Luminescent Aggregated Copper Nanoclusters Nanoswitch

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Luminescent Aggregated Copper Nanoclusters Nanoswitch Controlled by Hydrophobic Interaction for Real-time Monitoring of Acid Phosphatase Activity Yuanyuan Huang, Hui Feng, Weidong Liu, Yingying Zhou, Cong Tang, Hang Ao, Meizhi Zhao, Guilin Chen, Jianrong Chen, and Zhaosheng Qian Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02957 • Publication Date (Web): 31 Oct 2016 Downloaded from http://pubs.acs.org on November 1, 2016

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Analytical Chemistry

Luminescent

Aggregated

Copper

Nanoclusters

Nanoswitch Controlled by Hydrophobic Interaction for Real-time Monitoring of Acid Phosphatase Activity Yuanyuan Huang,† Hui Feng,† Weidong Liu, Yingying Zhou, Cong Tang, Hang Ao, Meizhi Zhao, Guilin Chen, Jianrong Chen and Zhaosheng Qian* * 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

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ABSTRACT: A reversible luminescence nanoswitch through competitive hydrophobic interaction among copper nanoclusters, p-nitrophenol and α-cyclodextrin is established, and a reliable real-time luminescent assay for acid phosphatase (ACP) activity is developed on the basis of this luminescence nanoswitch. Stable and intensely luminescent copper nanoclusters (CuNCs) were synthesized via a green one-pot approach. The hydrophobic nature of CuNCs aggregate surface is identified, and further used to drive the adsorption of p-nitrophenol on the surface of CuNCs aggregate due to their hydrophobic interaction. This close contact switches off the luminescence of CuNCs aggregate through static quenching mechanism. However, the introduction of α-cyclodextrin switches on the luminescence since stronger host-guest interaction between α-cyclodextrin and p-nitrophenol causes the removal of p-nitrophenol from the surface of CuNCs. This nanoswitch in response to external stimulus p-nitrophenol or α-cyclodextrin can be run in a reversible way. Luminescence quenching by p-nitrophenol is further utilized to develop ACP assay using 4-nitrophenyl phosphate ester as the substrate. Quantitative measurement of ACP level with a low detection limit of 1.3 U/L was achieved based on this specific detection strategy. This work reports a luminescence nanoswitch mediated by hydrophobic interaction, and provides a sensitive detection method for ACP level which is capable for practical detection in human serum and seminal plasma.

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INTRODUCTION Acid phosphatase (ACP, EC 3.1.3.2), a phosphatase widespread in many animal and plant species, can remove a phosphate group from its substrate by hydrolyzing phosphoric acid monoester into a phosphate ion and a molecule with a free hydroxyl group, and has the optimum catalysis ability at an optimal acidic pH.1 It has been proven that an abnormally elevated level of ACP in the human body is closely associated to some diseases including metastasized prostate cancer, hyperparathyroidism, Gaucher’s disease, kidney disease and multiple myeloma.2,3 As a result, precise monitoring of ACP level in blood is required for medical diagnosis and drug screening of related diseases because it is always regarded as an important serum marker and a useful prognostic indicator, and can be used as drug targets.4,5 In addition, ACP level in seminal plasma is also considered as an appropriate biomarker to evaluate prostate function.6 It is of great practical significance to develop reliable and sensitive assays for ACP since the level of ACP has been adopted as an auxiliary tool for preliminary pathologic diagnosis for many diseases. Traditional assays for ACP are based on colorimetric determination of hydrolysis products of its substrates, which varied from p-nitrophenylphosphate and phosphotyrosine to 2,6-dichloro-4acetylphenyl

phosphate.7-9

Although

several

assays

based

on

potentiometric

and

chromatographic approaches were established to detect ACP level,10-13 most efforts have been devoted to fluorometric assay because of its high sensitivity, convenience and accessible instrument requirement. To date, only a few fluorescent assays for ACP have been proposed, and most of them mainly adopted organic dyes or fluorescent polymers as the fluorogenic indicators. The initial assays based on organic dyes utilized fluorogenic substrates,14 but recent work demonstrated the evaluation of ACP level through aggregation-caused quenching of free organic dyes.15,16 Two types of fluorescent polymers were also used to determine ACP activity through

