Rhodol-conjugated polymersome sensor for visual and highly-sensitive detection of hydrazine in aqueous media

Journal Pre-proof Rhodol-Conjugated Polymersome Sensor for Visual and Highly-Sensitive Detection of Hydrazine in Aqueous Media Faran Nabeel, Tahir Rasheed

PII:

S0304-3894(19)31711-X

DOI:

https://doi.org/10.1016/j.jhazmat.2019.121757

Reference:

HAZMAT 121757

To appear in:

Journal of Hazardous Materials

Received Date:

2 October 2019

Revised Date:

13 November 2019

Accepted Date:

24 November 2019

Please cite this article as: Nabeel F, Rasheed T, Rhodol-Conjugated Polymersome Sensor for Visual and Highly-Sensitive Detection of Hydrazine in Aqueous Media, Journal of Hazardous Materials (2019), doi: https://doi.org/10.1016/j.jhazmat.2019.121757

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Rhodol-Conjugated Polymersome Sensor for Visual and Highly-Sensitive Detection of Hydrazine in Aqueous Media

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Faran Nabeel, Tahir Rasheed*

School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix

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Composites, Shanghai Jiao Tong University, Shanghai 200240, China;

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Graphical Abstract

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corresponding authors E-mail: [email protected] (T. Rasheed).

Highlights 

Development of a simple and efficient polymersome vesicle sensor. 1

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Bare eye detection of Hydrazine.

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Highly selective response accompanied by excellent quantifying ability.

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Real-time recognition of the environmental pollutants.

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Biological applications for live cell imaging.

ABSTRACT

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Hydrazine is a hazardous environmental pollutant, which contaminates land, air and water

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posturing a severe risk to human health. For the first-hand estimation, a qualitative approach

(colorimetric) for recognition of hydrazine could suffice. However, for accurate measurement,

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under the threshold limit value (TLV), a quantitative technique is desired. We report the

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polymersome-based sensor for visual detection and quantification of hydrazine in water. The rhodol-functionalized amphiphilic hyperbranched multiarm copolymer (HSP-RDL) was self-

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assembled into vesicles. The HSP-RDL vesicle probe exhibited high sensitivity and selectivity for hydrazine recognition in presence of various competitive species such as cations, anions, and

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neutral species. The fast responsive pink color change from colorless could be visualized with naked eye due to spirolactone ring opening by hydrazinolysis triggered strong fluorescence emission. The vesicle probe could detect hydrazine in water with a limit of detection (LOD) value of 2nM (0.0652 ppb), which is lower than TLV (10 ppb) given by USEPA (United States

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Environmental Protection Agency). Furthermore, the vesicle probe could quantify hydrazine (recovery ≥ 99%) in a wastewater sample collected from Huangpu river. The membranepermeable characteristics of HSP-RDL led hydrazine detection in live cells through confocal fluorescence microscopy. Keywords; Hyperbranched polymer; colorimetric vesicles; chromogenic visual detection; Intracellular monitoring; environmental pollutant; quantification. 2

INTRODUCTION In spite of its wide application in industry as a reducing and biological reagent, hydrazine is highly toxic and poses significant danger to public health, animal and aquatic life (Servo et al., 2014; Matsumoto et al., 2016; Fisher et al., 1980). When the amount of hydrazine exceeds its extremely low threshold value (10 ppb), it causes nausea, dizziness, and damages to the lungs, liver, body central nervous system, and DNA (Reja et al., 2016). Most of hydrazine present in

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the environment is in aqueous solution. Therefore, its selective and sensitive detection and

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monitoring in water becomes essential for environment and public health. Up to now, several conventional techniques such as chromatography, electrochemical analysis, chemiluminescence,

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capillary electrophoresis, and spectrophotometry have been developed for this purpose (Yu et al.,

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2017; Channon et al., 2015; Huang et al., 2014; Kosyakov et al., 2017). However, these methods generally rely on spectroscopic methods or special instrumentation, which limits their in-field

hydrazine are highly desired.

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applications. For these applications, facile and easy-to-handle techniques for visual detection of

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Several probes have been applied to visually detect hydrazine in aqueous solution (Tiensomjitr et al., 2018; Shia et al., 2019; Li et al. 2018; Zhu et al. 2013; Kong et al., 2018; Liu et al., 2019). Among these probes, colorimetric sensing probes based on protection-deprotection mechanism of functional groups could be ideal due to their high selectivity, sensitivity, biocompatibility, on-

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site and real-time detection (Rasheed et al., 2019a; Tang et al., 2015; Li et al., 2014). Different single-molecule fluorophores, such as triphenol, coumarin and rhodol (RDL), were prepared in the form of cleavable ester and applied for this purpose, as they can be cleaved in the presence of hydrazine (Ju et al., 2017; Yu et al., 2015; Zhu et al., 2013 ). For example, Chang et al. have

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developed levulinated coumarin probe for visual sensing of hydrazine via cleavable levulinoyl ester linkage (Choi et al. 2011). Compared to the above-mentioned colorimetric probes, sensors based on xanthene moiety and self-assembled nanoparticles are more advantageous, as the spatial display of multivalent probes on the outer surface of the nanoparticles can provide much larger access and extremely high

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sensitivity to the specific analytes (Rasheed et al., 2019b-f; 2018a-b; Chapman et al., 2015; Zheng et al., 2018; Kaushik et al., 2018; Huang et al. 2017; Hou et al. 2017). Vesicle-based

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chemosensors also have been developed, and by introducing different functional moieties onto

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vesicles, various analytes such as toxins, metal ions, proteins, biomolecules and enantiomers of organic molecules can be effectively detected in aqueous solution (Chen et al., 2017; Rasheed et

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al. 2019f; 2019g). For example, Stevens et al., (2015) have applied liposome substrate-assisted multivalent nanoparticle network for the visual detection of human phospholipase-A2 in serum.

