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A TP-FRET-based fluorescent sensor for ratiometric visualization of selenocysteine derivatives in living cells, tissues and zebrafish
Journal of Hazardous Materials 381 (2020) 120918
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A TP-FRET-based fluorescent sensor for ratiometric visualization of selenocysteine derivatives in living cells, tissues and zebrafish
T
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Xiongjie Zhaob, Gangqiang Yuana, Haiyuan Dinga, Liyi Zhoua, , Qinlu Lina a
Hunan Key Laboratory of Processed Food for Special Medical Purpose, National Engineering Laboratory for Deep Process of Rice and Byproducts, Hunan Key Laboratory of Grain-oil Deep Process and Quality Control, College of Food Science and Engineering, Central South University of Forestry and Technology, Changsha, Hunan, 41004, China b Department of Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Editor: D. Aga
Selenium is a biologically essential micronutrient element serving as an essential building block for selenoproteins (SePs), which is playing a key role in various cellular functions. Hence, it is of great significance to developing a reliable and rapid method for detection of Sec in biosystems. Compared with the previously reported probes that have been developed for selective detection of Sec, two-photon (TP) ratiometric Sec-specific probes would be advantageous for the NIR excitation and built-in correction of the dual emission bands. To quantitatively and selectively detect Sec over biothiols with rapid and sensitive response, we for the first time report a new fluorescence resonance energy transfer (FRET)-based TP ratiometric fluorescence probe CmNp-Sec, which was constructed by conjugating a TP fluorophore 6 (coumarin derivative with a D-π-A-structure) with a naphthalimide fluorophore 9 via a non-conjugated linker, and employed a 4-dinitrobenzene-ether (DNB) with a strong ICT effect as Sec responsive moiety. It exhibits quantitatively detect Sec in a wide range (0-50 μM) with a limit of detection of 7.88 nM within 10 min. More impressively, this probe can be conveniently used to detect Sec in living cells, tissues and zebrafish, demonstrating it has the latent capability in further biological applications.
Keywords: Two-photon bioimaging selenocysteine ratiometric imaging zebrafish
Abbreviations: Sec, selenocysteine; SePs, selenoproteins; GSH, glutathione; Cys, cysteine; Glu, glutamate; TP, two-photon; NIR, near-infrared; DTT, dithiothreitol); EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; DMAP, 4-dimethylaminopyridine; DCM, dichloromethane solution; OP, one-photon; 2PFM, TP laser confocal imaging; DNB, 4-dinitrobenzene-ether; ICT, intramolecular charge transfer; FRET, fluorescence resonance energy transfer ⁎ Corresponding author. E-mail address: [email protected] (L. Zhou). https://doi.org/10.1016/j.jhazmat.2019.120918 Received 13 March 2019; Received in revised form 16 July 2019; Accepted 23 July 2019 Available online 29 July 2019 0304-3894/ © 2019 Published by Elsevier B.V.
