A highly sensitive fluorescent probe for fast recognization of DTT and its application in one- and two-photon imaging

Author’s Accepted Manuscript A highly sensitive fluorescent probe for fast recognization of DTT and its application in oneand two-photon imaging Tong Sun, Lili Xia, Jinxin Huang, Yueqing Gu, Peng Wang www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(18)30539-3 https://doi.org/10.1016/j.talanta.2018.05.046 TAL18687

To appear in: Talanta Received date: 18 February 2018 Revised date: 7 May 2018 Accepted date: 11 May 2018 Cite this article as: Tong Sun, Lili Xia, Jinxin Huang, Yueqing Gu and Peng Wang, A highly sensitive fluorescent probe for fast recognization of DTT and its application in oneand two-photon imaging, Talanta, https://doi.org/10.1016/j.talanta.2018.05.046 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A highly sensitive fluorescent probe for fast recognization of DTT and its application in one- and two-photon imaging Tong Suna1, Lili Xiab1, Jinxin Huangb, Yueqing Gub, Peng Wangb,* a b

Shandong Institute for Food and Drug Control, Jinan 250101, China

Department of Biomedical Engineering, School of Engineering, China Pharmaceutical University, Nanjing 210009, China

*

Corresponding email address: [email protected]

Abstract As a widely used reducing agent, 1, 4-dithiothreitol (DTT) plays important roles in the fields of biology, biochemistry, and biomedicine. The development of facile and fast methods for DTT detection is urgent and necessary. In this article, we rationally constructed

a

novel

two-photon

fluorescent

probe

6-(methylsulfinyl)-2-phenyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (NC-DTT) for detecting DTT, which employed the 1,8-naphthalimide and sulfoxide as the fluorophore and receptor unit respectively. The sulfoxide group in probe NC-DTT can

be

reduced

by

DTT

to

compound

6-(methylthio)-2-phenyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (NC), which could emit strong fluorescence with large Stokes shift presumably due to the enhanced intramolecular charge transfer (ICT). This probe responded to DTT quickly (within 1000 s) and showed satisfactory selectivity. A good linearity between fluorescence intensity and the concentration of DTT in the range of 0 -700 μM was observed, and the detection limit towards DTT was 1.410-7 M. Furthermore, the probe was successfully employed in one- and two-photon imaging of DTT in HepG2 cells with low cytotoxicity. Graphical abstract

1

T. Sun and L. Xia contributed equally. 1

Keywords: DTT; fluorescent probe; bioimaging; two-photon.

Introduction 1, 4-dithiothreitol (DTT), a non-physiological synthetic molecule[1, 2], usually as a reducing agent, plays important roles in various fields of science including biology, biomedicine, and biochemistry[3-8]. Generally, low level of this reducing agent (around 300 M) can be used as an antidote to protect cells and organisms[9]. But it also noted that high concentration of DTT (over 10 mM) would cause oxidative damage to some biomolecules[10]. For this reason, DTT has become a serious threat in the laboratory and industrial accidents. Thus, it is necessary and urgent to develop efficient and fast methods for recognizing and monitoring DTT. Some traditional analytical methods have been developed for the detection of thiols, such as electrochemical assay, capillary electrophoresis, high-performance liquid chromatography, mass spectrometry and gas chromatography[11-14]. However, these methods are failed in extensive use to detect biomolecular thiols because of their high costs and tedious operations. Due to their distinct advantages of high sensitivity, noninvasive-ness and high spatiotemporal resolution for imaging in biological system, fluorescent probes are widely employed in the determination of thiols[15, 16]. Although some reported fluorescent probes can effectively discriminate specific thiols (such as Cys, Hcy, and GSH), few fluorescent indicators were developed to undertake the detection and discrimination of DTT[17, 18]. The reported fluorescent probes for DTT were limited by the low sensitivity or unfavorable selectivity. Thus, the desire of developing new fluorescent sensors that can efficiently differentiate DTT from other 2

biothiols and reducing agents has provided the incentive for researchers. Recently, a specific fluorescent probe utilizing sulfoxide as response group was discovered to reveal the compromised activity of methionine sulfoxide reductases, which can convert sulfoxides to the corresponding sulfides[19]. Inspired by the manipulation of the intramolecular charge transfer (ICT) process, we have successfully

designed

and

synthesized

a

novel

fluorescent

sensor

6-(methylsulfinyl)-2-phenyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (NC-DTT) for detecting DTT. This new turn-on fluorescent probe was mainly composed by the 1,8-naphthalimide fluorophore, which was a distinguished two-photon imaging group[20]. The sensor NC-DTT exhibited satisfactory advantages of fast response, excellent sensitivity and high selectivity for the recognition of DTT. Additionally, it performed well in cell imaging with low cytotoxicity.

