Facile Synthesis of Enhanced Fluorescent Gold–Silver Bimetallic

Aug 1, 2016 - Facile Synthesis of Enhanced Fluorescent Gold–Silver Bimetallic Nanocluster and Its Application for Highly Sensitive Detection of Inor...
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Facile Synthesis of Enhanced Fluorescent Gold-Silver Bimetallic Nanocluster and its Application for Highly Sensitive Detection of Inorganic Pyrophosphatase Activity Qian Zhou, Youxiu Lin, Mingdi Xu, Zhuangqiang Gao, Huang-Hao Yang, and Dianping Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02543 • Publication Date (Web): 01 Aug 2016 Downloaded from http://pubs.acs.org on August 2, 2016

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Analytical Chemistry

Facile Synthesis of Enhanced Fluorescent Gold-Silver Bimetallic Nanocluster and its Application for Highly Sensitive Detection of Inorganic Pyrophosphatase Activity

Qian Zhou, Youxiu Lin, Mingdi Xu, Zhuangqiang Gao, Huanghao Yang, and Dianping Tang*

Key Laboratory of Analysis and Detection for Food Safety (MOE & Fujian Province), Department of Chemistry, Fuzhou University, Fuzhou 350108, People's Republic of China

CORRESPONDING AUTHOR INFORMATION Phone: +86-591-2286 6125; fax: +86-591-2286 6135; e-mail: [email protected] (D. Tang)

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ABSTRACT: Herein, gold-silver bimetallic nanoclusters (Au-Ag NCs) with the high fluorescent

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intensity were first synthesized successfully and utilized for the fabrication of sensitive and specific

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sensing probes toward inorganic pyrophosphatase (PPase) activity with the help of copper ion (Cu2+)

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and inorganic pyrophosphate ion (PPi). Cu2+ was used as the quencher of fluorescent Au-Ag NC,

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whilst PPi was employed as the hydrolytic substrate of PPase. The system consisted of PPi, Cu2+

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ion and bovine serum albumin (BSA)-stabilized Au-Ag NC. The detection was carried out by

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enzyme-induced hydrolysis of PPi to liberate copper ion from the Cu2+-PPi complex. In the absence

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of target PPase, free copper ions were initially chelated with inorganic pyrophosphate ions to form

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the Cu2+-PPi complexes via the coordination chemistry, thus preserving the natural fluorescent

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intensity of the Au-Ag NCs. Upon addition of target PPase into the detection system, the analyte

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hydrolyzed PPi into phosphate ions, and released Cu2+ ion from the Cu2+-PPi complex. The

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dissociated copper ions readily quenched the fluorescent signal of Au-Ag NCs, thereby resulting in

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the decrease of fluorescent intensity. Under optimal conditions, the detectable fluorescent intensity

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of the as-prepared Au-Ag NCs was linearly dependent on the activity of PPase within a dynamic

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linear range of 0.1 – 30 mU/mL, and allowed the detection at a concentration as low as 0.03 mU/mL

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at the 3sblank criterion. Good reproducibility (CV < 8.5% for the intra-assay and interassay), high

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specificity and long-term stability (90.1% of the initial signal after a storage period of 48 days) were

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also received by using our system toward target PPase activity. In addition, good results with the

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inhibition efficiency of sodium fluoride were obtained in the inhibitor screening research of

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pyrophosphatase. Importantly, this system based on highly enhanced fluorescent Au-Ag NCs offer

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promise for simple and cost-effective screening of target PPase activity without the needs of sample

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separation and multiple washing steps.

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Analytical Chemistry

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Recent years have witnessed rapid development of metal nanoclusters composing of several

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to a few hundred atoms because of their molecule-like properties and attractive features, e.g.,

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ultasmall size (usually less than 2 nm), discrete energy levels and strong fluorescence.1-4 Sizes

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comparable to the Fermi wavelength of the electrons endow metal nanoclusters with the

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physicochemical properties that place in-between isolated atoms and larger nanoparticles.5-6

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These features make them as the ideal nanostructures for different applications in the field of

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biomedical and environmental science.7-9 In particular, ultrasmall noble metal nanoclusters

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(e.g., Au, Ag, Pd and Pt) have drawn considerable attention as a promising class of fluorescent

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probes since they have a high surface-to-volume ratio, a large proportion of surface atoms (i.e.,

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predominantly edge and corner atoms) and intense intrinsic fluorescence.10-12 Ongoing efforts

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have been devoted to the facile synthesis of gold nanoclusters and silver nanoclusters by using

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different methods with the increasingly use of biomolecules, such as protein, enzyme, peptide,

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and oligonucleotide, as the environmentally benign templates.13-16 Advances have been done

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on resolving structures of small-to-medium nanoclusters over the past few years. Bimetallic

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nanoclusters with the controlled atomic distributions offer an additional degree of freedom.

