A novel red-emissive probe for colorimetric and ratiometric detection of hydrazine and its application in plant imaging

Journal Pre-proof A novel red-emissive probe for colorimetric and ratiometric detection of hydrazine and its application in plant imaging Qiuqin Lai, Shufan Si, Tianyi Qin, Benjie Li, Hanxiang Wu, Bin Liu, Hanhong Xu, Chen Zhao

PII:

S0925-4005(19)31839-8

DOI:

https://doi.org/10.1016/j.snb.2019.127640

Reference:

SNB 127640

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

12 November 2019

Revised Date:

23 December 2019

Accepted Date:

27 December 2019

Please cite this article as: Lai Q, Si S, Qin T, Li B, Wu H, Liu B, Xu H, Zhao C, A novel red-emissive probe for colorimetric and ratiometric detection of hydrazine and its application in plant imaging, Sensors and Actuators: B. Chemical (2019), doi: https://doi.org/10.1016/j.snb.2019.127640

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A novel red-emissive probe for colorimetric and ratiometric detection of hydrazine and its application in plant imaging Qiuqin Lai,‡a Shufan Si,‡b Tianyi Qin,a, b Benjie Li,a Hanxiang Wu,a Bin Liu,b* Hanhong Xu,a* and Chen Zhaoa*

a

State Key Laboratory for Conservation and Utilization of Subtropical

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Agro-Bioresources and Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, 510642,

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China

Shenzhen Key Laboratory of Polymer Science and Technology, College of Materials

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Science and Engineering, Shenzhen University, Shenzhen 518060, China.

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‡These two authors contribute equally to this work.

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*Corresponding author:

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B. Liu: [email protected], H. Xu: [email protected], C. Zhao: [email protected]

Graphical abstract

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A novel red-emissive fluorescence probe was designed for colorimetric and

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ratiometric detection of hydrazine. 

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Highlights

This probe could rapidly, sensitively, and selectively respond to hydrazine with a distinct emission color change from red to cyan. The sensing mechanism was carefully studied based on DFT, HRMS and NMR spectra.

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This fluorescent probe was successfully employed for imaging of hydrazine in

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plants.

Abstract

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With the wide application in chemistry and pharmaceutical industries, the toxic effect of hydrazine on the ecological environment (e.g. inhibition on seed germination and seedling growth) as well as human health (carcinogen) arises extensive concerns. Although a lot of probes have been developed and used for fluorescence imaging of hydrazine in living cells and animals, to our best knowledge, there are rare reports on hydrazine probe successfully applied for

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plants imaging. In this work, we report a novel red-emissive fluorescent probe with hydrazine-sensitive dicyanovinyl group for colorimetric and ratiometric

detection of hydrazine. This newly reported probe was capable of rapidly,

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sensitively, and selectively detecting hydrazine with a low detection limit (2.88

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imaging of hydrazine in plant roots.

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ppb), and more importantly, this probe achieved the ratiometric fluorescence

Keywords: fluorescent probes, hydrazine, ratiometric, bioimaging, brassica

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chinensis

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1. Introduction

Since 1940s, hydrazine (N2H4) has been applied in the propellant of onboard

space vehicles, including the rockets, missiles, and fighter jets, due to its high heat of combustion and high gas production.[1] Nowadays, hydrazine with the high reducibility and alkalinity has also been widely used as the synthetic precursor to 3

various of insecticides, pharmaceuticals, blowing agents, textile dyestuff, and polymers industries.[2, 3] However, hydrazine itself can cause a serious hazard to ecological environment and human health. The toxic effects of hydrazine on human health are embodied as the irritation of eyes, temporary blindness, liver and kidney damage. Furthermore, since hydrazine can react with endogenous formaldehyde and produce the DNA-methylation agent,[4] hydrazine has also

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been categorized by the U.S. Environmental Protection Agency (EPA) and the European Commission’s Scientific Committee on Occupational Exposure Limits