Experimental section Chemical and Instruments All chemical reagents were purchased from commercial companies and used without any further purification. Water used throughout all experiments were acquired from double distillation of running water. The silica gel (200-300 mesh) was used for the chromatography purification. 1,4-dithiothreitol (DTT) was purchased from Shanghai Aladdin Reagent Co Ltd. The thin layer chromatography (TLC) analysis was performed on silica gel plates. MS spectra were obtained from quadruple mass spectrometry.

1

H-NMR and

13

C-NMR were performed using Bruker Advance

300-MHz Spectrometer. The UV-vis absorption spectra were recorded by One Drop spectrophotometer (OD-1000+, Nanjing, China). The fluorescence spectra were obtained on Fluorescence Spectrophotometer (LS55, PerkinElmer). The one-photon imaging and two-photon imaging of cells were performed with Laser confocal fluorescence microscopy (FluoViewTM, FV 1000, Olympus, Japan). Synthesis

and

characterization

of

compound

6-(methylthio)-2-phenyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (NC) 4-Bromo-1,8-naphthalic anhydride (554 mg, 2 mmol) and aniline (930 mg, 10 3

mmol) were mixed in acetic acid (CH3COOH, 30 mL) and heated under reflux for 12 h. The mixture was cooled to room temperature and poured into water. The solid was obtained by filtration and used for the next step without further purification. Then the obtained solid (700 mg, 2 mmol) was dissolved in dimethylformamide (DMF, 30 mL), and CH3SNa (20% in water, 2.1 g) was added slowly to the solution. The mixture was stirred for 12 h at room temperature and then poured into water. The compound NC was obtained by filtration in 88% yield. 1H-NMR (300 MHz, DMSO-d6) δ: 8.56 (s, 2H), 8.41 (s, 1H), 7.93 (s, 1H), 7.72 (s, 1H), 7.31-7.57 (m, 5H), 2.77 (s, 3H). 13

C-NMR (75 MHz, DMSO-d6) δ: 131.0, 130.5, 129.4, 129.0, 128.7, 128.1, 127.1,

121.5, 14.0. ESI-MS: m/z 342.0 [M+Na]+. Synthesis and characterization of probe NC-DTT Compound NC (320 mg, 1 mmol) was dissolved in dichloromethane (DCM, 20 mL). Then m-chloroperoxybenzoic acid (m-CPBA, 172 mg, 1 mmol) was slowly added to the solution. The mixture was stirred at room temperature for 0.5 h. After solvent evaporation, the residue was purified by chromatography on silica gel (petroleum ether:AcOEt = 3:1) to obtain desired compound NC-DTT in 60% yield. 1

H-NMR (300 MHz, DMSO-d6) δ: 8.69 (d, J = 7.5 Hz, 1H), 8.59 (d, J = 7.2 Hz, 1H),

8.51 (d, J = 8.4 Hz, 1H), 8.36 (d, J = 7.5 Hz, 1H), 8.01 (t, J = 7.8 Hz, 1H), 7.39-7.57 (m, 3H), 7.41 (d, J = 7.17 Hz, 2H), 2.93 (s, 3H).

13

C-NMR (75 MHz, DMSO-d6) δ:

163.2, 163.1, 149.8, 135.7, 131.1, 130.2, 129.0, 128.8, 128.4, 128.2, 127.9, 126.5, 124.8, 123.5, 122.6, 43.0. ESI-MS: m/z 358.0 [M+Na]+. Absorption spectrum studies of NC-DTT and NC All measurements of UV-vis spectrum were carried out on UV-vis spectrophotometer. Collecting the absorption spectrum from 300 nm to 500 nm with 5.0/5.0 nm slit widths. The absorption spectra of intermediate NC and the sensor NC-DTT (5 μM) in DMSO-PBS solution (10 mM, pH = 7.4, 1:9) were recorded. Moreover, the absorption spectrum of probe NC-DTT (5 μM) in the presence of DTT (5 mM) was also tested. Fluorescence response studies of NC-DTT to DTT Fluorescence spectrophotometer was applied to record the fluorescence intensity 4

