Highly Stable and NIR Luminescent RuâLPMSN Hybrid Materials for

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Biological and Medical Applications of Materials and Interfaces

Highly Stable and NIR Luminescent Ru-LPMSN Hybrid Materials for Sensitive Detection of Cu2+ in vivo Fangman Chen, Fangnan Xiao, Weibing Zhang, Chentao Lin, and Yunkun Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08887 • Publication Date (Web): 23 Jul 2018 Downloaded from http://pubs.acs.org on July 24, 2018

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ACS Applied Materials & Interfaces

Highly Stable and NIR Luminescent Ru-LPMSN Hybrid Materials for Sensitive Detection of Cu2+ in vivo Fangman Chen a, c†, Fangnan Xiao b†, Weibing Zhang a, c, Chentao Lin d, Yunkun Wu* b a

State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of

Matter, Chinese Academy of Sciences, Fuzhou 350002, China. b

Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation,

College of Life Science, Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, Fujian Normal University, Fuzhou 350119, China. c

School of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China.

d

Department of Immunology, Institute of Biotechnology, Fujian Academy of Agricultural

Sciences, Fuzhou 350003, China.

ACS Paragon Plus Environment

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ABSTRACT: Herein, new NIR luminescent ruthenium complexes were prepared for detecting Cu2+ ions. Then, ruthenium complex hybrid nanomaterial (Ru-LPMSN) was fabricated successfully by imbedding ruthenium complex into mesoporous silica nanoparticles. Benefiting from the novel large pore mesoporous structure and good adsorbility of LPMSN, Ru-LPMSN hybrid material showed significantly enhanced fluorescence intensity and stability. NIR fluorescence of Ru-LPMSN was rapidly quenched by Cu2+ ions. Ru-LPMSN also showed high Cu2+ ion selectivity and sensitivity as a sensor. The detection limit of Cu2+ ions was 10 nM with wide linear relationship between the fluorescence intensity of Ru-LPMSN and the concentration of Cu2+ ions. The mechanism of fluorescence quenching might be that the combination of ruthenium complex and Cu2+ constrained the photo-induced electron transfer (PET) process. Furthermore, Ru-LPMSN dramatically increased fluorescence signals in cells and achieved Cu2+ ions detection. Ru-LPMSN had different tissue affinities and could monitor distribution of exogenous Cu2+ ions in vivo. Moreover, Ru-LPMSN realized directly and rapidly detection of Cu2+ ion content in serum. These results indicated the potential applications of the prepared nanomaterials as Cu2+ detection agents.

KEYWORDS: Ruthenium complex; LPMSN; Cu2+; In vivo


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1. INTRODUCTION Ruthenium (Ru) polypyridine derivatives have excellent photo/electro luminescence emission, excited-state reactive capacity and stability,1 which allow them to be used as luminescent sensors,2-3 light-emitting devices,4 dye-sensitized solar cells5 and photocatalysts.6 As a luminescence probe, Ru complexes have a large Stokes Shift, long fluorescence lifetime, strong emission, high biocompatibility and easy metabolization, which make them a broad bioapplication. Various studies have been conducted on Ru complexes as biosensor materials.7-8 For bioimaging applications, near infrared (NIR) fluorescence has attracted more and more attention due to its deep tissue penetration, low background interference and minimal sample light damage. However, there are few studies on the synthesis of NIR Ru complexes, with little knowledge about their structural modification and structure-activity. Meanwhile, construction of Ru-based organic-inorganic hybrid materials could be a promising approach to prepare desirable chemical properties and performance of Ru complex, which has aroused great concern over the past decades.9 More recently, Ru complex was reported to exhibit an efficient anticancer activity encapsulated in liposome and the Ru-layered material showed significantly improved luminescent property.10-11 The traditional strategies to enhance the sensitivity of Ru-based luminescent sensors were to increase the loading density of Ru complexes in nanomaterials.12-13 Thus, nanomaterial containing nano-cavity structure is considered as model materials for the study of Ru luminescent sensors. Nano-cavity materials with negative charge would be a better choice because most Ru complexes are positively charged. In addition to sensitivity, absorption, accumulation and mass transfer of analyte are probably more important for nanomaterial sensor. These characters are related to the structure, surface state and morphology of nanomaterials. For