(SCOEL) as the genotoxic carcinogen. In considering of its high toxicity, a

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threshold limit value for hydrazine in environment was set to be as low as 10

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ppb.[5]

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Development of analytical methods for sensitive and selective detection of hydrazine in environmental and biological samples has attracted increasing

(GC),[6]

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attentions. Traditional analytical methods, including gas chromatography high-performance

liquid

chromatography

(HPLC),[7]

and

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electrochemical analysis,[8] are capable of detecting hydrazine in environment

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with the satisfied sensitivity and accuracy, but these methods often become inadequate under the specific conditions of analysis associated with hydrazine in biological samples. Owing to non-invasiveness, fast response, high temporal resolution, and good biocompatibility, the fluorescent method by utilizing small organic fluorophores has been considered as a powerful analytical tool for biological application.[9-11] To date, a great number of fluorescent probes for 4

hydrazine have been designed and recently were comprehensively reviewed by Liu and Bandyopadhyay. [12-51] As part of our efforts to develop the hydrazine probe,[52, 53] recent progress revealed that the fluorescent probes were capable of tracking hydrazine in living cells and living animals.

In fact, since the accidental discharge of hydrazine from related industries

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into environmental water system occasionally occurs, the crops and plants grown in polluted water will bear the brunt of it. The study by Wang, et. al. has demonstrated that the excessive concentration of hydrazine could effectively

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inhibit the germination of seeds and seedling growth, and lead to the wilting of

leaves.[54] Except for the hydrazine-induced crops output reduction, residual

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hydrazine in plants may also be accumulative through the food chain to an extent

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that become harmful to animals even human health. Thus, it is of particular importance to study the absorption and distribution of hydrazine in plants.

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However, to our best knowledge, there are very rare reports [55] on hydrazine

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probes applied for plants imaging.

In this work, we report a novel red-emissive fluorescent probe 1 based on

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hydrazine-sensitive dicyanovinyl group for colorimetric and ratiometric detection of hydrazine (Scheme 1). This newly reported probe could rapidly, sensitively, and selectively respond to hydrazine with a distinct fluorescence color change from red to cyan. Importantly, this probe was successfully applied for ratiometric fluorescence imaging of hydrazine in plant roots. 5

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Scheme 1 Probe 1 for ratiometric fluorescent detection of hydrazine.

2. Experimental Section

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2.1 Materials and instruments

All chemicals, including reactants, catalysts, and solvents, were purchased from

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Energy Chemical China without further purification. The 1H NMR and

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C NMR

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spectra were obtained on a Bruker AVANCE III 600-MHz spectrometer. Chemical shifts were reported in parts per million using tetramethylsilane as the internal

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standard. Fluorescence quantum yields were measured by HAMAMATCU Quantaurus-QY. The high-resolution mass spectra (HRMS) were measured by

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Thermo-Fisher Q Exactive spectrometer (ESI-TOF). UV-Vis absorption and

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fluorescence spectra were measured by the Thermo-Fisher Evolution 220 and Thermo-Fisher Lumina fluorometer, respectively. The fluorescence images were carried out using a confocal fluorescent microscopy (Leica TCS SP8 STED 3X).

2.2 Synthesis and characterization

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Synthesis of compound 2. Compound 3 (1 mmol), Pd(PPh3)4 (0.1 mmol), K2CO3 (6 mmol), and 2-(4-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (4 mmol) were placed into a Schlenk flask. Under a nitrogen atmosphere, 20 mL of toluene and 5 mL of methanol were added into the flask, and the mixture was stirred at 80 oC for 24 h. After the reaction was completed, the metal ions were removed by filtration, and the solvents was evaporated under vacuum. The crude product was

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purified by column chromatography using hexane/CH2Cl2 (v/v = 3/1). Yield: 89%. H-NMR (600 MHz, DMSO-d6): δ (ppm) 9.96 (s, 2H), 7.83 (d, 4H, J = 8.4), 7.34 (d,

4H, J = 8.4), 7.25 (d, 4H, J = 9.0), 7.06 (d, 4H, J = 9.0), 6.77 (s, 2H), 3.82 (s, 6H).