of samples. Emissions of solutions from 400 to 700 nm were collected with ex = 400 nm. The fluorescence response was observed when adding different concentrations of DTT to the solution of NC-DTT. The probe NC-DTT (5 μM) in DMSO-PBS solution (10 mM, pH = 7.4, 1:9) were incubated with different concentrations of DTT (0-5 mM) at 37 °C for 30 min. Then the fluorescence spectra of the solutions were collected. The selectivity of probe NC-DTT towards DTT was tested by comparing with other biorelevant species. The biorelevant species are as follows: physiologically important reducing agent ascorbic acid (Vc) and environmentally relevant species, including cysteine (Cys), glutathione (GSH), sodium sulfide (Na2S), sodium hydrosulfide (NaHS), 2-mercapto-ethanol (ME), 2-mercaptoacetic acid TGA (TGA). The sensor NC-DTT (5 μM) in DMSO-PBS solution (10 mM, pH = 7.4, 1:9) was incubated with DTT or other biorelevants at 37 °C for 30 min. Then all the fluorescence spectra were recorded by fluorescence spectrophotometer. Sensing mechanism study of NC-DTT with DTT NC-DTT (5 M) and DTT (5 mM) in DMSO-PBS solution (10 mM, pH = 7.4, 1:9) were in shaking table at 37◦C for 0.5 h. The mixture was extracted with dichloromethane for three times. The collected organic layers were removed under reduced pressure. The residue was purified with silica gel column. MS-ESI: 342.0 [M + Na]+. Cell culture HepG2 cells (liver hepatocellular carcinoma) were purchased from American Type Culture Collection and cultured in culture medium (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, containing 80 U/mL penicillin and 0.08 mg/mL streptomycin) for 24 h at 37 °C under a humidified atmosphere containing 5% CO2. MTT assay For MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay, the HepG2 cells were cultured in 96-well microtiter plates at a density of 5000-8000 cells/well and cultured at 37 °C in a 5 % CO2/ 95℅ air (v/v) incubator for 24 h. Then the medium was replaced with different concentrations of 5

NC-DTT (0, 5, 10, 20, 30, 40, 50 μM). After cultured for 24 h, 20 μL MTT solution (0.5 mg/mL) was added to each well. After 4 h, the MTT solution was abandoned, and 150 μL of DMSO was added to each well to dissolve the formed formazan. The plates were shaken for 15 min, and the absorbance at 490 nm was measured by a multi-well plate reader. The cell viability was calculated using the following formula: Cell viability = (the optical density of test wells – the optical density of medium control wells) / (the optical density of untreated wells – the optical density of medium control wells) × 100%[21]. Cell imaging For confocal fluorescence imaging, HepG2 cells were cultured in three group confocal dishes, for each dish is investigating the exogenous DTT cell imaging, control group and block DTT group, respectively. Before imaging, the HepG2 cells of exogenous DTT group were incubated with DTT (100 μM) for 1 h and further incubated with probe NC-DTT (10 μM) for 30 min. For the control group, the HepG2 cells were incubated with probe NC-DTT (10 μM) for 30 min. For DTT-block experiment, the cells were pretreated with N-ethylmaleimide (NEM, 1 mM) for 1 h, and then treated with NC-DTT (10 μM) for another 30 min. The cells were washed three times with PBS buffer before imaging. Single-photon cell imaging and two-photon cell imaging were performed on FV 1000 confocal laser scanning microscope. The single-photon imaging was excited at 405 nm and collected the fluorescence signal from 488 nm to 543 nm. For the two-photon imaging, obtaining the emission signal in the blue channel under the excitation of 750 nm.

Results and discussion Synthesis of probe NC-DTT As illustrated in Scheme 1, the two-photon fluorescent probe NC-DTT was successfully synthesized in three steps from 4-bromo-1,8-naphthalic anhydride and aniline. These two starting materials were transformed to the amide intermediate, which was reacted with sodium thiomethoxide to give compound NC at room temperature. Then the desired probe NC-DTT was obtained by the oxidation of 6

m-CPBA. The compounds NC and NC-DTT were characterized by 1H NMR,

13

C

NMR, and mass spectrum.

