ZnS QDs via host-guest interaction for ratiometric fluorescence sensing of metal ions

Accepted Manuscript Construction of β-cyclodextrin derived CDs-coupled block copolymer micelles loaded with CdSe/ZnS QDs via host-guest interaction for ratiometric fluorescence sensing of metal ions Siyao Zhu, Feifei Zhao, Mingxiao Deng, Tianyi Zhang, Changli Lü PII:

S0143-7208(19)30168-8

DOI:

https://doi.org/10.1016/j.dyepig.2019.04.051

Reference:

DYPI 7506

To appear in:

Dyes and Pigments

Received Date: 22 January 2019 Revised Date:

19 March 2019

Accepted Date: 21 April 2019

Please cite this article as: Zhu S, Zhao F, Deng M, Zhang T, Lü C, Construction of β-cyclodextrin derived CDs-coupled block copolymer micelles loaded with CdSe/ZnS QDs via host-guest interaction for ratiometric fluorescence sensing of metal ions, Dyes and Pigments (2019), doi: https://doi.org/10.1016/ j.dyepig.2019.04.051. 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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A novel CDs@QDs-loaded micelle-based ratiometric fluorescent sensor for metal ions was constructed by the host-guest interaction of azobenzene-terminated

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polymeric micelles and β-cyclodextrin derived CDs (β-CD-CDs).

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Construction of β-cyclodextrin derived CDs-coupled block copolymer micelles loaded with CdSe/ZnS QDs via host-guest interaction for

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ratiometric fluorescence sensing of metal ions

Siyao Zhu, Feifei Zhao, Mingxiao Deng, * Tianyi Zhang, Changli Lü *

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Institute of Chemistry, Northeast Normal University, Changchun 130024, P. R.China

* Corresponding author. E-mail addresses: [email protected]; [email protected] (C. Lü).

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ABSTRACT The nanostructured hybrid assembly has gained growing interest due to their unique electronic, optical and catalytic properties. Herein, a novel ratiometric fluorescent sensor was constructed by

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coupling blue-green emission carbon dots (CDs) to a block copolymer micelle-encapsulated red-emission CdSe/ZnS QDs through host-guest interaction. The carbon dots (CDs) were prepared

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by carbonization of β-cyclodextrin (β-CD) via a simple hydrothermal process. The amphiphilic diblock copolymer AZO-PNIPAM-b-P(St-co-MQ) containing azobenzene (AZO)-terminated

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hydrophilic poly(N-isopropylacrylamide) (PNIPAM) block and hydrophobic polystyrene (PS) block was synthesized by a typical RAFT polymerization. The 5-(2-methacryloylethyloxymethyl)-8quinolinol (MQ) units were introduced into PS block, which could make CdSe/ZnS QDs to be embeded into the core of the block polymer micelles via coordination interaction in the

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self-assembling process. The as-fabricated β-cyclodextrin derived CDs-coupled block copolymer hybrid micelles loaded with CdSe/ZnS QDs as a fluorescence sensor exhibited dual emission peaks

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of CDs centered at 450 nm and CdSe/ZnS QDs at 620 nm with a large peak separation of 170 nm upon excitation at 360 nm. And the as-prepared hybrid micelles as a ratiometric fluorescence sensor

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can be applied to detect Hg2+ and Cu2+ with the detection limits down to 1.6 and 2.74 µM, respectively.

Keywords: Carbon dots; β-Cyclodextrin; QDs; Block copolymer hybrid micelles; Ratiometric sensing

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1. Introduction The problem of environmental pollution has become increasingly critical along with the rapid

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expansion of industrialization, owing to the widespread use of pesticides and large quantities of sewage discharged from factories. It is noteworthy that heavy and transition metal (HTM) contamination is getting more and more severe, more importantly, due to its intense toxicity to plants,

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animals, and even human beings, the living beings in the world are constantly threatened. Therefore,

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it’s still a serious issue which has been focused on for a long time [1-3]. Both Cu2+ and Hg2+ ions have always attracted much attention among diverse HTM ions, which is due to their irreplaceable physiological functions in the life system, remarkable environmental impact, and their universal existence in nature. Consequently, developing a rapid and efficacious method for detection towards

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Hg2+ and Cu2+ simultaneously is extremely essential [4, 5].

Carbon dots (CDs) as a category of promising fluorescent nanomaterials have attracted tremendous attention increasingly since their identification by Xu and co-workers [6] in 2004. In

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addition, CDs are considered to be fascinating and versatile for their diverse applications in

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fluorescence sensor, photocatalysis, nanomedicine, cellular imaging and printing ink [7-12], etc. The above facts are in view of exceptional features of CDs such as low toxicity, size-tunable emission, superior photostability, easy preparation, biocompatibility, excellent water-solubility, and high sensitivity towards targets [13-25]. Moreover, the capping CDs with various stabilizing agents can protect their luminescence properties and enhance their dispersion stability in aqueous solution. But beyond that, certain of specific surface modifications of CDs further endow them with stronger affinity with target analytes. The above advantages of functionalized carbon dots (FCDs) endow

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ACCEPTED MANUSCRIPT them as excellent candidates for various applications. For example, Tang et al. reported FITC or TRITC modified hollow CDs for use in cell imaging [26]. Meng et al. demonstrated Au/N co-doped CDs as an excellent fluorescence probe for detecting mercury ions [27]. Luo et al. reported that

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β-cyclodextrin modified S, N codoped CDs were prepared for detecting testosterone [28]. Tang et al also developed β-cyclodextrin functionalized carbon quantum dots for determining alkaline phosphatase activity by means of host-guest interaction [29]. Nevertheless, the controlled surface

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modification of CDs is complex for further applications, this is due to the fact that not all CDs have

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surface reaction groups. Therefore, many functionalized CDs have been obtained via post-functionalization method based on covalent binding. For example, Tang et al. developed β-CD modified CQDs which were synthesized via B-O bonds from boronic acid-functionalized CQDs and β-cyclodextrins (β-CD) [30], and Luo et al. prepared β-CD-CDs by taking mono-(6-amino-6-

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deoxy)-β-cyclodextrin and carbon dots with carboxyl groups on the surface as raw materials via amidation reaction utilizing EDC, NHS as activators [28]. However, the above post-functionalization methods exist some insufficiencies, such as high cost, tedious operations, and harsh reaction

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conditions, etc. Thus, a simple, low cost and environmentally friendly method is highly desirable.