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Zhang et al. found that introduction of gold atoms on the surface of palladium nanoclusters

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considerably improved their activity for glucose oxidation.17 Gold-silver (Au-Ag) bimetallic

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nanoclusters have been also prepared aiming to achieve more attractive advantages over the

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monometallic ones. Recent reports indicated that introduction of silver into gold nanoclusters

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caused significantly enhanced fluorescence.18,19 These advantages in the fluorescent intensity

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and the synthesis process of gold-silver nanoclusters would contribute for the development of

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sensitive sensing platforms.

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Enzyme reactions are of great importance for the metabolism in almost all living organisms.

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Enzymes with a wide variety of functions are indispensable for signal transduction and cell

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regulation, often via kinases and phosphatase.20 Inorganic pyrophosphatase (PPase, a kind of

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hydrolytic enzyme), can catalyze the conversion of one-molecular inorganic pyrophosphate

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ion (PPi, P2O74−) into two orthophosphate ions (Pi, PO43−) specifically.21 The hydrolysis of

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PPi catalyzed by pyrophosphatase can release a large amounts of energy, which provides the

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thermodynamic impetus for the biosynthetic reactions.22,23 It has been demonstrated that the

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inorganic pyrophosphatase has direct relationship with many important biological processes, 3

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e.g., carbohydrate metabolism, lipid metabolism (including lipid synthesis and degradation),

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DNA synthesis, bone formation, as well as other biochemical transformation.24,25 Moreover,

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the activity of pyrophosphatase is connected to several clinical diseases including colorectal

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cancer, hyperthyroidism and lung adenocarcinomas.26-28 To this end, sensitive and convenient

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analysis of pyrophosphatase activity is of great importance and significance. To date, several

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methods including colorimetric, enzymatic, fluorometric and electrochemical techniques have

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been developed for the determination of PPase activity.29-32 Despite some advances in this

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field, there is still an urgent requirement for technically simple and effective method for PPase

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activity assay.

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Scheme 1 Schematic illustration of gold-silver bimetallic nanoclusters (Au-Ag NCs)-based fluorescent

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probes for quantitative monitoring of inorganic pyrophosphatase (PPase) activity coupling enzyme-induced

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hydrolysis of pyrophosphate ion (PPi, P2O74−) with the release of copper ion (Cu2+) from the Cu2+-PPi

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complexes.

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Herein, we report on the proof-of-concept of simple and powerful sensing strategy for the

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quantitative monitoring of PPase activity on the basis of high-efficiency gold-silver bimetallic

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fluorescent nanoclusters (Scheme 1). Gold-silver bimetallic nanoclusters are synthesized via

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utilizing bovine serum albumin (BSA) as both protecting and reducing agent in the alkaline

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aqueous solution. The formed Cu2+-PPi complex by the coordination chemistry between free

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copper ion and PPi effectively protects the gold-silver nanoclusters with the strong fluorescent 4

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intensity. Addition of target PPase induces the hydrolysis of pyrophosphate ions to liberate the

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chelated copper ion, and the released copper ion quenches the fluorescence of nanoclusters.

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The PPase activity is achieved by monitoring the fluorescence of gold-silver nanoclusters by

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varying with target-ruled competitive reaction in the Au-Ag NC/Cu2+-PPi system. In this case,

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the Au-Ag NC/Cu2+-PPi system exhibits a low fluorescent intensity in the presence of PPase,

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versus a high fluorescent intensity in the absence of PPase.

