SERS dual-sensing and imaging of intracellular Zn2+

Accepted Manuscript Fluorescent/SERS dual-sensing and imaging of intracellular Zn

2+

Dan Li, Yadan Ma, Huazhen Duan, Fei Jiang, Wei Deng, Xingang Ren PII:

S0003-2670(18)30871-7

DOI:

10.1016/j.aca.2018.07.020

Reference:

ACA 236121

To appear in:

Analytica Chimica Acta

Received Date: 25 January 2018 Revised Date:

10 June 2018

Accepted Date: 9 July 2018

Please cite this article as: D. Li, Y. Ma, H. Duan, F. Jiang, W. Deng, X. Ren, Fluorescent/SERS 2+ dual-sensing and imaging of intracellular Zn , Analytica Chimica Acta (2018), doi: 10.1016/ j.aca.2018.07.020. 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.

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Fluorescent/SERS dual-sensing and imaging of

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intracellular Zn 2+

Dan Li, a Yadan Ma, a Huazhen Duan, a Fei Jiang, a Wei Deng, *, a and Xingang Ren *, b, c

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a School of Chemical and Environmental Engineering, Shanghai Institute of Technology, 100

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Haiquan Road, Shanghai 201418, P. R. CHINA

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b Key Laboratory of Intelligent Computing & Signal Processing, Ministry of Education, Anhui

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University, No.3 Feixi Road, Hefei, Anhui Province 230039, P. R. CHINA

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c Institute of Physical Science and Information Technology, Anhui University, No.3 Feixi Road,

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Hefei, Anhui Province 230039, P. R. CHINA

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Keywords: Surface-enhanced Raman scattering (SERS); Fluorescence; Dipicolylamine

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derivative; Zinc ions.

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Abstract: A fluorescent and surface-enhanced Raman spectroscopy (SERS) dual-mode probe is

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developed

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ylmethyl)amino)ethyl)-2-mercaptoacetamide (MDPA) modified gold nanoparticles (MDPA-

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GNPs). Benefiting from the chelation-enhanced fluorescence (CHEF) between MDPA-GNPs

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and Zn2+, the fluorescent intensities of MDPA-GNPs are substantially enhanced with the

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increment of Zn2+ concentrations, which can be clearly observed by the naked eye. Under

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physiological conditions, the probe exhibits a stable response for Zn2+ from 1 µM to 120 µM,

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with a detection limit of 0.32 µM in aqueous solutions. The resultant MDPA-GNPs can be used

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for ultrasensitive SERS detection of Zn2+ because of the strong inter-particle plasmonic coupling

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generated in the process of Zn2+-triggered MDPA-GNPs self-aggregation, with a low detection

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limit of 0.28 pM, which is eight order of magnitude lower than the United States Environmental

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Protection Agency (US EPA)-defined limit (76 µM) in drinkable water. More importantly, the

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proposed probe can be applied for efficient detection of intracellular Zn2+ with excellent

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biocompatibility and cellular imaging capability. Therefore, a highly sensitive and selective

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nanosensor has been demonstrated for both reliable quantitative detection of Zn2+ in aqueous

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solution and real-time imaging of intracellular Zn2+, suggesting its significant potential utility in

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bioanalysis and biomedical detection in the future.

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of

intracellular

Zn2+

based

on

N-(2-(bis(pyridine-2-

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imaging

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for

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1. Introduction Zinc ion (Zn2+) is the second most abundant transition-metal ion in the human body and plays

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critical roles in biological processes such as facilitating enzyme regulation, gene expression and

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neural related signal transmission [1]. Due to its important biological roles, the ability to

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precisely detect Zn2+ at ultra-low concentrations is highly desirable in many fields such as

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healthy monitoring and environmental protection [2]. Dual-mode detection strategies have

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gained considerable attentions and have been proved to be more efficient than the single-mode

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method

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colorimetric/fluorescent and magnetic/fluorescent dual mode sensing, magnetic resonance

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computed tomography, and fluorescence/surface enhanced Raman scattering dual-mode imaging

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and so on [4-6]. Among these methods, the fluorescent and surface-enhanced Raman

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spectroscopy (SERS) dual-mode assay has received intensive attentions and has become an

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extremely sensitive analytic tool in biomedical applications. These dual mode sensors not only

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enable the visualization of the target analysts simply with naked eyes but also validate a reliable

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quantitative SERS analysis in a simple and rapid feature [7-9]. To date, most of the fluorescent

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and SERS dual-mode nanosensors are designed by integrating two optical parts together,

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including organic molecules [10], metal−organic frameworks (MOFs) [11], semiconductor

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quantum dots (QDs) [12], metal nanoclusters (NCs) [13, 14], and so on. These methods can

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realize the sensitive, selective and convenient detection of targets, but the complicated

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construction procedure, low biocompatibility and high cost hinder their widespread application.

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Therefore, it is desirable to develop novel fluorescent and SERS dual-mode nanosensors with

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more simple preparation procedures, low cost, and excellent biocompatibility.

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A

number

of

dual-mode

assays

have

been

developed,

such

as

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Recently, plasmonic nanoparticles, especially gold nanoparticles (GNPs), have attracted

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significant attention due to their unique properties, such as easy functionalization, good stability

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and biocompatibility [15, 16]. GNPs are also employed as carriers for fluorescent dyes for the

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intracellular fluorescence imaging. In addition, GNPs can be applied as excellent substrates for

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SERS detection to carry Raman signaling molecules [17, 18]. Although impressive progress has

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been made, the sensitivity and applicability of these protocols still require further substantial

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improvement. For example, fluorescent probes have the advantage of high specificity, while the

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fluorescence imaging method of living cells suffers from photo bleaching, non-ignorable

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background signal and instability. Differently, SERS method has the advantage to avoid photo

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bleaching due to the extremely short lifetimes of Raman scattering, which can provide abundant

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vibrational information of molecules, but its targeting ability is not as good as that of the

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fluorescent probes. Therefore, the development of novel nanoprobes combining the merits of

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both fluorescence and SERS is highly desired.