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Based on these requirements, here we developed a one-pot synthesis method for forming CDs with much more β-cyclodextrin groups on their surfaces via carbonization of β-cyclodextrin. However, we learned that there are few reports similar to ours. For instance, Hu et al. developed one-pot preparation of photoluminescent carbon nanodots by carbonization of cyclodextrin in aqueous solution pretreated with hydrochloric acid [31], and Choi et al. demonstrated that CDs were prepared via dehydrating carbohydrates taking α-cyclodextrin as reactant utilizing H2SO4 and HNO3 [32]. But the above methods require complicated subsequent operations, and the reaction should be conducted

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β-cyclodextrin in aqueous solution without any superfluous treatments. Furthermore, the β-cyclodextrin molecules on the surface of CDs were applied in host-guest recognition for fabricating hybrid assembly.

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Herein, we demonstrated for the first time a novel CDs@micelle-based ratiometric fluorescent

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sensor which was obtained by binding azobenzene-terminated polymeric micelles to β-cyclodextrin derived CDs (β-CD-CDs) via host-guest recognition for the detection of copper and mercury ions using dual emission fluorescence under single wavelength excitation (Scheme 1). Owing to polymeric micelles possess superiorities of easy preparation, high stability, and excellent

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water-solubility, we chose PNIPAM as a hydrophilic block while PS as a hydrophobic block with MQ ligands for further coordination with fluorescent quantum dots (QDs). Hydrophobic CdSe/ZnS QDs

with

high

quantum

amphiphilic

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azobenzene-terminated

yield

were

encapsulated

diblock

copolymer

into

the

polymeric

micelles

of

AZO-PNIPAM-b-P(St-co-MQ)

by

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self-assembly, which endows QDs with promising candidates for metal ion sensing due to their excellent water-solubility and stability. The results showed that the fluorescence of QDs embedded in micelles can be quenched dramatically by Cu2+ and Hg2+. The β-CD-CDs were then attached to the hybrid micelles with encapsulated QDs via host-guest recognition of β-cyclodextrin molecules on the surface of β-CD-CDs with the azobenzene moiety outside the micelles. In the ratiometric fluorescent sensor here, CDs act as a reference signal because there is a very small impact on the luminescence intensity of β-CD-CDs after adding diverse ions while the fluorescence of CdSe/ZnS QDs changed

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ACCEPTED MANUSCRIPT dramatically. And the ratiometric fluorescent sensor here exhibited dual emission peaks centered at 450 and 620 nm upon excitation by single-wavelength and is highly sensitive to Hg2+ and Cu2+.

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2. Experimental 2.1. Materials β-cyclodextrin

(β-CD),

4-hydroxyazobenzene,

dicyclohexylcarbodiimide

(DCC)

and

propanoic

acid

(RAFT

agent)

and

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2-Methyl-2-[(dodecylsulfanylthiocarbonyl)sulfany]

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4-dimethylaminopyridine (DMAP) were all offered by Sinopharm Chemical Reagent Co. Both of

5-(2-Methacryloylethyloxymethyl)-8-quinolinol (MQ) were synthesized according to previous literature [33]. N-isopropylacrylamide (NIPAM) was provided by Sigma-Aldrich. Styrene (St) was obtained from Aladdin, and underwent a washing treatment with 5% NaOH aqueous solution first,

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followed by washing with deionized water until the solution became neutral for removing polymerization inhibitor, the solution went through a drying process for at least 12 h with anhydrous sodium sulfate next, and the final product could be obtained by distillation under reduced pressure.

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Hydrophobic CdSe/ZnS QDs capped with octadecylamine ligand were purchased from Suzhou

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Xingshuo Nanotech Co. Ltd. Azobisisobutyronitrile (AIBN, 99%) was provided by Aladdin and went through a necessary recrystallization treatment with ethanol for at least three times before use. 2.2. Characterization instruments 1

H-NMR spectra of different samples were recorded in CDCl3 by using AVANCE Bruker

spectrometer (600 MHz). Fourier transform infrared (FTIR) spectra were obtained between 400 and 4000 cm-1 on a BrukerVector-22 FTIR spectrometer. Gel permeation chromatography (GPC) was applied on a Waters instrument (Waters 600E) for determination towards the molecular weights of

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ACCEPTED MANUSCRIPT various polymers at a flow rate of 1.0 mL min-1 under 25 oC with the DMF as eluent, and the final molecular weights would be confirmed vs. polystyrene standards. In addition, UV-Vis absorption testing from 200 to 600 nm was performed using SHIMADZU UV-2550 UV-visible

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spectrophotometer. The photoluminescence data of QDs-based or CDs-based materials were all obtained with Edinburgh FLSP920 spectrometer. X-ray photoelectron spectroscopy (XPS) was carried out by a Quantum 2000 spectrometer with non-monochromatized Al Ka excitation radiation.

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various samples on a JEM-2100F electron microscope.

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Transmission electron microscopy (TEM) was applied to determine the morphology and size of

2.3 Synthesis of carbon dots by carbonization of β-CD (β-CD-CDs)

We adopted the hydrothermal method to synthesize the β-CD-CDs by directly using β-CD as a raw material. Briefly, 0.3 g β-CD without any superfluous treatments was dissolved in 15 mL

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deionized water first. The mixture was then transferred into Teflon equipped with stainless-steel autoclave and kept heating for 6 h at 180 oC. And when the mixture cooled down to room temperature, it was subsequently filtered through filtration membrane (0.22 µm) which was used for

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the aqueous phase to remove large precipitate particles. And the as-obtained solution was then

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purified by dialysis, which was dialyzed against deionized water using a dialysis bag (MWCO=1000 Da) for 36 h, followed by drying via a freeze-dryer to obtain the final product (β-CD-CDs) as deep brown powder.