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Similarly, Palivan and coworkers have developed metal functionalized vesicles prepared from poly(butadiene)-b-poly(ethylene oxide) and CuII-trisnitrilotriacetic acid for the detection of

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protein (His6-tags) ( Tanner et al., 2012).

Recently, our group has developed polymersome-based sensors for SO2, and aqueous Hg2+ detection by naked eye (Huang et al., 2017; Rasheed et al., 2019f; Hou et al., 2017). These

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polymersomes are self-assembled by amphiphilic hyperbranched multiarm copolymers HBPOstar-PEO (hyperbranched poly(3-ethyl-3-oxetanemethanol)-star-poly(ethylene oxide)) that were prepared by one-pot synthesis, and exhibit large-scale functionalization capacity and good stability (Zhou et al. 2007). Herein, we report a highly sensitive visual detection probe for hydrazine, which was prepared by grafting RDL on the polymersomes self-assembled by HBPOstar-PEO (HSP), denoted HSP-RDL. The RDL was selected as fluorophore due to excellent 4

photochemical properties like high photostability, large excitation coefficient, strong fluorescence, and long excitation wavelength in visible range (Whitaker et al., 1992; Kamiya et al., 2011). The RDL functionalized vesicles for hydrazine sensing portrays a unique and advantageous approach as the multivalent probe molecules are spatially displayed onto the vesicle surface, which leads to enhanced sensitivity to hydrazine in aqueous solution (Jung et al.,

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2015; Yan et al., 2016; Kaushik et al., 2018) The main concept of the polymersomes-based hydrazine sensing is illustrated in Scheme 1. The

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RDL-functionalized hyperbranched polymer HSP-RDL self-assembles in water into colorless

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HSP-RDL vesicles. Upon addition of hydrazine the HSP-RDL vesicles suddenly change to pink color. This color change results from spirolactone ring opening of cleavable RDL moiety, which

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leads to the formation of cyclic 4,5-dihydro-6-methylpyridazin-3(2H)-one group. Such a fast

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color change can be used for visual detection of hydrazine in water-based systems. EXPERIMENTAL SECTION

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Reagents and Instrumentation.

Resorcinol (99% pure), 2-(4-diethylamino-2-hydroxybenzoyl)benzoic acid (DHBA) (>98% pure) and trifluoroacetic acid (TFA) (99%) were purchased from Adamas beta and used as received. All other solvents and chemicals were purchased from Adamas beta, Shanghai Chemical &

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Reagents Co. China and Aldrich USA and used as received. Tetrahydrofuran (THF) and triethylamine (TEA) were refluxed using CaH2 before use. The stock solution of cations, anion, and neutral species were prepared with deionized water. The UV/vis spectroscopy was performed on Schimadzu UV-2600 UV/vis spectrophotometer. Fluorescence observations were carried out on a PerkinElmer LS 55 fluorescence spectrophotometer. 1H and 13C NMR spectra were recorded on a Bruker AVANCE III HD 500 NMR spectrometer using deuterated DMSO or 5

chloroform with TMS (tetramethylsilane) as internal standard. TEM imaging was performed on a Tecnai G2 Spirit Biotwin with 120 kV/voltage. DLS measurements were carried out on Malvern Zetasizer ZS90 (Malvern Instruments, Ltd.) in aqueous solutions at 25 °C. AFM instrument (Multimode Nanoscope-IIIa Scanning Probe Microscope) from Digital Instruments Co., Ltd, USA was used for AFM imaging by applying TM tapping mode at 300 kHz. The high resolution electrospray ionization mass spectra (HR-ESIMS) were obtained from SolariX 7.0T Fourier

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Transform Ion Cyclotron Resonance Mass Spectrometer (FT-ICR-MS). Confocal Laser

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Scanning Microscopy (CLMS) observations were recorded on a Leica TCS SP8 STED 3X

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Super-resolution multiphoton confocal microscope. Synthesis of HSP and RDL.

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The hyperbranched copolymer (HSP) (DParm≈10) was prepared by previously reported method

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(Experiment detail in SI) (Zhou et al., 2004; 2007). The number-average molar mass (Mn) of the prepared HSP was 8125 Da with the dispersity (Mw/Mn) of 2.5. The molar fraction of the PEO arms (fEO) in HSP was 90.9%. The degree of branching (DB) of HBPO core was determined to

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be 38%. The RDL was synthesized according to the literature reported protocol (Scheme S1) with excellent yield (Dickinson et al., 2010). Briefly, in a round bottom flask the DHBA (4.0 mmol, 1.26 g) and resorcinol (4.0 mmol, 0.44 g) were dissolved using 20 mL of TFA. The

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reaction solution was refluxed at 90 ℃ for 12 hours. After completion of reaction, the solvent was evaporated, and the crude product was purified by silica gel column chromatography using a solvent mixture of dichloromethane, ethyl acetate, and methanol (4.5:4.5:1). The product was further purified by precipitating in ethyl acetate to yield a light reddish brown solid (1.2 g, 3.21 mmol, yield 77%). Synthesis of HSP-RDL. 6

The synthesis of HSP-RDL was performed in two steps as shown in Scheme 2. In the first step, HSP (1.901 g, 3.419 mmol of OH) dissolved in 70 mL of THF was charged in a round bottom flask of 250 mL. TEA (0.95 mL, 6.838 mmol) and butanedioic anhydride (683.8 mg, 6.838 mmol) was then added to the solution. The reaction solution was refluxed at 70 ℃ for 20 hours. The reaction mixture was then cooled to room temperature and precipitated three times using petroleum ether at 30-60 ℃. The product (HSP-COOH) was further dried under vacuum for 24

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hours at room temperature.