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In this work, by taking advantage of the unique optical properties of GNPs and strategically

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designing of the surface−interface chemistry, we will design and synthesize a compound,

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MDPA, which has a mercaptoacetic acid (MPA) and a di-(2-picolyl) ethylenediamine moiety

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(DPA) as a receptor that has a high affinity for Zn2+ with excellent sensitivity and selectivity

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(Scheme 1). MDPA can be used both as fluorescent and SERS sensor for detection of Zn2+

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because of the stable photoluminescence (PL), large Raman scattering cross section, strong

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binding with noble metal nanoparticles. The novel dual-mode sensor has four major advantages:

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(1) The preparation procedure is simple, the source materials are cheap, and the photostability

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and water-solubility are excellent. (2) Zn2+ can be detected selectively with high sensitivity in the

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range of 10-6−120 µM with a detection limit of 0.28 pM. Zn2+ can also be easily visualized via

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evident fluorescent color enhances under a UV lamp, and the detection limit can reach 5 µM. (3)

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The addition of different concentrations of Zn2+ induces corresponding aggregation degree of

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MDPA-GNPs, thus permitting the SERS detection of Zn2+ due to the formation of

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electromagnetic “hot spots” among the high-density GNPs. (4) The MDPA-GNPs can be used

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for the efficient detection of intracellular Zn2+ with excellent biocompatibility and cellular

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imaging capability. More importantly, it can distinguish Zn2+ from Cd2+ based on the distinct

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Raman characteristics, which is a significant improvement for SERS sensor as the Zn2+ and Cd2+

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is indistinguishable in SERS spectra in previous reports, thus allowing for selective and

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quantitative detection of Zn2+ in complex matrix samples.

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Scheme 1

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2. Experimental section

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2.1 Materials

Chloroauric acid (HAuCl4·4H2O, >99%), sodium citrate (Na3C6H5O7, 99%), sodium sulfate

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(Na2SO4, 99%), 2-(chloromethyl) pyridine hydrochloride (PicCl·HCl, 98%), N-BOC-

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ethylenediamine (98%), trifluoroacetic acid (TFA, 99%), N-ethyl-N´-(3-(dimethylamino) propyl)

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carbodiimide (EDC, 98%), N-hydroxysuccinimide (NHS, ≥97%) were obtained from Aladdin-

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Reagent (Shanghai, China). N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, ≥

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98 %), Methylthiazolyldiphenyl-tetrazolium bromide (MTT, 98%). Metal ions solutions were

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prepared from their nitrate salts. Ultrapure water with a conductivity of 18 MΩ·cm was used in

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all experiments. All the other chemical reagents were of analytical grade and used without

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further purification.

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2.2 Synthesis of gold nanoparticles (GNPs)

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GNPs (~50 nm in diameter) were synthesized by sodium citrate reduction of HAuCl4 in

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aqueous solutions [19]. Typically, a sodium citrate (1 mL, 10mg) was rapidly injected into a 5

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boiling solution of HAuCl4 (100 mL, 10mg) under vigorous stirring and the solution was kept

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continually boiling for another 30 min before cooled down to room temperature. The obtained

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GNPs colloid (0.5 nM) was stored at 4 oC before use, whose concentrations were estimated by

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Lambert-Beer Law [20].

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2.3 Synthesis of N-(2-(bis(pyridine-2-ylmethyl)amino)ethyl)-2-mercaptoacetamide (MDPA, 3)

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The MDPA was synthesized according to the following procedures. Firstly, N-tert-

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butoxycarbonyl-N’, N’- bis(pyridin-2-ylmethyl)ethane-1,2-diamine (1) was prepared, simply

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(2.24 g) N-BOC-ethylenediamine and (4.6 g, 2eq) 2-(chloromethyl) pyridine hydrochloride were

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suspended in NaOH aq (50 mL, 5 M) and stirred overnight. Distilled water (50 mL) was added

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under stirring and extracted with dichloromethane. The organic layer was dried over anhydrous

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Na2SO4 and evaporated. The crude product was further purified by silica gel column

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chromatography using dichloromethane/methanol (95:5, v/v) as the eluent, and then the product

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was obtained as brown oil. 1HNMR (270 MHz, CDCl3): δ 1.42 (s, 9H), 2.89 (t, 2H), 3.20 (m,

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2H), 3.84 (s, 4H), 7.09 (m, 2H), 7.38 (d, 2H), 7.63 (t, 2H), 8.52 (d, 2H).

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Secondly, N1,N1-bis(pyridin-2-ylmethyl)ethane-1,2-diamine (DPA, 2) was synthesized

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through removal of protecting tert-butoxycarbonyl (Boc) group. Typically, the compound 1 was

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treated with 45 mL of TFA/dichloromethane (3:1, v:v) for 1.5 h on ice-water bath and stirred for

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1.5 h. Then the extract was dissolved in NaOH solution (15 mL, 2 M) and washed with

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dichloromethane (20 mL), dried over anhydrous Na2SO4, and evaporated to give the product 2.

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Thirdly, compound 2 was reacted with mercaptoacetic acid via the EDC/NHS cross-linking

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method to obtain DPMA. Typically, amino compounds (1g) was suspended in 15 mL of

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EDC/NHS (1:1 molar ratio) and stirred for 12h. The crude product was further purified by silica

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gel column chromatography to afford 3 as a yellow colloid (0.817 g, yield: 87%). 1H NMR (270

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MHz, CDCl3): 2.62 (t, 2H), 3.28 (d, 2H), 3.50 (d, 2H), 3.86 (s, 4H), 7.12 (m, 2H), 7.30 (d, 2H),

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7.64 (t, 2H), 8.60 (d, 2H).

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2.4 Synthesis of MDPA-GNPs The MDPA modified GNPs (MDPA-GNPs) were obtained via a ligand exchange process.

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Namely, 5 mL of GNPs (0.5 nM) were centrifugation at 5,000 rpm for 10 min to remove excess

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sodium citrate, and it was dispersed in 5 mL of ethanol with the aid of sonication for 30 min.

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Then, 5 mL of GNPs and 0.1 mL of 0.1 mM MDPA were incubated in H2O at room temperature

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for 24 h. The mixture was centrifugation at 5,000 rpm for 5 min to remove the excess MDPA.

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After centrifugation, the precipitate was further re-dispersed in 5 mL of doubly distilled water

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and stored at 4oC before use. The concentration of as-prepared MDPA-GNPs) (0.45 nM) was

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estimated according to the concentration of GNPs core using Lambert-Beer Law [20].