Optical Response of Probe NC-DTT The spectral properties of probe NC-DTT were carried out in DMSO-PBS solution (10 mM, pH = 7.4, 1:9). Probe NC-DTT exhibited one peak shape absorption band around 345 nm (Figure 1a). When DTT (5 mM) was added to the solution of NC-DTT (5 M), the maximum absorption peak showed wider peak width with a 50 nm red shift. Moreover, the intermediate NC exhibited a similar absorption peak centered at 400 nm. Accordingly, in the fluorescence emission spectrum, probe NC-DTT showed very weak fluorescence emission around 510 nm under excitation at 400 nm. In the presence of DTT, a substantial increase (>1000 fold) of fluorescence emission intensity was as expected observed (Figure 1b). These absorption and emission spectrum results indicated that the probe NC-DTT had the capability to recognize DTT.

Further, we investigated the relationship between fluorescence intensity and the concentration of DTT. As shown in Figure 2, with the increase of the concentration of DTT, the fluorescence intensity at 510 nm (ex =

400 nm) was gradually enhanced.

And a good linearity was observed between fluorescence intensity I510nm and the concentration of DTT in the range of 0 -700 μM with a linear coefficient of 0.9958. Hence, the linear curve of the fluorescence intensity I510nm allowed for the efficient quantitative detection of DTT over this concentration range. According to the reported methods[22, 23], the detection limit of probe NC-DTT towards DTT was calculated to be 1.410-7 M (n=11, S/N =3), which was lower than the previously reported DTT probe[24]. Therefore, probe NC-DTT is a very promising probe for the detection of DTT. 7

Response Time Assay Then the response time of probe NC-DTT to DTT was evaluated by monitoring the fluorescent intensity of probe NC-DTT (5 μM) at 510 nm in the presence of 10 equivalent of DTT (Figure S1). The fluorescence signal showed that the I510nm was increasing fast and reached a plateau within 1000 s, indicating that probe NC-DTT possessed the advantages of fast response and high sensitivity toward DTT detection. Effect of pH pH is one of the most important factors in the sensing probes. To investigate the suitable pH value range for DTT determination, the fluorescence spectra of NC-DTT in the absence and presence of DTT at different pH values ranging from 3.0 to 11.0 were collected (Figure S2). Probe NC-DTT exhibited excellent spectra stability with no fluorescence signal in both acid and alkaline environments. After the response to DTT, the fluorescence intensity at 510 nm was an obvious increase from p H

6.0

to

11.0. These results demonstrated that probe NC-DTT could work efficiently in wide pH value environments, especially in physiological conditions. Selectivity of probe NC-DTT Selectivity test was performed for the free probe NC-DTT using various interfering substances (Figure 3). The fluorescence emission intensity at 510 nm of probe NC-DTT in the several interfering substances (Cys, GSH, Na2S, NaHS, Vc, ME and TGA) was measured. Compared with DTT, the addition of Na2S, NaHS, and Vc caused little change in the fluorescence signal. Although very weak fluorescence was observed in the presence of Cys, GSH, ME or TGA, the effect on the DTT detection was negligible. Consequently, the selectivity experiments confirmed that the probe NC-DTT exhibited great potential in specifically recognizing DTT in complex and changeable systems.

Proposed Mechanism To confirm the recognizing mechanism of NC-DTT with DTT, the reaction between probe NC-DTT and DTT was studied under the standard condition as 8

described above. As shown in Figure 4, the negative sulfhydryl ion from DTT can attack the sulfoxide group in NC-DTT, which had no fluorescence because of the lack of intramolecular charge transfer (ICT). After the cyclization reaction and release of water, the product with fluorescence was produced. The sulfide as electron donating group could strengthen the ICT process and lead to high fluorescence. The reaction product was achieved and characterized by One Drop spectrophotometer and Fluorescence spectrophotometer (Figure S3). The absorption spectrum showed that the solution of probe NC-DTT with DTT has a maximum absorption peak at 400 nm, which was the same as the solution of intermediate NC. And the similar result was observed in the fluorescence experiment. This indicated the release of compound NC, as further confirmed by the mass spectral analysis (m/z = 342.0 [M+Na]+, Figure S4).