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example, nanomaterials with large confined space can facilitate adsorption, accumulation of analyte, and are also favorable to the interaction between analyte and Ru complexes. Mesoporous silica nanoparticles (MSN) are porous compounds with adjustable surface charge and groups.14-15 The rigid structure of MSN allows them to load and protect various guests, and the combination of large specific surface area and variable porous size of MSN results in a relative large guest molecule load. Moreover, transparent MSN enables the utilization of the optical properties of the intercalated dyes. A number of studies have been conducted on the properties of dye/MSN hybrid materials.16-18 For Ru complex, its luminescence process is vulnerable to the surrounding environment, such as solvent, temperature and oxygen, which restricts its practical application. Reports have indicated that the luminous efficiency and stability can be improved by assembling dyes into mesoporous silica materials. Ru complexes had been loaded into mesoporous MCM-48 by dipping method.19 It was found that the fluorescence intensity of Ru-MCM enhanced obviously, while the loaded Ru complexes increased, and the maximum emission wavelength was red shifted with the solvent evaporation. Ogawa adopted similar method for assembling Ru complex with mesoporous C18-FSM.20 Ru complexes dispersed in the channel of the mesoporous and fluorescence intensity increased greatly in moist C18-FSM. Among different pore sizes of mesoporous silica, large-pore MSN (LPMSN) exhibit supremacy over smaller pore-MSN with regard to mass transfer, diffusivity and penetration ability for guests into or out of the pore system.21 LPMSN is easily functionalized to covalently bond or adsorb the guests more effectively.22 Moreover, LPMSN can also be more easily to degrade in living organisms and exhibited bio-safety.23 Few studies are found on NIR Ru-LPMSN nanomaterials, therefore the interactions between NIR Ru complexes and LPMSN remain to be elucidated.


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In this work, we report the synthesis of novel NIR Ru-LPMSN hybrid materials via selfassembly method (Figure 1). Firstly, a series of Ru1-Ru4 complexes containing 2,2’dimethylpyridine ethylenediamine (DPA) ligand were synthesized by coordinating method. These complexes showed red to NIR fluorescence emission property and exhibited excellent selectivity for Cu2+ detection, since DPA binds well with Cu2+ ion. Secondly, Ru4, the most suitable ruthenium complex considering NIR fluorescence deeply organize penetrating, was absorbed into the big mesoporous silica channels of LPMSN by hydrogen bonding and electrostatic adsorption etc. Benefiting from LPMSN absorbing ruthenium complex, keeping the environmental stability and resisting the outside invasion, fluorescence intensity of Ru-LPMSN was greatly enhanced. Meanwhile, Cu2+ sensitivity can be increased effectively with the large surface and big tunnel structure of LPMSN for accumulation and absorption of Cu2+ ion. The prepared Ru-LPMSN hybrid materials showed rapid, sensitive and selective detection of Cu2+ ion. The detection limit was 10 nM in vitro. Ru-LPMSN hybrid materials were potential application as a NIR Cu2+ detection agent both in vitro and in vivo, and the mechanism was investigated. Ru-LPMSN were proved to exhibit good biocompatibility, access cells rapidly, locate mainly in the cytoplasm and detect the intracellular Cu2+ level. Moreover, Ru-LPMSN was used to monitor the distribution of Cu2+ content in zebrafish. The present work provides an effective means for biodetection applications. 2. EXPERIMENTAL SECTION 2.1. Material Ethylenediamine,

2-chloromethylpyridine

hydrochloride,

Trifluoroacetate,

Anhydrous

tetrahydrofuran, Di-tert-butyl dicarbonate, 4,4-dicarboxy-2,2-bipyridine, RuCl3·3H2O and Ethyl orthosilicate

(TEOS,

98%)

are

purchased

from

Shanghai

Aladdin.


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Hexadecyltrimethylammonium toluene-p-sulphonate (CTATOS), Thionyl chloride, 2,2-bipyridyl and 1,10-phenanthroline are purchased from Aladdin (China) and used without further purification. 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute (RPMI) were purchased from sangon Biotech (Shanghai, China). 2.2. Synthesis of dicarboxyl pyridine ethylenediamine dimethyl pyridine (bpy– –dpa). A mixture of 2,2'-Bipyridine-4,4'-dicarboxylic acid (0.5 g) and 10 mL of thionyl chloride was refluxed for 8 h at 90 °C. The excess thionyl chloride was blow-dried by N2. Synthesize N, N - dimethyl pyridine 'ethylenediamine (dpa) based on reported articles.24 A mixture of dpa (988 mg) and triethylamine (1.85 mL) was solved in 21 mL of dry dichloromethane. The mixture was dropwise added to acylation carboxyl-bipyridine redisappered in 21 mL of dry dichloromethane at 0 °C with stirring for 12 h. Then solvent was evaporated and resolved in 3 mL of ethanol to recrystallize to get bpy-dpa. C40H40N10O2，1H NMR (300 MHz, d-DMSO): δ 9.02 (s, 2H), 8.78 (s, 2H), 8.50 (s, 2H), 7.90 (d, 4H), 7.53 (s, 2H), 7.51 (d, 4H), 7.35 (d, 4H), 7.05 (m, 4H), 3.90 (s, 8H), 3.60 (d, 4H), 2.86 (d, 4H). MS (ESI): calcd for [M+H]+, 693.34; found, 693.33. 2.3. Synthesis of Ru1-Ru4. Ru(dcbpy)2Cl2 synthesized according to the reported article.25 A mixture of bpy–dpa (165 mg) and Ru(dcbpy)2Cl2 (169 mg) was dissolved in 20 mL of ethanol and refluxed for 5 h at 80 °C under N2 atmosphere.26 The solvent was evaporated and purified by silica