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2.5 Fluorescent and SERS detection for Zn2+

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Fluorescent and SERS sensing for Zn2+ were performed under the following procedures. A

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total of 10 µL of Zn2+ with different concentrations ranging from 0 to 10 mM was added into the

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1 mL of MDPA-GNPs (0.45 nM). The resulting mixtures were incubated for 1 min at room

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temperature and then detected by both the fluorescent and SERS channel. For fluorescent

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detection, fluorescent spectra of the mixtures were recorded directly by fluorescence

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spectrophotometer and photographed with a digital camera under a 365 nm UV lamp. A

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calibration curve was built based on the concentrations of the standard solutions and their

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corresponding fluorescence intensities.

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The SERS measurements were performed using a portable Raman spectrometer (BWS415, B

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&W Tek Inc., USA) with a resolution of 5 cm-1 and a beam diameter of 10 mm. A 785 nm laser

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line with 10 mW was used as the excitation light source. The instrument background was

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deducted and baseline was corrected with multiple point linear curve fitting. 10 µL of the

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resultant mixture was dropped onto silicon wafer under ambient conditions and dried in the

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ambient environment, and the SERS signal was then collected. For application in SERS

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detection of Zn2+, a laser power of 10 mW was selected and spectra were recorded with an

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integration time of 30 s.

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2.6 MTT Assays

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The cell viability assessment was carried out using the MTT assay. Typically, 100 µL of

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macrophages were seeded in a 96-well plate with a density of 2 × 105 macrophages per mL and

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allowed to adhere overnight. After incubation with MDPA-GNPs for 24 h at 37 °C in a

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humidified atmosphere with 5% CO2, 20 µL of MTT (5 mg·mL-1) was added to each well. Three

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wells with MTT but no cells were used as control. After incubating for another 4 h at 37 °C, the

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media were removed, and 150 µL dimethyl sulfoxide was added to each well and shaken for 10

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min. Finally, the absorbance was measured at 490 nm using a microplate reader (BMG

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LABTECH, CLARIO star). The cell viability was defined as the ratio of the absorbance in the

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presence of MDPA-GNPs (A test) to that in the absence of MDPA-GNPs (A control).

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2.7 Intracellular imaging of Zn2+

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Macrophages grown on 18 × 18 mm2 glass coverslips were first cultured in Dulbecco’s

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modified Eagle’s medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal

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bovine serum (FBS), 100 mg·mL−1 glutamine, 100 mg·mL−1 sodium pyruvate, penicillin (100

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units·mL−1), and streptomycin (100 units· mL−1) at 37 °C in a humidified atmosphere of 5% CO2

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overnight followed by incubation with 300 µg·mL−1 MDPA-GNPs for 24 h. Finally, the culture

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media were discarded and the culture dish was gently washed with PBS before the fluorescent

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and SERS measurements.

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To demonstrate that the uptake of MDPA-GNPs is through the cooperative recognition of

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Zn2+, some competition experiments were performed. Macrophages were seeded at the same

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density in two chambers and incubated for 24 h, one of which was treated with 0.45 nM of

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MDPA-GNPs. The second chamber was cultured with 10 µM TPEN before incubation with 0.45

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nM of MDPA-GNPs and 1 µM Zn2+. The third chamber was only cultured with 1 µM Zn2+

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before incubation with 0.45 nM MDPA-GNPs. After six hours of incubation, all cells were

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washed with PBS and fixed for fluorescent and SERS measurements.

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2.8 Characterization

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UV–vis spectra were obtained on a UV-2100 spectrophotometer (Tokyo, Japan). Fluorescent

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spectra were recorded on a RF-5301PC FL Spectrophotometer (Tokyo, Japan) equipped with a

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Xe lamp and a plotter unit and a 1 cm quartz cell. The scanning electron microscopy (SEM) were

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carried out on an FEI-Sirion 200 field-emission scanning electron microscope (FEI Co. with 20

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kV operating voltage). Transmission electron microscopy (TEM) measurements were performed

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on a JEOL JEM-2010 with accelerating voltage 200 kV. All pH measurements were obtained

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using a pHS-25 pH meter (Shanghai, China). IR spectra were recorded using a Bruker Vertex 70

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spectrometer. The XPS measurements were performed on an Axis Ultra DLD photoelectron

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spectrometer (Shimadzu Corp., Japan) with a monochromatic Al Kα radiation (1486.6 eV). The ζ

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potentials of modified GNPs were measured by using a Zetasizer Nano (Malvern, ZS90, Malvern

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Instruments Ltd., Worcestershire, U.K.).

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The cell imaging was carried out an inverted microscope (eclipse Ti-U, Nikon, Japan) that was

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equipped with a dark-field condenser (0.8
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100 W halogen tungsten lamp was used for obtaining the scattering light. The true-color

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scattering images of cells were taken using a true-color digital camera (Nikon DS-fi). The image

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acquisition software is NIS-Elements provided by Roper Scientific. Fluorescent images of cells

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were recorded at 350 nm excitation and SERS spectra were obtained at 785 nm excitation. A 300

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mm focal-length and 300 grooves/mm monochromator (Acton SP2300i) with a 512×512-pixel

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cooled spectrograph CCD camera (CASCADE 512B, Roper Scientific) were used to obtain the

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fluorescence spectra and the Raman spectra. The obtained spectra of cells were corrected by

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subtracting the background spectra taken from the adjacent regions without the cells and dividing

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with the calibrated response curve of the entire optical system. The integration time used in all

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experiments spectral acquisitions is 20 second.

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2.9 Theoretical studies

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The monomer, dimer, trimer and tetramer gold nanoparticles are studied through rigorously

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solving Maxwell’s equations by three-dimensional finite-difference method [21, 22]. The

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dielectric constant of gold nanoparticle is adopted from reference [23]. In the simulation, the

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diameter of gold nanoparticle is set as 50 nm, the inter-particle distance is 2 nm and the mesh

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size is 0.5 nm. The perfect matched layer is adopted for truncating the simulation domain and

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absorbing the reflected wave. The polarization of the incident light is along the horizontal

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direction (indicated in the figure). The simulated SERS enhancement factor is defined as

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EF=|E|4/|Einc|4, here E is the electric field intensity at the 785 nm excitation wavelength and Einc

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is the intensity of the incident field.