Journal of Hazardous Materials 381 (2020) 120918
X. Zhao, et al.
1. Introduction
2. Experimental
Selenium is a biologically essential micronutrient element playing a key role in various cellular functions in biosystems [1–3]. Despite numerous forms of selenium exist, such as hydrogen selenide, selenocysteine (Sec), selenite, selenophosphate, selenodiglutathione, and charged Sec-tRNA, Sec appears to carry out the majority of the function of the various Se-containing species in living systems, and is recognized as an essential building block for selenoproteins (SePs) [4]. SePs are related to many physiologically and pathologically processes and involved in numerous human disorders, such as inflammations, cancers, cardiovascular and neurodegenerative diseases [4–6]. Hence, in order to investigate the physiological function of Sec, it is of great significance to developing some reliable and rapid methods for the determination of Sec in vivo. It is well-known that fluorescent probes have their potential applications to serve as ideal tools in the field of bioimaging analysis and monitoring biological related analytes in biosystems, since they can offer less invasiveness, fast response, high selectivity and sensitivity, as well as high spatial and temporal resolution [7–8]. However, the similar chemical properties of Sec and biological thiols (including glutathione (GSH), cysteine (Cys), and homocysteine (Hcy)) make the design of Sec-specific fluorescence probes challenging. Additionally, the thiols are more abundant than Sec in living systems, which may bring potential interference in the detection of Sec. Therefore, to date, many efforts are part of the development of fluorescence probes for the determination of biothiols progressed fast in biosystems, while the reported for imaging physiological relevant Sec-specific fluorescent probes is still rare. The first Sec-specific fluorescent probe was developed based on the strategy of using the difference of pKa values between Sec (∼5.8) and biothiols (∼8.3) [9], while its application in biosystems was restricted due to the poor selectivity of this probe in physiological conditions (pH > 5.8). Impressively, in 2015, the first Sec-specific fluorescence probe that can be applied in physiological environment was reported by Zhang et al. [10]. Thereafter, some Secspecific fluorescent probes were developed [11–21]. However, the practical applications of these probes were limited since most of them were excited by UV or visible light, which may induce the photobleaching effect and shallow tissue penetration depth. In addition, the reported most of the Sec-specific fluorescent probes with a single fluorophore for single emission intensity change may be affected by instrumental efficiency, probe concentration and detecting conditions. Compared to these fluorescent probes in the visible region and single emission intensity change, it is well known that TP ratiometric fluorescent probe is a more favorable tool for image in biosystems, since it is excited under near-infrared (NIR) region resulting in deeper penetration depth (> 500 μm), minimized fluorescence background, less light scattering and tissue injury [22–26]. In addition, ratiometric fluorescence probes have the capability to eliminate interference from instrumental efficiency, detecting conditions, and the concentration of the probe by a built-in correction of the dual-emission band [22–25,27]. Therefore, it is highly desirable to develop a Sec-specific fluorescent probe with TP ratiometric fluorescence response, which would be advantageous in the qualitatively and quantitatively detection of the accurate concentration of Sec in biosystems. To the best of our knowledge, until now, no TP ratiometric fluorescent probe for the determination of Sec over other biothiols in biosystems has been reported. The design of the TP ratiometric probe which can qualitatively and quantitatively detect the accurate concentration of Sec is more challengeable. Herein, for the first time, a novel TP ratiometric fluorescence probe (CmNp-Sec) has been developed for the selective detection of Sec over biothiols with rapid TP ratiometric fluorescence response under physiological environment. In addition, fluorescence imaging experiments of Sec in living cells, tissues, and zebrafish demonstrate that this probe has the latent capability to monitor Sec in biosystems.