C-NMR (150 MHz, DMSO-d6): δ (ppm) 192.9, 159.6, 148.3, 144.1, 135.7, 132. 2,

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130.9, 130.2, 128.5, 127.9, 116.2, 115.1, 114.6, 100.0, 55.6.

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Synthesis of compound 1. Compound 2 (0.2 mmol), malononitrile (2 mmol), and 5 drops of pyridine were added into 20 mL of CHCl3/toluene mixture (v/v = 3/1).

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The mixture was firstly stirred at room temperature for 6 h, and then gradually was heated to 60 oC for another 6 h reaction. After the reaction was completed, the mixture

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was washed with brine and water for 3 times, dried over anhydrous Na2SO4, filtered,

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and concentrated under vacuum. The crude product was purified by column chromatography using hexane/CH2Cl2 (v/v = 2/1 ~ 1/1) as eluent. Yield: 89%. 1

H-NMR (600 MHz, DMSO-d6/D2O (v/v = 9:1)): δ (ppm) 8.49 (s, 2H), 7. 90 (d, 4H, J

= 8.4), 7.83 (d, 4H, J = 8.4), 7.26 (d, 4H, J = 9.0), 7.08 (d, 4H, J = 9.0), 6.83 (s, 2H), 3.82 (s, 6H).

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C-NMR (150 MHz, DMSO-d6): δ (ppm) 161.1, 160.3, 149.8, 145.5,

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133.2, 132.1, 131.7, 131.5, 130.1, 129.9, 115.4, 115.3, 114.4, 82.8, 56.7. HRMS (m/z): calculated for [M + H]+: 571.21395, found: 571.21338.

Synthesis of compound 1-N2H4. Compound 1 (0.1 mmol) and hydrazine (5 mmol) were added into 10 mL of DMSO/acetone mixture (v/v = 1/1). The mixture was stirred at room temperature for 8 h. The mixture was added by 30 mL of DCM,

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then washed with brine and water for 3 times, dried over anhydrous Na2SO4, and concentrated under vacuum. The product was obtained without any further

purification. Yield: 96%. 1H-NMR (500 MHz, DMSO-d6): δ (ppm) 7.70 (s, 2H), 7.43

3.87 (s, 6H).

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(d, 4H, J = 8.5), 7.27 (d, 4H, J = 9.0), 7.11 (m, 8H), 6.88 (s, 4H, -NH), 6.68 (s, 2H),

C-NMR (125 MHz, DMSO-d6): δ (ppm) 159.3, 143.3, 141.5, 136.3,

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503.24415, found: 503.24362.

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132.1, 131.7, 127.9, 125.6, 125.5, 114.4, 56.5. HRMS (m/z): calculated for [M + H]+:

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2.3 Growth conditions of plants and fluorescent imaging

Chinese cabbage (Brassica chinensis) seeds were purchased from Guangzhou

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Changhe Seed Co., Ltd., China. These seeds were germinated in nutrient soil

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(purchase from Dutch JIFFY TREF) in the flowerpot (10 X 10 cm), which was placed in the plant growth chambers with a 16-h-light/8-h-dark regime at 20 oC. Moderated amount of water was administered.[56] For the root segments imaging, 5-day-old Brassica chinensis seedlings were harvested from culture medium and their root tissues were carefully washed by diluted water to remove the soil.

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Seeds of Arabidopsis thaliana were provided by South China Agricultural University. Seeds were firstly surface-sterilized by immersion in 70% (v/v) ethanol, then in 2% NaClO solution for 5 min. The seedlings were transferred into nutrient solution and vermiculite (3:1), which was held in a growth chamber delivering a photoperiod of 10-h light-14-h dark regime at 20 oC with a relative humidity of 70%. For the root segments imaging, 2-weeks-old Arabidopsis thaliana seedlings were

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harvested from culture medium and their root tissues were carefully washed by diluted water to remove the soil.

