A rapid-response and ratiometric fluorescent probe for nitric oxide: From the mitochondria to the nucleus in live cells

Journal Pre-proof A rapid-response and ratiometric fluorescent probe for nitric oxide: From the mitochondria to the nucleus in live cells Chen Li, Wen-Jian Tang, Wei Feng, Chao Liu, Qin-Hua Song PII:

S0003-2670(19)31274-7

DOI:

https://doi.org/10.1016/j.aca.2019.10.047

Reference:

ACA 237181

To appear in:

Analytica Chimica Acta

Received Date: 7 August 2019 Revised Date:

18 October 2019

Accepted Date: 19 October 2019

Please cite this article as: C. Li, W.-J. Tang, W. Feng, C. Liu, Q.-H. Song, A rapid-response and ratiometric fluorescent probe for nitric oxide: From the mitochondria to the nucleus in live cells, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.10.047. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier B.V.

Graphic Abstract

1

A rapid-response and ratiometric fluorescent probe for nitric oxide: From the mitochondria to the nucleus in live cells

Chen Li,a, † Wen-Jian Tang,b, †, Wei Feng,a Chao Liu,a and Qin-Hua Song*a a

Department of Chemistry, University of Science and Technology of China, Hefei 230026, P. R. China. School of Pharmacy, Anhui Medical University, Hefei 230032, P. R. China.

†

b

These authors contribute equally.

*Corresponding author. E-mail: [email protected]

Abstract Nitric oxide (NO) is a very important signal molecule implicated in numerous physiological and pathological processes, and its detection is the key to understand these processes. For this reason, various fluorescent probes have been developed for detection analysis of NO. However, few rapid-response (< 1 min) and ratiometric fluorescent probe are reported for real-time detection of short-time NO in biological systems. In this work, we report a rapid-response (within several seconds) and ratiometric fluorescent probe, RatioTr, which displays selective and sensitive detection of NO in solutions, and detections of exo- and endogenous NO in live RAW 264.7 cells. Unexpectedly, the probe RatioTr and its sensing product (p-Nus) display different cellular localizations, the mitochondria and the nucleus, which were demonstrated by co-stained experiments. The sensing process of RatioTr toward NO from mitochondria to nucleus was observed in live cells by confocal 1

fluorescence images. Furthermore, the subcellular localizations were demonstrated by measurements of pKa and interaction of p-Nus and DNA. In the presence of a natural DNA, calf thymus DNA, RatioTr is more sensitive to NO (LOD = 2.8 nM). Therefore, due to the nucleus localization together with a high fluorescence efficiency in the nucleus, p-Nus is a good candidate of cell-permeant nucleic acid stain or a fluorescent probe for the nucleus.

Keywords: Ratiometric fluorescent probes, Nitric oxide, Cell-imaging, cellular localization, Real-time detection, Nucleic acid stains

1. Introduction Nitrogen monoxide (NO) not only is the simplest radical molecule prompting various organic reactions [1], but also serves as secondary messenger in physiology. The endogenous NO produced from L-arginine by NO synthases in organism exhibits diverse physiological roles such as muscle relaxation, neurotransmission, regulation of immune and cardiovascular functions, and its effects of its concentration and spatial and temporal constraints of cell microenvirment [2, 3]. Abnormal level of NO in vivo can cause various pathophysiological processes such as Parkinson’s disease, Alzhieimer’s disease and liver cancer [4, 5]. Moreover, some NO donors such as sodium nitroprusside (SNP) or low concentrations of gaseous NO have been employed as regulars in preclinical treatments of cancers and other diseases [6, 7]. However, the mechanisms for NO exerting its diverse biological functions are still not entirely clear. For this reason, it is the key to develop real-time detection method of NO in biological systems with high selectivity and sensitivity.

2

Due to the unique advantages in sensitivity, visualization, and noninvasive detection, a number of fluorescent probes for NO have been developed in the last two decades [8-10], as two main categories, organic fluorescent probes [11-15] and metal-ligand complex probes [16-19]. Among the former, the o-phenylenediamine (OPD)-based fluorescent probes have shown great potentials for applications in biological systems, and some of them are commercially available [20]. Besides, based on organic reactions of NO, some new strategies have been developed including nitrosation reaction [21-23], the formation of diazo cyclic compounds [24-28], deamination reaction [29-30], formation of Se-NO bond [31], aromatization of Hantzsch dihydropyridines [32], and others [33-35]. Among these fluorescent probes, most probes are turn-on fluorescence response. Because endogenous NO exists in low concentrations (10-9-10-6 M) and short lifetime (seconds) in orgainisms [36], the rapid and highly selective reaction of fluorescent probes is necessary for a real-time detection of NO. Moreover, owing to its self-referencing capability in quantitative analysis, a ratiometric fluorescent probe is highly appealing, however, there is a few cases constructed with a single-fluorophore [37, 38] or two fluorophores, coumarin-rhodamine [39] or BODIPY-rhodamine [40] scaffold as fluorescence resonance energy transfer (FRET) dyads. In addition, fluorescence imaging of a ratiometric fluorescent probe can provide more information including both a probe (before) and its product (after the sensing reaction) in live cells, and a turn-on fluorescent probe can only achieve the latter fluorescence images. However, there is no one case with both rapid (< 1 min) and ratiometric fluorescence response toward NO so far. In this work, we have developed a ratiometric fluorescent probe, RatioTr, which can fast sense NO with a high sensitivity and a high selectivity. Moreover, RatioTr and its sensing product p-Nus display different localizations, the mitochondria and the nucleus, respectively, shown in Scheme 1. The 3

different localizations for RatioTr and p-Nus were demonstrated by interactions with DNA and their fluorescence efficiencies.

