Metal-enhanced fluorescence of gold nanoclusters as a sensing platform for multi-component detection

Accepted Manuscript Title: Metal-Enhanced Fluorescence of Gold Nanoclusters as a Sensing Platform for Multi-component Detection Authors: Dang-Dang Xu, Bei Zheng, Chong-Yang Song, Yi Lin, Dai-Wen Pang, Hong-Wu Tang PII: DOI: Reference:

S0925-4005(18)32078-1 https://doi.org/10.1016/j.snb.2018.11.122 SNB 25720

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

29 September 2018 22 November 2018 23 November 2018

Please cite this article as: Xu D-Dang, Zheng B, Song C-Yang, Lin Y, Pang D-Wen, Tang H-Wu, Metal-Enhanced Fluorescence of Gold Nanoclusters as a Sensing Platform for Multi-component Detection, Sensors and amp; Actuators: B. Chemical (2018), https://doi.org/10.1016/j.snb.2018.11.122 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Metal-Enhanced Fluorescence of Gold Nanoclusters as a Sensing Platform for Multi-component Detection Dang-Dang Xu a,b, Bei Zheng a, Chong-Yang Song a, Yi Lin a, Dai-Wen Pang a, Hong-Wu Tang a*

a

IP T

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of

Education), College of Chemistry and Molecular Sciences, and The Institute for

b

SC R

Advanced Studies, Wuhan University, Wuhan, 430072, People’s Republic of China

Key Laboratory for Special Functional Materials of Ministry of Education, Henan

N

U

University, Kaifeng, 475004, People’s Republic of China

ED

M

A

*Corresponding Author. E-mail: [email protected]. Phone: +86-27-68756759

Highlights

The core-shell nanocomposites Ag@SiO 2 -AuNCs provide 3.21-fold fluorescence

PT

•

CC E

enhancement and ~10% higher quantum yield than bare AuNCs. •

The metal-enhanced fluorescence is resulted from the interaction between the Ag core and the AuNCs conjugated on the outer silica shell.

A

•

•

The composite nanostructures were applied to develop a sensitive sensing platform for multi-component Cu2+, PPi and PPase. The platform was successfully applied to detect Cu2+, PPi and PPase in both buffer and living cells. 1

Abstract: Metal-enhanced fluorescence (MEF) has been applied to construct biosensing systems in the recent decades owing to its favorable optical properties.

IP T

Herein, silver nanoparticles (AgNPs) are used to enhance fluorescence of gold nanoclusters (AuNCs), by fabricating a core-shell composite nanostructure

SC R

Ag@SiO 2 -AuNCs consisting of a silver core, a silica shell and an outer conjugated

layer of AuNCs. The core-shell MEF-capable nanoparticles possess water dispersibility, high stability and good biocompatibility. The interaction between

U

AuNCs on the surface of the outer silica shell and the silver core, significantly

N

improves the excitation efficiency, and thus enhances the fluorescence emission,

A

photostability and quantum yield of the AuNCs. The composite nanoparticle

M

Ag@SiO 2 -AuNCs provides a fluorescence enhancement of up to 3.21-fold when the separation distance (the thickness of the silica shell) is about 10 nm. Finally, the

ED

composite nanostructures have further been applied to develop a sensing platform for multi-component detection based on the MEF capability and the OFF-ON-OFF

PT

switching nature of the fluorescence signal, and the detection limits for Cu2+,

CC E

inorganic pyrophosphate (PPi) and pyrophosphatase (PPase) are 39 nM, 78.7 nM and 0.976 mU, respectively. This platform has been applied to detect Cu2+, PPi and PPase

with satisfactory results in living cells.

A

Keywords: Metal-enhanced fluorescence (MEF); Gold nanoclusters (AuNCs); Core-shell composite nanostructure; Fluorescence sensor

2

IP T

1. Introduction

Metal enhanced fluorescence (MEF) has enriched a great variety of fundamental

SC R

research and technological innovation in recent years, owing to their favorable optical

properties and has shown great potential in the field of biomedicne [1-6]. The emission

U

of fluorescent materials close to metal nanostructures could be significantly enhanced

N

by near-field, which changes the radiative decay rate and increases coupling efficiency

A

of the fluorescence emission to the far-field [3, 7-9]. Consequently, the brightness,

M

quantum yield and photostability of fluorescent materials could be increased. The

ED

influences on nearby fluorescent materials by various types of metal nanoparticles and surfaces, e.g., Au, Ag and Cu, have been fairly well studied [8-17]. It has been found

PT

that the fluorescence emission is sensitive to the distance between the fluorophores

CC E

and the metal nanostructures. An ultra-short distance might lead to a strong quenching of the fluorophores, while a few nanometers distance might significantly enhance the emission of these fluorophores.

A

Fluorescent gold nanoclusters (AuNCs) have attracted considerable interests owing to its good biocompatibility and photostability [18, 19]. Gold nanomaterials are widely used as optical biosensing nanomaterials because of its low-toxicity [18-22]. As a kind

of gold nanomaterials, AuNC consists of several to tens of Au atoms with a size equal 3

to the Fermi wavelength of the conduction electrons [23-27]. Although the fabricated AuNCs exhibits fluorescence in the blue to near-infrared (NIR) region, the quantum yields (QYs) are either too low or the reaction if time-consuming and large particles are produced as byproduct. It is therefore very important to develop new approaches

IP T

to significantly improve fluorescence QY of AuNCs to broaden their applications. Moreover, the preparation of highly red or NIR emitting AuNCs with a simple,

SC R

one-pot, green synthesizing route is of great importance for us to develop biodetection

systems that offer reduced background fluorescence and improved depth of tissue

U

penetration depth.

N

Inorganic pyrophosphatase (PPase) is quite crucial in biosystems and is one of the

A

ubiquitous assayed enzymes. As a metal-dependent hydrolysis enzyme, it specifically

M

catalyses one molecule of inorganic pyrophosphate (PPi) into two orthophosphate (Pi)

ED

ions [28]. The enzymatic hydrolysis process provides a thermodynamic force to drive these biosynthetic reactions to be completed with much less energy [29-31]. Recently,

PT

much attention has been devoted to study the activity of PPase [32-36]. As a kind of

CC E

bio-functional anions, pyrophosphate ion PPi plays an important role in energy transduction in organisms and several metabolic processes [37-40]. As is known, the

level of PPi is highly related to the process of DNA replication and the expression of

A

genetic information. So the detection of PPi has been a real-time DNA sequencing method and became an important indicator in cancer diagnosis [41]. Abnormal PPi concentrations can help to identify some diseases such as calcium pyrophosphate dihydrate crytal deposition disease, arthritis, chondrocalcinosis and hypophosphatasis 4

[40, 42]. Therefore, PPi is important in biological systems and accurate determination of PPi is of great significance. In this contribution, we first fabricate a core-shell composite nanoparticle Ag@SiO 2 -AuNCs with AuNCs conjugated on its surface. These core-shell

IP T

nanoparticles possess MEF-capability, and have the abilities of strong fluorescence emission, good water dispersibility, low toxicity, biological compatibility, excellent

SC R

stability, and being environmentally abundant for easy conjugation. Thanks to the excellent MEF capability and OFF-ON-OFF switching nature of their fluorescence, a

U

highly sensitive and specific strategy using these nanoparticles has been applied for

N

the detection of Cu2+, PPi and PPase, respectively. Scheme 1 shows the preparation of

A

these nanoparticles and the detection principle for all the targets. Copper ions form

M

complexes with carboxyl groups due to cation-π of Cu2+ [42, 43], causing the

ED

fluorescence quenching of Ag@SiO 2 -AuNCs. The coordination between PPi and Ag@SiO 2 -AuNCs is stronger than that between Cu2+ and Ag@SiO 2 -AuNCs.