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3. Results and discussion

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3.1 Preparation and Characterization of MDPA-GNPs

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The synthesis procedure of the fluorescent/SERS dual-mode MDPA- GNPs nanoprobes is

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illustrated in Scheme 1. Initially, the DPA was prepared, followed by the covalent binding of

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mercaptoacetic acid to the amino-group of DPA using an EDC/NHS cross-linking procedure (Fig.

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S1-S4). Finally, MDPA was covalently adsorbed on the surfaces of GNPs via Au–S bond.

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MDPA is chosen as the reporter molecule because of its strong binding with GNPs, large Raman

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cross-section, and excellent Zn2+ chelating ability. Upon addition of Zn2+, the MDPA-GNPs

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aggregates immediately appear based on the coordination interaction between N atoms of MDPA

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and Zn2+, resulting in a quantitatively fluorescent and SERS response to the concentration of

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Zn2+.

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The as-prepared MDPA-GNPs exhibit an absorption peak at 526 nm at the localized surface

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plasmon resonance (SPR) (Fig. S5), red-shifted 6 nm relative to the citrate stabilized GNPs (Cit-

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GNPs) due to the higher refractive index of the MDPA coating [15]. The SPR peak remains

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unchanged in various buffer solutions such as phosphate buffered saline (PBS), Tris-HCl and

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carbonate buffer (Fig. S6), confirming the excellent stability of the MDPA-GNPs. This is crucial

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for nanoparticle-based probes because the optical signals incurred by non-specific aggregation

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can be effectively eliminated. Fig. 1A shows that the inter-particle self-aggregation is

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immediately turned on upon the addition of Zn2+ (10 µM) in the MDPA-GNPs dispersion (0.45

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nM). There are no visible absorbance peaks between 550 and 800 nm of GNPs in the absence of

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MDPA (Fig. S5), which indicates the appearance of a broad band absorbance peak around 680

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nm are attributed to the Zn2+-induced aggregation of MDPA-GNPs. In addition, the intensity of

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the SPR peak at 526 nm progressively decreases, which is accompanied by a colorimetric red-to-

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blue transition (Inset of Fig. 1A). Meanwhile, the emission peak originally at 435 nm for the

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MDPA shifts to the red curve by 3 nm after binding to GNPs and by a further 12 nm with an

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increased intensity after treatment with a 10 µM Zn2+ solution (Fig. 1B). This shift can be

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attributed to the modification of the refractive index on the surface of MDPA-GNPs by the

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binding process.

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To investigate the Zn2+-induced stimulus response of MDPA-GNPs, we study the morphology

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of Zn2+-treated MDPA-GNPs (Zn2+-MDPA-GNPs) by TEM. As shown in Fig. 1C, the MDPA-

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GNPs remained as dispersed single nanoparticle in the absence of Zn2+, while the addition of

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Zn2+ will induce clustering of the probe to form large aggregates. To obtain information on the

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surface composition of the MDPA-GNPs, FT-IR spectra were recorded from the obtained Cit-

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GNPs, MDPA and MDPA-GNPs. As shown in Fig. 1E, the spectrum for Cit-GNPs shows two

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distinct bands of 3430 cm-1 and 1381 cm-1, assigned to the -OH stretching vibrations and

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deformations vibrations, respectively [24]. The appearance of sharp strong bands at 1610 cm-1,

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owing to the C=N vibrations, indicate the presence of MDPA on the GNPs. In addition, the

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weakly basic characteristic vibrational peak at 2545 cm-1 can be assigned to S-H stretching,

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indicating that the MDPA-GNPs are successfully prepared by this facile method [25]. The

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appearance of broad band in the range of 3155–3425 cm−1, owing to the -NH stretching in the

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Zn2+-MDPA-GNPs, indicates the bonding of imine nitrogen atoms with the Zn2+ [26]. XPS data

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provide further information on the formation of MDPA-GNPs. As shown in Fig. S7, the

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expected peaks from C1s, O1s, S2p, N1s and Au4f core levels are clearly detected in the survey

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spectra of MDPA-GNPs, indicating that MDPA molecules adsorb on the surface of GNPs.

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Moreover, The N 1s signal of MDPA-GNPs molecules is centered at 398.8 eV. It is noticed that

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one peak located at 399.8 eV was observed for Zn2+-MDPA-GNPs (Fig. S8). The shift in binding

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energy of N 1s orbitals from MDPA-GNPs to Zn2+-MDPA-GNPs suggests that pyridyl nitrogen

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had interacted with Zn2+ [27]. To further confirm the successful modification of GNPs with

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MDPA, we measured the ζ potentials of Cit-GNPs, MDPA-GNPs with and without Zn2+ (Fig.

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S9). After modification, the results show the ξ-potential values of the MDPA-GNPs change from

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–37.5 mV to -10.3 mV, which indicates that the GNPs are successfully modified with MDPA.

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Compared to MDPA-GNPs, Zn2+-MDPA-GNPs shows a more positive ζ potential (+3.61 mV),

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which is mainly attributed to the positive charge carried by Zn2+ in aqueous solution [28]. To evaluate the SERS performance of the MDPA-GNPs towards Zn2+, the SERS behavior of

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MDPA-GNPs and Zn2+-MDPA-GNPs are investigated (Fig. 1F). Upon addition of Zn2+, the

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Zn2+-MDPA-GNPs displays a new Raman bands at 1024 cm-1 (blue curve in Fig. 1F), assigning

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to pyridine ring breathing [2, 27]. Clear changes in the intensity and Raman shifts occur

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suggesting binding of Zn2+ to the MDPA-GNPs with subsequent charge redistribution and

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structural modifications to the MDPA [2, 29]. These results substantially demonstrate the

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successful formation of Zn2+-MDPA-GNPs aggregates, which combined the inherent properties

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of both the MDPA and the GNPs, thus could be used both as fluorescent and SERS nanosensor

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for Zn2+, as demonstrated below.