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The seedlings were transplanted into the centrifuge tubes (40 mL), and were

incubated with the corresponding solution (water, dye solution, or hydrazine solution).

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Before imaging, the roots of seedlings were washed with water for three times, and

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then were cut into segments to be placed on the microscope slides. The fluorescence images were carried out using a confocal fluorescent microscopy (Leica TCS SP8

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STED 3X). Excitation source: 405 nm laser. Blue channel: 450 nm – 520 nm; Red

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channel: 600 nm – 700 nm.

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3. Results and discussion 3.1 Synthesis

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Scheme 2 The synthetic route to probe 1

The synthetic route to probe 1 was shown in Scheme 2. According to the

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literature [57], the intermediate 3 was firstly prepared through the dimerization of

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4-ethynylbenzaldehyde with a high stereoselectivity by using PdCl2 and CuBr2 as catalysts. Subsequently, the compound 2 was produced through the Suzuki coupling

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reaction [58] of 3 with corresponding borate ester. Finally, probe 1 was obtained based on the Knoevenagel condensation between 2 with malononitrile. In order to

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verify the sensing mechanism, we also prepared the 1-N2H4 by simply reacting probe

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1 and hydrazine. The details of synthesis and characterization including 1H NMR, 13C NMR and HRMS were described in experimental section and Figure S1-S8 in

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supporting information (SI).

3.2 Optical properties

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Figure 1 (a) The absorbance and (b) fluorescence spectra of probe 1 (1 μM) in the absence and the presence of hydrazine (10 μM) in DMSO/PBS mixture (v/v = 9:1, [PBS] = 1 mM). λex

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= 450 nm.

With compounds in hands, the optical properties of probe 1 were measured in

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DMSO/PBS mixture (v/v = 9:1, [PBS] = 1 mM, pH ~ 7.4). As shown in Figure 1a,

probe 1 itself exhibited two major absorption bands at 323 nm and 451 nm. Upon

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addition of hydrazine, both of the original two absorption bands decreased,

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accompanied by a new band centered at 396 nm. The spectral change in longer-wavelength absorption from 451 nm to 396 nm led to a color change from

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yellowish-orange to colorless (Insert in Figure 1a). Under the light excitation at 450 nm, probe 1 exhibited single red emission centered at 650 nm, which was blue-shifted

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into 515 nm upon reacting with hydrazine (Figure 1b). This ultra-large chromatic shift (135 nm) in emission provided a typical ratiometric signal response (red to cyan) for

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hydrazine, which could be easily observed by naked eyes (Insert in Figure 1b). The fluorescence quantum yield (QY) of probe 1 in solid state was determined as 10.55%. Moreover, continuous irradiation for 5 minutes under UV lamp did not cause severe photobleaching (< 10% intensity loss), indicating a good photostability of probe 1 (Figure S9 in SI). 11

In pure PBS buffer ([PBS] = 1 mM, pH ~ 7.4), the fluorescence of probe 1 exhibited the similar ratiometric response pattern towards hydrazine. As shown in Figure S10 in SI, the maximum emission wavelength of probe 1 changed from 630 nm into 490 nm upon addition of hydrazine with a larger chromatic shift (140 nm). However, the overall sensitivity towards hydrazine in pure buffer solution was not as good as that in DMSO/PBS mixture. It is understandable because the production of

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hydrazone from dicyanovinyl group requires a dehydration process, so the existence

of water will be destined to decrease the conversion rate.[59] Ratiometric fluorescent method normally possessed the self-calibration feature as its ratiometric signal was

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insusceptible to variation in excitation source power probe concentration, as well as

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the photobleaching the during detection, particularly suiting for biological application.