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■ EXPERIMENTAL SECTION

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Material and Chemical. HAuCl4·4H2O, AgNO3, NaOH and BSA were purchased from

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Sinopharm Chem. Re. Co., Ltd. (Shanghai, China). Na4P2O7·10H2O and CuSO4·5H2O were

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achieved from Beijing Chem. Re. Inc. (Beijing, China). Inorganic pyrophosphatase (PPase,

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EC 3.6.1.1, MW 71 kDa) from the baker’s yeast (S. cerevisiae) (≥ 90%, HPLC, lyophilized

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powder, ≥ 1000 units per mg protein) (note: One-unit PPase can liberate 1.0 µmol inorganic

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orthophosphate per min at pH 7.2 at 25 °C referring to Sigma’s unit definition) was acquired

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from Sigma-Aldrich (St. Louis, MO 63103 USA). All other reagents were of analytical grade

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and used as received without further purification. Ultrapure water obtained from a Millipore

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water purification system (18.2 MΩ cm-1, Milli-Q, Millipore) was used in all runs.

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Preparation of Gold-Silver Bimetallic Nanoclusters (Au-Ag NCs). All glassware used in

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this experiment was completely soaked for 30 min in aqua regia (HNO3 : HCl = 1 : 3, v/v)

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(Caution!!) and rinsed thoroughly with ultrapure water prior to use. Gold-silver nanoclusters

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were synthesized at the different molar ratios of HAuCl4 and AgNO3 by using BSA as both

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protecting and reducing agent.19 Initially, 5.0 mL of 50 mg/mL of BSA aqueous solution was

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mixed with 4.0 mL of 10 mM HAuCl4 aqueous solution. Thereafter, 1.0 mL of AgNO3

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aqueous solution with different concentrations was introduced to the resulting mixture under

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vigorous stirring, respectively. After stirring for 10 min at room temperature, 1.0 mL of 1.0 M

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NaOH aqueous solution was injected immediately into the mixture, and reacted for 12 h at

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37 °C under the same conditions. Finally, the resulting suspension was dialyzed in ultrapure

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water for 48 h with the water change every 6 h to acquire gold-silver bimetallic nanoclusters

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(i.e., Au-Ag NCs), and stored at 4 °C for further use.

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Fluorescent Measurement of PPase Activity. Before measurement, the above-prepared

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Au-Ag NCs were diluted to 10-fold with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

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(HEPES) buffer solution (10 mM, pH 7.2, containing 10 µM Mg2+). A 5-µL volume of PPase

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solution with different activities was initially added into 100 µL of the diluted Au-Ag NCs

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containing Cu2+ (3 µM) and PPi (60 µM), and then incubated at room temperature for 40 min.

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Following that, the fluorescent spectra of the suspension were recorded on a fluorescence

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spectrophotometer (F-4600, Hitachi, Japan) in a 1.0 × 1.0 cm quartz cuvette (λex = 270 nm,

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λem = 630 nm). The fluorescent intensity was collected and registered as the sensing signal

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relative to target PPase activity. All measurements were performed at room temperature (25 ±

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1.0 °C). Analyses were always made in triplicate.

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Inhibition Assay of PPase Activity. To investigate the inhibition effect of sodium fluoride

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(NaF) on PPase activity, a 5-µL volume of various-concentration NaF was initially added into

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the PPase solution (20 mU/mL). After a brief mixing, the NaF-treated PPase was injected into

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Au-Ag NC solution in the HEPES buffer containing Cu2+ (3 µM) and PPi (60 µM). After that,

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the mixture was incubated for 40 min at room temperature and the fluorescent spectrum was

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recorded as before.

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■ RESULTS AND DISCUSSION

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Characterization of Au-Ag Bimetallic Nanoclusters. In this work, BSA-protected Au-Ag

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NCs with the different molar ratios were facilely synthesized by adjusting the amount of the

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HAuCl4 and AgNO3. The fluorescence spectra of the as-prepared nanoclusters are shown in

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Figure 1A. It can be seen that Au-Ag hybrid nanoclusters exhibited much stronger fluorescent

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intensity than those of pure Au nanoclusters (curve 'a') and pure Ag nanoclusters (curve 'i'),

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which might be attributed to their synergistic effect.33 No fluorescent signal was observed at

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BSA-protected Ag nanoclusters (curve 'i'), which was accordance with the previous report.34

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Importantly, gold-sliver nanoclusters with the molar ratio of 4:1 could display the maximum

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fluorescent emission intensity [Note: The fluorescence intensities of Au:Ag = 5:1 and 6:1

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were lower than that of Au:Ag = 4:1 (data not shown)], which was chosen as the fluorescent