2.4 Synthesis of azobenzene-terminated block copolymer 2.4.1 Synthesis of azobenzene-functionalized RAFT agent (AZO-RAFT) AZO-RAFT was prepared based on the previous report [34]. Briefly, RAFT agent (0.36 g, 0.99 mmol) and 4-hydroxyazobenzene (0.2 g, 37.5 mmol) were dissolved into 8.0 mL dry

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ACCEPTED MANUSCRIPT dichloromethane in a 50 mL three-necked flask and degassed with nitrogen for 30 min, then the flask with reaction solution was put into an ice bath and kept stirring for an hour under nitrogen atmosphere. After that, both DMAP (0.024 g, 0.2 mmol) and DCC (0.42g, 2.04 mmol) were added

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into the solution before the reaction was allowed to proceed for 24 h under ambient temperature. Subsequently the solution was conducted with vacuum filtration and the filtrate was collected for the next step of concentration treatment, afterwards the filtrate was dissolved into trichloromethane and

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extracted with distilled water for three times. Followed by drying the organic phase with anhydrous

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sodium sulfate overnight, and the mixture was purified by filtration, concentration and drying process. Finally, the azobenzene-functionalized RAFT agent was obtained. 2.4.2 Synthesis of AZO-PNIPAM macro-RAFT agent

The typical synthesis procedure was as follows: AZO-RAFT (0.03 g, 0.055 mmol), AIBN (4.0 mg,

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0.033 mmol) and NIPAM (2.0 g, 17.7 mmol) were dissolved into dry THF (3.0 mL). After undergoing three freeze-pump-thaw cycles, the mixture was then heated up to 70 oC with stirring continuously for 12 h. After the reaction solution was cooled down to 25 oC, the mixture was

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precipitated three times with ether to remove superfluous monomers, and the resulting product was

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obtained as white powder.

2.4.3 Synthesis of azobenzene-terminated block copolymer AZO-PNIPAM-b-P(St-co-MQ) (AZO-BCP)

AZO-BCP was synthesized via a typical RAFT polymerization route using AZO-PNIPAM as the macro-RAFT agent. Briefly, AIBN (8.0 mg, 0.066 mmol), AZO-PNIPAM (0.6 g, 5.30 mmol), St (1.0 g, 9.60 mmol), and MQ (0.14 g, 0.488 mmol) were added into dry 1,4-dioxane (6.0 mL) and after three times freeze-pump-thaw cycles, the reaction was conducted at 70 oC for 12 h with

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2.5 Fabrication of AZO-BCP micelles encapsulated CdSe/ZnS QDs AZO-BCP (3.0 mg) was stirred in THF (3.0 mL) for 0.5 h, meanwhile, 10 µL CdSe/ZnS QDs (4.17 mg mL-1 in chloroform) was mixed with THF (2.0 mL) under ultrasonic irradiation for 0.5 h.

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The QDs solution was then added into above BCP solution under drastic agitation. And the mixture

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was kept stirring for 2 h to complete the coordination of CdSe/ZnS QDs with MQ units on the BCP. Then, deionized water (20 µL) was dropped into the solution every thirty seconds until the emergence of the Tyndall effect under irradiation by a laser pen. Subsequently, the reaction was continued stirring for 2 h, and deionized water (30 mL) was added into the solution for completely

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fixing the morphological structures of AZO-BCP micelles loaded with CdSe/ZnS QDs hybrid aggregates. The above solution system was dialyzed against deionized water using a dialysis bag (20 KDa) for 48 h and was stored for further use.

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2.6 Preparation of β-CD-CDs@AZO-BCP@QDs micelle inclusion via host-guest interaction

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To bind the β-CD-CDs with AZO-BCP micelles encapsulated CdSe/ZnS QDs, 8.0 mg β-CD-CDs was dispersed in the distilled water first, and then mixed with hybrid micelles aqueous solution. And the mixture was kept stirring under room temperature for 12 h for ensuring sufficient recognition behavior. Finally, the reaction solution was dialyzed using water in a dialysis bag (MWCO=50 KDa), and the inclusion was dispersed in water for further use and characterization. 2.7 Colorimetric sensing of Cu2+/Hg2+ A stock solution of β-CD-CDs@micelles@QDs with 0.25 mg mL-1 was first diluted with

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ACCEPTED MANUSCRIPT distilled water to 0.05 mg mL-1 for further sensing tests. For the colorimetric sensing experiments, different volumes of copper and mercury ions solution (1×10-2 M) were added into the diluted stock solution of β-CD-CDs@micelles@QDs to ensure that the total volume of solution was 5 mL, after

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that, the solution was incubated for 5 min under static condition at ambient temperature to reach equilibration, followed by recording the PL spectra of the mixture with different concentrations of Hg2+ and Cu2+. In order to evaluate the selectivity of β-CD-CDs@micelles@QDs as the ratiometric

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fluorescent sensor, other metal ions such as Fe3+, Fe2+, Cd2+, Co2+, Cr3+, Na+, K+, Al3+, Ni2+, Ca2+,

procedure.