2.1. Materials and instruments All chemical reagents were obtained from commercial suppliers and used without further purification. Water used in all experiments was doubly distilled and purified by a Millipore Milli-Q system (USA). Mass spectra were recorded by an LCQ Advantage ion trap mass spectrometer (Thermo Finnigan). NMR spectra were recorded on a Bruker DRX-400 spectrometer using TMS as an internal standard. UV-vis absorption spectra were recorded in 1.0 cm path length quartz cuvettes on a Hitachi U-4100 UV-vis spectrophotometer (Japan). The pH was measured with a PHS-3C pH meter. All fluorescence measurements with a G9800A fluorescence spectrometer with both excitation and emission slits fixed at 2.5 nm, respectively. Fluorescence imaging experiment was conducted on a confocal laser scanning microscope (Olympus, Japan) with 458 nm and 760 nm excitation. 2.2. Synthesis and characterization of fluorophore and probe 2.2.1. Synthesis of fluorophore CmNp-OH The obtained compound 6 and 9 were prepared according to the previous research [28,29,30,31,32] and used to synthesize TP fluorophore CmNp-OH, as showed in Scheme 1. Specifically, the dichloromethane solution (DCM, 10 mL) of 329 mg compound 6 (1 mmol), 271 mg compound 9 (1 mmol), 570 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 3 mmol), and 50 mg 4-dimethylaminopyridine (DMAP, 0.4 mmol) was stirred at ambient temperature for 4 h until the reaction was completed. The remaining solvent of DCM was then removed by a rotary evaporator. The residue was subsequently purified by column chromatography with DCM/MeOH = 50:1 (v/v) to obtain 273 mg yellow solid with a yield of 47%. 1 HNMR (400 MHz, d6-DMSO) δ(ppm): 9.02(s, 1 H), 8.58-8.55(d, J=12 Hz, 1 H), 8.43-8.42(d, J=4Hz, 1 H), 8.29-8.27(d, J=8Hz, 1 H), 8.05-8.04(d, J=4Hz, 1 H), 7.70-7.66(t, J=8Hz, 1 H), 7.52-7.51(d, J=4Hz, 1 H), 6.99-6.97(d, J=8Hz, 1 H), 6.76-6.74(d, J=8Hz, 1 H), 6.57(s, 1 H), 5.01-4.98(d, J=12 Hz, 2 H), 3.74-3.55(m, 12 H), 1.231.07(t, J=32 Hz, 6 H); 13 C NMR (100 MHz, d6-DMSO) δ(ppm):165.96, 164.53, 163.09, 158.99, 157.33, 152.06, 144.88, 131.68, 130.72, 130.02, 122.08, 116.30, 111.52, 110.08, 107.46, 96.66, 44.66, 12.77; LC-MS: m/z C32H30N4O7 calcd 582.21, found [C32H31N4O7]+ 583.3. 2.2.2. Synthesis of probe CmNp-Sec 100 mg CmNp-OH (0.17 mmol), 96 mg 2,4-dinitrofluorobenzene (0.51 mmol) and 70 mg potassium carbonate (0.51 mmol) were dissolved in 10 mL DCM and further reacted at 25 °C for 2 h. The reaction mixture was then rotary evaporated to remove the solvent. The crude product was subsequently purified with column chromatography by DCM/MeOH = 50:1 (v/v) to afford 69 mg yellowish solid with a yield of 54%. 1 HNMR (400 MHz, d6-DMSO) δ(ppm): 9.02(s, 1 H), 8.628.59(t, J=6.0 Hz, 2 H), 8.54-8.51(d, J=12.0 Hz, 2 H), 8.05-7.97(m, 2 H), 7.60-7.51(m, 3 H), 6.76-6.75(d, J=4.0 Hz, 1 H), 6.57(s, 1 H), 5.01-4.98(d, J=12.0 Hz, 2 H), 3.74-3.35(m, 12 H), 1.23-1.07(t, J=32.0 Hz, 6 H); 13 C NMR (100 MHz, d6-DMSO) δ(ppm):165.50, 163.46, 163.17, 155.84, 152.17, 140.66, 133.32, 124.34, 119.10, 115.63, 109.91, 108.01, 96.79, 44.67, 12.66; LC-MS: m/z C38H32N6O11 calcd 748.21, found [C38H33N6O11]+ 749.3. 2.3. Preparation of solutions and spectrophotometric measurements The preparation of a 1 × 10-3 M stock solution of probe is by dissolving right amount of CmNp-Sec in DMSO. The Sec was prepared by the reaction of equimolar selenocystine (Sec)2 with dithiothreitol (DTT) in 10 mM phosphate buffer solution (PBS) at 37 °C for 30 min and freshly used [9–10]. Other solutions of bio-relative analytes in selectivity and competition tests were prepared from dissolving 2
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Scheme 1. The synthetic route of TP-FRET-based ratiometric fluorophore CmNp-OH and fluorescent probe CmNp-Sec.
samples using a lens with a focus length of 3.0 cm. The emission was collected at an angle of 90° to the direction of the excitation beam to minimize the scattering. The emission signal was directed into a CCD (Princeton Instruments, Pixis 400B) coupled monochromator (IsoPlane160) with an optical fiber. A 750 nm short pass filter was placed before the spectrometer to minimize the scattering from the excitation light. The two-photon absorption (TPA) cross section (δ) of the sample (s) at each wavelength was calculated according to equation (2), and rhodamine B in CH3OH was used as the reference (r).
corresponding donors, including KCl, NaCl, CaCl2, ZnSO4, MgSO4, NaF, NaBr, KI, Na2CO3, NaNO3, Na2S2O3, NaClO, Na2SeO3, Ala, Glu, Arg, Ser, Lys, Asp, Gly, Leu, Ile, Gln, Tyr, His, Trp, Thr, Phe, Asn, Met, Val, Cys, Hcy and GSH in distilled water. The 10 mM PBS buffer was obtained by dissolving Na2HPO4 and NaH2PO4 in distilled water, and the pH was adjusted with NaOH and HCl solutions. Test samples of titration test were prepared by 2.0 mL volumetric flasks. Specifically, 0.20 mL of probe was firstly added into the volumetric flask and then diluted to approximately 1.80 mL by PBS solution. And then 0.20 mL of Sec solutions with different concentrations (0-2 mM) was added and finally fixed to 2.0 mL of liquid volume with PBS. The absorption and fluorescence spectra were recorded after reacting for 10 min at room temperature.