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probe for the detection of PPase activity. Also, we observed that gold-silver (4:1) nanoclusters

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exhibited orange color and orangered color under visible light and 365-nm ultraviolet light,

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respectively (Figure 1B, inset). The UV-vis absorption spectrum of the Au-Ag NCs in Figure

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1B (curve 'a') showed that most of the absorption of the as-synthesized nanoclusters appeared

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at UV region (partial absorption was at the visible region). The maximum fluorescent

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emission peak of the (4:1) Au-Ag nanoclusters was achieved at 630 nm (Figure 1B, curve 'c'),

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while that of maximum excitation peak was at 270 nm (Figure 1B, curve 'b'). High-resolve

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transmission electron microscopy (HRTEM, Model H-7650, Hitachi Instruments, Japan) was

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further applied to characterize the size and morphology of the Au-Ag nanoclusters. As shown

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in Figure 1C, the Au-Ag nanoclusters were mono-dispersed and the mean diameter was

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estimated to be ~1.9 nm. Moreover, no core-shell-like structure and obvious lattice mismatch

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could be observed from the crystallinity of the Au-Ag nanoclusters, as displayed in the inset

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of Figure 1C.

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Figure 1. (A) Fluorescence emission spectra of Au-Ag bimetallic nanoclusters with different molar ratios

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[insets: photographs under 365-nm UV light (top) and visible light (bottom), respectively]. (B) UV-vis

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absorption (a), fluorescence excitation (b) and emission (c) spectra of Au-Ag nanoclusters with 4:1 molar

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ratios [insets: photographs under visible light (a) and 365-nm UV light (b)]. (C) TEM image of Au-Ag

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nanoclusters (inset: HRTEM image). (D,E) XPS spectra of (D) Au 4f and (E) Ag 3d for Au-Ag nanoclusters.

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(F) FT-IR spectra of BSA (a) and BSA-stabilized Au-Ag nanoclusters (b).

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X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific, Model Escalab 250

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Spectrometer, Al Kα, 1486.6 eV) was applied to illustrate the oxidation states of the Au and

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Ag in the as-prepared Au-Ag nanoclusters. As shown in Figure 1D, gold in the Au-Ag

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nanoclusters showed a dominant Au0 metallic state with the binding energies of 83.75 eV for

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Au 4f7/2 and 87.65 eV for Au 4f5/2.34,35 Meanwhile, the binding energies of 367.4 eV for Ag

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3d5/2 and 373.6 eV for Ag 3d3/2 could be assigned to be Ag(I) and Ag0 in Au-Ag nanoclusters,

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respectively (Figure 1E).19 Fourier transform infrared spectroscopy (FT-IR) has become a

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useful tool for characterizing the protein conformational change and analyzing the secondary

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structure. It has been reported that the amide I in 1600-1700 cm-1 wavenumber region (mainly

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C=O stretch) and the amide II band at about 1550 cm-1 (C-N stretch coupled with N-H bend)

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are relevant to the secondary structure of protein.36 Moreover, the amide I band is more

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sensitive to the secondary structure change of protein owing to the transition dipole

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coupling.37 Figure 1F displays the FT-IR spectra of BSA (curve 'a') and BSA-stabilized

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Au-Ag nanoclusters (curve 'b') in the wavenumber range of 1300-1700 cm-1. It can be found

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an obvious shift of the amide I band to lower wavenumber after conjugation from 1651 cm-1

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to 1638 cm-1 and a slight shift of amide II band to higher wavenumber, indicating a secondary

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structure change of BSA caused by the labeling of Au-Ag nanoclusters.

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Principle and Feasibility Study of PPase Sensing Strategy. It has been demonstrated that

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PPi has a higher Cu2+ affinity than that of groups on protein.38,39 Moreover, Pi (the enzymatic

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hydrolysate of PPi in the presence of PPase) has a low affinity to Cu2+.29,40 Inspired by the

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different Cu2+ affinities with the PPi, Pi and BSA-stabilized Au-Ag nanoclusters, this work

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constructed a sensitive fluorescent sensing strategy for the PPase activity on the basis of

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competitive binding of Cu2+ between BSA-stabilized Au-Ag nanoclusters and PPi. Figure 2A

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displays the fluorescent spectra of Au-Ag nanoclusters and its mixtures after reaction with the