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two different detection strategies, but these assays often lack sufficient sensitivity due to the limited emission efficiency of the polymers.17,18 Recently, several novel luminescent nanomaterials including graphene quantum dots,19,20 gold nanoclusters21 and CuInS2 quantum dots22,23 were employed to quantify ACP level, and these assays showed good analytical performance in evaluation of ACP activity. However, most of these assays require the mediation of transition metal ions such as Ni(II), Cu(II) and Cr(VI), and their susceptibility to pH values and buffer solution may lead to instability of the sensing system and narrow application. As a result, the development of reliable and sensitive assays for ACP based on novel detection strategies and fluorogenic indicators is still a challenging task. Luminescent copper nanoclusters (CuNCs), as one member of metal nanoclusters, have been becoming an active research area owing to their inherent merits including excellent luminescence and significantly low cost in comparison to silver and gold nanoclusters.24 Current research progress focuses on the synthetic methods of CuNCs and the improvement of their optical property.25 It is found that the stabilizing agents on the surface significantly impact their size and optical property, and various capping ligands including DNA oligonucleotides,26-28 proteins,29,30 peptides31 and some small thiol-containing molecules can be used to prepare CuNCs. These thoil-containing molecules are more suited for synthesis of CuNCs because they can serve as both reductant and capping ligand, and have a much low cost compared to the others.32-34 More importantly, recent reports revealed that CuNCs synthesized from small thoil-containing molecules possess a specific aggregation-induced emission enhancement (AIEE) property because high compactness suppresses intramolecular vibration and rotation of the capping ligand and enhances the emission intensity of CuNCs.35-37 CuNCs with this AIEE property have been utilized for detection of some chemical species such as water, metal cations, anions and small

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molecules of interest.38-40 However, to our best knowledge, none of these CuCNs capped with small molecules has been used to assay biomolecules such as enzyme to date. In this study, we design a copper nanoclusters (CuNCs) aggregate based nanoswitch controlled by hydrophobic interaction. The brightly red luminescent CuNCs aggregate from a facile onestep synthesis was adopted as the indicator. Under acidic condition CuNCs severely aggregate to enhance the luminescence due to hydrophobic nature of the capping penicillamine in the protonated form, and a specific hydrophobic microenvironment on the surface of CuNCs aggregate is created. p-Nitrophenol with similar hydrophobicity is driven to adsorb on the surface of CuNCs aggregate in aqueous solution due to their strong hydrophobic interaction, and this close proximity results in luminescence quenching of CuNCs aggregate. Stronger hydrophobic interaction between α-cyclodextrin and p-nitrophenol leads to the removal of pnitrophenol from the CuNCs surface, and thus the introduction of α-cyclodextrin switches on the luminescence of CuNCs aggregate. As a result, this CuNCs aggregate nanoswitch can respond to external stimulus p-nitrophenol or α-cyclodextrin. The sensitive response of the nanoswitch to pnitrophenol can be used to design the detection of ACP level when 4-nitrophenyl phosphate ester (NPP) was chosen as the substrate. Therefore, a convenient and reliable assay for ACP can be established in terms of this correlation between quenching efficiency and ACP level. This assay for ACP level detection possesses sufficient sensitivity for practical samples including serum and seminal plasma, and is capable to monitor ACP level in a 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), acetic acid (HAc), sodium acetate (NaAc), α-cyclodextrin (αCD), p-nitrophenol (NP), p-nitrophenyl phosphate disodium hexahydrate (NPP) were purchased