Scheme 1. A ratiometric fluorescent probe for NO and its sensing product with different cellular localization.

2. Experimental 2.1. Materials and methods. All chemicals were obtained from commercial suppliers and used as received without further purification. Water for preparation of solutions was purified with a Millipore water system. Solvents of technical quality were distilled prior to use. The photophyical property was investigated by determining the UV-Vis absorption spectra on a Shimadzu 2450 UV/Vis spectrometer and fluorescence spectra on a Shimadzu RF-5301PC spectrofluorophotometer. The organic compounds were characterized with 1H and

13

C NMR spectra on a 400 MHz Brucker AV spectrometer, and

high-resolution mass spectrometry (HRMS) with a Thermo LTQ Orbitrap mass spectrometer. Agilent 1200 HPLC with a C-18 reversed-phase column was employed to analyze the mixture of the sensing reaction. All measurements were performed at room temperature unless otherwise stated. 2.2. Synthesis of 5-bromo-2-methylquinolin-6-amine (1). 6-Amino-2-methylqhanoline [41] (200 mg, 1.26 mmol) was brominated with NBS (235 mg, 1.33 mmol) by stirring with catalytic amount of normal phase silica gel (15 mg) in CH3CN (100 mL) for 16 hours at room temperature. The crude product after filtration and evaporation was purified by column chromatography (ethyl acetate/petroleum ether, 1:6) to afford compound 1 (222 mg, 74%). 1H NMR 4

(400 MHz, CDCl3, TMS): δ = 8.23 (d, J = 8.8 Hz, quinoline-H, 1H), 7.81 (d, J = 8.8 Hz, quinoline-H, 1H), 7.28 (d, J = 8.8 Hz, quinoline-H, 1H), 7.20 (d, J = 8.8 Hz, quinoline-H, 1H), 4.40 (s, 2H, NH2), 2.70 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3, TMS): δ = 155.3, 143.1, 141.8, 133.4, 128.9, 126.9, 123.2, 120.7, 102.5, 24.5. TOFMS (ESI) m/z calcd for C10H10N2Br 237.0027 [M+H]+, found 237.0028. 2.3. Synthesis of 5-(3-(dimethylamino)phenyl)-2-methylquinolin-6-amine (RatioTr). Under N2 atmosphere, using Pd(PPh3)4 (50 mg, 0.05 mmol) and Na2CO3 (1.6g, 15.1 mmol) as catalytic system, the coupling reaction of compound 1 (450 mg, 1.9 mmol) and 3-(N,N-dimethylamino)phenylboronic acid (315 mg, 1.9 mmol) was carried out in solvent mixture of H2O:EtOH:toluene (v/v 3:3:10, 32 mL) by reflux for 24 h. Water was added into the reaction mixture and extracted with CH2Cl2 (50 mL×3). Solvent was removed in vacuo to afford a crude product, and purified by column chromatography (ethyl acetate/petroleum ether, 1:3) to afford RatioTr (205 mg, 39%) as a light yellow powder. FTIR (KBr): ν˜= 3414 (m, N-H), 3299 (m, N-H); 1626 (s, N-H), 1599 (s, Ar), 1575 (s, Ar), 1501 (s, Ar), 1379 (s, N-CH3), 1359 (m, C-N) cm−1. 1H NMR (400MHz, CDCl3, TMS): δ = 7.86 (d, J = 8.8 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.40 (m, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 6.81 (m, 1H), 6.66 (m, 2H), 3.82 (br, 2H), 2.98 (s, 6H), 2.66 (s, 3H). 13C NMR (100 MHz, CDCl3, TMS): δ = 154.4, 151.2, 142.7, 140.7, 136.9, 133.1, 130.0, 128.7, 127.0, 122.0, 121.3, 120.2, 118.6, 114.4, 111.7, 40.5, 24.7. TOFMS (ESI) m/z calcd for C18H20N3 278.1657 [M+H]+, found 278.1656.