gel

column

with

eluent:

acetonitrile/water/saturated

potassium

nitrate

aqueous/trimethylamine: 40/4/1/1. Synthesis of Ru2-Ru4 was similar to Ru1. Ru1: C64H56F12N14O10P2Ru，1H NMR (400 MHz, D2O/NaOD): δ 11.13 (d, 6H), 10.89 (d, 2H), 10.58 (s, 2H), 10.02 (s, 2H), 9.73 (d, 12H), 9.60 (d, 6H), 7.16 (s, 2H), 6.08 (s, 2H), 5.95 (s, 4H), 4.25


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(s, 4H), 3.63 (d, 6H), 3.36(s, 4H). C64H56F12N14O10P2Ru: calcd. C 48.89, H 3.59, N 12.47; found C 48.91, H 4.16, N 12.53. Ru2: C64H56F12N14O2P2Ru, 1H NMR (300 MHz, d-DMSO)：δ 8.78 (d, 2H), 8.63 (d, 2H), 8.55 (d, 2H), 8.18 (t, 10H), 7.79 (d, 6H), 7.55 (t, 2H), 7.45 (d, 2H), 7.32 (s, 8H), 6.92 (d, 4H), 3.67 (s, 8H), 3.37 (s, 4H), 2.65 (s, 4H), 1.93 (d, 2H). C64H56F12N14O2P2Ru: calcd. C 53.23, H 3.91, N 13.58; found C 53.12, H 4.07, N 13.72. Ru3: C60H56F12N14O2P2Ru，1H NMR (300 MHz, d-DMSO): δ 9.05 (s, 4H), 8.87 (d, 4H), 8.42 (d, 4H), 8.21 (d, 4H), 8.19 (d, 2H), 7.95 (d, 4H), 7.82 (d, 2H), 7.80 (s, 2H), 7.75 (d, 2H)，7.62 (s, 4H), 7.57 (d, 4H), 7.17 (t, 4H), 3.85 (s, 8H), 3.10 (s, 4H), 2.74 (s, 4H). C60H56F12N14O2P2Ru: calcd. C 51.62, H 4.04, N 14.05; found C 51.57, H 4.28, N 13.96. Ru4: C60H60F12N18O2P2Ru, 1H NMR (400 MHz, D2O): δ 8.64 (d, 4H), 8.38 (d, 4H), 8.00 (d, 6H), 7.71 (d, 4H), 7.46 (s, 6H), 7.20 (s, 2H), 7.07 (s, 2H), 6.94 (s, 4H), 4.19 (s, 4H)，3.57 (t, 6H), 3.11 (t, 12H). C60H60F12N18O2P2Ru: calcd. C 49.49, H 4.15, N 17.31; found C 49.69, H 4.28, N 17.47. 2.4. Preparation of Ru-LPMSN nanoparticles. Hexadecyltrimethylammonium toluene-psulphonate (CTATOS, 0.9614 g) and triethanolamine (TEA, 0.14 mL) were dissolved in 50 mL distilled water at 80 °C under vigorous stirring for 1 h. Tetraethyl orthosilicate (TEOS, 2.5 mL) was added, and the mixture was continued to stir for another 2 h. The mesoporous silica nanoparticles (LPMSN) were collected by centrifugation and washed for seven times with ethanol and water to remove the residual reactants. Then the collected products were extracted for 12 h with 0.6wt % solution of ammonium nitrate (NH4NO3) ethanol solution at 60 °C to remove the template CTATOS. The extraction was carried out four times.


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Ru-LPMSN was synthesized through stirring mixture of Ru4 and LPMSN for 24 hours. The resulted products were collected by centrifugation, and gently washed with distilled water for 3 times. 2.5. Fluorescence response of Ru-LPMSN to copper ions. Titration with copper ion: Cu2+ ions (0-1 µM) was gradually titrated into Ru-LPMSN (1 µM), and the fluorescence intensity of Ru-LPMSN was determined at 500 nm excitation. Combination ratio: Total concentration of Cu2+ ions and Ru-LPMSN was 2 µM. Changing the concentration ratio, fluorescence intensity of Ru-LPMSN was determined. Ion selectivity: The fluorescence intensity of Ru-LPMSN was measured after various ions adding to Ru-LPMSN (1 µM) aqueous solution. Cu2+ ion concentration was 1 µM, and other ions concentration was 10 µM such as Na+, K+, My2+, Ag+, Ni2+, Fe2+, Hg2+, Cd2+, Ca2+, Co2+ , Mn2+, Pb2+, Cr2+, Li+ and Zn2+. Then adding Cu2+ ions (1 µM) to each experimental group, the fluorescence intensity was measured again. Repeatability: Cu2+ ions (1 µM) was added to Ru-LPMSN (1 µM) aqueous solution, and the fluorescence of Ru-LPMSN was quenched. Then adding EDTA (5 µM), fluorescence of RuLPMSN was recovery. Removing EDTA and Cu2+ ions by centrifugation, Ru-LPMSN was resuspended and repeated the appeal process 5 times. PH stability: In different pH buffer (including hydrochloric acid aqueous solution pH1.1, disodium hydrogen phosphate-citrate buffer pH2-4, PBS buffer pH 5.6-8 and Tris-HCl buffer pH 8.3-8.5), the fluorescence intensity of Ru-LPMSN (1 µM) was measured at 500nm excitation wavelength. After adding Cu2+ ions (1 µM), and fluorescence intensity of the mixture was determined.