PT

Therefore, Cu2+ is withdrawn from the Ag@SiO 2 -AuNCs-Cu2+ complexes, restoring

CC E

the fluorescence of the nanoparticles Ag@SiO 2 -AuNCs. Interestingly, when PPase is present, it drives one molecule of PPi to generate two molecules of Pi since PPi is a kind of natural substrates of PPase. As a result, the fluorescence of Ag@SiO 2 -AuNCs

A

is quenched due to the regeneration of the complexes Ag@SiO 2 -AuNCs-Cu2+.

2. Experimental Section 2.1 Materials Cupric sulfate anhydrous (CuSO 4 ), sodium hydroxide (NaOH) and sodium 5

pyrophosphate were obtained from Shanghai Chemical Reagent Co. Tetraethyl orthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTES), chloroauric acid (HAuCl 4 ), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), bovine serum

albumin

(BSA),

4-morpholineethanesulfonic

acid

(MES),

polyvinyl

IP T

pyrrolidone (PVP), N-hydroxysuccinimide (NHS), ammonium hydroxide (NH 4 OH, 25–28 wt%), 3-(4,5-dimethylthiahizol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), (EDC),

SC R

N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimidehydrochloride

Baker’s

yeast inorganic pyrophosphatase (PPase, EC 3.6.1.1), rhodamine 6G were obtained

U

from Sigma Chemical Co. (St. Louis, MO, USA). Ultrapure water (18.25 MΩ·cm)

M

2.2 Instrumentations

A

N

was applied throughout all experiments.

ED

A fluorescence spectrophotometer (F-4600, Hitachi) was employed to monitor the fluorescence emission spectrum. The fluorescence was measured with excitation and

PT

emission at 470 and 640 nm. UV–vis absorption spectra were acquired with a

CC E

spectrophotometer (UV-2550, Shimadzu). Transmission electron microscopy (TEM) observation was performed on a transmission electron microscope (JEM100CXII, JEOL, Japan) at an accelerating voltage of 200 kV. Fourier transform infrared (FT-IR)

A

spectra were obtained with a FT-IR spectrophotometer (Thermo Nicolet 360).

Fluorescence images were recorded using an inverted fluorescence microscope (Ti_U, Nikon, Japan) equipped with a CCD camera (Nikon DS-Ri1).

6

2.3 Synthesis of Au nanoclusters (AuNCs) Au nanoclusters were prepared according to a previous report [44]. The glassware was cleaned with aqua regia (V HCl /V HNO3 =3:1) and rinsed extensively with water prior to use. In a typical process, 5 mL HAuCl 4 solution (10 mM, 37 °C) was mixed with 5

IP T

mL BSA solution (50 mg/mL, 37 °C) under constant stirring for 2 min. Thereafter, 0.5 mL 1M NaOH solution was added, and the mixture was vigorously stirred at 37°C for

SC R

11 h, until the mixture changed its color from pale yellow to dark brown.

U

2.4 Synthesis of Ag nanoparticles (AgNPs)

N

Ag nanoparticles were synthesized by a seed-mediated method at room temperature

A

[45]. A 25 mL 40% glycerol in a flask was heated to 95°C while stirred constantly.

M

Once this temperature became stable, 0.5 mL 3% sodium citrate and 0.0045 g silver

ED

nitrate were added to the solution. The reaction solution was stirred at 95°C for one hour, then the seed solution was obtained. Afterwards, monodisperse AgNPs with

PT

diameter ~50 nm were synthesized by combining glycerol with ascorbic acid and PVP.

CC E

145 mg PVP and 5.75 mL glycerol were dissolved in 34.5 mL of water. After PVP was dissolved, 3.6 mL seed solution was introduced. The mixture was stirred for 1 min. 72.5 μL diamine silver complex and 23 mL 0.1 mg/mL ascorbic acid solution

A

were added to the solution. After 1 h, the AgNPs were stabilized with 0.5 g PVP and kept at 4°C for use.

2.5 Synthesis of Ag@SiO 2 nanoparticles 7

The Ag@SiO 2 core-shell nanoparticles with different thickness of silica shell were prepared using Stöber method [46]. The as-prepared AgNPs were purified by centrifugation and washed with ethanol three times, and then added to the mixture of 2 mL water, 0.1 mL ammonia and 8 mL ethanol under stirring. After 15 min, 15 μL

IP T

TEOS was slowly added under constant stirring. After 6 h reaction, the products were obtained upon centrifuging and washing the particles with ethanol and water thrice.

SC R

The thicknesses of silica shell could be adjusted by the dosage of TEOS.

U

2.6 Surface modification of Ag@SiO 2 nanoparticles

N

The as-prepared Ag@SiO 2 particles were dissolved with 5 mL isopropyl alcohol, and

A

then heated to 70°C under sealing stir with the introduction of 500 μL APTES for 6 h

M

[47]. Next, the mixture was purified by centrifugation, and washed with ethanol and

ED

DMF twice. After that, the product was dissolved with 5 mL DMF containing 0.06 g/mL succinic anhydride [48]. The reaction was kept for 18 h under sealing stirring.

PT

Finally, by centrifugation and washing with ethanol and water twice, the final product

CC E

was obtained and kept in water.

2.7 Conjugation of Ag@SiO 2 -AuNCs particles

A

200 μL Ag@SiO 2 particles were activated in 100 mM NHS and 100 mM EDC in 1 mL PB buffer (pH 6.8) at room temperature with 30 min shaking. Then the activated

Ag@SiO 2 -COOH nanoparticles were purified by refrigerated centrifugation, and washed with PB buffer (pH 7.2) thrice. Next, the product was resuspended in 1 mL 8

PB buffer (pH 7.2) to react with 2 μL 25.8 mg/mL AuNCs for 4 h with gentle shaking. The product was washed with PB and HEPES buffer to remove any unreacted AuNCs

2.8 Investigation of the feasibility of this approach

IP T

and was dispersed in 4 mL HEPES buffer (pH 7.4).

The as-prepared 20 µL Ag@SiO 2 -AuNCs in HEPES buffer with the addition of

SC R

following single or mixed components, to prepare different experimental groups for

comparison: (1) 100 µM Cu2+, (2) 100 µM PPi, (3) 100 mU PPase, (4) 100 µM

U

Cu2+ and 100 µM PPi, (5) 100 µM PPi and 100 mU PPase, (6) 100 µM Cu2+ and 100

N

mU PPase, and (7) 100 µM Cu2+ , 100 µM PPi and 100 mU PPase. Pure

A

Ag@SiO 2 -AuNCs solution was used as the control. The fluorescence spectra of the

ED

M

mixtures were obtained after a period of time with excitation at 470 nm.

2.9 Fluorescence detection of Cu2+ in buffer solution

PT

Firstly, the as-prepared 20 µL Ag@SiO 2 -AuNCs were resuspended in HEPES buffer

CC E

(pH 7.4) with the addition of various concentrations of Cu2+. After that, the mixture was incubated for 5 min to record fluorescence spectrum at the excitation at 470 nm.

A

2.10 Fluorescence detection of PPi in buffer solution 100 µM Cu2+ was introduced to 20 µL Ag@SiO 2 -AuNCs solution to quench the

fluorescence for 5 min. Afterwards, different concentrations of PPi were added with 10 min incubation to measure fluorescence spectrum. In order to investigate the 9

specificity of PPi detection, 100 µM PPi and 1000 µM other coexisting anions including PO 4 3-, HPO 4 2-, H 2 PO 4 -, NO 3 -, CO 3 2-, SO 4 2-, , F-, Cl-, Br-, and I- were added and the experiment was conducted under the same condition as in buffer solution, and

2.11 Fluorescence detection of PPase in buffer solution

IP T

then the fluorescence intensities were measured for comparison.

