Stimulus-responsive electrochemiluminescence from self-assembled block copolymer and nonpolar carbon quantum dot composite nanospheres

Accepted Manuscript Stimulus-responsive electrochemiluminescence from self-assembled block copolymer and nonpolar carbon quantum dot composite nanospheres Hangqing Xie, Fulan Bai, Yao Fu, Haogang Zhu, Kun Yan, Kaihui Mao, Paul K. Chu, Lizhe Liu, Xinglong Wu PII:

S0008-6223(19)30255-6

DOI:

https://doi.org/10.1016/j.carbon.2019.03.032

Reference:

CARBON 14037

To appear in:

Carbon

Received Date: 16 January 2019 Revised Date:

11 March 2019

Accepted Date: 12 March 2019

Please cite this article as: H. Xie, F. Bai, Y. Fu, H. Zhu, K. Yan, K. Mao, P.K. Chu, L. Liu, X. Wu, Stimulus-responsive electrochemiluminescence from self-assembled block copolymer and nonpolar carbon quantum dot composite nanospheres, Carbon (2019), doi: https://doi.org/10.1016/ j.carbon.2019.03.032. 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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Graphical Abstract

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Polarity

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PEO PPO PEO

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Stimulus-responsive electrochemiluminescence from self-assembled block copolymer and nonpolar carbon quantum dot composite

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nanospheres Hangqing Xie1, Fulan Bai1, Yao Fu1, Haogang Zhu1, Kun Yan1, Kaihui Mao1, Paul K. Chu2, Lizhe Liu1,*, and Xinglong Wu1,* 1

National Laboratory of Solid State Microstructures and School of Physics, Nanjing

2

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University, Nanjing 210093, People’s Republic of China

Department of Physics and Department of Materials Science and Engineering, City

*Corresponding author

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University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China

E-mail: [email protected] (X. L. Wu) E-mail: [email protected] (L. Z. Liu)

ABSTRACT

self-assembled

superstructure

composed

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A

of

block

copolymers

with

stimulus-response properties is attractive, but how to achieve electrochemiluminescence (ECL) with good stimulus responsiveness from biocompatible materials is a big

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challenge. Herein, F127/NPCD nanospheres comprising the block copolymer Pluronic F127 and nonpolar carbon quantum dots (NPCDs) are designed and prepared. The materials with hydrophobic interaction show excellent ECL stimulus response. The ECL

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mechanism is investigated by analyzing the cyclic voltammetry, ECL, photoluminescence, and electron paramagnetic resonance results. There are two secondary structures in the F127/NPCDs nanospheres, namely a hydrophobic layer and a hydrophilic layer. The hydrophobic layer contains a polyoxypropylene PPO moiety of F127 and NPCDs, and the hydrophilic layer contains polyoxyethylene PEO of F127. The hydrophilic layer as a repository of co-reactants can adsorb a large amount of SO4•– to excite the NPCDs in the nearby hydrophobic layers to produce ECL at a gentle voltage. In nonpolar solvents, separation of the NPCDs from F127 leads to insufficient contact between NPCDs and 2

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SO4•–, and hence, the NPCDs require excitation of a larger potential to produce ECL to exhibit the stimulus response via a unique turn-on mechanism.

The in vitro cytotoxicity

experiments indicate that the F127/NPCDs nanospheres have good cytocompatibility. The stimulus-responsive ECL nanospheres are broadly applied as biological

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microelectrode materials in biological monitoring, tissue component recognizing, drug

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distribution, and sustained release.

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1. Introduction Electrochemiluminescence (ECL) is different from photoluminescence (PL)[1], which requires light excitation to produce fluorescence subsequently affecting the detection of the emitted light. ECL proceeds in two steps.[2] In the first step, the materials

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are excited from the ground state to the excited state by a series of redox reactions, and in the second step, the excited state relaxes to the ground state via emission of a photon.[3] In ECL, a light source is not required in either stage, thus simplifying the instrumentation and avoiding interference due to the light source, such as scattering, reflection, and

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frequency doubling. The stimulation mechanism of ECL relies on several stimulation methods for quenching or enhancing ECL emissions, for example, consumption of co-reactants,[4,5] changes in the materials structure,[6,7] and utilization of energy resonance

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transfer.[8,9] The excitation voltage can be increased or decreased by changing the excitation mechanism, but the pertinent ECL mechanism is still not well understood. Nonetheless, it has been shown that changing the excitation mechanism affects the ECL intensity at the same voltage[10,11], and this phenomenon can be exploited in bioanalytical research.