2.4. Synthesis of p-product and o-product.

5

RatioTr (63.0 mg, 0.23 mmol) was dissolved in dilute HCl solution (30 mL) and cooled to 0°C with ice bath, and then NaNO2 (45mg, 0.63 mmol) aqueous solution was added slowly and stirred for 15 min. The reaction mixture was extracted using CH2Cl2 for three times (50 mL×3). The solvent was removed in vacuo to give the crude product, which was purified by column chromatography to afford o-product (11 mg, 16%) and p-product (41 mg, 64%). o-product: FTIR (KBr): ν˜= 2918 (w, C-H), 1614 (s, N=N), 1504 (m, Ar), 1293 (w, C-N) cm−1. 1H NMR (400MHz, CDCl3, TMS): δ = 9.33 (d, J = 8.8 Hz, 1H), 8.77 (d, J = 9.2 Hz, 1H), 8.32 (d, J = 9.2 Hz, 1H), 8.23 (d, J = 8.4 Hz, 1H), 7.78 (t, J = 8.4 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.18 (d, J = 8.0 Hz, 1H), 3.41 (s, 6H), 2.87 (s, 3H). 13C NMR (100 MHz, CDCl3, TMS): δ = 160.2, 151.6, 149.4, 144.0, 141.0, 136.5, 132.5, 131.3, 131.1, 123.4, 122.0, 121.9, 119.2, 114.7, 113.4, 45.0, 25.2. TOFMS (ESI) m/z calcd for C18H17N4 289.1453 [M+H]+, found 289.1447. p-product: 1H NMR (400MHz, CDCl3, TMS): FTIR (KBr): ν˜= 2925(w, C-H), 2846 (w, C-H), 1590 (s, N=N), 1552 (m, Ar), 1507 (m, Ar), 1319 (m, N-CH3) cm−1. δ = 9.36 (d, J = 8.8 Hz, 1H), 8.68 (d, J = 9.2 Hz, 1H), 8.56 (d, J = 9.6 Hz, 1H), 8.25 (d, J = 9.2 Hz, 1H), 7.70 (d, J = 2.8 Hz, 1H), 7.51 (d, J = 8.7 Hz, 1H), 7.43 (m, 1H), 3.28 (s, 6H), 2.85 (s, 3H). 13C NMR (100 MHz, CDCl3, TMS): δ = 159.4, 151.7, 149.2, 144.3, 142.6, 134.9, 132.7, 131.6, 131.0, 123.9, 122.3, 121.7, 117.6, 116.9, 101.3, 40.5, 25.0. TOFMS (ESI) m/z calcd for C18H17N4 289.1453 [M+H]+, found 289.1451. 2.5. Preparation of Sample Solutions. The stock solution of RatioTr dissolved in DMSO was diluted to a given concentration with 0.1 M PBS buffer (pH 7.4) in the ratio of DMSO to the buffer (v/v 1:100). Various analyte (36 mM) solutions were prepared in the phosphate buffer (pH 7.4) and a 10 µL of which was added a 3 mL of sample solution, giving a final concentration of 120 µM. NO gas bubbled into deoxygenated 6

deionized water with highly pure Argon for 30 min to attain NO saturated solution, and its concentration is 1.99 mM [1]. Peroxynitrite (ONOO−) solution was prepared in terms of the literature method [42]. Superoxide (O2−) solution was obtained from 10 min vigorous stirring after adding KO2 (1 mg) to dry dimethyl sulfoxide (1 mL). Using Fenton reaction, hydroxyl radical (HO•) was generated in situ. NO2− and ClO− solutions can be obtained from freshly prepared NaNO2 and NaClO aqueous solutions, respectively. Singlet oxygen (1O2) was generated from ClO− and H2O2. H2O2 diluted solution was obtained by adding deionized water to commercial H2O2 solution. Various analytes, NO, ONOO−, NO2−, ClO, H2O2, HO•, O2−, 1O2, AA, DHA and MGO, were added into the solution of RatioTr (20 µM) in PBS (0.1 M, pH 7.4, 1% DMSO), for spectral measurements. 2.6. Cell Culture and Cytotoxicity Assay. Raw 264.7 murine macrophage cells were seeded in 35 mm culture dish and cultured with Dulbecco’s modified Eagle’s medium (DMEM) including 10% fetal bovine serum (FBS) at 37°C under an atmosphere of 5% CO2 and 95% air. The in vitro cytotoxicity was measured using a standard methyl thiazolyl tetrazolium (MTT) assay in Raw 264.7 murine macrophage cells. Briefly, the cells were well placed in a 96-well cell-culture plate, followed by addition of various concentrations of RatioTr and p-Nus ranged from 0 to 50 µM. The cells were then incubated at 37°C in an atmosphere of 5% CO2 and 95% air for 24 h and permitted to adhere on the surface of plate. Subsequently, the culture medium was abandoned and the cells were treated with 100 µL of 1 mg mL−1 MTT reagent in PBS. Thereafter, growth medium was removed and a solvent of DMSO (100 µL) was added to dissolve MTT after incubation of 4 h. Finally, the absorption intensity of each sample was recorded through a microplate reader (Biorad, USA) at 630 nm. The cytotoxicity was expressed as percentage of cell viability relative to untreated cells. 7

2.7. Cell Imaging Experiments. Raw 264.7 murine macrophage cells cultured were employed for cell imaging experiments. The imaging of Raw 264.7 cells was performed by laser scanning confocal fluorescence microscope (Zeiss LSM 710 Meta NLO or Zeiss LSM 880 with Airyscan). Before imaging, the cells were washed with PBS buffer for three times. Exogenous NO detection performance was tested as follows: after the cells treated with 20 µM RatioTr for 1 h, aqueous solution of SNP or NO was added to the incubation medium, and incubated for 1 h or 30 min, respectively. For images of endogenous NO, some stimulants [43, 44], IFN-γ (400 U mL-1) and LPS (20 µg mL-1) as well as L-Arg (5 mg mL-1) were added to the cells, and incubated for 6 h or 12 h. The excitation wavelength was 405 nm and fluorescence signals were collected from a blue channel (408-500 nm or 408-475 nm) and a green channel (500–650 nm). For dynamic images of living cells, glass dishes were used for culture of Raw 264.7 cells. The Raw 264.7 cells were seeded with DMEM supplemented with 10% FBS in an atmosphere of 5% CO2 and 95% air at 37°C. The cells were taken out, removing the medium and adding 500 µl of cell culture medium in the bottom hole, and standing for 2 h in the incubator to allow the cell to settle on the wall. The cells were treated initially with 10 µM RatioTr for 1 h, and subsequently wished with PBS buffer for three times, and then culture medium (2.5 ml) was added for providing sufficient culture to reduce the osmotic pressure caused by water evaporation, and closed the lid to measure in an indoor environment. After acquiring images of above cells strained with RatioTr, NO aqueous solution was added to the incubation medium for imaging one time per minute. Confocal images were acquired from the blue channel (408-475 nm) and the green channel (500-650 nm) under 405 nm laser excitation. All the microscope settings were kept consistent in each experiment. 8