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Fig. 1

3.2 Fluorescent and SERS sensing of Zn2+ based on the MDPA-GNPs

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Considering the trace and ultra-trace amounts of the Zn2+ in environmental and biological

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samples, it is necessary to optimize the experimental parameters to obtain the optimal

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performance of the MDPA-GNPs. Therefore, a series of tests were performed to obtain the

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optimum conditions for the fluorescent and SERS detection of Zn2+. The molar ratio of the

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MDPA and the GNPs is rationally optimized at 1:4000 for the fluorescent and SERS sensing of

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Zn2+, as shown in Fig. S10. With the increasing amount of MDPA, the fluorescent intensity at

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425 nm (F425) and SERS intensity at 1024 cm-1 (I1024) remains stable in the range from 1:4000 to

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1:7000, owing to the saturated adsorption of MDPA on the GNPs. Moreover, the chelation

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reaction between MDPA-GNPs and Zn2+ can be completed quickly, and the F425 and I1024 of

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MDPA-GNPs reach the steady state within 1 min (Fig. S11). Therefore, we choose 1 min as the

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optimized reaction time in the sequent experiment. The effect of pH with buffer solutions on the

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fluorescent response is also investigated as displayed in Fig. S12. Under weak acidic (pH < 6)

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conditions or weak alkaline (pH 8-10) conditions, the F425 is found to decrease, whereas the

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SERS intensity at 1024 cm-1 (I1024) hits the climax at pH = 7.0 and remains stable in the range

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from 6.5 to 7.5. The F425 and I1024 values of MDPA-GNPs decrease below pH 6 can be attributed

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to the protonation of secondary amines of MDPA, hampers the formation of the Zn2+-MDPA-

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GNPs complex. Meanwhile, the precipitation of Zn2+ increases rapidly at pH>8. Therefore, the

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pH of reaction system is adjusted to 7.0 in all subsequent detection.

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In addition, the fluorescent response towards Zn2+ is almost the same from 20 °C to 30 °C (Fig.

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S13). Therefore, the temperature of 25 °C is selected as an optimization. Under the optimal

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conditions employed here, we evaluate the fluorescent and SERS performance of MDPA-GNPs.

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After the addition of different concentrations of Zn2+ into the dispersion of MDPA-GNPs at 25

13

°C for 1 min, the fluorescent and SERS spectra are investigated. As depicted in Fig. 2A, the

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successive addition of the Zn2+ to the MDPA-GNPs results in an increase in the enhanced

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fluorescent intensity at 425 nm (F425). The F425 values are linear with the Zn2+ within the range

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from 1 to 120 µM (Fig. 2B) (F425 = 1.26CZn2+ (µM) +101.6, R2 =0.9986). Moreover, SERS

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spectra of MDPA-GNPs with different concentrations from 10-6 to 20 µM are exhibited in Fig.

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2C. Upon addition of Zn2+, the MDPA-GNPs aggregates display the Raman band at 1024 cm-1,

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corresponds to the Raman characteristics of Zn2+-MDPA-GNPs chelates. As shown in Fig. 2C, it

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clearly demonstrates that the SERS intensity of MDPA-GNPs aggregates at 1024 cm-1 is

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gradually enhanced with the increasing concentrations of Zn2+, owing to the increase of “hot

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spots” among inter-particles aggregation (Fig. S14). The I1024 values linearly increase with

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increasing concentrations of Zn2+ ranging from 10-6 to 20 µM (I1024 = 1860 log[Zn2+] (µM) +

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12946, R2 = 0.9996) (Fig. 2D). Based on the 3σ rule, the limit of detection (LOD) for fluorescent

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and SERS methods are calculated to be 0.32 µM and 0.28 pM, respectively, which are much

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lower than the maximum allowable level of Zn2+ in drinking water (76 µM) recommended by

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U.S. Environmental Protection Agency (EPA) [30]. The performance of the dual-mode sensing

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strategy for Zn2+ is comparable with those of the classical SERS assay and fluorescent method

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(Table S1).

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3.3 Possible mechanism of the fluorescent and SERS response of MDPA-GNPs towards Zn2+ To investigate the mechanism of Zn2+ induced fluorescent and SERS enhancement, we

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examined the selectivity of MDPA-GNPs toward a variety of metal ions through FL and SERS

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spectra. As shown in Fig. 3A, 3B, the separate addition of these potential interfering substances,

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including metal-ions (K+, Ca2+, Al3+, Fe2+, Ni2+, Co2+ and Cu2+) and anions (Cl-, NO3-, CH3COO-

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), does not exhibit a significant response, either in the FL or the SERS intensity, which indicates

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the uniqueness of Zn2+ to the MDPA-GNPs. The multiple Zn salts with different anions do not

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cause significant changes in the SERS spectroscopic profiles and the enhancement factors,

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indicating the potential uses of the SERS sensor for the detection of metal ions from different

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origins (Fig. 3C). The Job’s continuous variation method is utilized to find out the stoichiometry

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between the Zn2+ and MDPA. As can be seen from Fig. S15, the maximum value was found at

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the mole fraction 0.31, which is indicative of the 1:2 binding stoichiometry between Zn2+ and

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MDPA. On the basis of fluorescence titration, the association constant (Ka) of MDPA+Zn2+ is

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computed to be 1.46×105 M-1 (Fig. S16), indicating that the probe can detect Zn2+ at the

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micromolar level. Thus the fluorescence enhancement by Zn2+ might be due to the effective

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coordination, which causes the Zn2+-MDPA chelates to be more coplanar structure than the

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MDPA itself, resulting in the chelation-enhanced fluorescence (CHEF) effect [31, 32]. Zn2+ has been the main source of interference for Cd2+ due to their similarity in terms of

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electronic structures, Zn2+ results in a slight shoulder at 425 nm in the FL spectrum and its

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intensity is considerably higher than that caused by Cd2+ at equivalent concentration. Despite the

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fact that Cd2+ exists in environmental and biological samples at extremely low concentrations

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because of its high toxicity, it is very challenging to translate the fluorescence changes for Zn2+

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and avoid interference from Cd2+ when used in complex medium. However, the ring breathing

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observed at 1005 cm-1 in the case of MDPA-GNPs shifts to 1024 cm-1 in the presence of Zn2+

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and 1014 cm-1 in the presence of Cd2+ (Fig. 3D). The appearance of new Raman bands enables

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distinguishing Zn2+ from Cd2+ separately and quantitative detection of Zn2+ in complex samples.

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The differences between the Raman characteristics of MDPA-GNPs with the presence of Zn2+

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and Cd2+ might be due to the effective coordination of Zn2+ with MDPA over Cd2+ [33].