SC R

100 µM Cu2+ was introduced to 20 µL Ag@SiO 2 -AuNCs solution to quench the

fluorescence for 5 min. Then 100 M PPi was added and kept for 10 min to restore the

U

fluorescence. After that different concentrations of PPase were added and the mixed

N

solutions were incubated for 5 min to measure fluorescence spectrum. In order to

A

investigate the specificity for PPase detection, 100 mU PPase and (5 µg/mL) other

M

interferences including BSA, HSA, GSH, Try, Thr, Lys, Gly were added and the

ED

experiment was conducted under the same condition as in buffer solution, and then

PT

the fluorescence intensities were measured for comparison.

CC E

2.12 Cytotoxicity assay and cellular imaging The cellular toxicity of the Ag@SiO 2 -AuNCs on HeLa cervical carcinoma cells was investigated with MTT assay. HeLa cells were plated in 96-well plates and kept at

A

37 °C for 24 h in a humidified incubator with 5% CO 2 atmosphere. After sucking out medium, different concentrations of Ag@SiO 2 -AuNCs were added and incubated for another 24 h. 100 μL PBS and 20 μL MTT 5 mg/mL were introduced to each well and

incubated at 37 °C for 4 h after pouring out the medium. Then, the MTT medium was 10

removed and replaced with DMSO. Thereafter the plate was incubated and viabilities of the cells were determined by measuring absorption at 490 nm. For studying feasibility of the scheme in living cells, the HeLa cells were cultured for 24 h with the introduction of 20 μL solution of Ag@SiO 2 -AuNCs and incubated for 6

IP T

h at 37 °C. The treated cells were washed thrice with PBS. Afterwards, 100 μM Cu2+ was added into the wells with 40 min incubation. Then 100 μM PPi was added with

SC R

40 min incubation. Finally, 8 μM Mg2+ and 100 mU PPase were added and the cells

U

were incubated for another 40 min.

N

3. Results and Discussion

A

3.1 Characterization of Ag@SiO 2 -AuNCs nanoparticles

M

Since 50 nm AgNPs offer higher fluorescence enhancement for nearby fluorophores

ED

[27]. We prepared AgNPs with this size as the metal cores to construct MEF-based composite nanoparticles. The morphology of AgNPs is characterized by TEM images,

PT

which show that it has uniform particle size and is well-dispersed in water (Fig. S1A).

CC E

The particle size distribution of a large of number particles also indicates that the size of AgNPs is uniform (Fig. S1B). Fig.S1C shows that the UV-vis absorption peak of AgNPs is located at ~425 nm, which is consistent with the known work [45]. To

A

investigate MEF effect using the nanocomposite Ag-core/SiO 2 -spacer/AuNCs, the

spacer thickness was changed to optimize the metal-fluorophore distance and increase the fluorescence enhancement. Fig. 1A is the TEM images of the Ag@SiO 2 particles with different spacer thickness (~7 nm, ~10 nm, ~12 nm, ~15 nm, ~20 nm, ~25 nm), 11

which are highly dispersible in water and fairly uniform size. The UV-vis absorption peak of Ag@SiO 2 exhibits gradual red shift while the thickness of spacer increases (Fig. S1C). We explain this red shift by Mie theory: the refractive index of the medium around AgNPs increases due to the silica coating [49].

IP T

The AuNCs were prepared according to the reported work at the physiological temperature [44]. BSA contains 21 tyrosine residues and 35 cysteine molecules, and

SC R

acts as both reducing and stabilizing agent in the preparation of AuNCs, and the reducing ability of BSA is enhanced after the addition of NaOH. The as-prepared

U

AuNCs possess fairly uniform size (1.7±0.3 nm) and nice monodispersity in water

N

(Fig. 1B). The solution of BSA-AuNCs shows dark brown color and exhibits red

A

fluorescence under UV irradiation. Fig. S2 shows the absorption and fluorescence

M

spectrum of AuNCs. The absorption spectra of AgNPs and Ag@SiO 2 are partly

ED

overlapped with absorption and fluorescence emission spectrum of AuNCs, thus Ag nanoparticles may influence the fluorescent emission of nearby AuNCs. In addition,

PT

the data demonstrate that there are rich carboxyl groups on the surface of

CC E

BSA-AuNCs since it is highly negative charged (Fig. S3A, curve a, −48.1 mV). Although the hydroxyl on the surface of Ag@SiO 2 is capable of coupling with

BSA-AuNCs through electrostatic interaction and hydrogen bond, the above

A

Ag@SiO 2 nanoparticles were modified with carboxyl in order to increase the

coupling efficiency between Ag@SiO 2 nanoparticles and BSA-AuNCs. To verify successful synthesis of the nanocomposite Ag@SiO 2 -AuNCs, FTIR spectra and zeta potentials of Ag@SiO 2 nanoparticles (before and after modification) and 12

Ag@SiO 2 -AuNCs nanocomposites were shown in Fig. S3. Fig. S3A shows the significant changes in zeta potentials of Ag@SiO 2 : before modification (b: −25.2 mV), after amination (c: +26.1 mV), after carboxylation (d: −34.5 mV), and after coupling with AuNCs (e: −51.4 mV). These data demonstrate that the modification of

IP T

Ag@SiO 2 nanoparticle and the construction of Ag@SiO 2 -AuNCs nanocomposite are successful. As shown in the FTIR spectra (Fig. S3B), 1100 cm-1 is the typical

SC R

absorption peak of Si-O stretching and it has subtle shifts upon the changes of

material structure. In Fig. S3B, curve b (Ag@SiO 2 nanoparticles after amination), to the stretching vibration of -CH 2 - in

U

2950 cm-1 and 2890 cm-1 should be ascribed

N

the APTES, and curve c (Ag@SiO 2 nanoparticles after carboxylation) and curve d

A

(Ag@SiO 2 -AuNCs) show typical absorption peak of C=O stretching vibrations (1715

M

cm-1). The results approve that the modification on the surface of Ag@SiO 2

ED

nanoparticles and the conjugation between particles Ag@SiO 2 and AuNCs are successful. The coupling efficiency was 83% (Fig. S4). The nanocomposites have

PT

been endowed water-dispersibility and reactive sites for conjugating biomolecules by

CC E

carboxyl groups on the nanocompostes, which are essential for bioanalytical applications. Furthermore, the Ag@SiO 2 -AuNCs was of good stability (Fig. S5). After conjugating AuNCs with Ag@SiO 2 , the interaction between AuNCs and the

A

metal core remarkably improves the excitation efficiency, thus increases the fluorescence quantum yield and the fluorescence emission of the AuNCs is greatly enhanced. In addition, the aqueous solution of Ag@SiO 2 -AuNCs exhibiting a fluorescence peak centered at about 640 nm is consistent with the fluorescence 13

emission of AuNCs under the excitation at 470 nm. The fluorescence enhancement factor is dependent on the thickness of silica shell. Here the enhancement factor is defined as the ratio of fluorescence intensities between equal quantities of AuNCs contained in Ag@SiO 2 -AuNCs nanocomposites and free AuNCs. The maximum of

IP T

3.21-fold enhancement of the Ag@SiO 2 -AuNCs fluorescence was obtained when the shell thickness is about 10 nm (Fig. 1C, Fig. S6). In addition, the QY of

SC R

Ag@SiO 2 -AuNCs is ~10% higher than that of bare AuNCs when rhodamine 6G in

ethanol (QY=95%) is used as the reference (Fig. S7). Therefore, we conclude that the

U

fluorescence enhancement is ascribed to the increase of the QY of AuNCs on the

A

N

surface of the shell.