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2.6. Lifetime measurement. The fluorescence lifetime of Ru1-Ru4 represented the fluorescence lifetime measurement results of the ruthenium mesoporous hybrid at room temperature. Fluorescence lifetime of Ru-LPMSN (10 µM) with and without Cu2+ ions (5 µM) was measured using the FLS980 at the optical excitation wavelength of 500 nm. The decay curves can be fitted by a double exponential model, which is expressed as I(t) = A1exp(−t/τ1) + A2exp(−t/τ2)

(1)

Where I(t) is luminescence intensity, τ1 and τ2 represent the rate constants, A1 and A2 represent the amplitudes of the components, respectively. The average lifetime τ value is calculated by the expression in equation: τ= (A1τ12 + A2τ22)/(A1τ1 + A2τ2)

(2)

2.7. Cell culture and fluorescence imaging. HeLa cells were cultured in DMED medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin−streptomycin at 37 °C, 95% humidity and 5% CO2. Hela cells incubated with Ru-LPMSN (10 µM) for 20-60 min. Remove the medium and wash the PBS three times. The treated Hela cells incubated with Cu2+ ion (20 and 50 µM) for another 30 min. The medium was removed and washed three times with PBS. DAPI stained for 10 min and PBS washed three times. Paraformaldehyde (4%) was used to fix, and the Laser confocal microscopy was used for fluorescence imaging of cells (The laser confocal imaging parameters DAPI: Ex = 405 nm, Em = 460 nm; Ru-LPMSN: Ex = 496 nm, Em = 727 nm). 2.8. Cell cytotoxicity assay. HeLa cells were seeded into 96-well cell culture plates (104 cells/well) and cultured for 24 h. In vitro cytotoxicity was evaluated by methyl thiazolyl tetrazolium (MTT) assays. HeLa cells were subsequently incubated with different concentrations of Ru-LPMSN (0, 20, 60, 80, 100 µM) for another 24 h. Then, MTT (10 µL, 5 mg/mL) was


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added to each well and incubated for an additional 4 h. Removing medium, 100 µL dimethyl sulphoxide (DMSO) was added to dissolve the formazan crystals. The optical density (OD) of each well was measured at 490 nm using microplate reader. The following formula was used to evaluate the activity of cell growth: Cell viability (%) = (mean OD value of treatment group / mean OD value of control group) x100. 2.9. Zebrafish fluorescence imaging. Zebrafish was kept at 28 °C and maintained at optimal breeding conditions. For mating, male and female zebrafish was incubated in one tank on a 12 h light/12 h dark cycle, then the spawning of eggs were triggered by giving light stimulation in the morning. The eggs were fertilized immediately. Two week old zebrafish were incubated with Ru-LPMSN (10 µM) for 3 h. The treated zebrafish were incubated with Cu2+ ion for 30 min. The zebrafish were imaged using fluorescence inversion microscope. (The imaging parameters RuLPMSN: Ex = 505 nm). 3. RESULTS AND DISCUSSION 3.1. Preparation of NIR Ru complex. We designed and synthesized 4 kinds of ruthenium complexes, all of which had Cu2+ ion response ligand bipyridine-DPA (bpy-dpa) (Figure 1A). The UV-visible spectrum of the ruthenium complex was showed as Figure 1B. Ru3 has absorption peaks at 246 nm, 286 nm, 350 nm and 480 nm. The absorption peaks belonged to the π → π * electron transition at 246 nm, 286 nm, to metal center electronic transition at 350 nm, assigned to metal-ligand charge transfer transitions (MLCT) at 480 nm. After bpy ligand modification by carboxyl (Ru1) or amino (Ru4), the absorption peak at around 280 nm was altered to 303 nm (Ru1) and 270 nm (Ru4) respectively, and the peak at 480 nm was blue-shifted to 465 nm (Ru1) and red-shifted to 502 nm (Ru4), respectively. Substituted bpy for