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Meanwhile, Zn2+ added into the MDPA-GNPs can specifically bind to pyridyl nitrogen of

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MDPA, decreasing the distance between GNPs and leading to aggregation of GNPs. To gain an

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in-depth understanding of MDPA-GNPs on Raman characteristics, the intensity profile of the

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near field has been obtained through rigorously solving the Maxwell’s equations [34]. As shown

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in Fig. 4, the near field of single GNPs is confined at the horizontal side. Due to the aggregation

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of GNPs, a strong inter-particle coupling can be readily obtained, which results in the highly

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localized near field i.e., several hot spots in between GNPs for enhancing the Raman intensity.

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As a consequence, the Raman intensity of reporter molecule MDPA is significantly intensified

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due to the formation of SERS “hot spots”.

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Fig. 4 3.4 Cytotoxicity of MDPA-GNPs and intracellular imaging of Zn2+ To evaluate the biological applications of the MDPA-GNPs nanosensor, their cytotoxicity to

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macrophages is determined using the MTT assay. The cell viability was shown to be more than

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91% upon the addition of MDPA-GNPs over a concentration range of 0-2 nM (Fig. S17). Due to

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the low cytotoxicity and excellent biocompatibility, the MDPA-GNPs shows great promise for

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monitoring Zn2+ in living cells. Hence, fluorescent and SERS dual-mode imaging of the Zn2+ in

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single cells are carried out to further demonstrate their feasibility in biological applications. After

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incubating the macrophages with MDPA-GNPs for 6 h at 37 °C, a large amount of probe

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particles appeared in macrophages (Fig. 5). When excited at 320 nm, strong fluorescence is

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obtained from these macrophages. However, no obvious fluorescence enhancement is observed

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from the macrophages without the probes inside, indicating that the strong fluorescence was not

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the cellular auto fluorescence (Fig. S18). As shown in Fig. 5, the probe appeared in the

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cytoplasm, which can be observed under a dark-field microscope (DFM), and the fluorescence of

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MDPA is quite weak under the observation of the fluorescent microscope. The corresponding

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SERS signal of MDPA is shown in Fig. 5E, where the predominant characteristic peaks of the

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probes at 1024 cm-1 are clearly displayed. The results suggest that the proposed probe is capable

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of keeping their distinct SERS and fluorescence performances after being taken up by living

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cells.

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Fig. 5

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To quantitatively evaluate the uptake levels of Zn2+, macrophages were incubated with the

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probes under three different conditions: (1) incubated with the probes for 6 h; (2) treated with 10

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µM TPEN before the probes and 1 µM Zn2+ were added and then incubated for 6 h; (3) treated

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with 1 µM Zn2+ prior to the addition of the probes and then incubated for 6 h. It can be observed

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that much stronger fluorescence and SERS signals are detected in the cells incubated with

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exogenous Zn2+ than that in the cells with TPEN (Fig. 5, a2-d2, a3-d3). As shown in Fig. 5F and

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5G, the intensities of the characteristic peak at 1024 cm-1 are plotted, revealing the same trends

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as the fluorescence does. It has been reported that TPEN can be used as a competitive ligand for

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binding with Zn2+ [35]. As a result, the bonding possibility of MDPA-GNPs to the Zn2+

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decreased. Therefore, the addition of free TPEN grabs Zn2+ from Zn2+-MDPA chelates but does

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not completely blocked the uptake of the probes. The results demonstrate that MDPA-GNPs

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nanosensors can serve as an effective probe for intracellular Zn2+ sensing.

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4. Conclusions

In summary, we have developed a high-selective and high-sensitive strategy for sensing of

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Zn2+ based on Zn2+-triggered MDPA-GNPs self-aggregation. This strategy integrates the

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advantages of fluorescence and SERS, resulting in the realization of the in situ observation of

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intracellular Zn2+ distribution at the single cell level. The effectiveness and practicality of the

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proposed strategy are confirmed by the successful quantitation of cellular uptake of the Zn2+.

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Compared with the reported methods, the proposed probe that prepared in a two-step

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derivatization process can provides a Zn2+-sensitive fluorescent/Raman dual-imaging strategy

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with good biocompatibility, high selectivity and low cytotoxicity. This synergetic targeting

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strategy is expected to have promising applications in nanomedicine or high-throughput drug

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screening in the future.

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Acknowledgements

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This research is supported by the Natural Science Foundation of China (No. 21507089,

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61701003), the Shanghai University Young Teacher Training Program (No. ZZyy15095), the

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Scientific Research Foundation for the Introduction of Talent of Shanghai Institute of

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Technology (YJ2015-6) and Shanghai Municipal Education Commission (Plateau Discipline

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Construction Program), this research is also supported by Natural Science Research Foundation

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of Anhui Province (No. 1808085QF179).

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7 Appendix A. Supplementary material

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Supplementary data associated with this article can be found on the online version at

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10 11 12 References

14

[1] H.G. Lee, J.H. Lee, S.P. Jang, H.M. Park, S.-J. Kim, Y. Kim, C. Kim, R.G. Harrison, Zinc

15

selective chemosensor based on pyridyl-amide fluorescence, Tetrahedron 67 (2011) 8073-

16

8078.

EP

TE D

13

[2] H. Zhuang, Z. Wang, X. Zhang, J.A. Hutchison, W. Zhu, Z. Yao, Y. Zhao, M. Li, A highly

18

sensitive SERS-based platform for Zn(ii) detection in cellular media, Chem. Commun. 53

19

(2017) 1797-1800.

AC C

17

20

[3] D. Li, Y. Ma, H. Duan, W. Deng, D. Li, Griess reaction-based paper strip for

21

colorimetric/fluorescent/SERS triple sensing of nitrite, Biosens. Bioelectron. 99 (2018) 389-

22

398.

23

[4] Y. Cao, R.C. Qian, D.W. Li, Y.T. Long, Raman/fluorescence dual-sensing and imaging of

19

ACCEPTED MANUSCRIPT

1

intracellular pH distribution, Chem. Commun. 51 (2015) 17584-17587. [5] Y. Liu, W. Duan, W. Song, J. Liu, C. Ren, J. Wu, D. Liu, H. Chen, Red Emission B, N, S-co-

3

Doped Carbon Dots for Colorimetric and Fluorescent Dual Mode Detection of Fe3+ Ions in

4

Complex Biological Fluids and Living Cells, Acs Appl. Mater. Interfaces 9 (2017) 12663-

5

12672.

RI PT

2

[6] S. Zong, Z. Wang, J. Yang, C. Wang, S. Xu, Y. Cui, A SERS and fluorescence dual mode

7

cancer cell targeting probe based on silica coated Au@Ag core-shell nanorods, Talanta 97

8

(2012) 368-375.