3. Results and Discussion 3.1. Synthesis of the probe and related compounds The synthetic procedure of the probe RatioTr was outlined in Scheme 2. Briefly, using 6-amino-2-methylquinoline [41] as a starting material, its brominzation with N-bromosuccinimide (NBS) carried out in acetonitrile at room temperature to afford compound 1 in a high yield, 74%. The Suzuki coupling reaction of compound 1 with 3-(N,N-dimethylamino)phenylboronic acid occurs under catalysis of tetrakis (triphenylphosphine) palladium (Pd(PPh3)4) at 110°C to afford the target compound, RatioTr, in the yield of 39%. RatioTr reacts with NaNO2/diluted HCl at 0°C to afford two product isomers, major p-product (p-Nus, 64%) and minor o-product (16%). These compounds were well characterized by 1H NMR, 13C NMR, and HRMS. In addition, the single crystals of p-Nus were obtained by solvent volatilization from p-Nus solution, and its structure was shown in Fig. 1. p-Nus is a flat molecule, and whole molecular skeleton is almost in a plane. N Br H2 N

NBS N

N

H2 N

CH3CN, r.t.

N

OH B OH

Pd(PPh3)4, 110oC

N NaNO2/HCl,

H2 N

0oC

N N

+

N

1

RatioTr

N

N

p-product

N N N

o-product

Scheme 2. Synthetic routes for RatioTr and its conversion under the diazotization condition.

Fig. 1. X-ray single-crystal structure of p-Nus. ORTEP diagrams of p-Nus with 10% thermal ellipsoids. Crystallographic data have been deposited as the number CCDC 1882691.

3.2. Photophysical property and spectral response toward NO

9

To evaluate a possible response mode, the photophysical properties of the probe and two products were investigated. Their UV/Vis absorption and fluorescence spectra in two solvents (PBS buffer and toluene) were determined and shown in Fig. 2. Both RatioTr and the proposed sensing product p-Nus are fluorescent, blue fluorescence for RatioTr (λmax ~428 nm) and strong green fluorescence (λmax ~538 nm) for p-Nus, and almost no fluorescence for o-products, in aqueous solutions (Fig. 2a). In contrast to those in aqueous solutions, fluorescence quantum yield (Φf) of RatioTr is the highest, and fluorescence maximum of o-product (545 nm) is longer over that of p-Nus (466 nm), in toluene (Fig. 2b). Their Φf values in two solvents were measured and listed in Table 1. Data show that their Φf values in toluene are much higher than those in aqueous solutions. Therefore, fluorescence efficiencies of these compounds strongly depend on their molecular environment. Further solvent effects were observed from their spectral measurements in other three solvents (methanol, DMSO and acetonitrile), and spectra and data were provided in Fig. S1 and Table S1. These observations support above conclusion from two classic solvents PBS aqueous solution and toluene.

0.5

0.6

a

500

b

600

RatioTr o-product p-product

400

0.2 200

Absorbance

0.3

400

0.4 300 200 0.2

Intensity (a.u.)

RatioTr o-product p-product

Intensity(a.u.)

Absorbance

0.4

100

0.1 0.0 300

400

500

600

Wavelength /nm

0 700

0.0 300

400

500

600

0 700

Wavelength /nm

Fig. 2. UV/vis absorption (bold) and fluorescence spectra (dash) of 20 µM RatioTr and its products in 0.1 M PBS buffer solution (pH 7.4) (a), or toluene (b), excitation at 350 nm for RatioTr, and 376 nm for two products.

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In our previous paper [26], a turn-on fluorescent probe NO-QA5 has been developed, and its non-fluorescent property may be ascribe to the rotation of the aniline at 6-position of the quinoline. In contrast to non-fluorescent NO-QA5, RatioTr emits blue fluorescence (Φf = 0.0053 in aqueous solution or 0.4 in toluene) possibly due to the rotation of 5-aniline unit is suppressed by C4-H. Actually, the distance between 5-aniline unit and C4-H is closer than that between 6-aniline unit and C7-H of NO-AQ5. Table 1 Photophysical properties of RatioTr and its products Compds

Φf a/10-3

abs

em

λmax

λmax

(PBS/Tol) b

(PBS/Tol)

(PBS/Tol)

RatioTr

5.3/400

345/356

428/420

p-product

16.0/90

440/418(π→π*)

538/466

o-product

0.64/82

438/452(n→π*)

515/545

a

Fluorescein in 0.1 M NaOH (Φf = 0.90) [45] for two products and quinine sulfate in 0.1 M H2SO4

(Φf = 0.546) [46] for RatioTr as references.

b

Measured in 0.1 M PBS buffer (pH 7.4) or toluene,

respectively. Besides notable solvent effects on the fluorescence efficiency, fluorescence maxima of p-Nus are 466 nm in toluene and 538 nm in the PBS buffer (a large bathochromic shift). Solvent effects reveal that p-Nus is a donor-acceptor (D-A) molecule. In contrast, fluorescence maxima of RatioTr in two solvents are similar, 428 nm vs. 420 nm, listed in Table 1. Moreover, UV/Vis absorption and fluorescence spectra of RatioTr and its products in other three solvents (acetonitrile, DMSO and methanol) were determined, and the results further support above conclusion (data were provided in