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different components. It can be seen that the fluorescent intensity of Au-Ag nanoclusters

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(curve 'a') could be immediately quenched by Cu2+ (curve 'b'), while PPi (curve 'c'), PPase

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(curve 'd') and their mixture (curve 'g') showed almost no influence on the fluorescence. With

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the addition of PPi into the mixture of Cu2+ and Au-Ag nanoclusters, an obvious recovery of

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the fluorescence was observed (curve 'e') due to the competitive bind process of Cu2+ between

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BSA-stabilized Au-Ag nanoclusters and PPi. Meanwhile, the addition of pure PPase showed

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almost no influence on the quenched fluorescence of the mixture consisting of Cu2+ and 8

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Au-Ag nanoclusters (curve 'f'). Finally, PPase was introduced to catalyze the hydrolysis of PPi

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to Pi, which has a low affinity with Cu2+, and the fluorescence was quenched again owing to

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the recombination of the as-released Cu2+ and Au-Ag nanoclusters (curve 'h'). In addition, the

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interferences from the phosphate-related anions including PO43−, HPO42−, and H2PO4− were

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also investigated. As shown in Figure 2B, the addition of all the phosphate-related anions

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could not recover the fluorescence of Au-Ag nanoclusters quenched by Cu2+ except for PPi.

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Therefore, a sensitive and selective sensing platform for PPase activity could be constructed

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by monitoring the fluorescent intensity of the Au-Ag nanoclusters solution containing Cu2+

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and PPi.

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Figure 2. (A) Fluorescent emission spectra of Au-Ag nanoclusters (a) and its mixtures with Cu2+ (b), PPi

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(c), PPase (d), Cu2+ + PPi (e), Cu2+ + PPase (f), PPi + PPase (g) and Cu2+ + PPi + PPase (h) (note: 20

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mU/mL PPase was used in this case). (B) Fluorescence emission spectra of Au-Ag nanoclusters in the

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presence of Cu2+ (a), Cu2+ + H2PO4- (b), Cu2+ + HPO42- (c), Cu2+ + PO43- (d) and Cu2+ + PPi (e). (C)

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Fluorescence decay curves and (D) FT-IR spectra of the as-prepared Au-Ag nanoclusters before (a) and

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after (b) the addition of Cu2+.

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Lifetime measurement was applied to infer the mechanism of Cu2+-induced quenching 9

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process. Figure 2C displays the fluorescence decay curves of the Au-Ag nanoclusters with and

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without the addition of Cu2+. The fluorescence decays of the Au-Ag nanoclusters in the

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absence and presence of Cu2+ could be fitted by a mono-exponential function with time

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constant of 0.9429 ns and 0.9897 ns, respectively. The results showed no obvious change in

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fluorescence lifetime of the Au-Ag nanoclusters before and after the addition of Cu2+. So the

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possibility of dynamic type of quenching could be ruled out, and the quenching mechanism

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could be considered to be a static type.41 Moreover, FT-IR was also applied to investigate the

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binding of Cu2+ with BSA-stabilized Au-Ag nanoclusters. As discussed above, the amide I

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and amide II bands are sensitive to the secondary structure of protein. Figure 2D displays the

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FT-IR spectra of BSA-stabilized Au-Ag nanoclusters in the absence (curve 'a') and presence

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of Cu2+ (curve 'b'). The peak at 1638 cm-1 for the amide I band and 1543 cm-1 for amide II

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band in BSA-stabilized Au-Ag nanoclusters shifted to 1647 cm-1 and 1551 cm-1, respectively.

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The observations showed that the binding of Cu2+ and BSA-stabilized Au-Ag nanoclusters

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could induce conformational changes in BSA, which indirectly suggested that Cu2+ interacted

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with the protein of the BSA-stabilized Au-Ag nanoclusters and resulted in the fluorescence

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quenching finally.

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Optimization of Experimental Conditions. Herein, the effect of Cu2+ concentration on

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the sensing system was first investigated. As seen from Figure 3A, the fluorescence intensity

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of Au-Ag nanoclusters was gradually quenched with the increasing Cu2+ concentration from 0

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to 10 µM. Meanwhile, it can be found that Cu2+ with a final concentration of 3.0 µM could

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quench 84% of the initial fluorescence, and the fluorescence intensity values showed a

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quasi-linear relationship along with the Cu2+ concentration from 0.1 to 3.0 µM. Therefore, 3.0

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µM was chosen as the final Cu2+ concentration in the following experiments.