M

3.2 Verification of the feasibility of the approach

ED

As one of the widespread metal, copper ions possess well-known efficient quenching capability of fluorescence because of its paramagnetic property through electron or

PT

energy transfer. Copper ions can form complexes with porphyrin molecules and

CC E

carboxyl groups due to cation-π of Cu2+, resulting in fluorescence quenching. Therefore, the interaction between Cu2+ and BSA on the particle surface can

effectively quench enhanced fluorescence of nanocomposite Ag@SiO 2 -AuNCs (BSA

A

possess abundant functional groups, such as carboxyl groups, which enable the interaction between Cu2+ and the nanocomposite) (Fig. 2). However, the coordination between PPi and Cu2+ is stronger than that between Ag@SiO 2 -AuNCs and Cu2+, hence Cu2+ is withdrawn from Ag@SiO 2 -AuNCs-Cu2+ complexes, and the 14

fluorescence of the Ag@SiO 2 -AuNCs is recovered (Fig. 2). Furthermore, this strong association between Cu2+ and PPi could be used for quantitative detection of PPase with PPi being used as the substrate (Fig. 2). As is known, PPi is a natural substrate of PPase which can drive one molecule of PPi to generate two molecules of Pi. Unlike

Ag@SiO 2 -AuNCs

fluorescence

due

to

the

IP T

PPi, Pi has lower affinity to Cu2+, which may also lead to quenching of formation

of

SC R

Ag@SiO 2 -AuNCs-Cu2+ complexes. As shown in Fig. 2, fluorescence intensity of

Ag@SiO 2 -AuNCs has negligible changes when PPi, PPase and mixture of PPi and

U

PPase are separately added into the solution of Ag@SiO 2 -AuNCs, demonstrating that

N

PPi and PPase have no obvious effects on the fluorescence emission of

A

Ag@SiO 2 -AuNCs. Meanwhile, the fluorescence of Ag@SiO 2 -AuNCs does not

M

restore obviously when PPase is added into the solution of Ag@SiO 2 -AuNCs-Cu2+,

ED

also proving that PPase has no obvious effect on fluorescence properties of the Ag@SiO 2 -AuNCs-Cu2+ complex. Therefore, sensitive and specific approach for the

PT

detection of PPase by competitive association of Cu2+ between Ag@SiO 2 -AuNCs and

CC E

PPi can be constructed.

A

3.3 Fluorescence detection of Cu2+ Clinically, the leak of Cu2+ will lead to serious health problems, such as cushing syndrome and Wilson’s disease. However, too much intake of Cu2+ by human body causes endocrine disease, gastrointestinal disorders, musculoskeletal diseases, etc. Therefore, sensitively monitoring the concentration of Cu2+ in biological systems has 15

been of great interests. In this work, the fluorescence of Ag@SiO 2 -AuNCs is significantly quenched with the increase of Cu2+ due to the formation Ag@SiO 2 -AuNCs-Cu2+ complexes under the optimal conditions (Fig. S8A, S8B). The reaction can be quickly accomplished in 5 min (Fig. S9A). As shown in Fig.

IP T

3, approximately 90% of the initial fluorescence intensity of Ag@SiO 2 -AuNCs can be quenched when 100 M Cu2+ is added, and no further fluorescence quenching occurs

SC R

when more than 100 M Cu2+ is present. Therefore, 100 M Cu2+ is applied for the detection of PPi and PPase in the following experiments. In addition, the fluorescence

U

intensity is linearly dependent on Cu2+ concentration in the range from 0.05 to 0.8 M.

N

The corresponding linear equation is F/ F 0 =1.0019-0.8125C (C is Cu2+ concentration,

A

F 0 is the fluorescence intensity of Ag@SiO 2 -AuNCs, and F represents the

M

fluorescence intensity of Ag@SiO 2 -AuNCs-Cu2+) with a regression coefficient 0.996.

ED

The detection limit reaches 39 nM, which is comparable with other detection methods

PT

for Cu2+ (Table S1) [50-54].

CC E

3.4 Fluorescence detection of PPi Because the affinity between PPi and Cu2+ is stronger than that between PPi and Ag@SiO 2 -AuNCs, when PPi was introduced, the fluorescence of the system was

A

dramatically restored, and the reaction was quickly accomplished in 10 min under the optimal conditions (Fig. S8A, S8C, S9B). Fig. 4A shows that fluorescence recovery achieves more than 80% of the initial fluorescence intensity of Ag@SiO 2 -AuNCs when 100 µM PPi was added, and the fluorescence recovery shows no more increase 16

when PPi concentration is higher than 100 µM (Fig. 4B). So, 100 µM PPi is applied for quantitative detection of PPase. The fluorescence intensity is linearly dependent on PPi concentration in the range from 0.5 to 60 µM (Fig. 4C). The linear equation is F/F 0 =0.075C+0.963 (C is the

IP T

concentration of PPi; F and F 0 are the fluorescence intensities in the presence and absence of PPi in the Ag@SiO 2 -AuNCs-Cu2+, respectively) with regression

SC R

coefficient 0.997. The detection limit is as low as 0.0787 µM, which is comparable

with other detection methods for PPi (Table S2) [28, 32-34]. Moreover, 100 µM PPi

U

and 1000 µM other anions including PO 4 3-, HPO 4 2-, H 2 PO 4 -, CO 3 2-, NO 3 -, SO 4 2-, F-,

N

Cl-, Br-, I-, Hg2+, Pb2+, Ni2+, Fe2+, Fe3+, GSH, Hcy and Cys were introduced under the

A

identical experimental conditions to evaluate the selectivity for PPi detection. The

M

existence of PO 4 3-, HPO 4 2- and H 2 PO 4 - causes slight recovery of fluorescence

ED

intensity, because these ions also form complexes with Cu2+ to some extent. The introduction of other ions may cause slight fluctuations of fluorescence intensity.

PT

However, these changes of fluorescence intensity are negligible, compared to the

CC E

change of fluorescence intensity by adding same amount of PPi to the complex Ag@SiO 2 -AuNCs-Cu2+. In conclusion, this method has shown good selectivity for

A

PPi quantitative detection.

3.5 Fluorescence detection of PPase PPase is a kind of matrix metalloproteinases, and its activity for the hydrolysis of PPi to Pi needs the participation of magnesium ions Mg2+ because (Mg2+) is essential in 17

this enzymatic reaction [55]. As shown in Fig. S10, the ratio F/F 0 (F and F 0 represent the fluorescence intensities in the presence and absence of PPase in the system Ag@SiO 2 -AuNCs+Cu2++PPi,

respectively)

significantly

changes

with

the

concentration of Mg2+ (0-20 µM), and the data show that the ratio becomes stable

IP T

when Mg2+ reaches 8 µM, thus 8 µM Mg2+ is applied in the quantitative detection of PPase. The detection of PPase was investigated by adding different concentrations of

SC R

PPase under the optimized conditions (Fig. S8A, S8D). The fluorescence emission

spectra of the system were recorded after adding different concentrations of PPase

U

(5-1000 mU). The reaction was carried out in 5 min (Fig. S9C). As is known, PPi is a

N

natural substrate of PPase, which can drive one molecule of PPi to generate two

A

orthophosphate ions (Pi), and Pi shows low affinity to Cu2+. This reaction is

M

demonstrated by our data that the fluorescence of the system is significantly decreased

ED

while PPase concentration increases because of the regeneration of the complex Ag@SiO 2 -AuNCs+Cu2+ (Fig. 5). The fluorescence intensity is linearly dependent on

PT

PPase concentration in the range from 5 to 100 mU (Fig. 5), and the linear equation is

CC E

F/F 0 =0.9943-0.0084C (C is the concentration of PPase, and F and F 0 are the fluorescence intensities in the presence and absence of PPase, respectively, in the mixture of Ag@SiO 2 -AuNCs, Cu2+, PPi and Mg2+) with regression coefficient 0.992.