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from J&K Chemical Company (Shanghai, China). D-penicillamine (PA), acid phosphatase (ACP, EC 3.1.3.2) from potato, bovine serum albumin (BSA), immunoglobulin G (IgG), acetylcholinesterase (AChE, EC 3.1.1.7), tyrosinase (TYR, EC 1.14.18.1), glucose oxidase (GOx, EC 1.1.3.4) and lysozyme (LYS, EC 3.2.1.17) were bought from Sigma-Aldrich Company (Shanghai, China). HAc-NaAc buffer solution (pH 5.0) was prepared by mixing stock solutions of NaAc (0.2 M) and HAc (0.2 M). All reagents were of analytical grade and without any further purification. Synthesis of CuNCs. In a typical synthesis, penicillamine-capped CuNCs were prepared by adding penicillamine (0.03 g) and Cu(NO3)2∙3H2O (0.006 g) into a 10 mL centrifuge tube with 5 mL of triple-distilled water followed vigorous stirring. After about 15 minutes, a milky and turbid solution was generated and exhibited a bright red luminescence under UV lamp irradiation (365 nm). The mixture was stirred for 90 min in air at room temperature. The precipitate was collected by centrifugation at 8000 rpm for 10 min followed by washing thoroughly with water for three times. Then the product was freeze-dried under the vacuum and stored in the refrigerator (-20 °C) for long-term preservation. Luminescence quenching of CuNCs by p-nitrophenol. For optimization of incubation time for luminescence quenching by 4-nitrophenol, 25 μL of p-nitrophenol solution (10.0 mM) was

added into 3.0 mL of CuNCs (0.28 mg/mL) buffer solution (HAc-NaAc, pH 5.0). Then, the luminescence of mixed solution was recorded after different incubation times of 2, 4, 6, 8, 10, 12, 14, 16 and 18 minutes respectively. Under the optimum incubation time, the solution (3.0 mL) containing CuNCs (0.28 mg/mL) and various amounts of p-nitrophenol (0.0 – 83.3 µM) in HAcNaAc (0.2 M, pH 5.0) solution was monitored by fluorescence spectroscopy at the optimal excitation wavelength. After a certain amount of p-nitrophenol is added every time, 10 min of

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incubation time is required. The emission spectra of solutions with different concentrations of pnitrophenol were recorded at the excitation wavelength of 302 nm. All experiments were

conducted at 37 °C. Luminescent assay of ACP activity. The ACP activity was first assessed as extending incubation time with a certain amount of ACP level. The sensing system containing CuNCs (HAc-NaAc, pH 5.0) solution, NPP (83.3 µM) and ACP (266.0 U/L) was monitored by fluorescence spectroscopy every minute. The luminescence spectra of the system were recorded from 0 to 24 minutes after the addition of ACP at the excitation wavelength of 302 nm. Under the optimal incubation time (16 min), the activity of the ACP assay was evaluated. The solution (3.0 mL) which contained CuNCs (0.28 mg/mL) and NPP (83.3 µM) was placed in a cuvette. Then, the luminescence of CuNCs (HAc-NaAc, pH 5.0) was recorded with continuous addition of ACP, where the activity of ACP ranged from 3.8 to 266.0 U/L. After adding a certain amount of ACP every time, 16 minutes of incubation time is allowed. The emission spectra of solutions with different amounts of ACP were recorded at an excitation wavelength of 302 nm. The temperature was maintained at 37°C. Characterization Methods. 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 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.

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RESULTS AND DISCUSSION

Scheme 1. Schematic Illustration of Detection Strategy for ACP Activity Based on the Luminescent CuNCs Nanoswitch Controlled by Hydrophobic Interaction.

Principle of ACP level detection based on luminescent CuNCs nanoswitch. The copper nanoclusters (CuNCs) were readily synthesized via a one-step approach using D-penicillamine and copper nitrate in aqueous solution. As-prepared CuNCs exist in the aggregated state, and possess many advantages including intense red luminescence, excellent pH stimuli-responsive and unique aggregation-induced emission enhancement (AIEE) behaviors. Under acidic condition, CuNCs severely aggregate due to the hydrophobic nature of protonated penicillamine and show bright red luminescence; however, they disperse in alkaline solution with weak emission owing to hydrophilic property of deprotonated penicillamine. As a result, the formation of CuNCs aggregates would create a specific hydrophobic surface microenvironment under acidic condition. p-Nitrophenol with low solubility in water has a similar hydrophobic property