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In the second step, HSP-COOH (215.2 mg, 1.43 mmol of COOH) was dissolved in

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dimethylformamide (DMF) (40mL) and charged in a 100 mL round bottom flask. The RDL (184 mg, 0.48 mmol), DCC (294.55 mg, 1.43 mmol) and 4-Dimethylaminopyridine (DMAP) (100

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mg, 0.82 mmol) were then added to the solution and mixture was stirred for 24 hours at room temperature. The solvent was removed under vacuum, and the crude product was purified by

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dialysis (MWCO 3.5 kDa) using DMF as a solvent to remove unreacted reagents. The polymer solution after dialysis was recovered and dried to obtain HSP-RDL. According to GPC the HSP-

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RDL has number-average molar mass (Mn) of 9620 Da with the dispersity (Mw/Mn) of 2.6 (Figure S7).

Self-assembly of HSPs and probe functionalized HSP-RDLs in water.

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Vesicles of HSP were obtained by slowly adding 10 mL of deionized water into 10 mg of HSP polymer in a 50 mL flask for half hour at room temperature. The concentration of polymer solution obtained was 1 mg/mL. Similarly, vesicles of probe functionalized HSP-RDL were prepared in water by slowly adding 10 mL of deionized water in 10 mg of the HSP-RDL polymer and stirred at room temperature for 30 minutes. The concentration of the polymer solution obtained was also 1 mg/mL. 7

The spectral investigation methodology. An aqueous solution of HSP-RDL (10 mg/mL) was taken as stock solution. The stock solutions of various cations (Na+, K+, Ag+, Li+, Mg2+, Ca2+, Ba2+, Cu2+, Zn2+, Ni2+, Co2+, Hg2+, Pd2+, Cd2+, Fe2+, Pb2+ and Fe3+), anions (NO3−, N3−, PO43−, HPO32−, HS−, I−, F−, CN−, Cl−, Br−, CH3COO−, HSO3−, HSO4−, SO42−, S2O32−, SO32-, CO32− and HCO3−), and other compounds (cysteine,

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CH3NH2, diaminopropane, urea, NH2OH, thiourea and ammonia) were prepared in deionized water with the concentrations of 10 mM, respectively. The fluorescence intensities for all the

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solutions were measured from 515 nm to 750 nm with an excitation wavelength, λex = 520 nm,

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and emission slit = 10.0 nm. RESULTS AND DISCUSSION

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Characterization of HSP-RDLs.

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The functionalization of HSP with carboxy groups was then carried out so as to facilitate the subsequent grafting of RDL onto the carboxylate functionalized HSP in the presence of a

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coupling agent DCC. The 1H NMR spectra of HSP-COOH, RDL and HSP-RDL are given in Figure 1 for comparison. Figure 1a shows the chemical resonances of HBPO core (protons A-E) and PEO (protons f-k) which are in good agreement with the protons of the HSP (Jin et al., 2011; 2012). The distinctive signal at 2.43 ppm (b, b’) is assigned to the proton of methylene group of

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butanedioic anhydride. The ratio of HSP functionalization with –COOH was estimated by comparing the integrals of peaks b, b’ and peak A using the relation (Sb,b’/4)/(SA/3) or (3Sb,b’/4SA), which is about 70 %. After grafting with RDL via esterification, the protons b and b’ were shifted to 2.70 and 2.87 ppm (l, l’). Together with the appearance of signals from RDL (δ 8.22 to 6.49 and 1.80 to 1.40 ppm), it confirms that the HSP was successfully functionalized with –COOH group (Figure 1c). The grafting percentage of RDL group is approximately 33.9%, 8

which was calculated by comparing the peak integral of RDL with that of –CH3 (A) from HSP core (Figure 1c) using the relation (SRDL/10)/(SA/3) or (3SRDL/10SA). Self-assembly of HSP-RDLs. Self-assembly HSP-RDL was performed by the previously reported protocol (Zhou et al., 2004; 2007) which is via direct hydration with the polymer concentration of 1 mg/mL. The self-

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assembled structures were characterized by dynamic light scattering (DLS), transmission

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electron microscopy (TEM) and atomic force microscopy (AFM). It confirms that HSP-RDL (Figure 2) self-assembled into vesicles. The TEM image of HSP-RDL in Figure 2a shows a

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vesicular morphology with a clear existence of dark rim. The vesicle size (hydrodynamic diameter, Dh) measured by DLS is ca. 326 nm with PDI of 0.22 (Figure 2b). Similarly, the AFM

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image shows that the vesicles are spherical with an average diameter of 347 nm and a height of

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1.8 nm, and their diameter to height ratio is 1:193. The HSP based vesicles in the range of 300 nm size are stable for at least six months (Tao et al., 2012; Jiang et al., 2015).

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Colorimetric Change of HSP-RDL Vesicle Probe Induced by Hydrazine. The sensing of hydrazine by HSP-RDL vesicle probe (10 mg/mL of HSP-RDL) was first ascertained by mixing with hydrazine (100 M) in deionized water. Before addition of hydrazine, the vesicle solution was colorless, and it showed negligible absorption in the visible

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region (400-700 nm, Figure S8a), while the color turned to pink instantly when hydrazine was introduced into the vesicle solution, which showed an absorption with a maximum at 520 nm (response times is about 5 seconds after addition of hydrazine). This was also confirmed by fluorimetry at an excitation wavelength of 520 nm, which showed little fluorescence before titration and a strong fluorescence with an intensity maximum at 545 nm (Figure S8b). This