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The block copolymer self-assembled micro-/nano-structure[12,13] has attracted a great deal of interest in medicine, biology, and engineering.[14-16] Flash nanoprecipitation (FNP) controls the size of the nanoparticles and polymer brush density, and the superstructure in the assembly shows a certain regularity. Prud’homme et al. studied the size control of the

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nanoparticles and polymer brush density by diffusion-limited aggregate assembly kinetics for sizes between 40 and 200 nm.[17] Priestley et al. theoretically and experimentally studied self-assembly of block copolymers and homopolymers with different molecular

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weights to form nanosheets with an internal layered pattern structure.[18] Stimulation of responsive microgels provides useful signals for external changes such as temperature, pH, illumination, and molecular recognition.[19] The block copolymer self-assembly superstructure is an important type of “smart” materials with the stimulus-responsive mechanism because of easy triggering of other responses with structural changes.[16,20] Several mechanisms for the stimulation of microgels have been reported. For example, Sukhishvili et al. reported temperature-induced expansion of block copolymer micelles for the release of molecules,[21] Ge et al. prepared drug-responsive sustained-release 4

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micelles with pH and glutathione response using a block copolymer,[14] and Yang et al. reported

CO2-stimulated

cytocompatibility.

[22]

organoplatinum(II)

metallacycles

with

good

Despite recent advances, research of the stimulation-responsive

mechanism of smart materials is still in the infancy stage.

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Most ECL stimulus-responsive polymeric materials rely on the temperature sensitivity or pH responsiveness of homopolymers. For example, Sojic et al. obtained stimulus-responsive

ECL

through

a

swelling–collapsing

transition

with

a

thermoresponsive microgel.[23] However, many functional homopolymers have potential

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cytotoxicity, poor structural controllability, colloidal instability, and aggregation-caused quenching[24] consequently limiting their application in the physiological environment. homopolymers,

some

block

copolymers

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Unlike

polyoxyethylene-polyoxypropylene-polyoxyethylene

(PEO-PPO-PEO,

such F127)[25]

as are

biocompatible, structurally controllable, and contain good ECL carriers. Block copolymers and hydrophobic quantum dots may self-assemble to form special structures because block copolymers and hydrophobic molecules have been reported to self-assemble with internal superstructures.[26,27] The hydrophobic and hydrophilic layers

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inside the self-assembly can be used to encapsulate the hydrophobic quantum dot emitters and hydrophilic co-reactants, respectively. According to previous investigations, substance exchange and electron transfer reactions can occur in polymer nanoparticle systems.[28] Oxidation or reduction of the hydrophobic and hydrophilic layer near the

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electrode may produce ECL emission, but the process has seldom been explored. Carbon quantum dots (CDs) are very competitive ECL materials because of their nontoxicity, water solubility, and flexible modification[29,30], and heteroatom-doped CDs

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are considered to be excellent ECL co-reactants.[31,32] Here, block copolymer F127 polyoxyethylene-polyoxypropylene-polyoxyethylene (PEO-PPO-PEO, Pluronic) and nonpolar carbon quantum dots (NPCDs) are used to form self-assembled F127/NPCDs nanospheres, and ECL is stimulated in nonpolar solvents. The alkane-modified NPCDs synthesized by one-step solvothermal method (180 °C) have good dispersibility in nonpolar solvents and exhibit a fluorescence quantum yield of 26%. The NPCDs and F127 solution are quickly mixed, and the transparent liquid becomes yellowish emulsion almost in one second. The strong fluorescence under illumination of the 360 nm UV light 5

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indicates that the NPCDs and F127 have produced an FNP self-assembly reaction in the aqueous solution. As shown in Figure 1, the hydrophobic portion of F127 is complexed with NPCDs by the hydrophobic interaction, and the hydrophilic portion of F127 is exposed to an aqueous solvent. This internally structured composite enables the

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co-reactants to enter the F127 hydrophilic portion and make contact with adjacent NPCDs to yield efficient ECL. The NPCDs are released when the nanostructure is in a nonpolar solvent, causing separation between the co-reactant and NPCDs. In this way, ECL excited by the same voltage excitation is almost completely quenched, and the

1000

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ECL (a.u.)

b

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mechanism can be exploited in designing switching devices.

ECL ON

500 0

0

5

ECL (a.u.)

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c

10 15 20 25 Time/s

400 200

ECL OFF

0 0

5

10 15 20 25 Time/s

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Figure 1. (a) Self-assembly and stimulated response of the F127/NPCDs nanospheres based on solvent polarity; (b) ECL spectrum of the F127/NPCDs nanospheres; (c) ECL

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spectrum of the F127/NPCDs nanospheres in a nonpolar solvent. The scan potential range is 0.0 to −1.2 V, and the scan rate is 100 mV s−1.

2. Experimental section 2.1 Chemicals

Pluronic F127 (average Mn, 2900), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), L-Arginine (Arg), and oleylamine were purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China). Potassium persulfate (K2S2O8), disodium hydrogen phosphate 6

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dodecahydrate, sodium dihydrogen phosphate dehydrate, sodium chloride, and cyclohexane were supplied by Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All the other reagents were of analytical grade and used without purification. All the

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aqueous solutions were prepared with ultrapure water.