3. Results and discussion

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Mn2+, Zn2+, Pb2+ with the same concentration of 33 µM have also been tested via the same

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3.1 Design and synthesis of β-CD-CDs-based hybrid micelles for colorimetric sensing The preparation route and detection mechanism of the ratiometric fluorescent sensor are shown

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in Scheme 1 and 2. The blue-green emissive β-CD-CDs with enough β-CD units on the surface were prepared by carbonizing β-CD simply via a facile hydrothermal method (Scheme 1a). Due to the

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introduction of MQ monomer units containing 8-hydroxyquinoline (HQ) ligand into the copolymer, and taking advantage of the strong coordination interaction between MQ and CdSe/ZnS QDs, we prepared the polymeric micelles encapsulated QDs by adding dropwise water into THF solution containing amphiphilic block copolymer and CdSe/ZnS QDs. And the obtained micelles containing QDs exhibit dual-emitting properties which come from intrinsic emission (620 nm) of CdSe/ZnS QDs and the coordination emission (526 nm) of MQ–Zn complex on the surface of QDs, respectively (Scheme 1b). And finally the AZO groups outside the hybrid micelles loaded with QDs 10

ACCEPTED MANUSCRIPT could interact with β-CD units on the surface of β-CD-CDs via host-guest complexation, thus the resulting hybrid assemblies exhibit unique triple-emitting properties with blue, green and red fluorescent emissions (Scheme 1c). The results show that the red fluorescence of QDs embedded in

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micelles can be quenched selectively by Cu2+ and Hg2+, while the blue fluorescence of β-CD-CDs is not much affected and green fluorescence of HQ–Zn complex on the surface of QDs is immune to the change. And the specific synthesis procedure of amphiphilic diblock copolymer

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AZO-PNIPAM-b-P(St-co-MQ) via RAFT polymerization is shown in Scheme 2.

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3.2 Characterization of block copolymer and corresponding QDs-loaded micelles The azobenzene-terminated block copolymer AZO-PNIPAM-b-P(St-co-MQ) was prepared via RAFT polymerization using AZO-RAFT as the chain transfer agent. The structure of AZO-PNIPAM and AZO-PNIPAM-b-(St-co-MQ) can be determined by 1H-NMR spectra as shown Fig. 1. The peaks

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at 7.46-7.54 and 7.87-7.96 ppm are ascribed to the protons of the azobenzene groups [34], and the characteristic protons of methylene in PNIPAM repeat units appear at 4.0 ppm [35]. The number-average degree of polymerization (DP) of AZO-PNIPAM is determined to be 106 by

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counting g (methylene on PNIPAM) and a (terminal methyl group of AZO-RAFT) in Fig. 1a.

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Correspondingly, the average molecular weight (Mn) of AZO-PNIPAM is determined as 12000, which is higher to the result determined by GPC (Mn,GPC=8200 with PDI=1.28). The structure of AZO-PNIPAM-b-P(St-co-MQ) is confirmed by 1H-NMR spectra as shown in Fig. 1b. Compared with AZO-PNIPAM, new characteristic proton signals in PS and MQ repeat units are obviously observed. The protons of quinoline ring in MQ units can be observed as the signals at 8.78 and 8.50 ppm. And the broad signal peaks of o, p, r and k at 6.00–7.50 ppm are assigned to the proton signals of the aromatic ring in PS repeating units and the quinoline ring in MQ units. The characteristic

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ACCEPTED MANUSCRIPT signals at 4.68 ppm are attributed to –CH2 in MQ repeat units. It can be demonstrated that the block copolymer AZO-PNIPAM-b-P(St-co-MQ) was synthesized successfully according to the above analysis. The DP of PMQ can be determined as 3 by calculating integral area proportion of peaks n

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(methene group of MQ) and a (terminal methyl group of AZO-RAFT), and DP of PSt block is calculated as 32 by cutting down the signals of protons of benzene ring in MQ and imino group in PNIPAM units after counting the ratio of integrated area of broad peak at 6.0-7.5 ppm and a

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(terminal methyl group of AZO-RAFT). Accordingly, the Mn of AZO-PNIPAM-b-P(St-co-MQ) is

Mn,GPC=13000 with PDI=1.17.

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calculated to be 16100 based on the 1H-NMR data in Fig. 1b, while the GPC result indicates a lower

The structure of amphiphilic diblock copolymer AZO-PNIPAM-b-P(St-co-MQ) was further confirmed by FTIR spectra (Fig. S1). The characteristic peak of AZO-BCP at 3454 cm-1 is originated

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from the stretching vibration of –OH in MQ units [36]. The stretching vibration for N–H in PNIPAM is observed at 3298 cm-1, and the absorption bands appearing at 2968, 2922 and 2864 cm-1 are attributed to C-H stretching vibration in copolymer. In addition, the characteristic vibration of

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–CONH– in PNIPAM appears at 1650 and 1538 cm-1 [37]. The peaks at 3078 and 710 cm-1 are

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originated from unsaturated C-H stretching vibration and bending vibration in aromatic rings of PS block [38]. The above results proved that AZO-BCP was synthesized successfully. The AZO-BCP micelles with MQ units located in the core were prepared via adding 20 µL deionized water every 30 s for about 70 times into 5 mL THF solution containing 3.0 mg AZO-BCP until the emergence of Tyndall effect upon irradiation of the laser pen, and followed with adding 30 mL deionized water 2 h later. The TEM image in Fig. 2a shows that the as-prepared empty micelles are uniformly dispersed with an average diameter of 21 nm. And the critical micelle concentration

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ACCEPTED MANUSCRIPT (CMC) of AZO-BCP micelles is estimated to be 2.2 mg mL-1 with the assistance of fluorescence spectroscopy utilizing pyrene as a reference (Fig.S2a). The QDs-loaded AZO-BCP micelles were obtained by mixing hydrophobic CdSe/ZnS QDs with amphiphilic copolymer AZO-BCP for 30 min

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to make sure that the coordination interaction between CdSe/ZnS QDs and MQ units on the copolymer in THF solution is complete before adding distilled water, and then followed by the procedure which was same as the next steps in preparation of empty micelles. Thus, the QDs

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encapsulated AZO-BCP micelles were prepared successfully through a coordination-driven

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self-assembly procedure. We prepared the sample of AZO-BCP micelles containing 10 µL QDs (4.17 mg mL-1 in chloroform), and their morphology is shown in Fig. 2c. Fig. 2b gives the HRTEM of CdSe/ZnS QDs in the micelles, which reveals that the lattice fringe of QDs is 0.24 nm [39], while the HRTEM of pure CdSe/ZnS QDs is shown in Fig. 2d. From the TEM images, we can obviously