11

Fig. S1 and Table S1 as Supporting Information (SI)). Hence, if RatioTr senses NO to form p-Nus, a ratiometric fluorescence response will be observed. As expected, upon the addition of NO, fluorescence spectra of RatioTr solution (pH 7.4) display a ratiometric fluorescence change. As shown in Fig. 3a, the peak at 424 nm decrease and a new peak at 530 nm appears and enhances gradually. The dose-dependent changes of the fluorescence intensity ratio (F530/F424) exhibit a good linear relationship with NO concentration in the range of 0–40 µM, shown in the Inset of Fig. 3a. Based on the fitting straight line, the limit of detection (LOD) of RatioTr was obtained to be 20 nM in terms of the formula, LOD = 3δ/k. 150

280

a

0.5 2

R = 0.9984 0.0

0

10

140

20

30

40

50

60

[NO] /µM

Intensity@530 nm

F 530 /F424

210

Intensity(a.u.)

b

1.0

30 µM 25

100

20 15

50

7

70

3 0 µM

0

0 400

450

500

550

600

650

700

750

0

50

Wavelength /nm

100

150

Time /s

Fig. 3. (a) Fluorescence spectra of 20 µM RatioTr solution after additions of different amounts of NO (0-5 equiv.) for 5 min. Inset: the plot of intensity ratio (F530/F424) vs. the concentration of NO, λex = 370 nm. (b) Time-dependent fluorescence intensities of 20 µM RatioTr upon additions of different concentration NO (0-1.5 equiv.), in PBS buffer solution (pH 7.4).

Furthermore, the response time of RatioTr toward NO was estimated by recording time-dependent fluorescence intensity of RatioTr with different concentrations of NO. As shown in Fig. 3b, most of fluorescence intensities increase (>80% of the total signal) are completed within ~20 s in a low NO concentration (20 µM). This rapid response allows to achieve a real-time detection of NO in cell-imaging experiments. 3.3. Confirmation of the sensing mechanism. 12

Two products p-Nus and o-product were obtained easily from RatioTr under the diazotization condition, NaNO2/HCl. To exam the ratio of two products in the reaction of RatioTr with a NO aqueous solution, HPLC was employed for analysis of the mixture of RatioTr with NO, shown in Fig. 4. With increasing amount of NO, the chromatographic peak at 16.6 min of RatioTr decreases gradually with a simultaneous increase for two new peaks at 9.3 and 13.1 min (ratio 6.1:1), which are in accordance with the retention times of neat p-Nus and o-product, which were further confirmed by co-injection experiments (Fig. S2). RatioTr

o-product

[N O ]

p-product

6

9

12

15

18

Time/ min

Fig. 4. HPLC profiles of RatioTr upon additions of different amounts of NO in PBS buffer (pH 7.4), mobile phase: methanol/water (70:30, v/v), monitored at 376 nm.

In addition, a HRMS provided evidence for sensing products from mixture of RatioTr with NO in the PBS buffer (pH 7.4). As shown in Fig. S3, the peak at m/z 278.1 accords with RatioTr, corresponding to calculation value of m/z [RatioTr+H]+ (278.1652), and another peak at m/z 289.3 is assigned to [o/p-product+H]+ (m/z, calcd: 289.1448). Therefore, a proposed sensing mechanism was suggested as illustrated in Scheme 3. N

N O NO,O2

H2 N H2 O

H

N

N

HO N N

N

H2 O

N N

H

N

+

N N

N

N

RatioTr H2O

N N N

N

N

+

N pN

N N

o-

H+

N

N

p-product

o-product

N

diazonium

13

Scheme 3. Proposed mechanism in the reaction of RatioTr with NO under aerobic conditions.

3.4. The selectivity To verify the specific detection of NO, fluorescence responses of RatioTr toward biologically relevant analytes were determined. As shown in Fig. 5, fluorescence spectra of RatioTr exhibits a remarkable ratiometric change for 3 equiv. NO, and no significant fluorescence change was observed for 6 equiv. of reactive oxygen species (ClO−, H2O2, OH•, O2•−, 1O2), intracellular molecules that can condense with OPD including dehydroascorbic acid (DHA), methylglyoxal (MGO) and ascorbic acid (AA)), or reactive nitrogen species (ONOO−, NO2−), as well as metal ions (Na+, K+, Ca2+, Zn2+) or biothols (Cys, Hcy and GSH) (Fig. S4a). Compared with blank and other analytes, the fluorescence ratio (F530/F425) value displays a 21-fold increment for NO-induced fluorescence response. Moreover, the interference of these analytes for detection of RatioTr was also estimated, that is, detections of RatioTr for NO were carried out in the presence of metal ions or biological relevant species (Fig. S4b). As shown in Fig. S4c, no significant difference for other analytes was observed relative to response of RatioTr toward only NO.

Intensity(a.u.)