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and thus is apt to enter hydrophobic environment from aqueous phase. As a result, hydrophobic interaction drives p-nitrophenol to be adsorbed on the surface of CuNCs aggregates when they co-exist in water. This close proximity could lead to an effective luminescence quenching of CuNCs aggregate via a static quenching mechanism. However, the recovery of the luminescence can be readily achieved by the addition of α-cyclodextrin (α-CD) because α-CD is capable to hold p-nitrophenol as the guest molecule to remove it from the surface of CuNCs aggregates. This specific nanoswitch based on CuNCs aggregates in response to external stimulus pnitrophenol or α-CD can be used to establish a fluorometric assay for ACP level as shown in Scheme 1. Under the catalysis of ACP, p-nitrophenyl phosphate disodium (NPP) as the substrate for ACP can be rapidly hydrolyzed into p-nitrophenol (NP). The similar hydrophobicity between p-nitrophenol and CuNCs aggregate drives p-nitrophenol to adsorb on the surface of CuNCs aggregate, and such a close proximity results in luminescence quenching. The ACP level is correlated to the luminescence quenching efficiency of CuNCs aggregates, which is used to establish a quantitative detection assay for ACP activity. Synthesis and characterization of CuNCs. A facile one-pot synthesis of CuNCs using Dpenicillamine as the reductant and capping agent was developed. Although similar methods have been reported, the use of different amounts of starting materials and distinction in reaction time in synthesis could generate CuNCs with different luminescent behaviors.37 As-prepared CuNCs exhibit shiny red luminescence under UV light indicating the formation of luminescent CuNCs aggregates. TEM images in Figure 1A display that the bright luminescent materials in the aggregation state are composed of small CuNCs with a mean diameter of 2 – 5 nm, which is consistent with CuNCs reported previously.41 The UV-vis absorption spectrum (Figure S1) of CuNCs with strong absorbance peaks at wavelengths below 300 nm is different from that of

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penicillamine, implying the formation of CuNCs from initial molecular precursors. X-ray photoelectron spectroscopy (XPS) analysis was conducted to determine the oxidation state of Cu in CuNCs. The survey spectrum of CuNCs illustrates that Cu nanoclusters are composed of C, O, N, S and Cu (Figure 1B). Two distinct peaks at 932.3eV and 952.1eV in Cu 2p spectrum are identified to be the binding energies of 2p3/2 and 2p1/2 electrons of Cu(0) or Cu(I), respectively (Figure S2). The absence of the characteristic satellite peak of Cu 2p3/2 at 942.0 eV from Cu(II) indicates copper atoms with high oxidation state are absent in CuNCs. As a result, copper atoms in CuNCs mainly consist of Cu(0) and Cu(I). The bright shiny red luminescence of as-prepared CuNCs aggregates was further confirmed by its photoluminescence spectrum as shown in Figure 2A. A narrow emission peak at 646 nm was recorded at the optimum excitation wavelength of 302 nm, which is slightly different with that of CuNCs prepared using similar methods.37,41 This difference in emission and excitation peaks clearly indicates the ratio of initial precursors and reaction time could exert an impact on the luminescence behavior of CuNCs. The quantum yield of CuNCs (0.28 mg/mL, pH 4.0) in aqueous solution at the optimum excitation wavelength was determined as 11.2% using rhodamine 6G (QY 0.95 in ethanol) as standard reference. The timeresolved luminescence decay curve of CuNCs in Figure S3A indicates that CuNCs have a long lifetime of 22.4 µs, suggesting that this luminescence can be classified as phosphorescence in terms of time duration of the emission. As-prepared CuNCs are very stable and its luminescence can be remained after several months’ storage in refrigerator. The pH effect on the luminescence of CuNCs was investigated as shown in the Figure 2B. It is found that the variation in pH significantly impact the luminescence intensity of CuNCs; the bright luminescence can be retained when the pH value is less than 4, but the luminescence intensity sharply declines to almost zero as the pH value increases from 5 to 8. This similar pH-

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Figure 1. TEM image (A) and XPS wide spectrum (B) of CuNCs. Inset: size distribution of CuNCs.