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3.3 Sensing properties

The titration experiments of probe 1 (1 μM) towards hydrazine were studied by

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measuring its UV-visible absorption and fluorescence spectra. As shown in Figure 2a,

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with addition of hydrazine, the intensity in absorption band peaked at 323 nm gradually decreased, meanwhile the absorption band at 451 nm was gradually

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blue-shifted into 396 nm. When the concentration of hydrazine reached 6 equivalents (equiv.) of the probe, the intensities in the absorption peaks kept relative stable (Figure 2b). As for the fluorescence spectra, with increasing concentration of hydrazine, the red fluorescence of probe 1 centered at 650 nm was quenched, accompanied by a gradually increased cyan fluorescence with the emission maximum 12

at 515 nm (Figure 2c). The fluorescent titration experiment suggested that the 6 equiv. of hydrazine could reach the saturated concentration threshold (Figure 2d), which was

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in good agreement with the absorption titration result.

Figure 2 (a) The UV-vis spectra and (b) absorbance at 323 nm, 396 nm, and 451

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nm of 1 (1 μM) with increasing concentration of hydrazine (0 ~ 10 μM). (c) The

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fluorescence spectra, (d) intensities at 515 nm and 650 nm, and (e) intensity ratios (I515/I650) of 1 (1 μM) with increasing concentration of hydrazine (0 ~ 10 μM). Insert in (e): The photographs of emission color changes of 1 to hydrazine under the irradiation of handheld UV lamp (λex ~ 365 nm). (f) Time-dependent intensity of probe 1 at 515 nm in the presence of hydrazine (10 μM). (g) The fluorescence spectra and (h) intensity ratios (I515/I650) of 1 (1 μM) upon addition of 10 μM of hydrazine or 13

other competitive compounds, including hydroxylamine, phenylhydrazine, ammonia, piperazine, trimethylamine (TEA), hexadecyl trimethyl ammonium bromide (CTAB), and dodecylamine. (i) The interference from common metal ions for fluorescent ratio response of 1 towards hydrazine. Detection solution: DMSO/PBS mixture (v/v = 9:1, [PBS] = 1 mM). λex = 450 nm. I515 and I650 represented for the fluorescence intensity

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at 515 nm and 650 nm, respectively.

As shown in Figure 2e, the intensity ratio (I515/I650) displayed a good linear

relationship (R2 = 0.98) with the concentration of hydrazine in the range from 0 to 6

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μM. Based on 3σ/s rule (σ is the standard deviation of the blank and s is the slope for the range of the linearity, n = 5), the limit of detection (LOD) for hydrazine was

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calculated to be as 90 nM (2.88 ppb), lower than the threshold limit value (10 ppb) set

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by EPA. The sensing dynamic of probe 1 for hydrazine was studied based on the time-dependent emission change at 515 nm (Figure 2f). The intensity increased with

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time and required 3 min to achieve the relative equilibrium at 3 minutes.

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The selectivity experiment of probe 1 was then studied for hydrazine over competitive amine-based compounds, including hydroxylamine, phenylhydrazine,

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ammonia, piperazine, trimethylamine (TEA), hexadecyl trimethyl ammonium bromide (CTAB), and dodecylamine. As shown in Figure 2g-2h, under identical testing conditions, hydrazine induced an approximately 40-fold increase in intensity ratio (I515/I650) of probe 1. Except for a small increase in fluorescence intensity at 515 nm by hydroxylamine, most of competitive compounds caused negligible ratio change. 14

Furthermore, when applying probe in environment water system, the anti-interference capacity should be evaluated, especially against the co-existence of metal ions. As shown in Figure 2i, common metal ions, including Na+, K+, Ca2+, Mg2+, Cu2+, Fe3+, and Zn2+ did not cause the obvious variation in intensity ratio (I515/I650) of probe 1 for hydrazine detection. All above results suggested that probe 1 possessed the fast response, high sensitivity, high selectivity, and good anti-interference capability for

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hydrazine detection.