δ = δr(SsΦrφrCr)/(SrΦsφsCs) (2) where S is the integrated fluorescence intensity, Φ is the fluorescence quantum yield, C is the concentration of sample (s) and reference (r), and φ is the collection efficiency of the experimental setup. The uncertainty in the measurement of cross sections is ∼15%. The detailed calculation is given in Fig. S5 in Supplementary Materials.
2.4. Fluorescent quantum yields and TP absorption active cross section of CmNp-Sec The quantum yield of CmNp-Sec was calculated by comparison with rhodamine 6 G (R = 0.95 in ethanol) as a reference using the following equation (1):
2.5. Selectivity and competition test Test samples of selectivity and competition assays were prepared in a series of 2 mL volumetric flasks. Specifically, 0.20 mL of probe (100 μM) was firstly added into the volumetric flask and then diluted to approximately 1.80 mL by PBS solution. Subsequently, 0.10 mL of 1.0 mM other bio-relative substance (including KCl, NaCl, CaCl2, ZnSO4, MgSO4, NaF, NaBr, KI, Na2CO3, NaNO3, Na2S2O3, NaClO, Na2SeO3, Ala, Glu, Arg, Ser, Lys, Asp, Gly, Leu, Ile, Gln, Tyr, His, Trp, Thr, Phe, Asn, Met, Val, Cys, Hcy and GSH) and 0.10 mL of Sec solutions (1.0 mM) was successively added into the flask. The solution mixture was finally fixed to 2.0 mL of solution volume with PBS buffer. All the fluorescence spectra were collected after reacting for 10 min at room
ΦF = IAR(n/nR)2ΦFR/IRA (1) Where F is the quantum yield, I is the integrated area under the fluorescence spectra, A is the absorbance, n is the refractive index of the solvent, and R refers to the reference rhodamine 6 G. The two-photon excited fluorescence was measured by using a Ti: sapphire femtosecond oscillator (Spectra Physics Mai Tai) as the excitation source. The output laser pulses have a tunable central wavelength from 690 nm to 1020 nm with pulse duration of less than 100 fs and a repetition rate of 80.5 MHz. The laser beam was focused onto the 3
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band of CmNp-OH centered at 419 nm, while the absorbance spectra of CmNp-Sec peaked at 406 nm under the experiment condition. After treated with 10 equivalent Sec, the probe CmNp-Sec exhibitd an absorption peak at 419 nm, which meant that the probe had reacted with Sec to release fluorophore. This phenomenon also could be confirmed by the HPLC analysis of CmNp-OH, CmNp-Sec and CmNp-Sec after treated with Sec. As shown in Fig.S1, the retention time of CmNp-OH (black line) and CmNp-Sec (pink line) were 3.4 min and 7.8 min respectively. Meanwhile, the HPLC spectrum of the sample of CmNp-Sec after treated with equivalent Sec (blue line) exhibited two absorbance peaks at 3.4 min and 7.8 min, which means that the CmNp-OH fluorophore has been generated upon added with Sec. In addition, we next tested the fluorescence response time of CmNpSec toward Sec. As shown in Fig. S2, upon added with 50 μM Sec into the solution of 1 μM probe, the emission at 545 nm was increased dramatically at first and gradually reached a stable state over 10 min. Moreover, the fluorescence spectra of CmNp-Sec with the addition of different concentration of Sec were next examined in pH 7.4 DMSO/ PBS (1:99, v/v). As shown in Fig. 1A(b) and B(a), the probe showed a strong blue fluorescence and exhibited an emission band peaked at 463 nm, which was ascribed to the fluorescence signal of cumarin moiety since the FRET process was blocked due to the ICT effect of DNB. However, upon added with Sec, the fluorescence signal of the probe changed to yellow and exerted a new center at 545 nm due to the release of naphthalimide fluorophore with the recovery FRET effect, and the signal at 545 nm gradually increased with the concentration of Sec in the range of 0-200 μM. Impressively, a good linear relationship (R2 = 0.9932) between fluorescence intensity ratios I545/I463 with the concentration of Sec in the range of 0-50 μM was achieved with a regression equation of I545/I463 = 0.064 [Sec] μM + 0.215. The limit of determination (LOD) was calculated as low as 7.88 nM. The titration experiment results indicated that the probe CmNp-Sec had the potentiality to quantitatively detect minute amount of Sec in aqueous solution. Furthermore, in order to verify the FRET process in the probe, we next examined the life time of the probe before and after reacted with Sec. As shown in Fig. S3, the life time of donor in probe was obvious longer than that of the donor in the solution of probe after addition of Sec. The results indicated that there was an efficient energy transfer of FRET process in the probe. In addition, we further measured the two-photon cross section (δ) of the probe solution to confirm the TP properties of this new probe by utilizing a Ti: sapphire femtosecond oscillator (Spectra Physics Mai Tai) as the excitation source. As shown in Fig. S4, the TP absorption cross action spectra of the probe exhibited δmax values of 182.2 GM at 760 nm under the experiment condition indicating the probe owned excellent TP properties and was potentially useful to imaging in deep tissue. The high selectivity to the target analyte over other potential competing species is an important index to evaluate the performance of a new probe, especially for a bioimaging analytical probe with potential application in complex biological systems. Therefore, we next examined the selectivity of the probe under simulative physiological condition. As depicted in Fig. 2a, representing cations (K+, Na+, Ca2+, Zn2+ and Mg2+), anions (F-, Br-, I-, CO32-, NO3-, S2O32-, ClO- and SeO32-), amino acids (Ala, Glu, Arg, Ser, Lys, Asp, Gly, Leu, Ile, Gln, Tyr, His, Trp, Thr, Phe, Asn, Met and Val) and biothiols (GSH, Cys and Glu) rendered no interference on the fluorescent response of CmNp-Sec in aqueous solutions. Meanwhile, the competition test was further conducted under simulative physiological environment. As showed in Fig. 2b, when the target analyte Sec coexisted with other potentially competing species, the ratio of fluorescence intensity I545/I463 exhibited no remarkable changes. Moreover, the selectivity and competition tests of probe CmNp-Sec toward nitroreductase (NTR), H2S and H2Sn were next conducted. As shown in Fig. S5, the addition of NTR and H2Sn could not cause remarkable interference in the selectivity experiments. Meanwhile, the addition of H2S for 10 min triggered only a small fluorescence enhancement that had nearly no interference to Sec detection.
temperature. 2.6. Cell cytotoxic assays and imaging Cytotoxicity of probe CmNp-Sec was evaluated by standard MTT assay. One-photon (OP) fluorescence images of HeLa cells were obtained by an Olympus FV1000-MPE multiphoton laser scanning confocal microscope (Japan). The OP excitation wavelength was fixed at 458 nm. The fluorescent emissions wavelength was recorded at (460495) nm, and (530-560) nm, individually. 2.7. Fluorescence imaging in tissues and living zebrafish Rat liver frozen slices were used for the tissue imaging, which was prepared by vibrating-blade microtome to get the tissue with a flat side. The slices were firstly incubated with 5 μM CmNp-Sec in an incubator at 37 °C for 30 min. Prior to TP laser confocal fluorescence imaging (2PFM), the slices were washed with PBS three times to remove the excessive amount of probe and incubated with Sec (25 or 50 μM) for 6 h. 2PFM images (with a magnification at 10×) were collected upon excitation at 760 nm with a pulse laser. The 5-d old wild type zebrafish was kindly gifted from the school of life sciences, Hunan Normal University. For the control group, zebrafish was only incubated with CmNp-Sec (5 μM) for 30 min, then imaged after washing by PBS buffer three times, and for the experimental group, zebrafish was pretreated with Sec (25 or 50 μM) for 6 h, subsequently incubated with CmNp-Sec for 30 min after washing by PBS buffer three times, then imaged with confocal fluorescence imaging