Figure 2. Luminescence excitation and emission spectra of CuNCs aggregate (A), and luminescence spectra of CuNCs aggregate at variable pHs (B). Inset: Photographs of CuNCs aggregate at pH 3 under the irradiation of white light (a) and UV light (b).

dependent luminescence behavior was also found on the CuNCs reported by Jia et al,37 and can be attributed to the change of aggregation state of CuNCs as a function of pH value. A little difference was also found that complete luminescence quenching is observed for CuNCs in this work at pH 8.0 but at pH 6.2 for CuNCs in the previous report,37 which implies that CuNCs in this work can emit light in a wider pH range. Luminescence stability of CuNCs at acidic pHs was examined taking pH 5.0 as the example, and the results in Figure S4 demonstrated that a slight 11



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decrease in luminescence intensity was recorded within the first six minutes but the luminescence remains almost unchanged after 6 min of incubation time, which suggests that the luminescence of CuNCs at pH 5.0 can rapidly reach stable within a short period of incubation. The reversibility of this luminescent switch upon variation of pH between pH 4.0 and 7.0 was further assessed as shown in Figure S5. The luminescence switch can be run more than five consecutive cycles without fatigue, indicating its excellent reversibility and potential to be used for luminescent pH chemosensors. Moreover, the influence of common salts on luminescence of CuNCs was also assessed at pH 5 using different concentrations of sodium chloride, sodium sulfate and sodium bromide. Figure S6 indicates that the presence of each of these sodium salts induces a very slight decrease in luminescence in the first ten minutes and then the luminescence keeps constant, and the ratios of declined luminescence to initial intensity for all three sodium salts are less than 9% even when their concentrations are up to 183.3 μM. These observations clearly suggest that co-existence of high concentrations of common salts has negligible impact on the luminescence of CuNCs at a fixed pH solution. Luminescent CuNCs aggregate nanoswitch controlled by hydrophobic interaction. The pH-dependent luminescence property of CuNCs reveals that CuNCs aggregate to enhance its luminescence in acidic solutions while they disperse at alkaline pHs accompanying the weakening or disappearance of the luminescence. The aggregation of CuNCs under acidic condition is due to the hydrophobic nature of protonated form of penicillamine at low pHs. The enhancement of the luminescence of CuNCs at low pHs also reveals the hydrophobicity of the surface of CuNCs aggregates. As a result, we speculated that this specific hydrophobic surface is able to adsorb and hold p-nitrophenol with the similar hydrophobic property under acidic condition in terms of the basic principle that the like prefers like. This point was verified by the

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Figure 3 (A) The change of luminescence spectra of CuNCs in the presence of different concentrations of pnitrophenol from 0.0 to 83.3 μM. (B) The changes of luminescence spectra of CuNCs in the presence of different concentration of α-CD from 0.0 to 166.7 μM. (C) The value (I0/I) and (τ0/τ) versus the concentration of p-nitrophenol in the range of 0.0-15.0 µM. The relative standard deviation (RSD) for all data varies from 0.5% to 1.2% (≥ 3 standard deviations). (D) The reversibility of CuNCs by introducing p-nitrophenol (83.3 μM) and α-CD (166.7 µM) respectively. The relative standard deviation (RSD) for all data varies from 0.2% to 1.1%. All the data have been repeated three times because no significant difference between data sets from three and seven parallel tests is observed.

severe luminescence quenching of CuNCs aggregates by p-nitrophenol as shown in Figure 3A. A gradual decline in luminescence intensity was observed as the increase of the concentration of pnitrophenol, and a very small amount of p-nitrophenol (83.3 µM) lead to almost an entire

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quenching of the luminescence. The pH effect on quenching efficiency by p-nitrophenol was also investigated as shown in Figure S7. It is found that quenching efficiency (I0-I)/I0 by 40.8 µM pnitrophenol progressively decreases as the pH value increases from 3.0 to 8.0, and varies from 90.0% to 5.1%. This observation provides another piece of evidence for the hypothesis that surface hydrophobicity of CuNCs aggregate controls the interaction between CuNCs and pnitrophenol. At lower pHs, the surface of CuNCs aggregate becomes more hydrophobic due to greater degree of aggregation, which further causes a stronger affinity to p-nitrophenol accompanying greater luminescence quenching; contrary situation occurs with less luminescence quenching at higher pHs because of the increase of hydrophilicity of the surface. In addition, salt effect on hydrophobic interaction between CuNCs aggregate and p-nitrophenol was examined. Figure S8 depicts that only very little decrease in luminescence intensity can be recorded after the addition of sodium chloride, sodium bromide or sodium sulfate with the amount as high as 183.3 µM despite of an influence-enhanced trend as the amount of salts increases. To explore the underlying nature for this quenching, the value I0/I versus the concentration of p-nitrophenol in the range of 0.0 – 15.0 µM was plotted in Figure 3C. The relationship between them can be expressed as I0/I = 1.00 + 0.050[Q] where [Q] is the concentration of pnitrophenol and R2 = 0.993, which is consistent with Stern-Volmer Equation. In addition to this Stern-Volmer plot, the values τ0/τ determined from Figure S3B as a function of the amount of pnitrophenol are nearly identical, which proves that this luminescence by p-nitrophenol belongs to static quenching mechanism. Since there is no evidence for the formation of possible groundstate complexes between CuNCs and p-nitrophenol, this quenching can be ascribed to the existence of a sphere of effective quenching due to the adsorption of p-nitrophenol on the surface of CuNCs aggregates according to Perrin’s model. A more suited regression equation between