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phenanthroline (phen), the absorption peak at 246 nm and 286 nm were moved to 255 nm. The absorption peak at 350 nm was red shifted to 384 nm, while the one at 480 nm was slightly blueshifted by 2 nm. The UV-Vis absorption spectra of Ru complex changed with the structure of the ligand (R). The changes can be related with the Ru complex molecular orbital decreases because of the impact of the HOMO energy level by replacing the auxiliary ligand bpy with the carboxyl group bpy, which do not affect the LUMO. The increased HOMO-LUMO energy gap, MLCT of Ru1 was blue-shifted. On the contrary, amino substitution decreased the HOMO-LUMO orbital energy gap, causing MLCT of Ru4 red-shifted.27-29 The emission wavelength of Ru complexes could be adjusted from orange to near infrared by changing the donor-acceptor electronic group of ligand R (Figure 1C). The results showed that four Ru complexes exhibited strong emission. Their fluorescence intensity was Ru1 > Ru3 > Ru4 under the same condition, and their emission peaks were red shift at the same time. The maximum emission wavelength was at 640 nm (Ru1), 682 nm (Ru3) and 727 nm (Ru4). There have been reported that quantum yield (QY) of dye increases by changing donor-acceptor electronic group.30-31 Carboxyl substitution of bpy (Ru1) decreases the electronic cloud density of bpy. The electronic charge shifting form Ru center to bpy is easilier than that of Ru3, which contributes to higher probability of metal-to-ligand charge-transfer.32 The emission intensity of Ru1 was stronger than Ru3. Replacing the auxiliary ligand bpy with phen (Ru2), the rigid structure of Ru complex was expanded, and its electron became more delocalization,33 resulting fluorescence emission intensity of Ru2 enhanced. In contrast, amino substitution of bpy (Ru4) increased the electronic cloud density of bpy, causing the fluorescence emission intensity of Ru4 decreased than Ru3. Furthmore, fluorescent lifetime of Ru complex showed that Ru1> Ru2 > Ru3 > Ru4, which is consistent with the change of fluorescence emission intensity (Table S1).


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Figure 1. (A) Synthesis of NIR Ru-LPMSN hybrid materials. (B) UV-visible spectra of Ru1Ru4 (10 µM), and the inserted figure was Ru3 UV-visible spectra. (C) Fluorescence emission spectra of Ru1-Ru4 (10 µM).

3.2 Characterization of Ru-LPMSN Hybrid. Ru4 emitted bright NIR fluorescence (Em = 727 nm) at 500 nm excitation, which is favorable for bioimaging and detection applications. The Ru-LPMSN had been prepared because LPMSN can provide large surface area, big tunnel structure for guest loading and protect the environmental stability and resist the outside invasion. As shown in TEM images (Figure 2A), the prepared Ru-LPMSN was high dispersed, spherical structure with diameter of 90 ± 10 nm. Dynamic light scattering (DLS) displayed that the


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average diameter of Ru-LPMSN was around 100 nm and they were in uniform size (Table S2), which was consistent with the result of TEM image. The potential was -27.14 ± 0.9 mv (LPMSN) and 24.57 ± 1.25 mv (Ru-LPMSN). The changes in potential also illustrated the successful assembly (Table S2). There was no obvious change in the particle size and the structure of the mesoporous channels after loading Ru4. The energy dispersive X-ray spectroscopy (EDX) further confirmed the loading of Ru4 by the element contents of RuLPMSN with Ru, C, Si and O (Figure 2B). N2 adsorption-desorption isotherm indicated that the average porous size of LPMSN were 11 nm and BET surface area was 535 m2·g-1 (Figure 2C). After loading Ru4, the pore diameter (9 nm) and BET surface area (294 m2·g-1) were significantly reduced. This further suggested that Ru4 was loaded successfully. The amount rate of Ru4 in Ru-LPMSN was 12%, according to the inductively coupled plasma mass spectrometry (ICP-MS) quantification. The emission performance of Ru-LPMSN was similar to Ru4 (Figure 2D, curve a and c), demonstrating that the emission properties of Ru4 in LPMSN were preserved. However, the intensity of Ru-LPMSN was increased significantly by 4.26 times, which indicated that the MSN protected Ru4 against quenching by oxygen and nonradioactive decay. Fluorescent stability of Ru-LPMSN had also been investigated. The results showed that the fluorescence intensity of RuLPMSN was almost constant within the pH range of 1-8.3 at room temperature (Figure S6 A). In contrast, fluorescence intensity of free Ru4 was highly stable in pH4-8.0, but fluorescence intensity was decreased in pH1.2-4.0 (Figure S6 B). These indicated that the fluorescence stability of Ru4 can be improved by assembling dyes into mesoporous silica materials and the strong interaction exists between Ru4 and LPMSN including electrostatic interaction (the negatively charged LPMSN and Ru4 anions) and hydrogen bounding (the hydroxyl group of


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LPMSN and the amino group of Ru4) etc. Moreover, the fluorescence intensity of Ru-LPMSN was not significantly attenuated after 90 days (Figure S7). These results demonstrated the high stable emission properties of Ru-LPMSN, which is favorable for investigation of biological events in vivo.34-35 We also investigated the fluorescence emission intensity of Ru4 confined in different porous size of MSN (Figure 2D). Fluorescence emission intensity of Ru-LPMSN was 2.5 times than that of Ru-MSN (porous size 2-3 nm, Figure S8), revealing the impact of porous size of MSN. The large pore size allows the dispersing of Ru4, avoiding the molecular interaction and fluorescence self-quenching.