2.2. Preparation of NPCDs

The NPCDs were synthesized by one-step solvothermal method. The specific steps were as follows. One gram (0.72 M) of Arg, 3 ml (1.14 M) of oleylamine, and 5 ml of

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ethanol were added to 25 ml of PTFE and stirred for 20 min to dissolve Arg as much as possible. The solution was sealed in a stainless steel reaction vessel and heated at 180 °C

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for 24 h. After allowing to cool to room temperature naturally, a brownish black product was obtained. The product was washed with n-hexane, and the precipitate was removed by centrifugation (8000 r min-1, 3 min) to obtain a wine red carbon dots dispersion. The solution was centrifuged thrice, and the supernatant was evaporated to remove n-hexane. The oleylamine-modified CDs, NPCDs, were concentrated in a vacuum oven at 50 °C for

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36 h. Finally, a viscous liquid of approximately 2.8 ml was obtained.

2.3 Preparation of the F127/NPCDs nanospheres The NPCDs were dispersed in a mixture of cyclohexane and ethanol (100 mg/mL, cyclohexane: ethanol 1:1, volume ratio). F127 (500 mg) was dissolved in 20 ml of

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deionized water and stirred until clear and transparent. The newly prepared 300 µL of NPCDs dispersion was quickly added, and the two clear transparent solutions immediately turned into a pale yellow milky dispersion with a pH of 6.5. After

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centrifugation (10000 r min-1, 30 min), the supernatant was dried in a vacuum oven for 36 h to obtain the F127/NPCDs nanospheres.

2.4. Characterization

Cyclic voltammetry (CV) was performed on an electrochemical workstation (CHI 760C, CHI Instruments, USA), and the ECL spectra were collected using an ECL detector (MPI-E, Xi'an Ruimai Analytical Instrument). A three-electrode system with a glassy carbon electrode (3 mm, Shanghai Chenhua Instrument) as the working electrode, 7

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a platinum wire as the auxiliary electrode, and a saturated calomel electrode as the reference electrode was used. In PBS (pH = 7), 100 mM of K2S2O8 was the co-reactant (100 mM s-1). The absorption spectra were acquired on an ultraviolet-visible spectrophotometer (Cary50 UV-vis, Varian, USA), and PL was performed on a molecular

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fluorescence spectrometer (Cary Bclipse, Varian, USA). The dried NPCDs, F127, and F127/NPCDs nanospheres were mixed with potassium bromide with a ratio of 1:200 and ground in an agate mill prior to acquiring the Fourier transform infrared spectroscopy (FTIR) on the FTIR spectrometer (NEXUS670, Nicolet, USA) in the range 400-4000

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cm-1. The F127/NPCDs nanocomposites were dispersed in deionized water and dropped on a carbon film copper mesh for transmission electron microscopy (TEM) (JEOL-2100F, Japan). The electron paramagnetic resonance (EPR) spectra were recorded on an EPR

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spectrometer (Bruker EMX-10/12, Germany). OH•– and SO4•– were produced by a Fenton-like reaction (Fe2+ and S2O82–) and captured by DMPO to form the DMPO-OH and DMPO-SO4 adducts. The cytotoxicity experiments on the F127/NPCDs nanospheres were performed by the standard 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The live cells were stained with Calcein-AM (green) and dead

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cells were stained with propidium iodide (red).

3. Results and discussion 3.1. Synthesis of NPCDs

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As a surfactant and surface ligand, oleylamine plays an important role in solvothermal synthesis of nanomaterials.[33] The boiling point of oleylamine is 348 °C,

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thus ensuring rapid growth of nanoparticles at the high temperature. Moreover, hydrophobic alkane of oleylamine improves the stability and oxidation resistance of the nanomaterials by segregating the nanomaterials.[34,35] The NPCDs are synthesized by a one-step solvothermal method, with oleylamine as the solvent and surfactant to obtain hydrophobic CDs, and the organic molecule Arg serves as a carbon source.[36,37] As shown in Figure S1, the CDs prepared by co-carbonization of Arg and oleylamine and functionalized by alkane are nonpolar surfaces and well dispersed in cyclohexane. In contrast, dispersion in an aqueous solution is poor despite stirring for 12 h. The NPCDs are characterized by FTIR (Figure S2a): the peak at 3324 cm-1 is the 8

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N-H stretching vibration and the two strong absorption peaks at 2921 and 2852 cm-1 correspond, respectively, to symmetric and asymmetric stretching vibrations of -CH2-. In the fingerprint area, the peak at 721 cm-1 is the rocking vibration of -CH2-. The results suggest that a large number of alkane is present on the NPCDs, and this is the reason why

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NPCDs can interact with hydrophobic oily solvents and be dispersed in nonpolar solvents. The absorption peaks at 1576-1645 and 1456 cm-1 are attributed to C=O, N-H, C-N, and C=C in NPCDs, and these groups have a large influence on the radiation centers in the NPCDs. The NPCDs are characterized by XPS (Figure S2b-e). The three strong peaks at