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I0/I and the amount of p-nitrophenol in the wider range of 0.0-50.0 µM was obtained in Figure S9 in terms of Perrin’s model I0/I = exp(VqNa[Q]): I0/I = exp(0.0518[Q]) with R2=0.998. The radius of the effective sphere was estimated to be around 2.7 nm, which is close to the mean diameter of CuNCs in the aggregate. To further verify the key role of hydrophobic interaction between CuNCs and p-nitrophenol on the luminescence quenching, a more hydrophobic solvent, ethanol, was used to assess the quenching ability of p-nitrophenol to CuNCs aggregates. Figure S10 showed that a much larger concentration of p-nitrophenol up to 500.0 µM in ethanol than in water is required to attain the same quenching efficiency to that in water. This low quenching efficiency of p-nitrophenol in ethanol is due to the fact that it is hard for p-nitrophenol to adsorb on the surface of CuNCs aggregate in ethanol because p-nitrophenol is easily dissolved in ethanol. The large difference in amount of p-nitrophenol to quench the luminescence of CuNCs aggregate clearly illustrates the key role of hydrophobic interaction in aqueous solution, which is the main driving force for creation of a sphere of effective quenching. To investigate possible interference from other aromatic compounds similar to p-nitrophenol, nine hydroxyl- or nitrocontaining phenyl derivatives including benzoic acid, aniline, phenol, o-aminophenol, catechol, m-nitrobenzoic acid, p-nitrobenzaldehyde, m-nitrophenylboronic acid and p-nitrophenylboronic acid were selected. The data for quenching efficiency induced by the preceding compounds with a fixed concentration (83.3 µM) in Figure S11 show that these hydroxyl-containing compounds exert very slight impact on the luminescence of CuNCs aggregate with no more than 20% of quenching efficiency, while those nitro-containing derivatives display a little higher quenching efficiency than the former where their quenching efficiencies are all close to 20%; however, the same amount of p-nitrophenol causes almost an entire luminescence quenching. This high specificity supports that p-nitrophenol has unique strong quenching ability to the luminescence

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of CuNCs, which is consistent with the finding in fluorescence quenching of carbon quantum dots and CdTe quantum dots.42,43 Since α-CD possesses a more hydrophobic cave and the size of the cave is suited for pnitrophenol, α-CD can perfectly hold p-nitrophenol via host-guest recognition. As a result, the presence of α-CD in the system containing CuNCs aggregate and p-nitrophenol would results in the competitive interaction among them, and stronger hydrophobic driving force between α-CD and p-nitrophenol would make α-CD preferentially hold p-nitrophenol. The departure of pnitrophenol from the CuNCs aggregate surface can cause the recovery of the luminescence of CuNCs. As shown in Figure 3B, the luminescence of CuNCs aggregate quenched by pnitrophenol progressively increases as the continuous addition of α-CD, which indicates that pnitrophenol has been stepwisely removed from the surface of CuNCs through the formation of the inclusion complex between α-CD and p-nitrophenol. Thus the luminescent CuNCs aggregate nanoswitch controlled by hydrophobic interaction can be run in this way: the luminescence of CuNCs aggregate is switched off by the addition of p-nitrophenol, and then switched on via the introduction of α-CD. The reversibility of this nanoswitch was also assessed in Figure 3D, and the results showed that five cycles of operation can be achieved despite of its imperfect reversibility. One can note that the luminescence only can be restored to around 75% of initial intensity after introducing double amount of α-CD into CuNCs solution containing 83.3 µM pnitrophenol, and the luminescence can not be completely recovered to its original value for every cycle. This imperfect recovery of the luminescence is due to small quenching effect of NP/α-CD inclusion complex to the luminescence of CuNCs as shown in Figure S12, which indicates that the presence of mere NP/α-CD complex can also result in luminescence quenching of CuNCs despite of its much lower quenching ability than p-nitrophenol, and quenching efficiency