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change in optical property is attributed to the ring opening of spirolactone moiety of RDL induced by hydrazine, leading to fluorescein-type spectroscopic characteristic. This was also confirmed by 1H NMR spectroscopy of HSP-RDL polymer before and after the addition of hydrazine (Figure 3). After addition of hydrazine, the resonances of HSP-RDL at 2.7 and 2.85 ppm (protons l and l’ in Figure 3a) disappeared; while a new peak at 2.25 ppm appeared instead, which corresponds to the resulting product HSP-4,5-dihydro-pyridazin-3(2H)-one 2(Geurnik et

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al., 2010; Xu et al., 2006). The HR-ESIMS analysis of HSP-RDL (Figure S9) before and after

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addition of hydrazine (Figure S10) were performed. The molecular ion of cleavable RDL [M + H]+ m/z 388.1531 (calcd. 388.1543) is observed in the presence of hydrazine (Figure S12) to

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support the reaction mechanism. In order to study the sensitivity of HSP-RDL probe for

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hydrazine, the titration was carried out with various concentrations of hydrazine. Into a vesicle solution of HSP-RDL (10 mg/mL) was introduced hydrazine (0-50 μM), and the color change

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was monitored by fluorometry with an excitation wavelength of 520 nm. Figure 4a presents the stepwise increase in fluorescence maximum at 545 nm with the increasing concentration of

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hydrazine from 0 to 50 μM. When the concentration of hydrazine was below 12 μM, the fluorescence intensity increased linearly, followed by a steady but slower increase when the concentration was higher than 12 μM (Figure 4b). The linear trend (inset Figure 4b) with hydrazine (0-12 μM) was used for LOD assessment. The LOD was determined by using the

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relationship 3SD/s (SD and s correspond to the standard deviation and the slope, respectively). The LOD was as low as 2 nM, which is significantly lower compared to other RDL or levulinate based single-molecule hydrazine detection probes (summarized in Table S1) and TLV (10 ppb) given by USEPA. The UV-Visible experiment were also conducted to estimate the detection performance of HSP-RDL for hydrazine by gradual addition of hydrazine (0-50 μM) in vesicle

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solution of HSP-RDL (10 mg/mL) as shown in Figure S11a and 11b. Moreover, the LOD was calculated by using the relationship 3SD/s of the linear curve fitting (Figure S11c) and it was 2.3 nM which is in well agreement to the fluorescence experiment. The little variation in LOD of UV-vis compared to fluorescence (i.e., 2nM) may be due to human error during experiment execution.

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pH and Temperature Effect on Hydrazine Detection by HSP-RDL.

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The pH conditions of HSP-RDL aqueous solutions were determined to be important for

detection of hydrazine. To study the pH effect, fluorescence of HSP-RDL (10 mg/mL) in the

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presence of hydrazine (10 M) were obtained at different pH values 2.0 to 12.0 as shown in Figure S12. No significant change in the fluorescence intensity was observed in the pH range of

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6.0-12.0. However, at pH≤5.0 the fluorescence intensity is decreased. The HSP-RDL, therefore

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can detect hydrazine efficiently in relatively wide range of pH from 6.0-12.0 especially the physiological pH range i.e. 6.0-9.0. The fluorescence emission intensities trend for reaction of

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HSP-RDL (10 mg/mL) solution with hydrazine (10 M) at different temperatures from 20-60 (°C) is shown in Figure S13. It is clear that fluorescence intensities decreases slightly from 20 to 50 (°C). Therefore, HSP-RDL probe can work efficiently in this temperature range. Selectivity of HSP-RDL for the Detection of Hydrazine.

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The HSP-RDL (10 mg/mL) was subjected to selectivity study against various analytes (10 M) such as cations (Na+, K+, Ag+, Li+, Mg2+, Ca2+, Ba2+, Cu2+, Zn2+, Ni2+, Co2+, Hg2+, Pd2+, Cd2+, Fe2+, Pb2+ and Fe3+), anions (NO3−, N3−, PO43−, HPO32−, HS−, I−, F−, CN−, Cl−, Br−, CH3COO−, HSO3−, HSO4−, SO42−, S2O32−, SO32-,CO32− and HCO3−), and other amines (cysteine, CH3NH2, diaminopropane, urea, NH2OH, thiourea and ammonia) as shown in Figure 5. The obtained 11

results suggest that the HSP-RDL is highly selective toward hydrazine among miscellaneous species. The observation revealed no substantial change in emission intensity of HSP-RDL for hydrazine when various species were present. Similar competitive species response was observed by UV-vis spectroscopy (Figure S14). Hence, it can be concluded that HSP-RDL can be considered as non-competitive recognition probe for hydrazine.

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In-Field Application Prospective of HSP-RDL.

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The chemical sensors for hydrazine generally work in solvents and require special equipment for detection which is inconvenient. For on-site analysis of hydrazine, however, it is practical if the

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HSP-RDL probe can be applied with test strip for naked eye detection. In the current study the HSP-RDL test strips were prepared by immersing the filter papers (dia. 2.5 cm) with HSP-RDL

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(10 mg/mL) solution and then dried in open air. The amount of HSP-RDL (ca. 0.2 mmol of

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RDL group) retained by filter paper was determined from the known parameters; volume of HSP-RDL solution consumed, the average molar mass of HSP-RDL and grafting percent of RDL. As the drops of the hydrazine solutions were loaded to the test strips, quick color change

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(in seconds) was observed. Figure 6 presents the change in colors of test strips (inset figure) from almost colorless to pink and corresponding absorbance curves when the concentration of hydrazine was increased (0-40 μM). Such a fast and clear response to hydrazine by change in

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color makes HSP-RDL test strip a promising tool for in-field application. Real Time Sensing of Hydrazine. The amount of hydrazine in the water samples taken from the Huangpu River in Shanghai city was monitored in real time. Before quantification, the standard curve was plotted (absorbance versus concentration, Figure S15) by adding a series of hydrazine solutions with various

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concentrations (0-5 μM) to the solution of HSP-RDL (10 mg/ mL). Raw water samples were passed through a microfiltration membrane prior to the quantification experiments. The HSPRDL (10 mg/mL) was added directly to prepare the aliquots of raw water samples. No significant absorbance was noticed. On the other hand, when the same samples were treated with the hydrazine solution with known concentration, a significant enhancement of absorption maximum was observed, which was used for the quantification of hydrazine by comparing it

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with the standard curve. The results demonstrate the estimation (recovery %) was ≥ 99 %. (Table

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1). It confirms the HSP-RDL vesicle sensor can be used as an effective tool for environmental

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monitoring of hydrazine at trace levels. Detection of Hydrazine in Live Cells.