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3.4 Sensing mechanism

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Figure 3 (a) The HOMO-LUMO energy levels and interfacial plots of the orbital for probe 1 and two possible products. (b) The 1H NMR spectra of probe 1 upon addition of hydrazine in

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DMSO-d6/D2O mixture (v/v = 9:1).

In order to verify the sensing mechanism, the density functional theory (DFT)

calculation was firstly utilized to explore the nature of optical response. Considering the hydrazone reaction between probe 1 and hydrazine, two possible products may be produced, the monosubstituted product 1-intermediate and disubstituted product 16

1-N2H4. As shown in Figure 3a, electrons mostly dispersed on the electron-donating methoxy-substituted π-conjugated skeleton in the HOMO (highest occupied molecular orbital) of probe 1, and in its LUMO (lowest unoccupied molecular orbital), electrons redistributed on the electron-withdrawing malononitrile π-conjugated skeleton. This electron redistribution from electron-donating moiety to electron-withdrawing moiety showed similar pattern in the S0-S1 transitions (HOMO-LOMO) of two possible

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products, which indicated the intramolecular charge transfer (ICT) process. By comparing the energy level between HOMO and LUMO, the energy gaps were

calculated as 2.28 eV, 2.29 eV, and 3.21 eV for probe 1, 1-intermediate, and 1-N2H4,

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respectively. The energy gap of 1-N2H4 was much larger than that of 1, and

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1-intermediate. Since larger energy gap corresponded to the shorter absorption wavelength, the blue-shift from 451 nm to 396 nm in absorption band of probe 1 with

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addition of hydrazine suggested that the main product may be 1-N2H4. The optical

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spectra of 1-N2H4 (Figure S11 in SI) were also in good accordance with the calculation result.

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As depicted in Figure S12-S13 in (SI), the possible products were then

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confirmed by the high-resolution mass spectrometry (HRMS). With addition of one equiv. of hydrazine, the mass peaks for 1-intermediate (C35H29O2N4, m/z = 537.22858) and 1-N2H4 (C32H31O2N4, found m/z = 503.24384) were both clearly observed. However, upon addition of excess amounts of hydrazine (10 equiv.), the mass peak for 1-Intermediate disappeared, and only one mass peak for 1-N2H4 could be found, suggesting the high reactivity of dicyanovinyl group with hydrazine. 17

To further confirm the proposed mechanism, the proton NMR spectra of probe 1 with titration of hydrazine were also performed in DMSO-d6/D2O mixture (v/v = 9:1). As shown in Figure 3b, with addition of 1 equiv. of hydrazine, two proton signals Ha and Hb on probe 1 were both shifted to Ha’ and Hb’ at the higher field, indicating the formation of hydrazone. A new single peak Hc’ (6.83 ppm) was found corresponding to the active proton signal of the primary amine on hydrazone. By applying 10 equiv.

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of hydrazine, the integral area of this Hc’ signal was calculated to be as four protons, indicating the fully conversion from probe 1 into 1-N2H4. All above evidences

consistently confirmed the reaction between probe 1 and hydrazine producing

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corresponding hydrazone, and at the test concentration ([hydrazine] = 10 μM), the

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3.5 Bioimaging application.

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main product was 1-N2H4.

Encouraged by the above-mentioned detection features, including fast response,

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high sensitivity and selectivity, the further application of probe 1 for fluorescence

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imaging of hydrazine in plants was evaluated. The uptake of probe 1 was firstly studied using 5-day-old Chinese cabbage (Brassica chinensis) seedlings incubated

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with different concentration of probe 1 (0, 1, 5, 20, and 50 μM) in water for 2 h. As shown in Figure 4a, upon laser excitation (405 nm), the signal intensity in red channel (600 - 700 nm) was gradually enhanced with the increasing concentration of probe 1. However, when concentration of probe 1 reached 50 μM, the aggregation of probe molecules was found in culture solution, impeding the uptake of probe from root-hair 18

and transport through vascular tissue in root. Therefore, 20 μM of probe 1 as stain

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solution was employed for further fluorescence imaging in plants.