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becomes higher as its concentration is larger. This nanoswitch in response to external stimulus pnitrophenol can be used to establish luminescent assay for ACP activity.

Figure 4 The change of luminescence spectra of CuNCs in HAc-NaAc buffer (pH 5.0) in the presence of different components. (a) mere CuNCs (0.28 mg/mL); (b) CuNCs, and NP (83.3 µM); (c) CuNCs, and NPP (83.3 µM); (d) CuNCs, NPP (83.3 µM) and ACP (266.0 U/L).

Real-time luminescent assay for ACP activity. The feasibility to assay ACP activity based on the CuNCs aggregate nanoswitch was first assessed as shown in Figure 4. The presence of 83.3 µM of 4-nitrophenyl phosphate disodium salt (NPP) results in only about 20% of luminescence quenching of CuNCs aggregate; however, the introduction of the same amount of p-nitrophenol to NPP leads to a quenching efficiency of more than 90%. This large difference in quenching efficiency provides the possibility to detect ACP level, which is confirmed by the sharp decrease in luminescence after the addition of 266.0 U/L of ACP into the system containing CuNCs and NPP. Thus, it is feasible to measure ACP level based this detection strategy in a real-time manner. The influence of the amount of the substrate NPP on the luminescence was evaluated as

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Figure 5 (A) The luminescence quenching efficiency versus incubation time at different ACP levels (166.0, 266.0, 366.0 U/L) in the mixture containing CuNCs and NPP (83.3 µM). (B) Luminescence spectra of the mixture containing CuNCs and NPP (83.3 µM) at varied ACP levels from 0.0 to 266.0 U/L. (C) The luminescence intensity of CuNCs versus ACP activity. (D) The fitting curve between quenched luminescence and ACP activity in the range from 3.8 to 22.8 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.

shown in Figure S13. It is found that the luminescence can also slightly impacted by NPP, and a smooth decrease in luminescence can also be observed in the range of 0.0 – 83.3 µM, but overall quenching efficiency caused by NPP was relatively small in comparison to that by p-nitrophenol. As a result, the concentration of 83.3 µM for NPP was chosen for the following detection. Figure 5A shows the optimization of incubation time for luminescence quenching in the presence of

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ACP and NPP. It is noted that quenching efficiencies using three different ACP levels including 166.0, 266.0 and 366.0 U/L are rapidly increasing within ten min of incubation time, and are almost unchanged after 16 min of incubation, suggesting 16 min is sufficient to attain the equilibrium. As shown in Figure 5B, the sensing performance of the system containing CuNCs and NPP (83.3 µM) was recorded with the introduction of different levels of ACP under optimum conditions, and the luminescence intensity is gradually decreased as ACP level is increased from 0.0 to 266.0 U/L. Figure 5C clearly demonstrates the decreasing trend as elevating the ACP level that a sharp decrease at the first stage from 0.0 to 76.0 U/L is followed by a smooth decline for the range over 76.0 U/L. Figure 5D shows that a good linear relationship between the luminescence and ACP level in the range from 3.8 U/L to 22.8 U/L. The regression equation can be expressed as y = -3494.9 x + 361458.8, where R2 is 0.997. The detection limit estimated from three times standard errors is 1.3 U/L, and the linear scope covers the range of 3.8 U/L - 22.8 U/L. Furthermore, another two concentrations for NPP were used to explore the influence of the amount of NPP on the analytical performance of the ACP assay. As shown in Figure S14 and S15, a similar decline trend in luminescence as a function of ACP level was observed for both of the assays using 40.0 and 120.0 µM of NPP. For the assay using 40.0 µM of NPP, the detection limit is determined to be 2.0 U/L, and the linear scope is 5.0 – 19.0 U/L; for the assay using 120.0 µM of NPP, the detection limit is determined to be 1.5 U/L, and the linear scope is 3.0 – 22.8 U/L. One can note that the amount of NPP used in the assays substantially exerts no impact on the analytical performance as long as the concentration of NPP is over 40.0 µM, and the detection limit in this work is better than that based on carbon quantum dots in our previous work (5.5 U/L).19 Although the detection limit of our method is inferior to those based on CuInS2 and graphene quantum dots (3.1 µU/L or 8.9 mU/L),20,22 our method is sufficiently