A

The detection limit is 0.976 mU. It is a quite competitive performance compared with

other reported methods (Table S3) [32-34, 36, 56, 57]. By examining the influences of some interferents in the assay of PPase, this approach shows excellent specificity (Fig. 5D). 18

100 mU PPase and 5 µg/mL other coexisting interferents including BSA, HSA, GSH, Try, Thr, Lys, Gly, Hcy, Cys, Hg2+, Pb2+, Ni2+, Fe2+, Fe3+ were operated under the same experimental condition. As shown in Fig. 5D, much higher contents of interferents than that of PPase show no obvious influences on PPase

IP T

detection. .Therefore, the above results demonstrate that this method can be applied

3.6 Cytotoxicity assay and cellular imaging

SC R

for sensitive and specific detection of PPase.

U

The cytotoxicity of Ag@SiO 2 -AuNCs was investigated by MTT viability assay, in

N

which HeLa cell was used as the model. The viability of the cell significantly changes

A

with the concentration of Ag@SiO 2 -AuNCs (Fig. S11). The Ag@SiO 2 -AuNCs

respectively,

confirming

low

cytotoxicity

and

high

ED

Ag@SiO 2 -AuNCs,

M

possess more than 90% cell viability for the doses of 5, 10, 15, 20, 25, 30 µL

biocompatibility of the nanocomposite. Then we conducted the fluorescence switch

PT

OFF-ON-OFF strategy for the sensing of multiple targets inside the living cells

CC E

(Fig.6). The cells have complex biological environment and contain a variety of proteins and other complex interfering substances, thus they may have an impact on

the reaction [58-68]. In order to ensure successful entry into cells and participation in

A

the reaction, the reaction time of Cu2+, PPi and Pase in the living cells were longer

than that in buffer. Red fluorescence was observed by a fluorescence microscope, when Ag@SiO 2 -AuNCs nanocomposites were incubated with the living HeLa cells. After introducing Cu2+, the fluorescence of the cells was quenched by Cu2+ because of 19

the affinity between Cu2+ and carboxyl groups on the nanocomposites. Subsequently, the introduction of PPi led to fluorescence resurgence of the cell samples. Ultimately, the fluorescence of cell sample was quenched again when PPase was added into the cell samples. These results are consistent with that in the pure buffer solution,

IP T

indicating that the Ag@SiO 2 -AuNCs nanoprobe can be applied for the assay of

SC R

different targets in both solution and living cells.

4. Conclusion

U

We have shown the successful synthesis of core-shell fluorescent nanocomposites

N

Ag@SiO 2 -AuNCs, which provide fluorescence enhancement factor 3.21 for

A

fluorescent AuNCs as the thickness of silica shell is ~10 nm. Moreover, a sensitive

M

and specific approach for the detection of multi-compontent Cu2+, PPi and PPase

ED

based on MEF and fluorescence switch OFF-ON-OFF strategy was successfully conducted. Owing to the remarkable fluorescence enhancement, there is a good linear

PT

relationship between the fluorescence intensity and PPase concentration with limit of

CC E

detection 0.976 mU. More importantly, this nanocomposite was applied to detect PPase in living cells successfully. To conclude, this work not only provides a new strategy for PPase detection, but also provides the potential of MEF nanocomposites

A

being used as novel sensing platforms.

Acknowledgement This work was supported by the National Natural Science Foundation of China 20

(81572086, 81772256 and 21827808).

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at

IP T

XXX.

SC R

References

[1] P. Anger, P, P. Bharadwaj, L. Novotny, Enhancement and Quenching of Single-Molecule Fluorescence, Phys. Rev. Lett, 96(2006) 113002.

U

[2] K. Aslan, I. Gryczynski, J. Malicka, E. Matveeva, J. R. Lakowicz, C. D. Geddes,

N

Metal-Enhanced Fluorescence: an Emerging Tool in Biotechnology, Curr. Opin.

A

Biotechnol, 16(2005) 55–62.

M

[3] P. P. Pompa, L. Martiradonna, A. D. Torre, F. D. Sala, L. Manna, M. De Vittorio, F. Calabi, R. Cingolani, R. Rinaldi, Metal-Enhanced Fluorescence of Colloidal

ED

Nanocrystals with Nanoscale Control, Nat. Nanotechnol, 1(2006) 126–130. [4] J. Zhang, Y. Fu, M. H. Chowdhury, J. R. Lakowicz, Metal-Enhanced

PT

Single-Molecule Fluorescence on Silver Particle Monomer and Dimer: Coupling Effect Between Metal Particles, Nano. Lett, 7(2007) 2101–2107.

CC E

[5] D. D. Xu, Y. L. Deng, C. Y. Li, Y. Lin, H. W. Tang, Metal-Enhanced Fluorescent Dye-Doped Silica Nanoparticles and Magnetic Separation: A Sensitive Platform for One-Step Fluorescence Detection of Prostate Specific Antigen, Biosens. Bioelectron,

A

87(2017) 881–887. [6] D. D. Xu, C. Liu, C. Y. Li, C. Y. Song, Y. F. Kang, C. B. Qi, Y. Lin, D. W. Pang, H.

W. Tang, Dual Amplification Fluorescence Assay for Alpha Fetal Protein Utilizing Immunohybridization Chain Reaction and Metal-Enhanced Fluorescence of Carbon Nanodots, ACS Appl. Mater. Interfaces, 9(2017) 37606−37614.

[7] E. Dulkeith, A. C. Morteani, T. Niedereichholz, T. A. Klar, J. Feldmann, S. A. 21

Levi, F. van Veggel, D. N. Reinhoudt, M. Moller, D. I. Gittins, Fluorescence Quenching of Dye Molecules near Gold Nanoparticles: Radiative and Nonradiative Effects, Phys. Rev. Lett, 89(2002), 203002. [8] K. Aslan, J. R. Lakowicz, C. D. Geddes, Rapid Deposition of Triangular Silver Nanoplates on Planar Surfaces: Applications to Metal-Enhanced Fluorescence, J. Phys. Chem. B, 109(2005) 6247–6251.

IP T

[9] K. Aslan, Z. Leonenko, J. R. Lakowicz, C. D. Geddes, Fast and Slow Deposition of Silver Nanorods on Planar Surfaces: Application to Metal-Enhanced Fluorescence,

SC R

J. Phys. Chem. B, 109(2005) 3157–3162.

[10] L. Zhang, Y. Song, T. Fujita, Y. Zhang, M. Chen and T. H. Wang, Large Enhancement of Quantum Dot Fluorescence by Highly Scalable Nanoporous Gold,

U

Adv. Mater, 26(2013) 1289–1294.

N

[11] S. L. Capehart, M. P. Coyle, J. E.Glasgow, M. B. Francis, Controlled Integration

A

of Gold Nanoparticles and Organic Fluorophores Using Synthetically Modified MS 2 Viral Capsids, J. Am. Chem. Soc, 135(2013) 3011–3016.

M

[12] R. Bardhan, N. K. Grady, N. J. Halas, Nanoscale Control of Near-Infrared

ED

Fluorescence Enhancement Using Au Nanoshells, Small, 4(2008) 1716–1722. [13] R. Bardhan, N. K. Grady, J. R. Cole, A. Joshi, N. J. Halas, Fluorescence

744–752.