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observe that the sample with 10 µL QDs still has some empty micelles and their average diameter is around 28 nm, which is larger than the empty micelles. Instead of traditional homogeneous incorporation of nanoparticles into micelles, herein, taking advantage of both hydrophobic and

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coordination interactions, the CdSe/ZnS QDs were introduced into the micelles successfully. And it

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can be observed that the arrangement of QDs is in the core of the QDs-loaded AZO-BCP micelles, revealing the tendency on forming radial arrays which is in accordance with the previous study [40]. Dynamic light scattering (DLS) measurement was also conducted to determine the diameter variation of AZO-BCP micelles containing 10 µL QDs with altering temperature in Fig. S2b. It is found that with increasing temperature before 28 oC, the hydrodynamic diameter for the QDs-loaded AZO-BCP micelles first increased, then decreased after closing to the lower critical solution temperature (LCST) of PNIPAM block due to the shrinkage of temperature-sensitive polymer

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ACCEPTED MANUSCRIPT segments which would go through a reversible phase transition above this critical temperature [41]. 3.3 Characterization of β-CD-CDs and micelle-based β-CD-CDs. Carbon dots (CDs) have been considered as a class of promising nanomaterials in numerous

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fields, owing to their outstanding physicochemical properties. Among these peculiarities, fluorescence stability and multiple surface structures of CDs attract us to apply them in ion detection. Inspired with the previous work by Wang’s group [31], we prepared β-CD-CDs utilizing

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β-CD as a single raw material via a facile hydrothermal method. To the best of our knowledge, our

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group is the first to synthesize β-CD-CDs in pure deionized water only employing β-CD without any pretreatments, and make full use of the superiorities of β-CD molecules on β-CD-CDs for applying them in host-guest interaction.

The structure of β-CD-CDs was further confirmed via FTIR spectra. And the typical peaks at

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3401, 2926 and 1643 cm-1 in Fig. S1a are originated from the O–H, C–H and C=O stretching vibrations, which proves that there are numerous residual hydroxyl groups. The absorption bands arising at 1418 and 1028 cm-1 indicate that there exist enough C–O groups on β-CD-CDs. The

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aromatic C-H bending vibration can be observed at 850 cm-1, which confirms that the existence of

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aromatic structure [31]. To further identify that there exist enough β-CD molecules on the surface of β-CD-CDs for the next host-guest interaction with azobenzene moieties, the β-CD-CDs@AZO-BCP was prepared by dissolving β-CD-CDs (1.0 mg) and AZO-BCP (10 mg) in THF solution with continuously stirring under ambient temperature for 12 h. Owing to superfluous unreacted AZO-BCP can be dissolved in acetone while β-CD-CDs@AZO-BCP is insoluble in acetone, the product as dark grey powder can be obtained by washing with acetone for 3 times following with drying in a vacuum oven at 50 oC. The formation of β-CD-CDs@AZO-BCP was preliminarily confirmed by FTIR

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ACCEPTED MANUSCRIPT spectrum in Fig. S1c. It can be clearly seen that the characteristic bands at 3454 and 3298 cm-1 of AZO-BCP are covered by the broad absorption band of O–H on the surface of β-CD-CDs appearing at around 3401 cm-1. Compared with the FTIR spectrum of β-CD-CDs in Fig. S1a, it is obviously

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noticed that there arise two new peaks at 3015 and 2850 cm-1 originating from PS and PNIPAM blocks, which indicates that β-CD-CDs@AZO-BCP was successfully obtained. As a contrast experiment, we also studied the host-guest interaction between β-CD-CDs and azobenzene-free

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copolymer (BCP) which was synthesized by utilizing 2-methyl-2-[(dodecylsulfanylthiocarbonyl)-

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sulfany] propanoic acid as the azobenzene-free RAFT agent, PNIPAM as the hydrophilic block, while PSt containing MQ units as the hydrophobic block via a typical RAFT polymerization. 1.0 mg β-CD-CDs and 10 mg azobenzene-free copolymer (BCP) were used to prepare β-CD-CDs@BCP assembly in the same way as above-mentioned. And the structure of the β-CD-CDs@BCP was

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studied by FTIR (Fig. S3b). It can be found that both peak shape and peak position of β-CD-CDs@BCP are relatively similar to the pure β-CD-CDs. Compared with β-CD-CDs@AZOBCP, we can clearly observe that there are no absorption peaks located between 1600-1200 cm-1

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which are corresponding to the BCP in Fig. S3c, indicating that the β-CD-CDs@BCP assembly was

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not formed because there is almost no interaction between BCP and β-CD-CDs. The above results further prove that the formation of β-CD-CDs@AZO-BCP is surely through host-guest interaction between β-CD molecules on β-CD-CDs and azobenzene moieties on the AZO-BCP. XPS measurement was conducted to verify the surface element composition of the as-prepared β-CD-CDs, and the results exhibit that there exist only two elements as C and O in Fig. 3a. The C 1s spectrum (Fig. 3b) of β-CD-CDs exhibits three peaks at 284.6, 286.2 and 288.4 ev, indicating that there are sp2 C=C, C–O, and C=O respectively, further revealing that there are abundant oxygen

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ACCEPTED MANUSCRIPT groups on the surface of β-CD-CDs [31]. The morphology of β-CD-CDs was confirmed by TEM measurement. It can be obviously observed that the as-prepared β-CD-CDs are uniformly dispersed (Fig. 4a) and exhibit an average diameter around 3.2 nm within a range from 1.5 to 4.5 nm in Fig.

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4c. The lattice fringe of β-CD-CDs is determined as 0.25 nm which is corresponding to that of graphite carbon based on the HRTEM image in Fig. 4b. The TEM image in Fig. 4d appears to show that the β-CD-CDs located in the shell of AZO-BCP micelles which are marked by red circles, and

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this demonstrates that β-CD-CDs@QDs-loaded AZO-BCP micelles are formed through host-guest

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complexation.