10

[7] G. Wang, Y. Wang, L. Chen, J. Choo, Nanomaterial-assisted aptamers for optical sensing,

M AN U

9

SC

6

Biosens. Bioelectron. 25(2010) 1859-1868.

[8] J. Yan, S. Su, S. He, Y. He, B. Zhao, D. Wang, H. Zhang, Q. Huang, S. Song, C. Fan, Nano

12

Rolling-Circle Amplification for Enhanced SERS Hot Spots in Protein Microarray Analysis,

13

Anal. Chem. 84 (2012) 9139-9145.

TE D

11

[9] Y. Shi, H. Wang, X. Jiang, B. Sun, B. Song, Y. Su, Y. He, Ultrasensitive, Specific,

15

Recyclable, and Reproducible Detection of Lead Ions in Real Systems through a

16

Polyadenine-Assisted, Surface-Enhanced Raman Scattering Silicon Chip, Anal. Chem. 88

17

(2016) 3723-3729.

EP

14

[10] M. Li, X. Wu, Y. Wang, Y. Li, W. Zhu, T.D. James, A near-infrared colorimetric

19

fluorescent chemodosimeter for the detection of glutathione in living cells, Chem. Commun.

20

50 (2014) 1751-1753.

AC C

18

21

[11] X.-Y. Xu, B. Yan, Fabrication and application of a ratiometric and colorimetric fluorescent

22

probe for Hg2+ based on dual-emissive metal–organic framework hybrids with carbon dots

23

and Eu3+, J. Mater. Chem. C 4 (2016) 1543-1549.

20

ACCEPTED MANUSCRIPT

1 2

[12] F. Yang, Q. Ma, W. Yu, X. Su, Naked-eye colorimetric analysis of Hg2+ with bi-color CdTe quantum dots multilayer films, Talanta 84 (2011) 411-415. [13] J. Deng, W. Ma, P. Yu, L. Mao, Colorimetric and Fluorescent Dual Mode Sensing of

4

Alcoholic Strength in Spirit Samples with Stimuli-Responsive Infinite Coordination

5

Polymers, Anal. Chem. 87 (2015) 6958-6965.

RI PT

3

[14] F. Zou, H. Zhou, T.V. Tan, J. Kim, K. Koh, J. Lee, Dual-Mode SERS-Fluorescence

7

Immunoassay Using Graphene Quantum Dot Labeling on One-Dimensional Aligned

8

Magnetoplasmonic Nanoparticles, Acs Appl. Mater. Interfaces 7 (2015) 12168-12175.

9

[15] Z. Krpetic, L. Guerrini, I.A. Larmour, J. Reglinski, K. Faulds, D. Graham, Importance of

10

nanoparticle size in colorimetric and SERS-based multimodal trace detection of Ni(II) ions

11

with functional gold nanoparticles, Small 8 (2012) 707-714.

M AN U

SC

6

[16] T. Yang, X. Guo, H. Wang, S. Fu, Y. Wen, H. Yang, Magnetically optimized SERS assay

13

for rapid detection of trace drug-related biomarkers in saliva and fingerprints, Biosens.

14

Bioelectron. 68 (2015) 350-357.

TE D

12

[17] J. Duan, M. Yang, Y. Lai, J. Yuan, J. Zhan, A colorimetric and surface-enhanced Raman

16

scattering dual-signal sensor for Hg2+ based on Bismuthiol II-capped gold nanoparticles.

17

Anal. Chim. Acta 723 (2012) 88-93.

19

[18] H. Xu, Y. Wang, X. Huang, Y. Li, H. Zhang, X. Zhong, Hg2+-mediated aggregation of gold

AC C

18

EP

15

nanoparticles for colorimetric screening of biothiols, Analyst 137 (2012) 924-931.

20

[19] J. Storhoff, R. Elghanian, R. Mucic, C. And, R. Letsinger, One-Pot Colorimetric

21

Differentiation of Polynucleotides with Single Base Imperfections Using Gold Nanoparticle

22

Probes. J J. Am. Chem. Soc. 120(1998) 1959-1964.

23

[20] W. Haiss, N. Thanh, J. Aveyard, D. Fernig, Determination of size and concentration of gold

21

ACCEPTED MANUSCRIPT

1 2 3

nanoparticles from UV-vis spectra, Anal. Chem. 79(2007) 4215-4221. [21] Z. Huang, T. Koschny, C.M. Soukoulis, Theory of pump-probe experiments of metallic metamaterials coupled to a gain medium, Phys. Rev. Lett. 108 (2012) 187402. [22] X. Ren, Z. Huang, X. Wu, S. Lu, H. Wang, L. Wu, S. Li, High-order unified symplectic

5

FDTD scheme for the metamaterials, Comput. Phys. Commun. 183 (2012) 1192-1200.

6

[23] P.B. Johnson, R.W. Christy, Optical Constants of the Noble Metals, Phys. Rev. 6 (1972) 4370-4379.

SC

7

RI PT

4

[24] S. Jia, D. Li, E.K. Fodjo, H. Xu, W. Deng, Y. Wu, Y. Wang, Simultaneous preconcentration

9

and ultrasensitive on-site SERS detection of polycyclic aromatic hydrocarbons in seawater

10

using hexanethiol-modified silver decorated graphene nanomaterials, Anal. Methods 8

11

(2016) 7587-7596.

M AN U

8

[25] N. M. Aghatabay, A. Neshat, T. Karabiyik, M. Somer, D. Haciu, B. Dülger, Synthesis,

13

characterization and antimicrobial activity of Fe(II), Zn(II), Cd(II) and Hg(II) complexes

14

with 2,6-bis(benzimidazol-2-yl) pyridine ligand, Eur. J. Med. Chem. 42(2007) 205-213.

15

[26] J. J. Yuan, D. C. Wan, Z. H Yang, A Facile Method for the Fabrication of Thiol-

18

EP

17

Functionalized Hollow Silica Spheres, J. Phys. Chem. C 112(2008) 17156-17160. [27] L. Jin, G. She, L. Mu, W. Shi, Highly uniform indicator-mediated SERS sensor platform for the detection of Zn2+, RSC Adv. 6 (2016) 16555-16560.