PT

Enhancement by Au Nanostructures: Nanoshells and Nanorods, ACS. Nano, 3(2009)

[14] Y. Chen, K. Munechika, D. S. Ginger, Dependence of Fluorescence Intensity on

CC E

the Spectral Overlap Between Fluorophores and Plasmon Resonant Single Silver Nanoparticles, Nano Lett, 7(2007) 690–696. [15] K. Ray, J. R. Lakowicz, Metal-Enhanced Fluorescence Lifetime Imaging and

A

Spectroscopy on a Modified SERS Substrate, J. Phys. Chem. C, 117(2013)

15790–15797. [16] H. Naiki, A. Masuhara, S. Masuo, T. Onodera, H. Kasai, H. Oikawa, Highly Controlled Plasmonic Emission Enhancement from Metal-Semiconductor Quantum Dot Complex Nanostructures, J. Phys. Chem. C, 117(2013) 2455–2459. [17] K. Sugawa, T. Tamura, H. Tahara, D. Yamaguchi, T. Akiyama, J. Otsuki, Y. 22

Kusaka, N. Fukuda and H. Ushijima, ACS. Nano, 7(2013) 9997–10010. [18] G. Li, R. Jin, Atomically Precise Gold Nanoclusters as New Model Catalysts, Acc. Chem. Res, 46(2013) 1749–1758. [19] H. Qian, M. Zhu, Z. Wu, R. Jin, Quantum Sized Gold Nanoclusters with Atomic Precision, Acc. Chem. Res, 45(2012) 1470–1479. [20] Y. C. Shang, C. C. Huang, W. Y. Chen, P. C. Chen, H. T. Chang, Fluorescent

IP T

Gold and Silver nanoclusters for Analysis of Biopolymers and Cell Imaging, J. Mater. Chem, 22(2012) 12972–12982.

SC R

[21] L. Y. Chen, C. W. Wang, Z. Q. Yuan, H. T. Chang, Fluorescent Gold Nanoclusters: Recent Advances in Sensing and Imaging, Anal. Chem, 87(2015) 216–229.

[22] X. D. Zhang, F. G. Wu, P. D. Liu, N. Gu, Z. Chen, Enhanced Fluorescence of

U

Gold Nanoclusters Composed of HAuCl 4 and Histidine by Glutathione: Glutathione

N

Detection and Selective Cancer Cell Imaging, Small, 10(2014) 5170-5177.

A

[23] Y. Negishi, Y. Takasugi, S. Sato, H. Yao, K Kimura, T. Tsukuda, Magic-Numbered Aun Clusters Protected by Glutathione Monolayers （n = 18, 21, 25,

ED

126(2004) 6518-6519.

M

28, 32, 39）:  Isolation and Spectroscopic Characterization, J. Am.Chem. Soc,

[24] Y. Shichibu, Y. Negishi, H. Tsunoyama, M. Kanehara, T. Teranishi, T. Tsukuda, Extremely High Stability of Glutathionate-Protected Au25 Clusters Against Core

PT

Etching, Small, 3(2007) 835-839.

[25] G. L. Wang, T. Huang, R. W. Murray, L. Menard, R. G. Nuzzo, Near-IR

CC E

Luminescence of Monolayer-Protected Metal Clusters, J. Am.Chem. Soc, 127(2005) 812-813.

[26] H. W. Duan, S. M. Nie, Etching Colloidal Gold Nanocrystals with

A

Hyperbranched and Multivalent Polymers: A New Route to Fluorescent and Water-Soluble Atomic Clusters, J. Am. Chem. Soc, 129(2007) 2412-2413. [27] W. I. Lee, Y. J. Bae, A. J. Bard, Strong Blue Photoluminescence and ECL from OH-Terminated PAMAM Dendrimers in the Absence of Gold Nanoparticles, J. Am. Chem. Soc, 126(2004) 8358-8359. [28] J. Sun, F. Yang, D. Zhao, X. R.Yang, Highly Sensitive Real-Time Assay of 23

Inorganic Pyrophosphatase Activity Based on the Fluorescent Gold Nanoclusters, Anal. Chem., 2014, 86, 7883−7889. [29] P. Heikinheimo, J. Lehtonen, A. Baykov, R. Lahti, B. S. Cooperman, A. Goldman, The Structural Basis for Pyrophosphatase Catalysis, Structure, 4(1996) 1491−1508. [30] E. Oksanen, A. K. Ahonen, H. Tuominen, V. Tuominen, R. Lahti, A. Goldman, P.

Pyrophosphatase, Biochemistry, 46(2007) 1228−1239.

IP T

Heikinheimo, A Complete Structural Description of the Catalytic Cycle of Yeast

[31] R. A. Terkeltaub, Inorganic Pyrophosphate Generation and Disposition in

SC R

Pathophysiology, Am. J. Physiol. Cell Physiol, 281(2001) C1-C11.

[32] L. L. Zhang, M. Li, Y. F. Qin, Z. D. Chu, S. L. Zhao, A Convenient Label Free Colorimetric Assay for Pyrophosphatase Activity Based on a Pyrophosphate-Inhibited

U

Cu2+–ABTS–H 2 O 2 Reaction, Analyst, 139(2014) 6298–6303.

N

[33] X. L. Zhu, J. W. Liu, H. Y. Peng, J. H. Jiang, R. Q. Yu, A Novel Fluorescence

A

Assay for Inorganic Pyrophosphatase Based on Modulated Aggregation of Graphene Quantum Dots, Analyst, 141(2016), 251–255.

M

[34] H. H. Deng, F. F. Wang, X. Q. Shi, H. P. Peng, A. L. Liu, X. H. Xia, W. Chen,

ED

Water-Soluble Gold Nanoclusters Prepared by Protein-Ligand Interaction as Fluorescent Probe for Real-Time Assay of Pyrophosphatase Activity, Biosens. Bioelectron, 83(2016) 1–8.

PT

[35] S. Yang, G. Feng, N. H. Williams, Highly Selective Colorimetric Sensing Pyrophosphate in Water by a NBD-Phenoxo-Bridged Dinuclear Zn（II） Complex,

CC E

Org. Biomol. Chem, 10(2012) 5606−5612. [36] J. J. Deng, Q. Jiang, Y. X. Wang, L. F. Yang, P. Y, L. Q. Mao, Real-Time Colorimetric Assay of Inorganic Pyrophosphatase Activity Based on Reversibly

A

Competitive Coordination of Cu2+ between Cysteine and Pyrophosphate Ion, Anal. Chem, 85(2013) 9409-9415. [37] S. K. Kim, D. H. Lee, J. Hong, J. Yoon, Chemosensors for Pyrophosphate, Acc. Chem. Res, 42(2009) 23-31. [38] S. Lee, K. Y. Yuen, K. A. Jolliffe, J. Yoon, Fluorescent and Colorimetric Chemosensors for Pyrophosphate, Chem. Soc. Rev, 44(2015) 1749-1762. 24

[39] N. Shao, H. Wang, X. D. Gao, R. H. Yang, W. H. Chan, Spiropyran-Based Fluorescent Anion Probe and Its Application for Urinary Pyrophosphate Detection, Anal. Chem, 82(2010) 4628-4636. [40] S. Bhowmik, B. N. Ghosh, V. Marjomaki, K. J. Rissanen, Nanomolar Pyrophosphate Detection in Water and in a Self-Assembled Hydrogel of a Simple Terpyridine-Zn2+ Complex, J. Am. Chem. Soc, 136(2014) 5543-5546.

IP T

[41] A. Gogoi, S. Mukherjee, A. Ramesh, G. Das, Aggregation-Induced Emission Active Metal-Free Chemosensing Platform for Highly Selective Turn-On Sensing and

SC R

Bioimaging of Pyrophosphate Anion, Anal. Chem, 87(2015) 6974-6979.

[42] L. J. Fan, W. E. Jones, A Highly Selective and Sensitive Inorganic/Organic Hybrid Polymer Fluorescence “Turn-on” Chemosensory System for Iron Cation, J.

U

Am. Chem. Soc, 128(2006) 6784–6785.