Figure 2. Characterization of Ru-LPMSN about (A) TEM images, (B) EDX spectra，(C) N2 adsorption-desorption isotherm. (D) Fluorescence emission spectra of Ru-LPMSN (curve a), RuMSN (curve b) and Ru4 (curve c) were comparison in the same concentration (10 µM).


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3.3 Detection of Cu2+ ion in water. We performed a Cu2+ ion titration experiment to investigate the interaction of Ru-MSN with Cu2+ ion in water. As shown in the fluorescence emission spectrum of Ru-LPMSN (Figure 3A), NIR fluorescence gradually quenched with the increase of the Cu2+ concentration. The fluorescence of Ru-LPMSN was linearly quenched as increase of Cu2+ concentration from 50 to 500 nM and 500 to 800 nM with a correlation coefficient of 0.999 and 0.995 (Figure 3B). The typical calibration curve was shown in Figure 3B. The detection limit for Cu2+ was 10.0 × 10-9 M (S/N = 3). The results demonstrated that RuLPMSN can be promising for sensitively monitoring trace amounts of Cu2+. The ruthenium complex has two Cu2+ ion binding sites, and so further titration experiments were performed to study the binding mode of the ruthenium complex to the Cu2+ ion. When the concentration ratio changed, the fluorescence intensity of Ru-LPMSN was measured before (FL0) and after (FL1) titration of copper ion. When the concentration ratio of Cu2+: Ru was 1, the FL0 / FL1 value was the largest showing the combination ratio of Ru-LPMSN to Cu2+ ion (Figure S9). The tests for ion detection specificity of Ru-LPMSN were carried out with various metal ions. As shown in Figure 3C, other metal ions had no significant effect on the fluorescence emission except that Ni2+ and Co2+ ions (10 µM) showed slightly the fluorescence quenching. Then the fluorescence intensity of Ru-LPMSN exhibited a significant quench after adding Cu2+ ion (1 µM). The results indicate that these metal ions cannot affect the combination between the Cu2+ ions and Ru complex. Moreover, the concentration of other ions (10 µM) was much higher than that of Cu2+ ions, indicating that the Ru-LPMSN had a good selectivity for detection of Cu2+ ions. So it had little effect on the detection of Cu2+ ion in the organism supporting the biological detection applications of Ru-LPMSN.


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We performed reproducibility experiments to further study the fluorescence reliability of the probes. After the quenching of fluorescence, the emission could return to the original intensity at about 98% by the addition of metal chelating agent ethylenediaminetetraacetic acid disodium (EDTA). Fluorescence emission of Ru-LPMSN was found to recover after repeating several times (Figure 3D), indicating its great reliability. Besides light quenching fluorescence, the fluorescent probes were also affected by pH value. The pH values of the external environment or different biological tissues can be quite different. Therefore, the fluorescent probes should be stable under different pH conditions. From Figure 3E, the fluorescence of Ru-LPMSN (curve a) was not changed in pH value 1.0-8.3. After adding Cu2+ions (curve b), the fluorescence of RuLPMSN was quenched, but not affected by pH value. These results demonstrated that RuLPMSN is suitable for the detection of Cu2+ ions under physiological conditions.


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Figure 3. Fluorescence properties of Ru-LPMSN. (A) Fluorescence titrated by Cu2+ ion (0-1 µM). (B) Fluorescence intensity with different concentrations of Cu2+ ion, mean SD, n = 3. The inserted figure was fluorescence linear quenching spectra of Ru-LPMSN with the concentration of Cu2+ from 50 to500 nM and 500 to 800 nM. (C) The selectivity of Ru-LPMSN to metal ions. Various metal ions (red bars) and Cu2+ ion (green bars). (D) Fluorescence repeatability titrated by Cu2+ ion and EDTA. (E) Fluorescence was stable in different pH buffer without (curve a) and with (curve b) Cu2+ ion.