Bioimaging of Probe NC-DTT The above results encouraged us to explore the practical application of NC-DTT in living cells. We supposed that the probe NC-DTT could provide fast response, high sensitivity and two-photon imaging for DTT in living cells. Before the cell imaging studies, MTT assay was carried out for the probe NC-DTT in different concentration (0-50 M) to evaluate its effects on HepG2 cells (Figure S5). The living cells grew very well and showed satisfactory cell viability after treatment with NC-DTT for 24 h even up to 50 M. The MTT assay demonstrated that our probe displayed low cytotoxicity to the living cells. Following, the cell imaging of probe NC-DTT in HepG2 cells was investigated (Figure 5 and S6). When HepG2 cells were incubated with NC-DTT (10 μM) for 0.5 h, slight green fluorescence was observed under the excitation at 405 nm. In the blocking experiment, the cells were performed with NC-DTT (10 μM) for 0.5 h after treated with N-ethylmaleimide (NEM, 1 mM) for 1 h. There was almost no emission in the green fluorescence channel. In contrast, when the HepG2 cells were pretreated with DTT (100 μM) before incubating with NC-DTT. Obvious and strong fluorescence signal was recorded in accordance with expectation. Moreover, the capability of NC-DTT in this aspect of two-photon fluorescence imaging was 9

investigated (Figure 6 and S7). The fluorescence intensity of the HepG2 cells with exogenous DTT was much stronger than the control group. And the blocking group pretreated with NEM exhibited almost no fluorescence.

Conclusion In summary, a novel turn-on two-photon fluorescence probe for DTT detection was successfully developed according to our reasonable design strategy. Upon the addition of DTT, the probe NC-DTT exhibited maximum absorption peak at 400 nm (redshift around 55 nm) and the maximum emission peak at 510 nm. The kinetic studies indicated that the response rate between NC-DTT with DTT was satisfactory. The probe exhibited high selectivity towards DTT over other biorelevants and high sensitivity with the detection limit of 1.410-7 M. Additionally, the probe NC-DTT displayed a desirable cellular imaging of DTT in living cells under single photon and two-photon confocal microscopy. Further applications of this two-photon probe in environmental monitoring and biochemical analysis of DTT are in progress.

Acknowledgments This work was financially supported by the National Natural Science Foundation of China (NSFC 81501529, 81671745 and 81220108012) and the 973 Key Project (2015CB755504).

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at…..

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13

Scheme 1. Synthetic route for probe NC-DTT

14

Figure 1. (a) The absorption spectra of NC-DTT (5 M) in the absence and presence of DTT (5 mM). (b) The fluorescence spectra of NC-DTT (5 M) in the absence and presence of DTT (5 mM). Inset: photographs of fluorescence changes under UV light (365 nm).

15

Figure 2. (a) Fluorescence response of NC-DTT (5 μM) toward different concentration of DTT (0-5 mM). (b) The linear relationship between fluorescence intensity at 510 nm with the concentration of DTT (0-700 μM)

16

Figure 3. Fluorescence response of NC-DTT (5 μM) to different biorelevants. Cys (20 mM), GSH (20 mM), Na2S (20 mM), NaHS (20 mM), Vc (20 mM), ME (10 mM) , TGA (10 mM) and DTT (5 mM). (λex = 400 nm, λem = 510 nm).

17

Figure 4. Possible reaction mechanism of NC-DTT with DTT

18

Figure 5. Single-photon confocal fluorescence images of living HepG2 cells. HepG2 cells incubated with the probe NC-DTT (10 μM) for 0.5 h after preincubated with DTT (100 μM) for 1 h: (a) green channel, (b) bright-field image, (c) overlay image of (a) and (b); HepG2 cells incubated with probe NC-DTT (10 μM) for 0.5 h: (d) green channel, (e) bright-field image, (f) overlay image of (d) and (e); HepG2 cells incubated with NC-DTT (10 μM) for 0.5 h after incubated with NEM (1 mM) for 2 h: (g) green channel, (h) bright-field image, (i) overlay image of (g) and (h). Excitation at 405 nm. Scale bar: 20 m.

19

Figure 6. Two-photon confocal fluorescence images of living HepG2 cells: (a) green channel, (b) bright-field image, and (c) overlay image; HepG2 cells incubated with the probe NC-DTT (10 μM) for 0.5 h after preincubated with 100 μM DTT for 1 h (d) green channel, (e) bright-field image, and (f) overlay image; HepG2 cells incubated with probe NC-DTT (10 μM) for 0.5h (g) green channel, (h) bright-field image, and (i) overlay image HepG2 cells incubated with NC-DTT (10 μM) for 0.5 h after incubated with 1 mM NEM for 2 h. Excitation at 750 nm

Highlights 

A fast response fluorescent probe for 1,4-dithiothreitol (DTT) has been developed.



NC-DTT exhibits excellent selectivity and high sensitivity towards DTT with LOD of 1.410-7 M.



NC-DTT can be used for one- and two-photon imaging of DTT in living cells with low cytotoxicity. 20