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Figure 4 (a) The fluorescence images of root segments (mature region) in Brassica chinensis incubated with different concentration of probe 1 (0, 1, 5, 20, and 50 μM) in water for 2 h. (b) Fluorescence imaging of hydrazine in root segments (mature region). Top row: plants were incubated in water for 4 h without any staining as the control. Middle row: plants were firstly stained with probe 1 (20 μM) for 2 h, and then were incubated in water for another 2 h. Bottom row: plants were firstly stained with probe 1 (20 μM) for 2 h, and then were incubated in water containing hydrazine (100 μM) for another 2 h. (c) Fluorescent intensity 19

profiles across root segments along the yellow circle indicated in (b). (d) The intensity ratio (Iblue/Ired) of root segment. Iblue and Ired represented for the average fluorescence intensity in blue and red channel, respectively. Error bars are ± SD. λex = 405 nm (Laser). Scale bar = 300 μm.

Next, we pretreated the seedlings with 20 μM of probe 1 for 2 h. After washing process, these seedlings were divided into two groups, which were continuously

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incubated with water or hydrazine solution (100 μM) for another 2 h, respectively. As shown in Figure 4b, the root segments only stained by probe 1 displayed a very weak

fluorescence in blue channel (450 - 520 nm) and a strong fluorescence in red channel.

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After the treatment of hydrazine, the root segments displayed an intense blue

fluorescence and a suppressed red fluorescence. The average intensity ratio (Iblue/Ired)

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between blue and red channel was calculated to be as 0.23 for the root segments only

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stained by probe 1, while this ratio was greatly increased to be as 3.46 upon treatment of hydrazine (Figure 4b-4c). This fluorescence ratio change was well corresponding to

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the reaction between probe 1 with hydrazine. Furthermore, in order to reveal the imaging ability of probe 1, 2-weeks-old model plants Arabidopsis thaliana were

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cultured and subsequently applied for hydrazine imaging. As shown in Figure S14 in

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SI, the ratiometric fluorescence response towards hydrazine could also be observed. All above results clearly demonstrated that probe 1 could be utilized for ratiometric fluorescence imaging of hydrazine in plants.

4. Conclusions

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In summary, we designed and synthesized a novel fluorescence probe for the colorimetric and ratiometric detection of hydrazine. This newly reported probe could rapidly, sensitively, and selectively respond to hydrazine with a distinct fluorescence color change from red to cyan, which could be easily observed by naked eyes. The sensing mechanism was verified by using DFT, HRMS, and NMR spectra. Significantly, fluorescence imaging experiments in Brassica

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chinensis and Arabidopsis thaliana demonstrated that this probe could be utilized for tracking hydrazine in plant roots. We expected this work could shed new light

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Conflict of interest

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fluorescent analytical method for hydrazine.

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on ratiometric probe design and promote the biological application of the

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The authors declare no conflict of interest.

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Acknowledgment

We would like to thank the financial support from the National Key R&D Program

of China (Grant No. 2017YFD0200504) and the National Natural Science Foundation of China (No. 31701826 and 21602139).

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Appendix A. Supplementary data

Supplementary data associated with this article can be found in the online version.

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Author Biographies Qiuqing Lai received her B. S. degree in plant protection and is now a M. S. candidate in South China Agricultural University. She is currently working on the distribution and transloction of pesticide in plants and insects. Shufan Si is now a M. S. candidate in the College of Materials Science and Engineering at Shenzhen University. He is currently working on the fluorescent probes and imaging agents for

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bioapplications.

Tianyi Qin received a Ph. D degree from Chinese Academy of Science. He currently works as postdoctoral researcher and his research interest is to design the ratiometric fluorescent probes.

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Benjie Li is a PhD candidate in South China Agricultural University majoring in pesticide toxicity,

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electrophysiology and confocal imaging.