300

200

NO

100 other analytes

0 400

450

500

550

600

650

Wavelength /nm

Fig. 5. Fluorescence spectra of 20 µM RatioTr in the presence of 3 equiv. NO and 6 equiv. other analytes in the PBS buffer solution (pH 7.4) recorded after 10 min. Inset: Photos for above solutions under the indoor light (upper) or 365 nm light (bottom): 1: Blank, 2: NO, 3: ClO−, 4: H2O2, 5: OH•, 6: O2•−, 7: 1O2, 8: MGO, 9: DHA, 10: AA, 11: NO2−, 12: ONOO−. 14

Meanwhile, the selectivity of RatioTr can also be observed from the color change of solutions under the indoor light and under 365 nm light by naked eyes (Inset of Fig. 5). These results show that RatioTr has a high selectivity for NO over other relevant biological species. 3.5. Cell images of NO. 3.5.1. pH effects and cell cytotoxicity. To investigate pH effects on RatioTr and its sensing reaction, we monitored the reaction of 20 µM RatioTr with 3 equiv. NO by fluorescence spectroscopy in various pH solutions. As shown in Fig. S5, RatioTr shows a stable and small ratio (~0.05) of F530/F424, and large and stable ratiometric response (F530/F424 ~1.4) for reaction system of RatioTr with NO in the pH region of 6–11. Thus, the probe and its sensing reaction are insensitive to pH change in the large pH range. Before the application of RatioTr for imaging NO in living cells, cytotoxicities of the probe and the sensing product were evaluated with standard cell viability protocols (MTT assay) and Raw 264.7 murine macrophage cells. Raw 264.7 cells were incubated with various concentrations (0–50 µM) of RatioTr or p-Nus for 24 h. MTT assays revealed that the survival rates are high, over 61% for RatioTr and over 72% for p-Nus at the highest concentration (50 µM) (Fig. S6). According to literature [47, 48], the safe dosages of both RatioTr and p-Nus for cell-imaging are less than 50 µM as cell viability is more than 80%. These results show low cytotoxic effects of RatioTr and p-Nus on Raw 264.7 cells. 3.5.2. Detections of exogenous and endogenous NO.

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Fig. 6. Confocal fluorescence images of RAW 264.7 cells stained with 20 µM RatioTr for 1 h (a–c), or cells stained with 20 µM RatioTr for 1 h and then incubated with 50 µM NO for 0.5 h (e–g). (a, e) blue fluorescence image; (b, f) green fluorescence image; (c, g) brightfield image; (d, h) merge of (a, e), (b, f) and (c, g), respectively. Fluorescence signals were collected from a blue channel (408-500 nm) or a green channel (500–650 nm).

Based on above assessments, RatioTr shows promise detection of NO in living cells. By confocal fluorescence images of RAW 264.7 cells, detections of exo- and endogenous NO in live cells were achieved. The fluorescence signals were collected from the blue channel 408–500 nm or the green channel 500–650 nm. The NO saturated solution was employed as exogenous NO in Raw 264.7 cells. As shown in Fig. 6, the cells treated with only RatioTr display blue fluorescence (Fig. 6a), very weak green fluorescence (Fig. 6b), and two emissive images can overlap nicely (Fig. 6c). In contrast, after further incubated with NO, the cells display weaker blue fluorescence (Fig. 6e), stronger green fluorescence (Fig. 6f), and two-chanel emissions aren’t the same position of the cells (Fig. 6h). This implies that the probe RatioTr and its sensing product p-Nus could locate at different positions of cells.

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Fig. 7. Confocal fluorescence images of RAW 264.7 cells incubated without (a–c) or with L-Arg (5 mg mL-1), IFN-γ (400 U mL-1) and LPS (20 µg mL-1) for 6 h (e-g), 12 h (i-k) then stained with 20 µM RatioTr for 1 h. (a, e, i) blue fluorescence image; (b, f, j) green fluorescence image; (c, g, k) brightfield image; (d, h, l) merge of (a, e, i), (b, f, j) and (c, g, k), respectively. Fluorescence signals were collected from a blue channel (408-500 nm) and a green channel (500-650 nm) under excitation at 405 nm.

Furthermore, the detection of RatioTr to endogenous NO in living cells was estimated. Endogenous NO generation can be induced by LPS and IFN-γ in RAW 264.7 cells [43, 44]. Likewise, RAW 264.7 cells treated with only RatioTr display bright florescence in the blue channel (Fig. 7a), very weak fluorescence in the green channel (Fig. 7b), and a nice overlap shown in Fig. 7d. However, RAW 264.7 cells pre-treated with L-Arg, IFN-γ and LPS for 6 h or 12 h, then incubated with RatioTr for 1 h, the cells show a gradual decrease in blue-channel fluorescence (Fig. 7a, e, i), and a gradual increase in green-channel fluorescence (Fig. 7b, f, j). Similarly, the image in the blue channel don’t well overlap with those in the green channel after sensing NO (Fig. 7h, l). In both Fig. 6 and Fig. 7, before the sensing reaction, blue fluorescent RatioTr seem to locate in motichondria, and after the 17

sensing reaction, green fluorescence appears on the nucleus. Namely, the probe RatioTr and the sensing product could locate in mitochondria and nucleus, respectively. To confirm this inference, RatioTr and p-Nus were used to treat cells for fluorescence images, respectively. As shown in Fig. 8, the cells with RatrioTr emit blue fluorescence from the mitochondria (Fig. 8a-d), and the cells with p-Nus give green fluorescence from the nucleus (Fig. 8e-h). Thus, these images show that the sensing process could undergo from the probe in the mitochondria to p-Nus in the nucleus.

Fig. 8. Fluorescence images RAW 264.7 cells incubated with (a-d) RatioTr (20 µM, 1 h) or (e-h) p-Nus (10 µM, 1 h). (a, e) Blue-channel fluorescence image, (b, f) green-channel fluorescence image, (c, g) brightfield image, (d, h) overlap of (a, e), (b, f), and (c, g). The images were collected from the blue channel (408–475 nm) and the green channel (500–650 nm) under excitation at 405 nm.