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To investigate the potential biological application of HSP-RDL for the detection of hydrazine in

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living cells, A549 human lung cancer cells were employed for CLMS study (experimental details are provided in SI) as shown in Figure 7. There was no fluorescence emission for A549 cells incubated with HSP-RDL (1 mg/mL) for 1 h at 37 °C (Figure 7b). While a distinguishing

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intracellular fluorescence emission was witnessed for A549 cells incubated with HSP-RDL (1 mg/mL) for 0.5 h followed by treatment with hydrazine (50 μM) for 0.5 h (Figure 7e). These investigations revealed that HSP-RDL is capable for visual detection of hydrazine in live cells

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by membrane permeation. Furthermore, the cytotoxicity of HSP-RDL was evaluated by using conventional MTT assay (Supporting Information Figure S16). It can be seen from the results that the viability of the cells is more than 82 %. upon exposure to a 50 μM concentration of HSP-RDL for 24 h. This ruled out the possibility of any substantial cytotoxic influence of the probe on A549 cells. CONCLUSIONS 13

Conclusively, an efficient chemical sensor for hydrazine detection with its application in environmental monitoring in aqueous medium was developed. The HSP-RDL vesicle probe displays high sensitivity and selectivity with real-time "turn-on" fluorometric response towards hydrazine recognition in aqueous solution, which is due to fluorescein-type spirolactone ring opening mechanism. The spatial display of multivalent RDL fluorophore on the surface of the vesicle makes the self-assembled probe a highly sensitive chemical sensor for hydrazine with

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LOD of 2 nM. Additionally, the HSP-RDL probe was successfully applied to monitor hydrazine

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in raw water samples and live cells.

Author Contribution

Declaration of interests

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Both the authors F.N. and T.R. have contributed equally

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

ASSOCIATED CONTENT

Supporting Information. Supplementary figures and experimental data showing the

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characterization of materials and analysis of the analytes.

Acknowledgement Authors are grateful to the State key laboratory of metal matrix composites, Shanghai Jiao Tong University, Shanghai 200240, China for providing the experimental facilities.

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Notes Authors declare that they do not have a conflict of interest in any capacity including competing

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or financial.

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REFERENCES Serov, A.; Padilla, M.; Roy, A. J.; Atanassov, P.; Sakamoto, T.; Asazawa, K.; Tanaka, H. Anode Catalysts for Direct Hydrazine Fuel Cells: from Laboratory Test to an Electric Vehicle. Angew. Chem. Int. Ed. 2014, 53, 10336–10339. Matsumoto, M.; Kano, H.; Suzuki, M.; Katagiri, T.; Umeda, Y.; Fukushima, S. Carcinogenicity and Chronic Toxicity of Hydrazine Monohydrate in Rats and Mice by Two-Year Drinking Water Treatment. Regul. Toxicol. Pharmacol. 2016, 76, 63–73.

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Fisher, J. W.; Harrah, C. B.; Berry, W. O. Hydrazine: Acute Toxicity to Bluegills and Sublethal Effects on Dorsal Light Response and Aggression. Tran. Am. Fish. Soc. 1980, 109, 304-309.

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Reja, S. I.; Gupta, N.; Bhalla, V.; Kaur, D.; Arora, S.; Kumar, M. A Charge Transfer Based Ratiometric Fluorescent Probe for Detection of Hydrazine in Aqueous Medium and Living Cells. Sensor Actuat. B Chem. 2016, 222, 923–929.

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Yu, G. Cao, Y. Liu, H.Wu, Q. Hu, Q. Jiang, B. Yuan Z. A spirobenzopyran-based multifunctional chemosensor for the chromogenic sensing of Cu2+ and fluorescent sensing of hydrazine with practical applications. Sensors and Actuators B 245 (2017) 803–814.

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Channon, R. B.; Joseph, M. B.; Bitziou, E.; Bristow, A. W. T.; Ray, A. D.; Macpherson J. V. Electrochemical Flow Injection Analysis of Hydrazine in an Excess of an Active Pharmaceutical Ingredient: Achieving Pharmaceutical Detection Limits Electrochemically. Anal. Chem. 2015, 87, 10064-10071.

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Huang, Y.; Zhang, W.; Wang, J.; Wei. Z. Probing the Sensory Property of Perylenediimide Derivatives in Hydrazine Gas: Core-Substituted Aromatic Group Effect. ACS Appl. Mater. Interfaces 2014, 6, 9307-9313.

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Kosyakov, D. S.; Amosov, A. S.; Ul'yanovskii, N. V.; Ladesov, A. V.; Khabarov, Y. G.; Shpigun, O. A. Spectrophotometric Determination of Hydrazine, Methylhydrazine, and 1,1Dimethylhydrazine with Preliminary Derivatization by 5- Nitro-2-Furaldehyde. J. Anal. Chem. 2017, 72, 171–177.

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Tiensomjitr, K.; Noorat, R.; Chomngam, S.; Wechakorn, K.; Prabpai, S.; Kanjanasirirat, P.; Pewkliang, Y.; Borwornpinyo, S.; Kongsaeree, P. A Chromogenic and Fluorogenic RhodolBased Chemosensor for Hydrazine Detection and its Application in Live Cell Bioimaging. Spectrochim. Acta. A Mol. Biomol. Spectrosc. 2018, 195, 136–141.