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The effect of PPi on the sensing system was also investigated by adding PPi with different

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concentrations into the mixture containing Au-Ag nanoclusters and 3.0 µM Cu2+. As shown in

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Figure 3B, the fluorescence intensity of the mixture showed a great increase with further

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addition of PPi, and displayed a quasi-linear relationship with PPi concentration from 1.0 to

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60 µM, which would be beneficial to the subsequent quantitative PPase activity assay. Thus,

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the optimized concentration of PPi was chosen at 60 µM.

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In this work, the evaluation of PPase activity was achieved by using Au-Ag nanoclusters 10

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containing 3.0 µM Cu2+ and 60 µM PPi, and the reaction time was another important factor

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need to be explored. As shown in Figure 3C, both the Cu2+ induced fluorescence quenching

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and PPi caused fluorescence recovery could be finished completely within 5 minutes. As for

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PPase-catalyzed hydrolysis process, the fluorescence intensity decreased with the increasing

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hydrolytic time initially, and tended to reach a plateau after 40 min. Considering time saving,

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40 min was chosen as the optimized time for the whole reaction process including competitive

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and hydrolytic reactions.

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Figure 3. Fluorescence emission intensity of (A) Au-Ag nanoclusters toward various Cu2+ concentrations,

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and (B) Au-Ag NC/Cu2+ complex toward different PPi concentrations. Effects of (C) reaction time for Cu2+

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induced fluorescence quench process (a), PPi caused fluorescence recover process (b), PPase regulated

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hydrolytic process (c), and (D) pH of HEPES buffer on the fluorescence intensity of Au-Ag nanoclusters by

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using 20 mU/mL PPase as an example.

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In addition, it is of great importance to maintain the bioactivity of PPase, and effect of

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the pH of HEPES buffer solution adopted in the dilution of Au-Ag nanoclusters was also

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studied. As seen in Figure 3D, an obvious fluorescence quench was acquired at pH 7.2

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HEPES buffer solution diluted Au-Ag nanoclusters, which indicated a good bioactivity of 11

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PPase. A higher or lower pH would cause lower fluorescence quench. Therefore, HEPES

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buffer at pH 7.2 was used for the determination of PPase activity throughout this work.

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Figure 4. (A) Fluorescence emission spectra and (B) the corresponding calibration curve of the system

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toward different PPase concentrations. (C,D) The specificity of the proposed sensing strategy toward PPase

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against GOx, HSA, Lysozyme, PSA and Exo III (PPase: 20 mU/mL; GOx: 0.2 U/mL; HSA: 0.2 U/mL;

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Lysozyme: 0.2 U/mL; PSA: 0.2 U/mL; ExoIII: 0.2 U/mL).

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Fluorescent Assay for PPase Activity. Under the optimal conditions, PPase activity was

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monitored by coupling with Au-Ag NC/Cu2+-PPi system in 10 mM HEPES buffer, pH 7.2.

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Figure 4A displays the fluorescence spectra of the Au-Ag nanoclusters after incubating Au-Ag

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NC/Cu2+-PPi system with target PPase at different activities from 0 to 30 mU/mL. Obviously,

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the restored fluorescence caused by PPi was quenched again with the addition of PPase, and

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the fluorescence intensity decreased with the increasing of PPase activities in the sample.

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Moreover, a linear correlation between fluorescence intensity and PPase activity was observed

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over the range of 0.1 to 30 mU/mL (Figure 4B). The fitted linear data could be expressed as F

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= -22.964 × C[PPase] + 957.18 (mU/mL, R2 = 0.9991, n = 9). The detection limit (LOD) was

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calculated to be 0.03 mU/mL at the 3sblank criterion, which could be comparable with the 12

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previous works, e.g., nanogold-based colorimetric assay (10 mU/mL),29 click chemistry-based

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fluorescent method (0.2 mU/mL),31 magnetic graphene nanosheet-based electrochemical

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method (0.05 mU/mL),32 gold nanocluster-based fluorescent assay (1.0 mU/mL),40 and

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graphene quantum dots-based fluorescent assay (1.0 mU/mL).42

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Specificity, Reproducibility and Stability. The specificity of the sensing strategy was

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investigated by challenging the sensors against other proteins including glucose oxidase

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(GOx), human serum albumin (HSA), lysozyme, prostate-specific antigen (PSA) and Exo III

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under the same conditions. As shown in Figure 4C, only PPase achieved an obvious decrease

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in the fluorescence intensity while others alone caused no obvious change. Moreover, we

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further challenged the sensing platform with PPase in the coexistence of other proteins. As

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seen from Figure 4D, the coexistence of other tested protein had almost no effect on the

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response of this sensing strategy to PPase. These results clearly indicated that the specificity

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of this sensing system was reasonably satisfactory for target PPase.