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284.0, 398.1, and 530.5 eV are assigned to C 1s, N 1s, and O 1s, respectively. The C 1s spectrum shows three peaks at 284.6, 285.0, and 287.8 eV, associated with C=C,

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C-C/C-H/C-N, and C=O, respectively. The peaks at 398.6, 399.3, 400.1 eV stem from C=N-H, N-H, and C-N, respectively, and those at 531.0 and 531.8 eV in the O 1s spectra are assigned to C=O and C-O, respectively. The concentrations of N and O are 9.19% and 4.30%, respectively, and the NPCDs have abundant sp2 and N-doped structures.

3.2. Self-assembly of F127/NPCDs

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Pluronic F127 is a triblock copolymer with hydrophobic polyoxypropylene (PPO) in the middle and hydrophilic polyoxyethylene (PEO) on the other two sides. In an aqueous solution, when the concentration is less than the critical micelle concentration, Pluronic F127 moves freely as a single-chain form in water. When the concentration is higher than

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the critical point, Pluronic F127 forms a colloid and has a solubilizing effect on the hydrophobic molecules. Unlike NPCDs in a water agglomerate, the NPCDs are dispersed

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uniformly in deionized water containing F127 during the FNP process. After the addition of NPCDs, the F127 solution changes from colorless and transparent to a milky pale yellow. Centrifugation at 10,000 rpm produces no precipitate, and the self-assembled F127/NPCDs nanospheres are synthesized (Figure 2). TEM and dynamic light scattering (DLS) are performed to determine the structure of

the F127/NPCDs self-assembly as shown in Figure 2. TEM shows that the self-assembled F127/NPCDs are nanospheres with black and white interiors with internal structures (Figure 2a). The size of the F127/NPCDs nanospheres is approximately 180 nm, which is similar to that of the self-assembly of block copolymers and hydrophobic molecules 9

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reported previously.[17,38,39] DLS shows that the average diameter of the F127/NPCDs nanospheres in the aqueous solution is approximately 225.3 nm in line with TEM (Figure 2d). The internal structure of the F127/NPCDs nanospheres is further examined by HR-TEM, as shown in Figure 2b. The positions with more NPCDs are darker, as shown

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in Figure 2a. HR-TEM (Figure 2c) and SAED (Figure S3) clearly show the lattice fringes and diffraction rings of the graphene structure. The size of the NPCDs is approximately 3 nm. TEM and DLS show that Pluronic F127 encapsulates NPCDs, and so the

b

c

0.1 0.0

10

NPCDs F127/NPCDs Stimulated Dh=13.5 nm Dh=225.3 nm D =11.6 nm h

Transmittance (a.u.)

20

e

10 15 Size/nm

20

f

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30

5

F127/NPCDs NPCDs

0 -1 0 1 2 3 4 10 10 10 10 10 10 4000 3000 2000 1000 -1 Wave number/cm Size/nm

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Number (Percent)

d

0

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Percent

0.2

Transmittance (a.u.)

0.3

a

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water-insoluble NPCDs are well dispersed in an aqueous solution.

F127/NPCDs F127

4000 3000 2000 1000 -1 Wave number/cm

Figure 2. (a) TEM image of the F127/NPCDs nanospheres (scale bar = 100 nm); (b)

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TEM image of a single F127/NPCDs nanosphere (scale bar = 30 nm) with the inset showing the size distribution of NPCDs in the F127/NPCDs nanospheres; (c) TEM image of a single NPCD in the F127/NPCDs nanosphere (scale bar = 2 nm); (d) DLS data for the size of NPCDs in cyclohexane (light blue line), F127/NPCDs nanospheres in aqueous solution (red line), and cyclohexane-treated F127/NPCDs nanospheres in cyclohexane (dark blue line, the corresponding TEM image is presented in Figure S4 for comparing the NPCD sizes). The distribution data of F127/NPCDs nanospheres are number weighted. (e) FTIR spectra of the NPCDs (blue) and F127/NPCDs nanospheres (red); (f) 10

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FTIR spectra of the F127 (blue) and F127/NPCDs nanospheres (red). All the FTIR data are obtained by mixing the sample with potassium bromide powder without solvent participation.

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c

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Diameter (nm)

200

100

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b

a

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0 0.0 2.5 5.0 -1 NPCDs concentration (mg ml )

Figure 3. TEM images of a single F127/NPCDs nanosphere to show the state of F127 and distribution of NPCDs outside the boundary: (a) scale bar = 50 nm and (b) scale bar

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= 30 nm; (c) Effects of NPCDs concentration on the F127/NPCDs nanospheres.