N

[43] H. Yorita, K. Otomo, H. Hiramatsu, A. Toyama, T. Miura, H. Takeuchi,

A

Evidence for the Cation−π Interaction between Cu2+ and Tryptophan, J. Am. Chem. Soc, 130(2008) 15266–15267.

M

[44] J. P. Xie, Y. G. Zheng, J. Y. Ying, Protein-Directed Synthesis of Highly

ED

Fluorescent Gold Nanoclusters, J. Am. Chem. Soc, 131(2009) 888–889. [45] D. Steinigeweg, S. Schlucker, Monodispersity and Size Control in the Synthesis of 20–100 nm Quasi-Spherical Silver Nanoparticles by Citrate and Ascorbic Acid

PT

Reduction in Glycerol–Water Mixtures, Chem. Commun, 48(2012) 8682-8684. [46] J. P. Yang, F. Zhang, Y. R. Chen, S. Qian, P. Hu, W. Li, Y. H. Deng, Y. Fang, L.

CC E

Han, M. Luqmanb, D. Y. Zhao, Core-shell Ag@SiO2@mSiO2 Mesoporous

Nanocarriers

for

Metal-Enhanced

Fluorescence,

Chem. Commun, 47(2011)

11618-11620.

A

[47] Y. H. Deng, Y. Cai, Z. K. Sun, J. Liu, C. Liu, J. Wei, W. Li, C. Liu, Y. Wang, D. Y. Zhao, Multifunctional Mesoporous Composite Microspheres with Well-Designed Nanostructure: A Highly Integrated Catalyst System, J. Am. Chem. Soc, 132(2010) 8466-8473. [48] D. Brouard, M. L. Viger, A. G. Bracamonte, D. Boudreau, Label-Free Biosensing based on Multilayer Fluorescent Nanocomposites and a Cationic Polymeric 25

Transducer, ACS. Nano, 5(2011) 1888-1896. [49] K. M. Mayer, J. H. Hafner, Localized Surface Plasmon Resonance Sensors, Chemical. Reviews, 111(2011) 3828-3857. [50] N. Shao, J. Y. Jin, H. Wang, Y. Zhang, R. H. Yang, W. H. Chan, Tunable Photochromism of Spirobenzopyran via Selective Metal Ion Coordination: An Efficient Visual and Ratioing Fluorescent Probe for Divalent Copper Ion, Anal. Chem,

IP T

80(2008) 3466-3475.

[51] W. Y. Lin, L. L. Long, B. B. Chen, W. Tan, W. S. Gao, Fluorescence Turn-on

SC R

Detection of Cu2+ in Water Samples and Living Cells Based on the Unprecedented Copper-Mediated Dihydrorosamine Oxidation Reaction, Chem. Commun, 46(2010) 1311-1313.

U

[52] C. Y. Lei, Z. Wang, Z. Nie, H. H. Deng, H. P. Hu, Y. Huang, S. Z. Yao,

N

Resurfaced Fluorescent Protein as a Sensing Platform for Label-Free Detection of

A

Copper(II) Ion and Acetylcholinesterase Activity, Anal. Chem, 87(2015) 1974-1980. [53] M. H. Cui, Q. Liu, Q. Fei, Y. Q. Fei, Y. M. Liu, Y. F. Huan, A Novel UV-Visible

M

Chemosensor Based on the 8-Hydroxyquinoline Derivative for Copper Ion Detection,

ED

Anal. Methods, 7(2015) 4252-4256.

[54] W. Cao, X. J. Zheng, D. C. Fang, L. P. Jin, Metal Ion-Assisted Ring-Opening of a Quinazoline Based Chemosensor: Detection of Copper(II) in Aqueous Media, Dalton

PT

Trans, 44(2015) 5191–5196.

[55] D. H. Vance, A. W. Czarnik, Real-Time Assay of Inorganic Pyrophosphatase

CC E

Using a High-Affinity Chelation-Enhanced Fluorescence Chemosensor, J. Am. Chem. Soc, 116(1994) 9397-9398. [56] S. Yang, G. Feng, N. H. Williams, Highly Selective Colorimetric Sensing

A

Pyrophosphate in Water by a NBD-Phenoxo-Bridged Dinuclear Zn（II） Complex, Org. Biomol. Chem, 10(2012) 5606−5612. [57] L. Z. Zhao, L. Zhao, Y. Q. Miao, C. Y. Liu, C. X. Zhang, Construction of a Turn Off-On-Off Fluorescent System Based on Competitive Coordination of Cu2+ between

6,7-Dihydroxycoumarin

and

Pyrophosphate

Ion

for

Sensitive

Pyrophosphatase Activity, J. Anal. Methods Chem, 1(2016) 1-10. 26

Assay

of

[58] G. Sivaraman, M. Iniya, T. Anand, N. G. Kotla, O. Sunnapu, S. Singaravadivel, A. Gulyani,

D.

Chellappa,

Chemically

Diverse

Small

Molecule

Fluorescent

Chemosensors for Copper Ion, Coordin. Chem. Rev, 357(2018) 50-104. [59] M. Maniyazagan, R. Mariadasse, M. Nachiappan, J. Jeyakanthan, N. K. Lokanath, S. Naveen, G. Sivaraman, P. Muthuraja, P. Manisankar, T. Stalin, Synthesis of rhodamine based organic nanorods for efficientchemosensor probe for Al (III) ions

IP T

and its biological applications, Sensor. Actuat. B-Chem, 254 (2018) 795–804.

[60] S. J. Ranee, G. Sivaraman, A. M. Pushpalatha, S. Muthusubramanian, Quinoline

SC R

based Sensors for Bivalent Copper Ions in Living Cells, Sensor. Actuat. B-Chem, 255 (2018) 630–637.

[61] G. G. V. Kumar, M. P. Kesavan, G. Sivaraman, J. Rajesh, Colorimetric and NIR

U

Fluorescence Receptors for F−ion Detection Inaqueous Condition and Its Live Cell

N

Imaging, Sensor. Actuat. B-Chem, 255 (2018) 3194–3206.

A

[62] G. G. V. Kumar, M. P. Kesavan, G, Sivaraman, J, Annaraj , K, Anitha , A, Tamilselvi, S, Athimoolam, B, Sridhar, J. Rajesh, Reversible NIR Fluorescent Probes

M

for Cu2+ Ions Detection and Its Living Cell Imaging, Sensor. Actuat. B-Chem,

ED

255(2018) 3235–3247.

[63] G. G. V. Kumar, M. P. Kesavan, A. Tamilselvi, G. Rajagopal, J. D. Raja, K. Sakthipandi, J. Rajesh, G. Sivaraman, A Reversible Fluorescent Chemosensor for the

PT

Rapid Detection of Hg2+ in an Aqueous Solution: Its Logic Gates Behavior, Sensor. Actuat. B-Chem, 273 (2018) 305–315.

CC E

[64] C. A. swamy P, J. Shanmugapriya, S. Singaravadivel, G. Sivaraman, D. Chellappa, Anthracene-Based Highly Selective and Sensitive Fluorescent “Turnon” Chemodosimeter for Hg2+, ACS Omega, 3(2018) 12341-12348.

A

[65] S. A. A. Vandarkuzhali, S. Natarajan, S. Jeyabalan, G. Sivaraman, S. Singaravadivel, S. Muthusubramanian, B. Viswanathan, Pineapple Peel-Derived Carbon Dots: Applications as Sensor, Molecular Keypad Lock, and Memory Device, ACS Omega, 3(2018) 12584-12592. [66]

P.

Sakthivel,

K.

Sekar,

G.

Sivaraman,

S.