3.4 Mechanism for the quenching fluorescence. Time-correlated single-photon counting (TCSPC) experiments have been used to test the charge transfer and exciton recombination process of Ru-LPMSN in the presence and absence of Cu2+ ions. The fluorescence decay of RuLPMSN at room temperature was showed in Figure 4A. The average lifetime of Ru-LPMSN was 283.73 ns and 281.77 ns in the absence and presence of Cu2+ ions, respectively. The fluorescence lifetime remained unchanged before and after titrating Cu2+ ion, indicating that the mechanism involved complexation (static quenching) rather than collision deactivation (dynamic quenching). The UV-vis absorption spectra of Ru-LPMSN in water exhibited an absorption band at 244 nm and 270 nm, which was significantly enhanced with Cu2+ ion (Figure 4B). At the same time, the absorption peak at 500 nm decreased obviously. Besides, a new peak appeared at 642 nm. These phenomena should be attributed to the combination of Cu2+ ion with Ru complex in Ru-LPMSN as other models.36-37 Moreover, the recovery of fluorescence intensity by adding EDTA (Figure 4C, curve b) indicated that EDTA, a metal ion chelator, can prevail over Ru complex and chelated with Cu2+ ions. Both the UV–vis spectra and the fluorescence intensity


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recovery test suggested that the quenching of Ru-LPMSN by Cu2+ ion was in a static quenching mechanism.

Figure 4. Fluorescence of Ru-LPMSN quenches mechanism. (A) Fluorescence decay curves with (red line) and without (black line) Cu2+ ion. (B) UV–vis spectra with (black line) and without (red line) Cu2+ ions. (C) Fluorescence (curve a) was quenched (curve b) and recovered (curve c).

3.5 Detection of copper ions in cells. HeLa cell line was chosen to investigate the cytotoxicity of Ru-LPMSN. The standard MTT assays were performed to evaluate the cell viability. The MTT result indicated that Ru-LPMSN was only slightly toxic against HeLa cells with IC50 values > 100 µM (Figure S10). The IC50 values were much higher than the concentrations of Ru-LPMSN employed (10 µM), thereby the impact on cell growth can be neglected. After HeLa cells were incubated with Ru-LPMSN, an intense intracellular red luminescence images were observed upon excitation at 496 nm. The absorption kinetic of Ru-LPMSN in HeLa cells was further studied. The images of Ru-LPMSN and free Ru4 were bright field (Figure 5A, a-c and gi) at different time points. After incubation for 20 min, weak-flourescent intensity was observed (Figure 5A, d), indicating that Ru-LPMSN had penetrated into the cell. After 40 min incubation,


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most of the HeLa cells showed strong red fluorescence (Figure 5A, e), and the intensity did not increase apparently after 60 min (Figure 5A, f), illustrating that Ru-LPMSN could penetrate and accumulate in HeLa cells rapidly. Compared to free Ru4 (Figure 5A, j-l), Ru-LPMSN showed significant enhancement of intracellular fluorescence intensity. The intracellular fluorescence intensity was quantified by Microplate Reader (Figure 5B). The fluorescence intensity of RuLPMSN was 9.64 times (Figure 5C) higher than that of free Ru4 after 60 min incubation. The fluorescence intensity enhancement in cells is higher than that in aqueous solution (Figure 5C), indicating that the mesoporous materials could facilitate Ru4 accumulation within cells.

Figure 5. (A) Cellular uptake kinetics of HeLa cell incubated with Ru-LPMSN for (a, d) 20 min, (b, e) 40 min, (c, f) 60 min and with free Ru4 for (g, j) 20 min, (h, k) 40 min, (i, l) 60 min. (B) Intracellular fluorescence intensity incubated with free Ru4 (red line) and Ru-LPMSN (back line) for 20 min, 40 min and 60 min, respectively. (C) Fluorescence enhancement of Ru-LPMSN compare to free Ru4 extracellularly and intracellularly.


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HeLa cells were incubated with Ru-LPMSN (10 µM) for 60 min and then washed three times with PBS to remove excess Ru-LPMSN. The cells were then replaced with new media containing different concentration of Cu2+ ions for another 30 min. As shown in Figure 6, the red fluorescence was gradually quenched as the concentration of Cu2+ ions increasing. After incubating with 50 µM Cu2+ ions for 30 min, the intracellular fluorescence was quenched completely (Figure 6k). These suggested that Ru-LPMSN can respond rapidly to Cu2+ ions in living cells. 4,6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei of HeLa cells to investigate the distribution of Ru-LPMSN. No fluorescent signals were observed in the nuclei, indicating that Ru-LPMSN could not pass through the nuclear membrane (Figure 6a-d). The results indicated that the probe was distributed in the cytoplasm.

Figure 6. HeLa cell fluorescence images soaked in Ru-LPMSN solution. Bright field imaged (a, e, i). DAPI fluorescence images excited at 405 nm (b, f, j). Ru-LPMSN fluorescence images excited at 496 nm with Cu2+ ions (c) 0 µM, (g) 20 µM and (k) 50 µM. Merge images(d, h, l).