Hydrophobic molecules such as Vitamin E and polystyrene (PS) have been reported to be in the core of the self-assembled structure and hydrophilic structures such as PEG

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occupy the outside of the self-assembly.[17] However, hydrophobic NPCDs are used instead of hydrophobic molecules in block copolymer self-assembly. The advantage of NPCDs compared to hydrophobic molecules is that NPCDs can be observed by TEM because the size and internal structure of the block copolymers prepared by the FNP can be monitored. The F127/NPCDs nanospheres have clear boundaries (Figure 2a), and the thin coating outside the boundaries is not obvious. Figure 3 shows that there are polymer coatings outside the boundaries of the nanospheres similar to previously reported self-assembled external polymer brushes. HR-TEM also discloses a large number of 11

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NPCDs in the polymer coating. The size distribution of the F127/NPCDs nanospheres is presented in Figure 3c and Figures S5 and S6. The F127/NPCDs nanospheres have good dispersibility in an aqueous solution, indicating that the outer portion of the nanosphere is occupied by the hydrophilic portion PEO of Pluronic F127. The hydrophilic portion of

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Pluronic F127 as a medium for NPCDs and co-reactants reduces the distance between them, and aggregation of NPCDs to the outer nanostructures facilitates ECL.

3.3. Stimulated response of F127/NPCDs nanospheres

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To explore the stimulated response mechanism of the F127/NPCDs nanospheres, FTIR is performed before and after self-assembly, and all the data are taken by mixing

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the sample with potassium bromide powder without solvent participation. Figure 2e compares the FTIR spectra of the NPCDs and F127/NPCDs nanospheres. After self-assembly of NPCDs, the N-H stretching vibration peak at 3324.0 cm-1 is overlapped by the absorption peak of 3429.5 cm-1 and the peak broadens, indicating that F127 introduces a large number of hydrogen bonds to improve the solubility of the F127/NPCDs nanospheres in water. The absorption peaks of -CH2- at 2921.0 and 2852.0

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cm-1 of the F127/NPCDs nanospheres weaken because NPCDs are coated with F127. Figure 2f compares the FTIR spectra of the F127 and F127/NPCDs nanospheres. The broad absorption peak at 3424.2 cm-1 stems from the terminal hydroxyl group of the hydrophilic end PEO of F127, indicating the presence of hydrogen bond. The peak at

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2888.3 cm-1 is the vibration of -CH2- in F127, and the intensity is reduced after compounding with NPCDs. The peak of C-O-C in the PPO of the hydrophobic part of

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F127 is near 1100.0 cm-1, and the intensity decreases after formation of the F127/NPCDs nanocomposite. All the hydrophobic moieties in the F127/NPCDs nanospheres, including the hydrophobic portion of F127 and hydrophobic NPCDs, are encapsulated inside the hydrophilic end of F127, resulting in a significant decrease in the intensity of some of the absorption peaks.[40] Based on the above results, the structure of the F127/NPCDs nanosphere is presented in Figure 1. The F127 hydrophobic portion and hydrophobic NPCDs are encapsulated in the nanospheres, and the hydrophilic portion of F127 is exposed to the surface in contact with water. The F127/NPCDs nanospheres are dispersed in the aqueous solution by intermolecular forces. 12

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F127 is hardly dispersible in a nonpolar solvent such as petroleum ether, cyclohexane, or n-hexane, but NPCDs have excellent dispersibility in a nonpolar solvent. The effects of nonpolar solvents on the F127/NPCDs nanospheres are studied. After the addition of cyclohexane to the NPCDs nanospheres, DLS reveals 11.2 nm nanoparticles

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in the cyclohexane solution (Figure 2d). After treatment with cyclohexane, the F127/NPCDs release NPCDs. The released NPCDs have sizes comparable to that from the DLS data in cyclohexane (Figure 2d and Figure S4). The kinetic diameter of the eluted nanoparticles is consistent with the results of the NPCDs cyclohexane dispersion.

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Meanwhile, the released NPCDs also have morphology and size similar to those of the NPCDs in F127/NPCDs nanospheres. The nonpolar solvent in the F127/NPCDs

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nanospheres exhibits strong blue fluorescence under 360 nm UV illumination, and the PL spectrum coincides with that of the NPCDs dispersion (Figure S7), thereby showing clear

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structural changes in the F127/NPCDs nanospheres in nonpolar solvents.

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3.4. ECL and response of F127/NPCD nanospheres

b F127/NPCDs NPCDs

400

-0.05

200

-2.0

-1.5

-1.0 E/V

-0.5

0 -2.0

0.0 d

c SO4

SO4

•–

SO4

2–

SO4

+

SO4

•–

SO4

S2O8

2–

S2O8

•–

0.0

2–

SO4

*

–

Electrode

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Electrode

-0.5

–

*

–

2–

2–

-1.0 E/V

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2–

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*

*

ECL (a.u.)