Singaravadivel,

Rhodamine–Benzothiazole Conjugate as an Efficient Multimodal Sensor for Hg2+ 27

Ions and Its Application to Imaging in Living Cells, New J. Chem, 42(2018) 11665-11672. [67] E. Ramachandran, S. A. A. Vandarkuzhali, G. Sivaraman, R. Dhamodharan, Phenothiazine Based Donor–Acceptor Compounds with Solid-State Emission in the Yellow to NIR Region and Their Highly Selective and Sensitive Detection of Cyanide Ion in ppb Level, Chem. Eur. J. 24(2018) 11042 – 11050.

IP T

[68] M. Iniya, B. Vidya, T. Anand, G. Sivaraman, D. Jeyanthi, K. Krishnaveni, D.

Chellappa, Microwave-Assisted Synthesis of Imidazoquinazoline for Chemosensing

A

CC E

PT

ED

M

A

N

U

SC R

of Pb2+ and Fe3+ and Living Cell Application, ChemistrySelect, 3(2018) 1282 – 1288.

28

470nm

640nm

470nm

470nm

640nm

+

+ Cu2+

AuNCs

640nm

+

+

640nm

Ag@SiO2

+ Pi

PPase

SC R

PPi

IP T

470nm

Scheme 1. Schematic illustration for multi-component detection procedure with

A

CC E

PT

ED

M

A

N

U

metal-enhanced fluorescence of AuNCs.

29

IP T SC R

N

U

Fig. 1 (A) TEM micrographs of composite nanoparticles Ag@SiO 2 with different

A

SiO 2 spacer thickness: (a)~7 nm, (b)~10 nm, (c)~12 nm, (d)~15 nm, (c)~20 nm,

M

(d)~25 nm. (B) The TEM micrographs of AuNCs (C) The dependence of the

A

CC E

PT

ED

fluorescence enhancement ratio on the spacer thickness.

30

Ag@SiO2-AuNCs

2500

Ag@SiO2-AuNCs+Cu2+ Ag@SiO2-AuNCs+PPi Ag@SiO2-AuNCs+PPase

2000

Ag@SiO2-AuNCs+Cu2++PPi Ag@SiO2-AuNCs+PPi+PPase Ag@SiO2-AuNCs+Cu2++PPase Ag@SiO2-AuNCs+Cu2++PPi+PPase

IP T

1500 1000 500 0 500

600 650 700 Wavelength (nm)

750

800

U

550

SC R

Fluorescence Intensity

3000

A

N

Fig. 2 Fluorescence emission spectra of Ag@SiO 2 -AuNCs, Ag@SiO 2 -AuNCs+Cu2+,

Ag@SiO 2 -AuNCs

+

PPi

M

Ag@SiO 2 -AuNCs + PPi, Ag@SiO 2 -AuNCs + PPase, Ag@SiO 2 -AuNCs + Cu2+ + PPi, + PPase,

Ag@SiO 2 -AuNCs

+ Cu2+ + PPase,

A

CC E

PT

ED

Ag@SiO 2 -AuNCs + Cu2+ + PPi + PPase in 10mM HEPES buffer (pH 7.4).

31

B

1.0

2500

1.0

2000

0.8

1000

0.6

0.6 0.4

0.4 0.0

0 500

0.2

0.0 550

600

650

700

750

800

0.4

0.6

0.8

100

120

Cu2+(µM)

0.2

500

0

20

40

60

Cu2+(µM)

Wavelength (nm)

80

IP T

0-120µM

1500

F/F0

0.8

F/F0

Fluorescence Intensity

A

SC R

Fig. 3 (A) Fluorescence emission spectra of the system upon the addition of

increasing amount of Cu2+. (B) Relative fluorescence intensity of the system upon the

A

CC E

PT

ED

M

A

N

the as-proposed detection platform (RSD < 5%).

U

addition of increasing amount of Cu2+. Inset: calibration curve for Cu2+ detection by

32

B

A

8 7 6

1500 1000

F/F0

5 0-200µM

4 3 2

500

1

0 500

0

7 6 5

F/F0

4 3 2

PO43- H2PO4-

SO42- Hg2+ Ni2+ Fe3+ Hcy

1 0 PPi

550

600

650

700

HPO42-F- Cl- Br- I- NO3- CO32- Pb2+ Fe2+ GSH Cys Blank

750

0

50

D

Wavelength (nm)

C

800

100

PPi (µM)

150

200

7

6

IP T

Fluorescence Intensity

2000

6

5

5 3 2

3 2

1

PO43- H2PO4

SO42- Hg2+ Ni2+ Fe3+ Hcy

1 0

10

20

30

PPi (µM)

40

50

60

0 PPi

HPO42-F- Cl- Br- I- NO3- CO 2- Pb2+ Fe2+ GSH Cys Blank 3

N

U

0

4

SC R

F/F0

F/F0

4

A

Fig. 4 (A) Fluorescence emission spectra of the system upon the addition of

M

increasing amount of PPi to Ag@SiO 2 -AuNCs-Cu2+ complex. (B) Relative fluorescence intensity of the system upon the addition of increasing amount of

ED

PPi to Ag@SiO 2 -AuNCs-Cu2+ complex. (C) Calibration curve for PPi detection by the

PT

as-proposed detection system (RSD < 5%). (D) Fluorescence intensities of the detection system in the presence of PO 4 3-, HPO 4 2-, H 2 PO 4 -, F-, Cl-, Br-, I-, NO 3 -,

A

CC E

SO 4 2-, CO 3 2-, Hg2+, Pb2+, Ni2+, Fe2+, Fe3+, GSH, Hcy, Cys (RSD < 5%).

33

A

B 1.0 0.8

1500 0-1000mU

F/F0

1000 500

0.6 0.4 0.2

0 500

0.0

550

600

650

700

750

0

800

Wavelength (nm)

200

1.0

600

800

0.8

0.4

0.6

SC R

F/F0

F/F0

0.8 0.6

0.4

0.2

0.2 0.0 0

20

40

60

80

100

0.0

PPase (mU)

1000

PPase (mU)

D 1.0

C

400

IP T

Fluorescence Intensity

2000

N

U

Blank BSA HSA GSH Try Thr Lys Gly Hcy Cys Hg2+ Pb2+ Ni2+ Fe2+ Fe3+ PPase

A

Fig. 5 (A) Fluorescence emission spectra of the system upon the addition of

M

increasing amount of PPase to Ag@SiO 2 -AuNCs+Cu2++PPi system. (B) Relative fluorescence intensity of the system upon the addition of increasing amount of

ED

PPase to Ag@SiO 2 -AuNCs+Cu2++PPi system. (C) Calibration curve for PPase

PT

detection by the as-proposed detection platform (RSD < 5%). (D) Fluorescence intensities of the detection system in the presence of BSA, HSA, GSH,Try, Thr, Lys,

A

CC E

Gly, Hcy, Cys, Hg2+, Pb2+, Ni2+, Fe2+, Fe3+ (RSD < 5%).

34

C

E

G

100 µm

100 µm

100 µm

100 µm

B

D

F

H

100 µm

100 µm

100 µm

100 µm

IP T

A

SC R

Fig. 6 Sensing of different targets in living HeLa cells with MEF-based Ag@SiO 2 -AuNCs nanocomposites. The bright-field images of the cells treated with (A) Ag@SiO 2 -AuNCs, (C) Ag@SiO 2 -AuNCs + Cu2+, (E)

U

(G) Ag@SiO 2 -AuNCs + Cu2+ + PPi + PPase, and the corresponding

N

Cu2+ + PPi,

Ag@SiO 2 -AuNCs +

A

fluorescence images of the cells treated with (B) Ag@SiO 2 -AuNCs , (D)

A

CC E

PT

ED

+ Cu2+ + PPi + PPase.

M

Ag@SiO 2 -AuNCs + Cu2+, (F) Ag@SiO 2 -AuNCs + Cu2+ + PPi, (H) Ag@SiO2 -AuNCs

35