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Hanxiang Wu is a postdoctoral researcher in South China Agricultural University. He works on the uptake, translocation, and distribution of agrochemicals in plants, with a special focus on

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carrier-mediated transport and agrochemical vectorization strategy in plants. Bin Liu received a Ph. D degree in polymer chemistry from University of Science and

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Technology of China. He is currently the associate professor in Shenzhen University. His research interest is to design novel fluorescent sensors and imaging agents.

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Hanhong Xu is a professor in South China Agricultural University, and also the director of the Key laboratory of Natural Pesticide & Chemical Biology, Ministry of Education of China. His research interests include the development of agrochemicals, botanical insecticides, and pesticide application technologies. Chen Zhao received her Ph. D degree in chemistry from University of South Carolina. She is 27

currently the associate professor in Key Laboratory of Natural Pesticide and Chemical Biology at South China Agricultural University. Her research interest is to study the small-molecular-based

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pesticides and fluorscence imaging of biohazard in plants.

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Scheme and Figures

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Scheme 1 Probe 1 for ratiometric fluorescent detection of hydrazine.

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Scheme 2 The synthetic route to probe 1

Figure 1 (a) The absorbance and (b) fluorescence spectra of probe 1 (1 μM) in the absence and the presence of hydrazine (10 μM) in DMSO/PBS mixture (v/v = 9:1, [PBS] = 1 mM). λex = 450 nm. 29

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Figure 2 (a) The UV-vis spectra and (b) absorbance at 323 nm, 396 nm, and 451

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nm of 1 (1 μM) with increasing concentration of hydrazine (0-10 μM). (c) The fluorescence spectra, (d) intensities at 515 nm and 650 nm, and (e) intensity ratios

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(I515/I650) of 1 (1 μM) with increasing concentration of hydrazine (0 ~ 10 μM). Insert

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in (e): The photographs of emission color changes of 1 to hydrazine under the irradiation of handheld UV lamp (λex ~ 365 nm). (f) Time-dependent intensity of

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probe 1 at 515 nm in the presence of hydrazine (10 μM). (g) The fluorescence spectra and (h) intensity ratios (I515/I650) of 1 (1 μM) upon addition of 10 μM of hydrazine or other competitive compounds, including hydroxylamine, phenylhydrazine, ammonia, piperazine, trimethylamine (TEA), hexadecyl trimethyl ammonium bromide (CTAB), and dodecylamine. (i) The interference from common metal ions for fluorescent ratio 30

response of 1 towards hydrazine. Detection solution: DMSO/PBS mixture (v/v = 9:1, [PBS] = 1 mM). λex = 450 nm. I515 and I650 represented for the fluorescence intensity

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at 515 nm and 650 nm, respectively.

Figure 3 (a) The HOMO-LUMO energy levels and interfacial plots of the orbital for probe 1 and two possible products. (b) The 1H NMR spectra of probe 1 upon addition of hydrazine in DMSO-d6/D2O mixture (v/v = 9:1).

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Figure 4 (a) The fluorescence images of root segments (mature region) in Brassica chinensis

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incubated with different concentration of probe 1 (0, 1, 5, 20, and 50 μM) in water for 2 h. (b) Fluorescence imaging of hydrazine in root segments (mature region). Top row: plants were

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incubated in water for 4 h without any staining as the control. Middle row: plants were firstly stained with probe 1 (20 μM) for 2 h, and then were incubated in water for another 2 h. Bottom row: plants were firstly stained with probe 1 (20 μM) for 2 h, and then were incubated in water containing hydrazine (100 μM) for another 2 h. (c) Fluorescent intensity profiles across root segments along the yellow circle indicated in (b). (d) The intensity ratio (Iblue/Ired) of root segment. Iblue and Ired represented for the average fluorescence intensity in

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blue and red channel, respectively. Error bars are ± SD. λex = 405 nm (Laser). Scale bar = 300

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μm.

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