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Figure 5. Inhibition of PPase activity (20 mU/mL PPase used in this case) toward NaF standards with

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different concentrations (NaF: 2.0 µM; NaF1: 0.4 µM; NaF2: 0.8 µM; NaF3: 1.2 µM; NaF4: 1.6 µM) by

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using Au-Ag NCs/Cu2+-PPi system.

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The reproducibility of the developed system was investigated by using the coefficients of

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variation (CV) of the intra- and inter-assay. Experimental results indicated that the CVs of the

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assays using the sensor with the same batch were 2.3, 4.2 and 3.5% at 0.1, 1.0 and 10 mU/mL

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PPase, respectively. The batch-to-batch reproducibility was also investigated and the CVs

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were 6.7, 8.3 and 7.4% at the mentioned-above levels, respectively. So, the reproducibility of

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this method was satisfactory. In addition, the stability of the Au-Ag NC/Cu2+-PPi sensing

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system was studied on a 60-day period. The mixture was stored at 4 °C and measurements

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toward PPase were taken intermittently (every 3-5 days). The result showed that it retained

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90.1% of initial signal after a storage period of 48 days, which suggested a good stability of

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the sensing strategy.

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Inhibition Assay of PPase. Typically, sodium fluoride (NaF), a kind of effective inhibitors

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toward PPase, can usually cause an instant decrease of the activity because of formation of

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[PPase-F-] intermediate.43,44 Considering this, NaF could be used to inhibit the bioactivity of

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PPase by spiking NaF standards with various concentrations into the detection solution during

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the fluorescent measurement. As shown in Figure 5, with the increasing NaF concentrations

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from 0 to 1.6 µM in the analytical system, the PPase activity was gradually inhibited and the

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fluorescence of the mixture exhibited a gradual recovery. Moreover, pure NaF was directly

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introduced into the Au-Ag NC/Cu2+-PPi system in the absence of PPase, and there was no

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obvious fluorescence change compared with the background signal, which indicated that NaF

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could not cause the dissociation of the Cu2+-PPi complex. These results demonstrated that

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NaF could be used for evaluating the inhibitor efficiency of PPase activity, and the bioassay

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system proposed in this work could be adopted to screen potential PPase inhibitors.

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■ CONCLUSIONS

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In summary, we have successfully synthesized the significantly enhanced fluorescent Au-Ag

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nanoclusters using BSA as both reducing and protecting agent in alkaline aqueous solution,

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and developed a rapid and simple sensing strategy for PPase activity based on the highly

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fluorescent Au-Ag nanoclusters and PPase-regulated competitive reaction with Cu2+ between

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PPi and BSA-stabilized Au-Ag nanoclusters. The Au-Ag nanoclusters-based sensing strategy

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for PPase activity exhibited several advantages, such as good sensing properties including low

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detection limit, wide linear range, high sensitivity and satisfactory specificity, simple

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operation at one step without the needs of multiple washing and sample separation steps, and

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the time saving of the whole detection process within 50 minutes. The enhanced fluorescence 14

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of Au-Ag nanoclusters may open a new door toward developing various highly fluorescent

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bimetallic nanoclusters or quantum dots in bioassay constructions. Nevertheless, one major

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limitation of using Au-Ag NC/Cu2+-based fluorescent sensing system lies in the fact that the

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concentration of free copper ions in the detection solution should be capable of be controlled

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through the PPi in order to ensure the generation of natural fluorescent intensity.

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■ ACKNOWLEDGEMENT

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This work was financially supported by the National Natural Science Foundation of China (41176079 and

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21475025), the National Science Foundation of Fujian Province (2014J07001), and the Program for

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Changjiang Scholars and Innovative Research Team in University (IRT15R11).

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