AC C

16

TE D

12

19

[28] J. Gao, X. Y. Huang, H. Liu, F. Zan, J. C. Ren, Colloidal Stability of Gold Nanoparticles

20

Modified with Thiol Compounds: Bioconjugation and Application in Cancer Cell Imaging,

21

Langmuir 28(2012) 4464-4471.

22 23

[29] Y. Zhao, J.N. Newton, J. Liu, A. Wei, Dithiocarbamate-coated SERS substrates: sensitivity gain by partial surface passivation, Langmuir 25 (2009) 13833-13839.

22

ACCEPTED MANUSCRIPT

1 2

[30] W.C. Lin, Z. Li, M.A. Burns, Drinking Water Sensor for Lead and Other Heavy Metals, Anal. Chem. 89 (2017) 8748-8756. [31] G.J. Park, D.Y. Park, K.M. Park, Y. Kim, S.J. Kim, P. S. Chang, C. Kim, Solvent-dependent

4

chromogenic sensing for Cu2+ and fluorogenic sensing for Zn2+ and Al3+: a multifunctional

5

chemosensor with dual-mode, Tetrahedron 70 (2014) 7429-7438.

RI PT

3

[32] K. Velmurugan, A. Raman, D. Don, L. Tang, S. Easwaramoorthi, R. Nandhakumar,

7

Quinoline benzimidazole-conjugate for the highly selective detection of Zn(II) by dual

8

colorimetric and fluorescent turn-on responses, RSC Adv. 5(2015) 44463-44469.

SC

6

[33] L. Zhu, Z. Yuan, J.T. Simmons, K. Sreenath, Zn(II)-coordination modulated ligand

10

photophysical processes-the development of fluorescent indicators for imaging biological

11

Zn(II) ions, RSC Adv. 4 (2014) 20398-20440.

M AN U

9

[34] X. Li, WCH. Choy, X. Ren, D. Zhang, H. Lu, Highly Intensified Surface Enhanced Raman

13

Scattering by Using Monolayer Graphene as the Nanospacer of Metal Film–Metal

14

Nanoparticle Coupling System, Adv. Funct. Mater. 24(2014) 3114-3122.

17 18 19 20 21

endogenous zinc ions in live cells and organisms, Biomaterials 33 (2012) 7818-7827.

EP

16

[35] Z. Guo, G.H. Kim, I. Shin, J. Yoon, A cyanine-based fluorescent sensor for detecting

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Figure Captions

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Scheme 1. Schematic illustration of the preparation and application of dual-mode MDPA-GNPs

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probe for detection of intracellular Zn2+.

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Fig. 1. (A) UV–vis spectra of aqueous dispersions (25oC, pH 7) of MDPA-GNPs after the

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addition of different concentrations of Zn2+ (10 µL, from 0 to 200 µM), and the inset shows

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photographs of the aqueous dispersion of MDPA-GNPs before and after Zn2+ addition. (B)

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Fluorescent emission spectra and photographs (inset, excited by a 365 nm UV lamp) of MDPA

6

(black curve), MDPA-GNPs (vial 1, red curve) and Zn2+-MDPA-GNPs (vial 2, green curve).

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TEM image of MDPA-GNPs probes in (C) absence and in (D) presence of 50 µM Zn2+. (E) FT-

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IR spectra of (a) Cit-GNPs, (b) MDPA, (c) MDPA-GNPs, (d) Zn2+-MDPA-GNPs. (F) Raman

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spectra of 1M MDPA and SERS spectra of MDPA-GNPs with and without 50 µM Zn2+.

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Fig. 2. (A) Fluorescent spectra of MDPA-GNPs after the addition of different concentrations of

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Zn2+ (10 µL, from 0 to 200 µM). (B) Plot of F425 versus concentration of Zn2+ in the range of 0-

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200 µM. (C) SERS spectra of MDPA-GNPs in the presence of different concentrations of Zn2+

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(10 µL, from 0 to 20 µM). (D) SERS intensities at 1024 cm-1 as a function of Zn2+ concentration

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in the range of 10-6 to 100 µM.

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Fig. 3. (A) FL spectra and (B) SERS spectra of the dispersion of the MDPA-GNPs with the

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presence of different metal ions (K+, Ca2+, Al3+, Fe2+, Ni2+, Co2+, Cu2+, Cd2+ and Zn2+) at 50 µM.

4

(C) SERS spectra of MDPA-GNPs in aqueous solutions of different Zn salt (50 µM). (D) SERS

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spectra of MDPA-GNPs treated with the metal ions of Zn2+, Cd2+ and mixed Zn2+/Cd2+.

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Fig. 4. (A-D) The simulated near-field intensity for the MDPA-GNPs monomers, dimers, trimers

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and tetramers under the excitation wavelength with a 785 nm laser. The insets in (A-D) show the

4

characteristic TEM images of the corresponding nanostructures. (E) The measured and

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calculated Raman enhancement factors of the SERS signals at 1024 cm-1 for the corresponding

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nanostructures in (A-D).

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Fig. 5. Fluorescence microscopy images (A, B) and their corresponding DFM images (C, D) of

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macrophages. a1)-a3): the detailed view of macrophages fluorescence images in b1)-b3). c1)-c3):

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the detailed view of macrophages DFM images in d1)-d3). (E) Mean fluorescence intensity per

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cell and (F) average SERS intensity on the basis of the probe's characteristic peak at 1024 cm-1.

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(G) The average SERS spectra obtained from 10 randomly selected cells. a1)-d1) incubated with

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0.45 nM of MDPA-GNPs for 6 h at 37 °C; a2)-d2) first incubated with 10 µM TPEN for 24 h

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and then incubated with 0.45 nM MDPA-GNPs and 1 µM Zn2+ for 6 h at 37 °C; a3)-d3) first

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incubated with 1 µM Zn2+ for 24 h and then incubated with 0.45 nM of MDPA-GNPs for 6 h at

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37 °C.

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Highlights

(1) The MDPA-GNPs can not only be used as a highly selective fluorescence ‘‘turn-on’’ Zn2+ probe, but also as a portable SERS substrates for reliable quantitative

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analysis of Zn2+.

(2) The MDPA-GNPs enables distinguishing Zn2+ from Cd2+ based on the distinct Raman characteristics.

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(3) The MDPA-GNPs can be used for quantitative detection of intracellular Zn2+ with

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excellent biocompatibility and cellular imaging capability.