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sensitive for practical detection of ACP level in human blood and seminal plasma because it is reported that the normal ACP levels in blood serum and seminal plasma are 35-123 U/L11 and 30-36 U/mL.6 Moreover, this is first time to achieve accurate measurement of ACP level based on CuNCs in a real-time manner, and this assay is by means of competitive hydrophobic interaction instead of the involvement of transition metal cations in the previous work. To investigate the specificity of constructed assay for ACP activity, six possibly interfering biological species including immunoglobulin G (IgG), acetylcholinesterase (AChE), bovine serum albumin (BSA), glucose oxidase (GOx), tyrosinase (TYR) and lysozyme (LYS) were assessed using standard assay solution. It is found from Figure S16 that the separate introduction of BSA, IgG, GOx, LYS, TYR and AChE into buffer solution (HAc-NaAc, pH 5.0) containing CuNCs aggregate and NPP results in a small decline or slight increase in luminescence intensity in comparison with the blank; however, a sharp luminescence quenching is observed after the same amount of ACP to the others was added in the assay solution, and quenching efficiency by ACP is up to 6 while the value of I0/I for most of the others is around 1 as the blank. In addition, 50-fold and 10-fold dilutions of newborn calf serum were also used to evaluate the interference from complex matrix. This assay shows a very close performance in 50-fold dilution to that in standard assay solution, and a small decrease in specificity coefficient I0/I for ACP in 10-fold dilution is observed while the values for the others remain almost unchanged, suggesting that complex matrixes such as real calf serum sample hardly exert any substantial interference to ACP detection. Matrix effect was further quantitatively evaluated by standard addition method using 50-fold dilution of calf serum as the matrix. Table S1 displays that the recovery ratio of measured value to added value for three samples varies from 97.79% to 98.77%, indicating that this assay is capable to accurately quantitate ACP level in such a complex matrix as real samples.

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CONCLUSION In summary, we design a luminescence nanoswitch based on copper nanoclusters aggregate which can be respond to external stimulus via competitive hydrophobic interaction, and further develop a real-time assay for acid phosphatase based on this nanoswitch. Similar hydrophobic nature of the CuNCs aggregate surface at acidic pHs and p-nitrophenol is utilized to quench the luminescence of CuNCs aggregate via static quenching mechanism, and stronger host-guest interaction between α-cyclodextrin and p-nitrophenol switches on the luminescence, which is responsible for the different responses of this nanoswitch to p-nitrophenol and α-cyclodextrin. The fact that the substrate 4-nitrophenyl phosphate ester can be rapidly hydrolyzed to pnitrophenol under the catalysis of ACP is used to design the ACP assay based on the nanoswitch. The correlation between quenched luminescence and ACP level is established to achieve sensitive measurement of ACP level. The detection limit can be lowered to be 1.3 U/L depending on the amount of the substrate. Although our method is not the most sensitive among the reported methods, the sensitivity is sufficient to detect ACP level in human serum and seminal plasma, and our method employs a novel detection strategy to avoid the involvement of transition metal cations. This work demonstrates an example of hydrophobic interaction mediated detection strategy, broadens the application of copper nanoclusters in bioanalysis, and provides a reliable and sensitive ACP assay without the involvement of transition metal ions.

ASSOCIATED CONTENT Supporting Information. 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 CuNCs aggregate and data for ACP assay.

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AUTHOR INFORMATION 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. 21675143, 21405142 and 21275131) and Natural Science Foundation of Zhejiang Province (Grant No. LY17B050003).

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For TOC only

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Luminescent Aggregated Copper Nanoclusters Nanoswitch

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