3.4 Optical properties of β-CD-CDs and micelle-based β-CD-CDs

The UV-vis absorption spectra of β-CD and β-CD-CDs in water are shown in Fig. 5a. It can be observed that compared with pure β-CD molecules, there exists a characteristic absorption peak of

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β-CD-CDs at about 287 nm, originating from the n–π* transition for C=O bond and the π–π* transition of the conjugated C=C band [42]. Fig. 5b is the photoluminescent (PL) spectra of β-CD-CDs under various excitation wavelengths, where we can observe that β-CD-CDs in water

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exhibit a blue-green emission centered at 450 nm upon the excitation at 360 nm and the emission

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peak position gradually red-shifted with increasing excitation wavelength. The excitation wavelength dependent phenomenon of the emission spectra indicates that the as-prepared β-CD-CDs may possess various sizes, as well as there are quite a few distributions of emission trap sites on the surface of each β-CD-CDs [43, 44]. The defects on the CDs endow the as-synthesized β-CD-CDs with a high fluorescence quantum yield as 16% without any passivation and doping. And the lifetime of β-CD-CDs was also determined as 2.79 ns by PL measurement as displayed in Fig. 5c. However, to further confirm that the β-CD-CDs@micelles@QDs can be prepared through

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ACCEPTED MANUSCRIPT host-guest interaction between the β-CD molecules on the surface of β-CD-CDs and the azobenzene molecules outside of QDs-loaded AZO-BCP micelles, we adopted RhB molecules to identify enough β-CD molecules existing on the β-CD-CDs which possess the ability to recognize specific guest

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molecules to form stable inclusion complexes via fluorometric assay. As shown in Fig. S4, there is a clear separation between the emissions at 450 nm of β-CD-CDs (blue curve) and 578 nm of RhB (red curve). When 5 µM RhB was added into β-CD-CDs solution, the RhB emission intensity (black

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curve) is about 49.6% of original emission intensity (red line) of 5 µM RhB, which should be

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attributed to the photoinduced electron transfer (PET) between RhB and β-CD-CDs when RhB enter into the cavity of β-CD through specific host-guest interaction [45]. Therefore, the above results indicate that β-CD-CDs@QDs-loaded AZO-BCP micelles can be constructed via host-guest interaction and developed as a sensor for further ion detection.

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Based on the above facts, we prepared the β-CD-CDs@micelles@QDs successfully. And Fig. 5d shows the PL spectra of β-CD-CDs, 10 µL QDs-loaded AZO-BCP micelles and β-CD-CDs@ micelles@QDs upon excitation of 365 nm. Comparing with the blue-green fluorescence of pure

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β-CD-CDs, QDs-loaded AZO-BCP micelles display a dual emission with a weak green light and a

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strong red light, which is resulting from the original emission (620 nm) of CdSe/ZnS QDs and the coordination emission (526 nm) between the MQ-Zn complex on the surface of the CdSe/ZnS QDs in the core of the micelles [46]. The β-CD-CDs@micelles@QDs assembly exhibits both emissions of β-CD-CDs and the QDs-loaded AZO-BCP micelles centered at 450, 526 and 620 nm with blue, green and red tri-channel fluorescence emissions with relative weak green light emission, revealing that the β-CD-CDs@micelles@QDs hybrid assembly can be used as a ratiometric fluorescent sensor. In addition, the large separation (170 nm) between the emission peaks of β-CD-CDs (450 nm) and

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ACCEPTED MANUSCRIPT QDs-loaded AZO-BCP micelles (620 nm) can be helpful in distinguishing accurate variation of intensities and ratios on fluorescence emissions for ratiometric fluorescent sensing application. To obtain the optimal fluorescence properties, we adjusted the proportion of β-CD-CDs and

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QDs-loaded AZO-BCP micelles by altering the amounts of QDs which were loaded in AZO-BCP micelles after ensuring the contents of β-CD-CDs fixed. As shown in Fig. 6a, 5, 10 and 20 µL CdSe/ZnS QDs were loaded into the AZO-BCP micelles, and the as-prepared QDs-loaded micelles

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interacted with a certain amount of β-CD-CDs (8.0 mg). It can be clearly observed that when the

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amount of QDs was adjusted to 10 µL, the emission intensity of CDs (450 nm) and QDs-loaded micelles (620 nm) could be better matched upon 365 nm excitation. Surprisingly, the luminescence of hybrid assembly containing 10 µL QDs is close to standard white light (CIE: 0.33,0.33) under excitation at 365 nm, which is favorable for further detection due to the fluorescence variation upon

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UV-lamp can be clearly observed with naked eyes. The CIE coordinates of three samples of β-CD-CDs@micelles@QDs with 5, 10 and 20 µL QDs are shown in Fig. 6b, and the corresponding photographs

are

shown

in

Fig.

6c.

Thus,

in

the

next

study,

we

adopted

the

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β-CD-CDs@micelles@QDs containing 10 µL QDs as our sensor for ion detection.

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The fluorescence properties of β-CD-CDs@micelles@QDs (10 µL) with an excitation range from 340 to 500 nm were also studied as given in Fig. 6d. Notably, the emission peak of the as-synthesized β-CD-CDs is still red-shifted with increasing excitation wavelength, while the emission peak at 620 nm coming from QDs-loaded micelles has no interference with altering excitation wavelength, showing high photostability. 3.5 Metal ion sensing properties of β-CD-CDs@micelles@QDs Before evaluating the sensing behavior of β-CD-CDs@micelles@QDs as a ratiometric

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ACCEPTED MANUSCRIPT fluorescent sensor for metal ions, we also studied the fluorescence intensity change of β-CD-CDs after addition of different metal ions with the same concentration (33 µM) as shown in Fig. 7a. For β-CD-CDs, there exist various fluorescence responses to different metal ions, however, both of

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fluorescence quenching effects and fluorescence enhancement effects are not remarkable enough, indicating that it is impossible to selectively detect specific ions by pure β-CD-CDs. Thus, the ratiometric fluorescent sensor for β-CD-CDs@QDs-loaded micelles was prepared for further specific

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ion detection.

