Facile Synthesis of Enhanced Fluorescent GoldâSilver Bimetallic

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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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ACS Paragon Plus Environment


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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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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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260

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

261

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

262

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

263

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

264

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

265

investigated by challenging the sensors against other proteins including glucose oxidase

266

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

267

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

268

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

269

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

270

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

271

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

272

of this sensing system was reasonably satisfactory for target PPase.

273 274

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

275

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

276

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

277

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

278

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

279

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

13



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280

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

281

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

282

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

283

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

284

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

285

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

286

the sensing strategy.

287

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

288

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

289

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

290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

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

298

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

299

■ CONCLUSIONS

300

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

301

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

302

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

303

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

304

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

305

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

306

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

307

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

308

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

310

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

311

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

312

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

313

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

314

■ ACKNOWLEDGEMENT

315

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

316

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

317

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

318

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Facile Synthesis of Enhanced Fluorescent GoldâSilver Bimetallic

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