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3.6 In vivo imaging in zebrafish larvae. The tissue distributions and detection application of Ru-LPMSN were investigated in zebrafish larvae considering its excellent cytocompatibility. Two-week-old zebrafish larvae were incubated with Ru-LPMSN (10 µM) for 2 h. Then the treated zebrafish were incubated with Cu2+ ions (50 µM) for 30 min. As images shown in Figure 7, the zebrafish incubated with Ru-LPMSN had bright red fluorescence, which suggested that Ru-LPMSN had penetrated into them. Ru-LPMSN penetrated into the zebrafish via swallowing and skin-absorption.38 Ru-LPMSN accumulated selectively in in head, yolk sac, intestine and tail, showing the tissue-dependent affinity (Figure 7b, e, h). The eyes were not observed with red luminescence (Figure 7b). On the contrary, when the zebrafish were incubated with free Ru4 complexes, the eyes were the brightest part of head and the lens can be readily distinguished from the eyeball (Figure S11). It revealed that free Ru4 complexes can enter the eye across the blood–ocular barrier but Ru-LPMSN cannot. The Ru-LPMSN accumulated in yolk sac and intestine indicated that it entered the digestive system (Figure 7e). The red luminescence of the dorsal blood vessel suggested the entry of Ru-LPMSN into the circulatory system (Figure 7h), which was a main route for Ru-LPMSN transport in zebrafish. Ru-LPMSN was transferred throughout the whole body by the cardiovascular system. The absorption and excretion routes of Ru-LPMSN were also revealed by its fluorescence distribution in zebrafish, in agreement with the report that Ru-LPMSN entered into the zebrafish through swallowing and skin-absorption, and was eliminated through the gut.39 Fluorescence of Ru-LPMSN in zebrafish was quenched after incubated in Cu2+ ion solution (Figure 7). Fluorescence of head, yolk sac, intestine and tail decreased obviously (Figure 7c, f, i). These results indicated that Cu2+ ion mainly accumulate in yolk sac, intestine and muscle of


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zebrafish. Resultantly, Ru-LPMSN can be used for florescence imaging in vivo and monitoring the distribution of exogenous Cu2+ ion in zebrafish.

Figure 7. Fluorescence images of zebrafish incubated with Ru-LPMSN. Fluorescence imaged in bright field (a, d and g), fluorescence field incubating without (b, e and h) and with Cu2+ ions (50 µM) for 30 min (c, f and i). EY, eye; YS, yolk sac; IN, intestine; and VE, vessel.

3.7 Determination of copper in human serum. To demonstrate the reliability of the present assay in real sample, the proposed sensor was used to detect copper in human serum. The serum samples was diluted 100-fold with PBS and directly used for experiment test. Ru-LPMSN was used as a fluorescence detecting probe consistent with the standard value provided by atomic absorption spectrometry (Table S3). Therefore, the analytical performance of the Ru-LPMSN proved it as a promising alternative for accurate Cu2+ measurement in real samples. 4 Conclusions In summary, a series of Ru1-Ru4 complexes were synthesized and showed orange red to NIR fluorescence property. These complexes exhibited excellent selectivity for Cu2+ detection. The


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fluorescence results suggested that the fluorescence property of Ru4 significantly improved in Ru-LPMSN hybrid material, changing with the pore size of MSN. Large pore size strengthened the dispersion of Ru4 in pore system and decreased self-quenching properties. More interestingly, Ru-LPMSN showed stable fluorescence property rarely influencing by light and pH value. As a sensor, the material exhibited excellent sensitivity, selectivity, rapid and NIR fluorescence detection of Cu2+ ions, and the detection limit was 10 nM. The process was proved to be static quenching mechanism. Moreover, the developed Ru-LPMSN sensor was successfully used for monitoring Cu2+ ions in cell and live zebrafish. The intracellular fluorescence responses of Ru-LPMSN were obviously amplified. Ru-LPMSN sensor was successfully applied to detection of Cu2+ ions in human serum with accurate results. The present work provides a new effective means for biodetection applications.

ASSOCIATED CONTENT Supporting Information Supplementary Information contains methods, MTT, DLS measurement, detection of serum copper ions and bioimaging of zebrafish. (PDF) AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (Y.Wu) Author Contributions


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† Fangman Chen and Fangnan Xiao contributed equally to this work. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Nature Science Foundation of China (31270790, 31470741), the Key Project of Fujian Province (2017N0101), the National Thousand Talents Program of China, and the China Postdoctoral Science Foundation (2016M592096). ABBREVIATIONS MSN, mesoporous silica nanoparticles LPMSN, large-pore mesoporous silica nanoparticles QY, quantum yield TEM, high-resolution transmission electron microscopy EDX, energy dispersive X-ray spectroscopy DLS, dynamic light scattering ICP-MS, inductively coupled plasma mass spectrometry TCSPC, time-correlated single-photon counting UV-vis, Ultraviolet-visible REFERENCES (1) Gill, M. R.; Thomas, J. A. Ruthenium(II) Polypyridyl Complexes and DNA-From Structural Probes to Cellular Imaging and Therapeutics. Chem. Soc. Rev. 2012, 41 (8), 31793192. (2) Hu, L. Z.; Xu, G. B. Applications and Trends in Electrochemiluminescence. Chem. Soc. Rev. 2010, 39 (8), 3275-3304.


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