Shia, X. Yin, C. Zhang, Y. Wen, Y. Huo F. A novel ratiometric and colorimetric fluorescent probe for hydrazine based on ring-opening reaction and its applications. Sensors & Actuators: B. Chemical 285 (2019) 368–374 Li, Z. Ren, M. Wang L. and Lin W.. An ethyl cyanoacetate based turn-on fluorescent probe for hydrazine and its bio-imaging and environmental applications. Anal. Methods, 2018, 10, 4016–4019.

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Zhu, S. Lin W. and Yuan L. Development of a near-infrared fluorescent probe for monitoring hydrazine in serum and living cells. Anal. Methods, 2013, 5, 3450–3453 Kong, X.Dong, B. Wang, C. Zhang, N. Song, W. Lin, W. A novel mitochondria-targeted fluorescent probe for imaging hydrazine in living cells, tissues and animals. JournalofPhotochemistryandPhotobiologyA:Chemistry 356 (2018)321–328 Liu, C. Liu, K.Tian, M. Lin. W. A ratiometric fluorescent probe for hydrazine detection with large fluorescence change ratio and its application for fluorescence imaging in living cells. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 212 (2019) 42–47

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Rasheed, T. Nabeel, F. Li, C. Zhang,Y. Rhodol assisted alternating copolymer based chromogenic vesicles for the aqueous detection and quantification of hydrazine via switch-on strategy Journal of Molecular Liquids 274 (2019a) 461–469.

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Tang, Y.; Lee, D.; Wang, J.; Li, G.; Yu, J.; Lin, W.; Yoon, J. Development of Fluorescent Probes Based on Protection-Deprotection of the Key Functional Groups for Biological Imaging. Chem. Soc. Rev. 2015, 44, 5003–5015.

-p

Li, X.; Gao, X.; Shi, W.; Ma, H. Design Strategies for Water-Soluble Small Molecular Chromogenic and Fluorogenic Probes. Chem. Rev. 2014, 114, 590–659.

re

Ju, Z.; Li, D.; Zhang, D.; Li, D.; Wu, C.; Xu, Z. An ESIPT-Based Fluorescent Probe for Hydrazine Detection in Aqueous Solution and its Application in Living Cells. J. Fluoresc. 2017, 27, 679–687.

lP

Yu, S.; Wang, S.; Yu, H.; Feng, Y.; Zhang, S.; Zhu, M.; Yin, H.; Meng, X. A Ratiometric TwoPhoton Fluorescent Probe for Hydrazine and its Applications. Sens. Actuat. B Chem. 2015, 220, 1338–1345.

ur na

Zhu, S.; Lin, W.; Yuan, L. Development of a Near-Infrared Fluorescent Probe for Monitoring Hydrazine in Serum and Living Cells. Anal. Methods 2013, 5, 3450–3453. Choi, M. G.; Hwang, J.; Moon, J. O.; Sung, J.; Chang, S. K. Hydrazine-Selective Chromogenic and Fluorogenic Probe Based on Levulinated Coumarin. Org. Lett. 2011, 13, 5260–5263.

Jo

Rasheed, T. Nabeel, F. Bilal, M. Rhodamine-assisted fluorescent strategy for the sensitive and selective in-field mapping of environmental pollutant Hg(II) with potential bioimaging. Journal of Luminescence. 2019b, 208, 519–526. Rasheed, T. Nabeel, F. Adeel, M. Bilal, M. and Iqbal, H. M. N. Aqueous monitoring of toxic mercury through a rhodamine-based fluorescent sensor. Mathematical Biosciences and Engineering, 2019c, 16(4): 1861–1873. Rasheed, T. Nabeel, F. Adeel, M. Bilal, M. and Iqbal, H. M. N. Turn-on” fluorescent sensorbased probing of toxic Hg(II) and Cu(II) with potential intracellular monitoring. Biocatalysis and Agricultural Biotechnology 17 (2019d) 696–701

17

Rasheed, T. Nabeel, F. Shafi, S. Chromogenic Vesicles for Aqueous Detection and Quantification of Hg2+/Cu2+ in Real Water Samples. Journal of Molecular Liquids. 2019e, 282 489–498 Rasheed, T.; Li, C.; Nabeel, F.; Huang, W.; Zhou, Y. Self-Assembly of Alternating Copolymer Vesicles For The Highly Selective, Sensitive and Visual Detection and Quantification of Aqueous Hg2+. Chem. Eng. J. 2019f, 358, 101–109. Rasheed, T. Li, C. Nabeel, F. Qi, M. Zhang, Y. and Yu, C. Real-time probing of mercury using an efficient “turn-on” strategy with potential in-field mapping kit and in live cell imaging. New J. Chem., 2018a,42, 10940-10946

ro

of

Rasheed, T. Li, C. Fu, L. Nabeel, F. Yu, C. Gong, L. Zhou. Y. Development and characterization of newly engineered chemosensor with intracellular monitoring potentialities and lowest detection of toxic elements. Journal of Molecular Liquids 272 (2018b) 440–449

-p

Chapman, R.; Lin, Y.; Burnapp, M.; Bentham, A.; Hillier, D.; Zabron, A.; Khan, S.; Tyreman, M.; Stevens M. M. Multivalent Nanoparticle Networks Enable Point-of-Care Detection of Human Phospholipase-A2 in Serum. ACS Nano 2015, 9, 2565–2575.

re

Zheng, H.; Li, X.; Jia, Q. Design of pH-Responsive Polymer Monolith Based on Cyclodextrin Vesicle for Capture and Release of Myoglobin. ACS Appl. Mater. Interfaces 2018, 10, 5909–5917.