-2

Current (mA cm )

0.00

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a

Figure 4. (a) CV curve of the F127/NPCDs nanospheres (green) and cyclohexane-treated F127/NPCDs nanospheres (red); (b) ECL spectra of the F127/NPCDs nanospheres (green) and cyclohexane-treated F127/NPCDs nanospheres (red); (c) Schematic diagram of the

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F127/NPCDs nanospheres based on mechanism Ⅰ to generate ECL; (d) Schematic diagram of the cyclohexane-treated F127/NPCDs nanospheres based on mechanism Ⅰ to generate ECL. The illustrations are schematic representations of the F127/NPCDs

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nanospheres and NPCDs, respectively.

Considering that NPCDs have excellent optical properties (Figures S8 and S9) and

quantum yield of 26% (Figure S10), surface states and defects greatly contribute to ECL emission. Electrochemical scanning and ECL monitoring are performed on the F127/NPCDs nanospheres, with S2O82– as the co-reactant in a PBS buffer solution (pH = 7). Figure 4a shows the CV curve of the F127/NPCDs nanospheres in the presence of 100 mM S2O82-, and the irreversible cathodic peak at -1.10 V is due to the reduction of S2O82–. 14

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After the F127/NPCDs nanospheres are treated with a nonpolar solvent, the self-assembled structure is destroyed. The NPCDs are separated from F127, and therefore, the current decreases and initial potential increases. The peak potential shifts to -1.29 V. Because SO4•– diffuses readily into the F127/NPCDs nanospheres and NPCDs are

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involved in the reduction process of S2O82–, it can be seen from the CV curve that the current of the F127/NPCDs nanospheres is larger than that of the NPCDs. After the F127/NPCDs nanospheres collapse in a nonpolar solvent, the probability of contact between NPCDs and SO4•– is reduced and the current in the CV curve decreases. The

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reaction from the ground state to an excited state through a redox reaction requires the interaction of an electrode and a co-reactant.[11] Previous studies have shown that the

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ground state may undergo oxidization or reduction by the electrode, or it may undergo direct oxidization or reduction by the radical generated by the co-reactant to generate an excited state.[41] In general, the voltage required for oxidation or reduction of the co-reactant is generally low (absolute value), and therefore, the potential required by the materials to be excited by free radicals to produce ECL is generally low.

(eq. 1)

NPCDs + SO4•– → NPCDs+ + SO42–

(eq. 2)

NPCDs+ + e– → NPCDs*

(eq. 3)

NPCDs* → NPCDs + hv

(eq. 4)

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S2O82– + e– → SO42– + SO4•–

As shown in Figure 4b, the F127/NPCDs nanospheres produce strong ECL signals

at -1.0 V. The NPCDs in the F127/NPCDs nanospheres are rinsed with cyclohexane to reduce the hydrophilicity of the electrode. Under the same conditions, the cyclohexane-treated F127/NPCDs nanospheres require a potential of -1.7 V to generate the ECL signal. Accordingly, we propose two possible mechanisms. In mechanism I (Figure 4b, eq.1-4), S2O82– is reduced by the electrode to form a sulfate radical with a higher potential, SO4•– (2.5 ~ 3.1 eV). The NPCDs are oxidized by SO4•– radicals or OH•– 15

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radicals (2.8 eV, SO4•– turning into OH•– in the aqueous solution), and the excited states NPCDs* are generated by electrode reduction. In mechanism II (Figure 4d, eq.5-8), S2O82– and NPCDs are reduced by the electrode and NPCDs– are oxidized by SO4•– to generate excited state NPCDs*. In mechanism I, the voltage required to generate ECL is

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determined by the reduction voltage of S2O82–. Because the reduction voltage required for S2O82– is relatively low, ECL can be excited at lower voltages. Reduction of NPCDs in mechanism II requires a large voltage, and so, ECL requires a high voltage. The abundant SO4•– ions inside the F127/NPCDs nanospheres are in full contact with the NPCDs.

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According to mechanism I, strong ECL can be generated at a small voltage (-1.0V). Free NPCDs cannot contact sufficient number of SO4•–, and a higher voltage (-1.70 V) is

S2O82– + e– → SO42– + SO4•– NPCDs + e– → NPCDs•–

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required to generate ECL.

