0 U/L), and it is found that the luminescence is rapidly decreasing within 4 min of incubation time and are almost unchanged after 6 min of incubation, suggesting 6 min is the sufficient to attain the equilibrium. The sensing performance of the system containing CuNC AIE particles and NPGal (150.0 µM) was shown in Figure 5C with the addition of varying levels of β-galactosidase under optimum conditions, and the luminescence intensity is progressively declined as β-galactosidase level is increased from 0.0 to 332.0 U/L. Figure 5D clearly reveals a good linear relationship between the luminescence and β-galactosidase level in the range from 2.5 to 212.0 U/L, and the regression equation can be expressed as y = -460.8x + 132335.9, where R2 is 0.997. The detection limit using three times


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standard deviations is determined to be 0.9 U/L. The influence of the amount of NPGal on the analytical performance of the developed β-galactosidase assay was further examined by using another three amounts of NPGal (50.0, 100.0 and 200.0 µM). Similar decline trends in luminescence as the rise of β-Gal level were observed for the assays using 50.0, 100.0 µM and 200.0 µM of NPGal shown in Figure S10, Figure S11 and Figure S12. Table S1 summarizes the detection limits and linear scopes of sensing systems using four different concentrations of substrate. It is noted that the detection limits using 50.0, 100.0 and 150.0 µM substrate are slight lower than that using 200.0 µM substrate, and the linear scope is broadest when 150.0 µM substrate was used. As a result, 150.0 µM of NPGal is best suited to quantify β-galactosidase level in the wide scope of 2.5 – 212.0 U/L. Table S2 summaries that the detection limit of this assay is comparable to that based on carbon quantum dots (0.6 U/L),28 but is much lower than those based SA-βGal probe (14.0 U/L)38 and quinolinium-based enzymatic probes (5.0 or 100.0 U/L).39 Several possibly interfering substances that are dominantly present in serum were selected to test the specificity of the β-Gal assay such as typical organic ions (K+, Na+, Ca2+, Mg2+, Fe2+, Cl-, PO43-), glutathione, cysteine, serum albumin and α-glucosidase. The results in Figure S13 show that these possible interferents with a large concentration (1.0 mM) only cause quenching efficiencies of less than 10%, but the presence of a small level of β-Gal leads to a high quenching efficiency of more than 80%. The tremendous difference in fluorescence response indicates the excellent specificity of the developed assay to β-Gal. In addition, human serum was used to quantitatively assess the interference from complex matrix by standard addition method. Eight standard added values in the range of 0.0 – 80.0 U/L were selected according to the β-Gal assay using 150 µM of NPGal. Table S3 lists the recovery ratios of measured value to added value for nine samples, and the recovery ratio varies from 101.2% to 103.8%, indicating that the


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complex matrix such as human serum exerts an insignificant interference on this assay, and this assay is capable to accurately measure β-Gal level in such a complex matrix as real samples.

CONCLUSION In summary, a facile synthesis of copper nanoclusters with significant aggregation-induced emission enhancement characteristic was proposed using hydrophobic 4-methylthiophenol as the capping ligand, and stable copper nanocluster AIE particles with intense red luminescence were prepared via self-assembly mediated by hydrophobic interaction. In comparison with CuNCs protected by hydrophilic molecules, CuNC AIE particles in this work can retain bright luminescence in various pH solutions including neutral and alkaline solutions. This property has enabled CuNC AIE particles to be served as a stable luminogen at physiological condition. It is found that 4-nitrophenol can effectively quench the luminescence of CuNC AIE particles through close proximity controlled by their similar hydrophobic nature. This specific quenching effect was utilized to design a luminescent assay for β-galactosidase in a real-time way. This demonstration of the use of CuNC AIE particles in assay enzyme activity has proven that CuNC AIE particles are capable to retain its bright luminescence at physiological condition, and can be effectively quenched by electron acceptors such as 4-nitrophenol. Although a long preservation of bright luminescence for CuNC AIE particles is still needed to be improved, reliable detection of β-galactosidase based on this material is achieved.

ASSOCIATED CONTENT Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Details of UV-visible, XPS, luminescence spectra of CuNC AIE particles and data for β-galactosidase assay.

AUTHOR INFORMATION


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Corresponding Author *E-mail: [email protected]. Tel: +86-579-82282269. Fax: +86-579-82282269. †

These authors contributed to this work equally.

ACKNOWLEDGMENT We gratefully acknowledge financial support from the National Natural Science Foundation of China (Grant Nos. 21775139 and 21675143) and Natural Science Foundation of Zhejiang Province (Grant No. LY17B050003).

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Fabrication of Stable and Luminescent Copper Nanocluster-Based

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