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To investigate the selective fluorescence sensing behaviors towards metal ions by using the as-prepared sensor, the quenching performance was proved in the presence of 15 diverse metal ions (Cr3+, Al3+, Cd2+, Co2+, Hg2+, Cu2+, Fe2+, Fe3+, Pb2+, Ca2+, Mn2+, Zn2+, K+, Na+, Ni2+) with a concentration of 33 µM upon 365 nm excitation (Fig. 7b). It can be clearly observed that the

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fluorescence of β-CD-CDs@QDs-loaded micelles at 620 nm is quenched around 88% by Hg2+ and Cu2+ as compared with the blank sample, while there is minimal or no interference with the addition of other metal ions (Fig. 7c). Although the fluorescence of β-CD-CDs at 450 nm is slightly affected

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by various metal ions, even among them, Cu2+, Fe2+, and Fe3+ have extremely high quenching ability

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to the PL emission of β-CD-CDs which is reduced to about 72% compared with the original. The results may be due to there exists strong affinity between β-CD-CDs and Cu2+, Fe2+ or Fe3+ ions, leading to a disruption of the radiative transition owing to the electron transfer between the carbon dots and responsive ions [47, 48]. However, the change of the fluorescence coming from β-CD-CDs is negligible compared with QDs in micelles, furthermore, Fe2+ and Fe3+ cannot quench the fluorescence of QDs, which indicates that the sensor cannot selectively detect them. Thus, the sensor can be utilized for selective detection for Hg2+ and Cu2+.

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ACCEPTED MANUSCRIPT We further studied the potential of the as-prepared sensor for Hg2+ and Cu2+ detection. Fig. 8a shows that the response of the ratiometric fluorescent sensor towards Cu2+ within a concentration range from 0 to 100 µM. Owing to the efficient charge transfer [49] between Cu2+ and CdSe/ZnS

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QDs, the fluorescence of QDs-loaded micelles serves as response signal for Cu2+, whereas the fluorescence emission peak of β-CD-CDs is utilized as the reference signal. When 10 µM Cu2+ was added into the sensor solution at the beginning, it can be obviously found that the extent of

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quenching effect on the fluorescence emission intensity of β-CD-CDs was even a little more than

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QDs-loaded micelles, which may be that tiny amounts of Cu2+ added at first is much easier to coordinate with β-CD-CDs, and the quenching effect on the fluorescence emission of QDs-loaded micelles would be dominant until the coordination between Cu2+ and β-CD-CDs reached saturation. Next, with the addition of Cu2+, the red emission coming from QDs (620 nm) diminished rapidly.

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Furthermore, there is no obvious interference to the emission peak centered at 450 nm coming from β-CD-CDs. As illustrated in Fig. 8c, the PL intensity ratio (Fβ-CD-CDs/FQDs) increases linearly with the concentration of Cu2+ in the range from 4.0 to 40 µM. The limit of detection (LOD) is calculated to

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be about 2.74 µM of the as-synthesized sensor towards Cu2+ using 3σ/S method, where σ represents

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a standard deviation of blank signal and S denotes the slope of the calibration curve [50]. The potential of the sensor for quantitive analysis of mercury ions was also studied via recording the fluorescence signal change with the increase of Hg2+ within a concentration range from 0 to 100 µM in Fig. 8b. The LOD towards Hg2+ can be calculated to be 1.6 µM from Fig. 8d. To prove the validity and outstanding detection performance of the as-synthesized β-CD-CDs@micelles@QDs as sensor, as a control experiment, we further studied the sensing abilities of pure β-CD-CDs and QDs-loaded micelles towards both Cu2+ and Hg2+. By evaluating the

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ACCEPTED MANUSCRIPT detection behaviors of pure β-CD-CDs, the fluorescence spectra were collected with the concentration of Cu2+ and Hg2+ ranging from 0 to 40 µM in Fig. S5a and b respectively, where we can observe that the fluorescence intensity of the β-CD-CDs is just slightly disturbed by the addition

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of Cu2+ and Hg2+, which indicates that the carbon dots cannot be utilized for the concentration determination towards Cu2+ and Hg2+. As for the detection behavior of pure QDs-loaded micelles towards Cu2+ and Hg2+ with the concentration ranging from 0 to 40 µM in Fig. S5c and d, it can be

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found that the emission intensity of CdSe/ZnS QDs in micelles at 620 nm is quenched rapidly with

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the increase of Cu2+ and Hg2+, and the LOD for Cu2+ can be calculated as 3.04 µM from Fig. S5e, whereas the LOD towards Hg2+ was calculated to be 2.88 µM based on Fig. S5f. And all above results show that our sensor possesses much lower LOD towards Cu2+ and Hg2+, which also demonstrates the dominant position of the ratiometric fluorescent sensor in detection.

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The mechanism of Cu2+ and Hg2+-induced fluorescence quenching of QDs-loaded micelles was further studied. Generally, the fluorescence quenching behavior is divided into two classes of processes, namely dynamic and static quenching respectively [51]. Specifically, dynamic quenching

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process occurs owing to the direct collision of the luminescent photons in excited state with

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quenching analytes, leading to a nonradiative transition process appearing as a transfer of photons from the excited state to the ground state, for instance, electron transfer and energy transfer [52]. Whereas, static quenching refers to forming a complex substance in static state with non-luminance through ground-state interaction between quenching analytes and fluorophores [53]. The quenching efficiency of above two quenching modes can be expressed by the Stern-Volmer (S-V) equation [54]: I0/I-1=KsvCA In the formula, I0 and I respectively represent the emission intensity of the fluorophore without and

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ACCEPTED MANUSCRIPT with the quenching species, and CA is the concentration of the quencher, while Ksv refers to the fluorescence quenching constant. When the concentration range of the Cu2+ is 4-40 µM, the relationship between the variation of fluorescence intensity at 620 nm of sensor versus the

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concentration of Cu2+ is conformed to be a linear S-V curve with the value of R2 as 0.993, which indicates that there exists only one quenching mode in the concentration range of 4-40 µM for Cu2+ showing in Fig. 8e, and the fluorescence quenching constant is calculated to be 3.1×104 M-1. As for

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Hg2+ in Fig. 8f, the relationship between the change of emission intensity of QDs at 620 nm against

of quenching constant is 5.32×104 M-1.