(eq. 5)

(eq. 6)

(eq. 7)

NPCDs* → NPCDs + hv

(eq. 8)

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NPCDs•– + SO4•– → NPCDs* + SO42–

Mechanisms I and II basically explain the ECL process of the F127/NPCDs nanospheres and NPCDs. The mechanism is studied further. As the scanned range of the

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CV curve of the F127/NPCDs nanospheres is increased, two ECL peaks are observed at -1.1 and -1.5 V (Figure S11), corresponding to the two mechanisms, respectively. The F127/NPCDs nanospheres produce ECL by the two mechanisms at a low and high potential, respectively, consistent with the coexistence of various mechanisms of ECL reported in the literature.[10,11] To investigate the stimulus-responsive ECL performance of the F127/NPCDs nanospheres, Figure 5a shows the CV scans of the F127/NPCDs nanospheres before and after cyclohexane treatment under the same conditions (0 ~ -1.2). Both CV curves show a 16

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clear reduction current, and the reduction current before the treatment is larger than the posttreatment current, indicating that the F127/NPCDs nanospheres consume a relatively large amount of S2O82–. Stimulation by nonpolar solvents has a dramatic effect on the ECL signal of the F127/NPCDs nanospheres (Figure 5b). The NPCDs in the

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F127/NPCDs nanospheres are reduced at low potentials (Figure 4) yielding strong ECL. Because the NPCDs and free radicals are close within the F127/NPCDs nanospheres, SO4•– can enter the nanospheres to oxidize NPCDs and ECL is generated by mechanism I. The F127/NPCDs nanospheres are destroyed in a nonpolar solvent resulting in separation

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of F127 from the NPCDs. The NPCDs and SO4•– are blocked by intermolecular interactions and are not in close contact with each other. The NPCDs, thus, require a high

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voltage in mechanism II to produce ECL, and there is no ECL at this voltage from mechanism I. The ECL signal changes because of the different ECL mechanisms before and after self-assembly of hydrophobic NPCDs.

-0.05

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F127/NPCDs NPCDs

-0.10 -1.2

ECL (a.u.)

400

-0.8

-0.4

200 0 -1.2

0.0

ECL (a.u.)

b

0.00

-2

Current (mA cm )

a

-1.2 -1.0 -0.8 -0.6 E/V

-0.8

0.0

d

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ECL (a.u.)

400

100

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c

ECL (a.u.)

-0.4 E/V

E/V

200

0 0

50

100 Time/s

50

0 0

150

50

100 Time/s

150

Figure 5. (a) CV curves of the F127/NPCDs nanospheres (red) and cyclohexane-treated F127/NPCDs nanospheres (black); (b) ECL from the F127/NPCDs nanospheres (red) and cyclohexane-treated F127/NPCDs nanospheres (black) with the inset showing the 17

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enlarged ECL spectrum; (c) ECL generated by scanning of F127/NPCDs nanospheres between 0 and -1.2 V; (d) ECL generated by scanning of the cyclohexane-treated F127/NPCDs nanospheres between 0 and -1.2 V.

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To rule out the effect of F127 on ECL, control experiments are conducted on surface-covered F127, F127/NPCDs nanospheres of glassy carbon electrodes in PBS, and F127-coated glassy carbon electrodes in the S2O82– solution. Under these conditions, there is no ECL except for the baseline at a potential of 0 ~ -1.2 V. The current and ECL

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intensity of the glassy carbon electrode coated with the F127/NPCDs nanospheres in the S2O82– solution do not change substantially before and after the addition of F127 to the

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solution. Hence, it can be inferred that F127 itself has no direct influence on ECL from the NPCDs in the potential window, and the impact of F127 itself on ECL can be excluded thus supporting mechanisms I and II. The nonpolar solvent causes a sharp drop in ECL from the NPCDs because F127/NPCDs nanosphere dissociation leads to NPCDs stacking on each other and the hydrophobic NPCDs do not sufficiently contact the hydrophilic SO4•–. The ECL response is different from that of the thermo-responsive

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microgel. We establish a stimulus-responsive mechanism by changing the luminescent environment to include different ECL mechanisms that result in different excitation voltages. To investigate the stability of ECL produced by the F127/NPCDs nanospheres by mechanism I, several scanning cycles are adopted (Figure 5c). The F127/NPCDs

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nanospheres are stable for the 8 cycles. Similarly, the cyclohexane-treated F127/NPCDs nanospheres produce weak ECL but are stable after 8 scans (Figure 5d). Here, we

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would like to mention that the saturated calomel electrode with timely maintenance does not degrade easily. The standard potential of the chlorine electrode is 1.358 V. At high negative voltages, chloride ions may be oxidized to chlorine. Hence, high concentrations of chloride ions are not suitable in this system. In low chloride ion concentrations, as adopted in our experiments, the membrane potential hardly changes and has negligible effect on the ECL system.

3.5. Adsorption of co-reactants by F127/NPCDs nanospheres 18

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♦ ♦ ♦

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♦

♦ ♦ ♦ ♦ ♦

Intrnsity (a.u.)