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Kaushik, R.; Sakla, R.; Ghosh A,; Selvan, G.T.; Selvakumar, P.M.; Jose, D.A. Selective Detection of H2S by Copper Complex Embedded in Vesicles through Metal Indicator Displacement Approach. ACS Sens. 2018, 3, 1142−1148.

ur na

Huang, T.; Hou, Z.; Xu, Q.; Huang, L.; Li, C.; Zhou, Y. Polymer Vesicle Sensor For Visual And Sensitive Detection of SO2 in Water. Langmuir 2017, 33, 340−346. Hou, Z. L.; Huang, T.; Cai, C. Y.; Rasheed, T.; Yu, C. Y.; Zhou, Y.; Yan, D. Y. Polymer Vesicle Sensor Through the Self-Assembly of Hyperbranched Polymeric Ionic Liquids for the Detection of SO2 Derivatives. Chin. J. Polym. Sci. 2017, 35, 602.

Jo

Chen, H. W.; Fang, Z. S.; Chen, Y. T.; Chen, Y. I.; Yao, B. Y.; Cheng, J. Y.; Chien, C. Y.; Chang, Y. C.; Hu C. M. J. Targeting and Enrichment of Viral Pathogen by Cell Membrane Cloaked Magnetic Nanoparticles for Enhanced Detection. ACS Appl. Mater. Interfaces 2017, 9, 39953−39961. Rasheed, T. Nabeel, F. Bilal, M. Self-Assembly of Artificial Peroxidase Mimics from Alternating Copolymers with Potential Chromogenic and Biocatalyst Potentialities. Journal of Industrial and Engineering Chemistry 78 (2019g) 315–323. Tanner, P.; Ezhevskaya, M.; Nehring, R.; Van Doorslaer, S.; Meier, W.; Palivan, C. Specific His6-Tag Attachment to Metal-Functionalized Polymersomes Relies on Molecular Recognition. J. Phys. Chem. B 2012, 116, 10113–10124.

18

Zhou, Y.; Yan, D. Supramolecular Self-Assembly of Amphiphilic Hyperbranched Polymers at All Scales and Dimensions: Progress, Characteristics, and Perspectives. Chem. Commun. 2009, 10, 1172−1188. Whitaker, J. E.; Haugland, R. P.; Ryan, D.; Hewitt, P. C.; Haugland, R.P.; Prendergast, F. G. Fluorescent Rhodol Derivatives: Versatile, Photostable Labels and Tracers. Anal. Biochem. 1992, 207, 267–279. Kamiya, M.; Asanuma, D.; Kuranaga, E.; Takeishi, A.; Sakabe, M.; Miura, M.; Nagano, T.; Urano Y. β-Galactosidase Fluorescence Probe with Improved Cellular Accumulation Based on a Spirocyclized Rhodol Scaffold. J. Am. Chem. Soc., 2011, 133, 12960–12963.

ro

of

Jung, S. H.; Jang, H.; Lim, M. C.; Kim, J. H.; Shin, K. S.; Kim, S. M.; Kim, H. Y.; Kim, Y. R.; Jeon, T. J. Chromatic Biosensor for Detection of Phosphinothricin Acetyltransferase by Use of Polydiacetylene Vesicles Encapsulated within Automatically Generated Immune Hydrogel Beads. Anal. Chem. 2015, 87, 2072−2078.

-p

Yan, Q.; Sang, W. H2S Gasotransmitter-Responsive Polymer Vesicles. Chem. Sci. 2016, 7, 2100−2105.

re

Zhou, Y.; Yan, D. Supramolecular Self‐ Assembly of Giant Polymer Vesicles with Controlled Sizes. Angew. Chem. 2004, 116, 5004–5008; Angew. Chem. Int. Ed. 2004, 43, 4896–4899.

lP

Zhou, Y.; Yan, D.; Dong, W.; Tian, Y. Temperature-Responsive Phase Transition of Polymer Vesicles:  Real-Time Morphology Observation and Molecular Mechanism. J. Phys. Chem. B 2007, 111, 1262–1270.

ur na

Dickinson, B. C.; Huynh, C.; Chang, C. J. A Palette of Fluorescent Probes with Varying Emission Colors for Imaging Hydrogen Peroxide Signaling in Living Cells. J. Am. Chem. Soc. 2010, 132, 5906–5915. Jin, H.; Zheng, Y.; Liu, Y.; Cheng, H.; Zhou, Y.; Yan, D. Reversible and Large-Scale Cytomimetic Vesicle Aggregation: Light-Responsive Host–Guest Interactions. Angew. Chem. Int. Ed. 2011, 50, 10352–10356. Jin, H.; Liu, Y.; Zheng, Y.; Huang, W.; Zhou, Y.; Yan D. Cytomimetic Large-Scale Vesicle Aggregation and Fusion Based on Host-Guest Interaction. Langmuir 2012, 28, 2066-2072.

Jo

Tao, W.; Liu, Y.; Jiang, B.; Yu, S.; Huang, W.; Zhou Y.; Yan, D. A Linear-Hyperbranched Supramolecular Amphiphile and Its Self-Assembly into Vesicles with Great Ductility, J. Am. Chem. Soc. 2012, 134, 762–764. Jiang, W.; Zhou, Y.; Yan, D. Hyperbranched Polymer Vesicles: from Self-assembly, Characterization, Mechanisms, and Properties to Applications. Chem. Soc. Rev. 2015, 44, 3874–3889. Geurink, P. P.; Florea, B. I.; Li, N.; Witte, M. D.; Verasdonck, J.; Kuo, C. L.; van der Marel, G. A.; Overkleeft, H. S. A Cleavable Linker Based on the Levulinoyl Ester for Activity-Based Protein Profiling. Angew. Chem. Int. Ed. 2010, 49, 6802–6805. 19

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ro

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Xu, H.; Zou, X. M.; Zhu, Y. Q.; Liu, B.; Tao, H. L.; Hu, X. H.; Song, H. B.; Hu, F. Z.; Wang, Y.; Yang, H. Z. Synthesis and Herbicidal Activity of Novel α,α,α- Trifluoro-m-Tolyl Pyridazinone Derivatives. Pest Manag. Sci. 2006, 62, 522–530.