Furthermore, the mitochondria localization of RatioTr and the nucleus localization of p-Nus were further demonstrated by co-localization experiments of RAW 264.7 cells stained respectively with RatioTr and a mitochondrial dye, MitoTracker@ Deep Red FM [20] or p-Nus and a nucleus specific dye, 4’,6-diamidino-2-phenylindole (DAPI) in succession. The confocal images of these cells display an excellent overlap between the blue-channel image for RatioTr and the red-channel images for the mitochondrial dye (Fig. 9a-c). Their fluorescence profile is excellently consistent (Fig. 9d). Similarly, 18

the confocal images of these cells display an excellent overlap between the green-channel image for p-Nus and the blue-channel images for the nucleus dye (Fig. 9e-g), and their fluorescence profile is excellently consistent (Fig. 9h). Moreover, the two images between the green channel and the blue channel display a very high overlap coefficient, 0.96 (Fig. S7). The perfect overlap indicates that p-Nus is a nucleus-targetable fluorescence dye.

Fig. 9. Co-localization fluorescence images of RAW 264.7 cells stained with RatioTr (20 µM, 30 min) and MitoTracker@ Deep Red FM (0.5 µM, 1 h) (a-d) or p-Nus (10 µM, 30 min) and DAPI (5 min) (e-h) in succession. (a, e) Blue-channel fluorescence image, (b) red-channel fluorescence image, (c) overlap of (a), (b) and brightfield image, (d) Fluorescence profile of a given region [yellow line in (c)], (f) green-channel fluorescence image, (g) overlap of (e), (f) and brightfield image, (h) fluorescence profile of a given region [yellow line in (g)]. The images were collected from the blue channel (408–475 nm) and the green channel (500–650 nm) under excitation at 405 nm, and the red channel (650-750 nm) under excitation at 633 nm.

3.5.3. Direct observation of the sensing process to NO in living cells To further observe the sensing process, a dynamic NO imaging in living cells with RatioTr strained RAW 264.7 cells were performed under room environment and shown in Fig. 10. Before the addition of 50 µM NO aqueous solution, the cells stained by RatioTr emit blue fluorescence, which locates in the mitochondria. After the addition of NO, the blue fluorescence weakens gradually and concomitantly green fluorescence appears and enhances gradually in the nucleus. The process is 19

better revealed by the plot of the total fluorescence intensities of two channels vs the time from Fig. 10, that is, blue fluorescence intensity decreases gradually implying consume of RatioTr, and increase in green fluorescence shows the formation of p-Nus in the sensing reaction, shown in Fig. S8. These observations clearly reveal the NO sensing process from the mitochondria (RatioTr) to the nucleus (p-Nus) in live cells.

Fig. 10. Dynamic images of live RAW 264.7 cells stained with 10 µM RatioTr upon the addition of 50 µM NO aqueous solution. Fluorescence images were collected from the blue channel 408-475 nm and the green channel 500-650 nm. Scale bar, 10 µm.

3.6. The reasons of cellular localizations of RatioTr and p-Nus 3.6.1. pKa values of RatioTr and p-Nus. To explore the reason of the subcellular localization, the pKa values for the probe RatioTr and the sensing product p-Nus were determined to 6.5 and 4.0, respectively (Scheme S1 and Fig. S9). Thus, RatioTr could be protonated and accumulate at the negatively charged surface of mitochondria, and as it converts into p-Nus, which would leave mitochondria because of non-protonated p-Nus in physiological condition (pH 7.4). 3.6.2. Fluorescence enhancement of p-Nus in the presence of natural DNA. 20

For the nucleus localization of p-Nus, we investigated the interaction between p-Nus and natural DNA, including calf-thymus DNA (ctDNA), or fish sperm DNA (fsDNA). Upon additions of different amounts of natural DNA (0–100 µM bp), the fluorescence intensity of 10 µM p-Nus solution enhances drastically, and fluorescence maxima from 535 nm without DNA to about 518 nm with different natural DNA (Fig. 11ab). In the presence of ctDNA, the fluorescence intensity at 518 nm enhances up to 20-fold increment, thus, the fluorescence quantum yield of p-Nus with DNA should be 0.32. However, only 2.5-fold fluorescence increment and no change in fluorescence maximum (515 nm) were observed for o-product in the presence of 1 mM fsDNA (Fig. S10). Based on the titration experiment, LODs of p-Nus for two neutral DNA were obtained, 28 nM bp for ctDNA and 46 nM bp for fsDNA, in terms of LOD = 3δ/k (Fig. 11c). 480

400

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[fsDNA]

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Fig. 11. Fluorescence spectra of 10 µM p-Nus solutions upon additions of different concentrations of natural DNA (0–100 µM bp), fsDNA (a) or ctDNA (b), and fluorescence intensities (c) from (a) and (b), excitation at 370 nm, determination at 25°C.

Furthermore, the titration experiment was performed again with 5 µM p-Nus solution in the presence of different amounts of ctDNA or fsDNA (0–250 µM bp), shown in Fig. S11ab. The change in fluorescence intensity of p-Nus in the presence of DNA can be used to determine binding constants (K) of p-Nus and DNA [49]. The plots of fluorescence intensity vs the concentration of DNA were showed in Fig. S11c. The fluorescence titrations reveal that p-Nus binds to ctDNA duplex with a K 21

value of 1.2×104 M-1 (25°C), which is close to that of proflavine to 3’,5’-DNA oligonucleotides (7×104 M-1 at 25°C) [50]. The value of p-Nus with fsDNA is K= 3.4×103 M-1 at 25°C. Therefore, localization of p-Nus at the nucleus could ascribe to the interaction between p-Nus and DNA, thereby, the fluorescence of p-Nus is enhanced by DNA. In the view of its molecular shape, p-Nus may intercalate between DNA bases from its diazo cycle. The specific binding mode between p-Nus and DNA awaits further investigation. 3.6.2. The fluorescence response of RatioTr toward NO in the presence of ctDNA. 20

1000

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R2 = 0.9841

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Fig. 12. (a) Fluorescence spectra of 20 µM RatioTr solution upon additions of ctDNA (0–100 µM bp) and subsequent addition of NO solutions (0–5 equiv.), inset: photos of above solutions before and after the addition of 100 µM DNA. (b) Plot of fluorescence ratio (F517/F425) vs the concentration of NO, and fitting straight line.