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the concentration of Hg2+ is also proved to be a linear S-V curve with the R2 as 0.995, and the value

As we all know, dynamic quenching can make the excited lifetime of fluorophores shorter than original, while there is no change in fluorescence lifetime during the static quenching process which

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is due to the formation of the complex with non-luminance between the fluorophore and quenchers, leading to a “dead” state for fluorescence lifetime [55]. Thus for further proving the quenching mode, the fluorescence decay curves of QDs in the sensor with and without Cu2+ and Hg2+ are displayed in

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Fig. 9. The lifetime of the sensor without metal ions is calculated to be 2.49 ns, whereas this value

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reduced to 1.83 and 2.11 ns in the presence of Cu2+ and Hg2+ respectively, appearing a faster decay rate in the presence of Cu2+ and Hg2+, which demonstrates that only dynamic quenching mode exists in the sensing system. The quenching behavior may be attributed to the donor–acceptor electron-transfer (ET) which refers to the transfer from electron-rich units (–OH, –NH2 of CdSe/ZnS QDs) to electron-deficient analytes (free cations such as Cu2+ and Hg2+ in solution) here [56].

4. Conclusions 22

ACCEPTED MANUSCRIPT We developed a facile strategy to construct a novel ratiometric fluorescent sensor based on β-CD-CDs@QDs-loaded micelles via host-guest interaction between β-CD units on the surface of β-CD-CDs and azobenzene moieties outside the shell of micelles for the detection towards both of

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Hg2+ and Cu2+. The fluorescence of β-CD-CDs at 450 nm serves as a reference signal and the emission peak of CdSe/ZnS QDs encapsulated in the core of micelles at 620 nm can be quenched significantly by Hg2+ and Cu2+. Under the optimal conditions, the white light-emitting sensor even

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can be obtained with CIE coordinate (0.33, 0.33) by adjusting the ratio of β-CD-CDs to CdSe/ZnS

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QDs, which is favorable for clearly observing fluorescence changes of the as-synthesized sensor. The novel strategy we proposed here for the construction of double-channel emission hybrid fluorescent nanomaterials based on host-guest recognization can not only be applied for diverse sensing systems,

Acknowledgements

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but also can broaden their applications in various fields like optoelectronics.

The authors would like thank the National Natural Science Foundation of China (21574017) for

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the financial support of this work.

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Figure captions: Scheme 1. Synthetic procedure of β-CD-CDs, CdSe/ZnS QDs loaded AZO-BCP micelles, and β-CD-CDs@micelles@QDs. Scheme 2. Schematic route of the preparation for amphiphilic diblock copolymer AZO-PNIPAM-b-

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P(St-co-MQ).

Fig. 1. 1H NMR spectra of AZO-PNIPAM (a) and AZO-PNIPAM-b-(St-co-MQ) (b).

CdSe/ZnS QDs in micelles (b) and pure CdSe/ZnS QDs (d).

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Fig. 2. TEM images of AZO-BCP micelles (a) and QDs-loaded AZO-BCP micelles (c); HRTEM of

Fig. 3. Wide region XPS spectra (a) and C 1s spectra (b) of β-CD-CDs.

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Fig. 4. TEM (a) and HRTEM images (b) of β-CD-CDs and their corresponding size-distribution histograms (c); TEM images of β-CD-CDs@AZO-BCP micelles (d).

Fig. 5. Uv-vis absorption spectra of β-CD-CDs and β-CD in water (a); PL spectra of β-CD-CDs in water under various excitation wavelengths (b); PL decay curves of β-CD-CDs (c); and PL emission

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spectra of QDs-loaded micelles, β-CD-CDs and the corresponding hybrid assembly (d). Fig. 6. PL spectra (a) and corresponding CIE coordinates (b) of β-CD-CDs based micelles with 5, 10 and 20 µL QDs (under 365 excitation); photographs of three samples under 365 nm UV-lamp (c); PL

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spectra of β-CD-CDs based micelles with 10 µL QDs upon different excitation (d). Fig. 7. Fluorescence response of β-CD-CDs with various metal ions (33 µM) (a); Fluorescence

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emission spectra (b) and corresponding histogram (c) of β-CD-CDs@QDs-loaded micelles with various metal ions (33 µM).

Fig. 8. Evolution of the fluorescence intensity of the sensor upon the exposure to different concentrations of Cu2+ (a) and Hg2+ (b). Linear relationship of I450/I620 versus different concentrations of Cu2+ (c) and Hg2+ (d) ranging from 4.0 to 40 µM; Stern-Volmer plots corresponding to the sensor with various concentration of Cu2+ (e) and Hg2+ (f). Fig. 9. Fluorescence decay curves for the sensor without (red curve) and with Cu2+ (green curve); Hg2+ (blue curve). 31

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Scheme 1.

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Scheme 2.

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Fig. 1.

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Fig. 2.

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Fig. 8.

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Fig. 9.

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► Blue-green emitting β-cyclodextrin derived carbon dots (β-CD-CDs) were synthesized. ► Azobenzene-terminated block copolymer containing MQ ligand was prepared.

► β-CD-CDs@QDs-loaded polymer hybrid micelles were fabricated via host-guest interaction.

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► White-light hybrid micelles were obtained by adjusting proportions of different emission centers.

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► β-CD-CDs@micelles@QDs as ratiometric fluorescent sensor can be used to detect Hg2+ and Cu2+.

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► Our strategy for hybrid fluorescent nanohybrid can be applied for diverse sensing systems.