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Scheme 1. Vesicle Probe Self-assembled by Fluorescent RDL-Conjugated Hyperbranched Copolymer (HSP-RDL) and Its Sensing of Hydrazine. In HSP and HSP-RDL the red, blue and grey color shows the hydrophobic core, hydrophilic arms, and the functionalized probe respectively. In vesicles, the grey color probe (1) is detached by hydrazine attack and turns into pink color (3).

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Scheme 2. The synthesis of HSP-RDL by esterification reaction from RDL and HSP-COOH produced from carboxylation of HSP. The percent yields of HSP-COOH and HSP-RDL were approximately 85 and 73 % respectively.

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Figure 1. 1H NMR spectra of (a) HSP-COOH (b) RDL and (c) HSP-RDL in DMSO-d6.

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Figure 2. Characterization of self-assembled HSP-RDL. (a) TEM image, (b) DLS histogram, (c)

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AFM image and (d) height profile of self-assembled vesicles of HSP-RDL. Figure 3. 1H NMR spectra of HSP-RDL in DMSO-d6 (a) before and (b) after addition of hydrazine

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(hydrazine / HSP-RDL = 1:1 molar).

Figure 4. (a) Fluorescence titration of HSP-RDL (10 mg/ mL) and hydrazine (0-50 M) in water

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at excitation wavelength of 520 nm, (b) plot of intensity versus hydrazine concentration with linear curve fitting (inset).

Figure 5. (a) Fluorescence emission spectra of aqueous HSP-RDL (10 mg/mL) solution at

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excitation wavelength of 520 nm in the existence of various analytes (10 M) and hydrazine (10 M). (b) Represents different cations (blue bars) , (c) anions (green bars) and (c) other amines (grey bars) before and after introduction of hydrazine (red bar) versus fluorescence intensities of HSP-RDL + other ions, HSP-RDL + hydrazine + all ions (black bar) and free HSP-RDL, respectively: 1) HSP-RDL, 2) HSP-RDL + hydrazine + all ions (black bar), 3) Na+, 4) K+, 5) Ag+, 6) Li+, 7) Mg2+, 8) Ca2+, 9) Ba2+, 10) Cu2+, 11) Zn2+, 12) Ni2+, 13) Co2+, 14) Hg2+, 15) Pd2+, 16) 21

Cd2+, 17) Fe2+, 18) Pb2+, 19) Fe3+, 20) NO3−, 21) N3−, 22) PO43−, 23) HPO32−, 24) HS−, 25) I−, 26) F−, 27) CN−, 28) Cl−, 29) Br−, 30) CH3COO−, 31) HSO3−, 32) HSO4−, 33) SO42−, 34) S2O32−, 35) SO32−, 36) CO32−, 37) HCO3−, 38) cysteine, 39) CH3NH2, 40) diaminopropane, 41) urea, 42) NH2OH and 43) thiourea, 44) Ammonia. Figure 6. Colorimetric responses of HSP-RDL test papers (inset figure) to hydrazine and

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corresponding absorbance curves with different concentrations of hydrazine.

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Figure 7. Confocal fluorescence images of A549 cells for hydrazine detection in cellular environment. (a-c) A549 cells incubated with HSP-RDL (1 mg/mL) for 1 h; (d-f) images of cells

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treated with HSP-RDL (1mg/ mL) for 0.5 h followed by treatment with hydrazine (50 μM) for 0.5 h. (a) and (d) are the fluorescence images of the cells nuclei counterstained by Hoechst 33342

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(excitation wavelength = 360-400 nm, emission wavelength = 410-480 nm). (b) and (e) are the

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images of the cells in fluorescence field (excitation wavelength = 520-550 nm, emission wavelength = 560-630 nm) and (c and f) are the merged images of (a,b) and (d,e). Scale bar: 50

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μm.

Scheme 1 22

j OH k

A

BC

O

g

i'

O

O

i

f

O

b'

O

OH

b

O

E,k

C,D

1

(b)

3

N

6

6 O

1

O

OH

O

O

9

O O

8

O

7

DMSO

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OH

O

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2

7

5

10

OH

8

O

(c)

5

4

O

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2

8 3,4

7

b,b' A B

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f

DMSO

O

O

g,h,i,i',j

i'

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D OH

h

O

i

E

DMSO

(a)

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Scheme 2

O O

l'

O O

O

l

O

N

l,l'

6

5

(ppm)

Figure 1

23

4

3

1

0

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Figure 2

(a) OH

O

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OH O

DMSO

O

O

O

O

O

O

l'

O

O

O

l

O

N

O

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1

l,l'

(b)

OH

O

OH

O

O

OH

O

O

O

O N

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2

10

9

8

7

N H

DMSO

O

e o O

O

O

e,o

N

3

6

5

(ppm)

Figure 3

24

4

3

1

0

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Figure 5

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Figure 6

Figure 7 26

Table 1. N2H4 determination in raw water samples from Huangpu River. Hydrazine Recovered

0

Not detected

-

1

0.99 ± 0.014

99

2

1.99 ± 0.013

99.5

3

2.96 ± 0.016

99.6

4

3.97 ± 0.12

99.3

5

4.95 ± 0.11

99

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(μM)

Percentage Recovery (%)

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Huangpu River

Hydrazine Introduced (μM)

27