The influence of DNA on fluorescence of RatioTr and p-Nus has been investigated by measuring their fluorescence spectra in the presence of ctDNA and shown in Fig. 12 and Fig. S12. Upon additions of ctDNA (0–100 µM bp), the fluorescence change (λmax 425 nm) of 10 µM RatioTr is no significant (Fig. S12a). However, further additions of NO (0–110 µM), the fluorescence intensity enhances sharply centered at 518 nm (Fig. 12a and Fig. S12bc). After addition of 3 equiv. NO, the fluorescence ratio (F518/F425) in the presence of 100 µM ctDNA displays a 15-fold increment relative to the ratio (F530/F424) from Fig. 2a. Thus, the natural DNA plays a role of amplifier for fluorescence 22

of p-Nus. From this fluorescence titration, a new LOD of RatioTr for NO was obtained to be 2.8 nM, in terms of LOD = 3δ/k (Fig. 12b). This value is much lower than that in the absence of DNA (20 nM). Hence, in the presence of neutral DNA, RatioTr exhibits a more sensitive fluorescence response to NO. The notable fluorescence response was also observed easily by naked eyes (Inset of Fig. 12a). In other word, RatioTr is more sensitive for the detection of NO in the nucleus. Based on above results, we can further understand the sensing process from Fig. S8. The green fluorescence intensity from p-Nus increases initially in slow rate (0-20 min) and subsequently in fast rate (20-25 min). The green fluorescence increases in two rates could imply the formation of p-Nus in the sensing reaction (small increase), and the interaction of p-Nus with DNA after it enters into the nucleus (large increase). In addition, this work also reveals that a fluorescent chemosensor may have distinct property from the sensing product, such as specific interaction with organelles and fluorescence efficiencies in the organelles. It is no way that this result is obtained from a turn-on fluorescent probe.

4. Conclusions In summary, we have developed a single-fluorophore ratiometric fluorescent probe, RatioTr, which can fast sense NO within seconds to generate a main fluorescent product, p-Nus. The sensing reaction is sensitive and specific for NO over some ROS/RNS and reactive species to OPD (MGO, DHA and AA), and RatioTr can achieve detection of exo- and endogenous NO in living RAW 264.7 cells. Intriguingly, RatioTr and its sensing product (p-Nus) exhibit the mitochondria localization and the nucleus localization respectively. There would be a remarkable interaction between p-Nus and DNA due to as high as 20-fold fluorescence enhancement in the presence of neutral DNA. Hence, in the presence of natural DNA or in the nucleus, RatioTr is more sensitive to NO (LOD=2.8 nM for 100 23

µM bp ctDNA). Therefore, p-Nus is a very good candidate as a cell-permeant nucleic acid stain or a green-fluorescent probe for the nucleus. Notes The authors declare no competing financial interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21772188), and Anhui Provincial Natural Science Foundation (No. 1708085MB33) and Anhui Province Key Research and Development Program Project (No. 201904d07020012). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca. References [1] J. Hartung, Organic radical reactions associated with nitrogen monoxide, Chem. Rev. 109 (2009) 4500-4517. [2] Solomon H. Snyder, Nitric oxide: First in a new class of neurotransmitters? Science 257 (1992) 494-496. [3] D. Fukumura, S. Kashiwagi, R.K. Jain, The role of nitric oxide in tumour progression, Nat. Rev. Cancer 6 (2006) 521-534. [4] S. Taysi, C. Uslu, F. Akcay, M.Y. Sutbeyaz, Malondialdehyde and nitric oxide levels in the plasma of patients with advanced laryngeal cancer, Surg. Today 33 (2003) 651-654. [5] G. P. Biro, Adverse HBOC-endothelial dysfunction synergism: A possible contributor to adverse clinical outcomes? Curr. Drug Discov. Technol. 9 (2012) 194–203. [6] P. Wang, M. Xian, X. Tang, X.Wu, Z. Wen, T. Cai, A. J. Janczuk, Nitric oxide donors:  chemical activities and biological applications, Chem. Rev. 102 (2002) 1091–1134. [7] J. P. Kinsella, G. R. Cutter, W. F. Walsh, D. R. Gerstmann, C. L. Bose, C. Hart, K. C. Sekar, R. L. Auten, V. K. Bhutani, J. S. Gerdes, T. N. George, W. M. Southgate, H. Carriedo, R. J. Couser, M. C. Mammel, D. C. Hall, M. Pappagallo, S. Sardesai, J. D. Strain, M. Baier, S. H. Abman, Early inhaled 24

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Highlights 1. A rapid- and ratiometric fluorescent probe was developed to detect endogenous NO in live cells. 2. The probe and its sensing product display different cellular localizations, i.e. at the mitochondria and at the nucleus, respectively. 3. The sensing product is a good candidate of cell-permeant nucleic acid stain or a fluorescent probe for the nucleus.

Declaration of interests ☒ 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. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Declarations of interest: none