♦

2-

F127/NPCDs-S2O8 2-

3460 3480 3500 Magnetic Field (G)

3520

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3440

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NPCDs-S2O8

Figure 6. EPR spectra of the F127/NPCDs nanospheres treated with S2O82– (red) and NPCDs treated with S2O82– (blue). The red symbols represent the characteristic signal from DMPO-OH and blue ones represent the characteristic signal from DMPO-SO4. To further validate mechanisms I and II for the F127/NPCDs nanospheres and

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NPCDs, an EPR is monitored to characterize the radicals. DMPO is added to the mixture as the spin-trapping agent for the SO4•– and OH•– radicals. As shown in Figure 6 and Figure S12, an EPR spectrum (red line) with a typical peak intensity ratio of 1:2:2:1 can be observed. This feature corresponds to the generation of the DMPO-OH adduct, which

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indicates the presence of OH•– radicals in the sample. However, the peak of DMPO-SO4 is weak because SO4•– reacts with water to form OH•–. It is a self-purifying effect of

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SO4•–.[42] In addition to the peaks of these two radicals, oxidized DMPO radicals can be observed.[43] The F127/NPCDs nanospheres that absorb S2O82– show a strong EPR signal (curve a), whereas the NPCDs in the S2O82– solution exhibit a weak one (curve b). The EPR signal of the F127/NPCDs nanospheres that absorb S2O82– is stronger than that for the same concentration of S2O82. These phenomena confirm the attraction of F127/NPCDs nanospheres to S2O82–, which is beneficial to NPCDs in the generation of ECL at a low voltage.

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3.6. Biocompatibility of the F127/NPCDs nanospheres b

0 0.05 0.125 0.25 0.5 Concentration (mg/ml)

c

d

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100 80 60 40 20 0

Cell Viability (%)

a

Control

0.05 mg/ml

0.25 mg/ml

Figure 7. (a) Viability of the MC-3T3 cells co-cultured with the F127/NPCDs

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nanospheres at concentrations of 0, 0.05, 0.125, 0.25, and 0.5 mg/ml for 24 h; Laser confocal microscopy images of the MC-3T3 cells stained with the live/dead cells kit after

(d) 0.25 mg/ml for 24 h.

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co-culturing with the F127/NPCDs nanospheres at concentrations of (b) 0, (c) 0.05, and

The F127/NPCDs nanospheres do not contain heavy metals and organic toxins, and the surface of the nanospheres is the hydrophilic part of F127 containing abundant hydrophilic -OH. The biocompatibility of F127/NPCDs nanospheres is assessed by the standard MTT assay employing MC 3T3 cells. The MC 3T3 cells are co-cultured with the

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F127/NPCDs nanospheres at concentrations of 0.05, 0.125, 0.25, and 0.5 mg/mL for 24 h. When the concentration of F127/NPCDs nanospheres is raised to 0.5 mg/mL, the cell viability is still 90%, thus demonstrating low cytotoxicity. The MC 3T3 cells are stained

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with the live/dead cell kit, and the confocal microscopy shows living cells (green) with very little dead ones (red) (Figure 7 and Figure S13) corroborating the excellent cell

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compatibility.

4. Conclusion

F127/NPCDs nanospheres having different ECL characteristics in solvents with

different polarities are designed and prepared. NPCDs with a strong fluorescence quantum yield of 26% are synthesized by co-carbonization of amino acids and oleylamine. There are multiple emission centers in NPCDs, including sp2 hybrid π-π* electronic states, with size effects, surface states, and defect states that are not affected by size. The F127/NPCDs nanospheres synthesized by incorporation of Pluronic F127 can 20

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be excited to generate strong ECL at a low voltage (-1.0 V, SCE) under excitation of the electrode and co-reactants. ECL is quenched in a nonpolar solvent due to the destruction of aggregation of radicals. There are two secondary structures in the F127/NPCDs nanospheres, namely a hydrophobic layer and a hydrophilic layer. The hydrophobic layer

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consists of a polyoxypropylene PPO moiety of F127 and NPCDs, and the hydrophilic layer contains polyoxyethylene PEO of F127. The hydrophilic layer as a repository of co-reactants can adsorb a large amount of SO4•– to excite the NPCDs in the nearby hydrophobic layers to produce ECL at a low voltage. In nonpolar solvents, separation of

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the NPCDs from F127 leads to inseparable contact between NPCDs and SO4•–, and hence, the NPCDs require excitation of a larger potential to produce ECL to exhibit the stimulus

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response via a unique turn-on mechanism. In addition, the F127/NPCDs nanospheres show high biocompatibility in vitro. Our results provide insights into the design and fundamental understanding of ultra-sensitive detection in real-time online imaging and other applications.

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ASSOCIATED CONTENT Supporting Information

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The Supporting Information is available free of charge on the website at DOI:.

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AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (X.L. Wu)

ORCID Xinglong Wu: 0000-0002-2787-3069 Notes

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The authors declare no competing financial interest

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*E-mail: [email protected] (L.Z. Liu)

ACKNOWLEDGMENTS

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This work was supported by National Key R & D Program of China (2017YFA0303200), and National Natural Science Foundation (61521001 and 11674163).

Partial support

was from the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX17_0036), as well as Hong Kong Research Grants Council (RGC) General Research

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Funds (GRF) No. CityU 11205617.

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