upon excitation at 458 nm. 3. Results and discussion 3.1. Design and synthesis In the present work, the design approach of TP ratiometric fluorescence probe CmNp-Sec is by introducing 4-dinitrobenzene-ether (DNB) to a novel fluorophore (CmNp-OH). DNB has been selected as the Sec response site due to its chemoselectivity to Sec and the intramolecular charge transfer (ICT) effect to quench the fluorescence of naphthalimide. As for the design of the fluorophore, CmNp-OH is constructed base on the strategy of fluorescence resonance energy transfer (FRET) by conjugating a coumarin moiety with a naphthalimide fluorophore via a non-conjugated linker. The response mechanism is shown in Scheme 1. Specifically, at first, the fluorescence of naphthalimide moiety has been quenched by DNB, and the fluorescence of CmNp-Sec exhibits only blue emission which is ascribed to the fluorescent signal of cumarin moiety. Upon addition of Sec, the fluorescence signal of naphthalimide has been recovered, and a bright yellow emission can be obtained. Additionally, the 7-(diethylamino) cumarin3-carboxylic acid fluorophore exerts excellent TP properties and is selected as a two-photon fluorophore scaffold, because a 7-(diethylamino) coumarin-based fluorophore has been reported to possess TP properties with the maximum absorption cross section (δmax) value of 164.7 GM at 760 nm [33]. Thus, by using the Sec-dependent nucleophilic ability to release CmNp-O-, a concentrate-dependent TP ratiometric fluorescence response can be observed when the probe is treated with Sec. The CmNp-OH and CmNp-Sec are synthesized according to the route depicted in Scheme 1, and fully characterized by 1H NMR, 13C NMR and mass spectrometry (details see experimental section and Supplementary Materials). 3.2. Spectroscopic properties and selectivity of CmNp-Sec With prepared fluorophore and probe in hand, we firstly investigated the spectral properties of CmNp-OH and CmNp-Sec in pH 7.4 DMSO/PBS (1:99, v/v). As showed in Fig. 1A(a), the absorption 4
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Fig. 1. A: (a) Normalized absorption spectra of CmNp-OH, CmNp-Sec and CmNp-Sec after treatment with 10 equivalent Sec, (b) Fluorescence emission spectra (excitation at 385 nm) of the probe (1 μM) with varied concentrations of Sec (0, 2, 4, 8, 10, 15, 20, 25, 30, 40, 50, 75, 100, and 200 μM, respectively), and (c) The linear relationship between fluorescence intensity ratios I545/I463 of 1 μM CmNp-Sec and Sec concentrations (0-50 μM) in 10 mM PBS buffers (1% DMSO); B: Sensing mechanism of probe CmNp-Sec for Sec and the fluorescence photographs of 1 μM CmNp-Sec before (a) and after (b) treatment 10 equivalent Sec in 10 mM PBS buffers (1% DMSO), λex = 365 nm.
tissues and zebrafish. HeLa cells were chosen as the model cell line. At first, the cytotoxicity experiment showed that the CmNp-Sec was nearly nontoxic to living cells under our experimental conditions (Fig. S6). Next, after incubation with CmNp-Sec (5 μM) at 37 °C for 30 min, followed by excitation at 458 nm for imaging, the cells showed weak fluorescence intensity in the yellow channel (Fig. 3b) and bright fluorescence signal in the cyan channel (Fig. 3a). At the same time, tested group with addition of 25 or 50 μM Sec (6 h) and 5 μM CmNpSec for another 30 min, showed brighter fluorescence intensity in yellow channel (Fig. 3f & j, respectively) accompanying with weaker cyan emission intensity (Fig. 3e & i, respectively). The higher resolution of pseudo color mode for ratiometric images (Fig. 3h & k) and quantified relative fluorescence ratio of IYellow/ICyan of images analyzed using Nikon NIS Element software (Fig. 3l) were obtained indicating the
However, in the competition tests, NTR and H2Sn could render an attenuated enhancement of emission intensity after addition of Sec, which might be caused by the consumption of probe due to the reduction of nitro group by NTR or nucleophilic substitution of F atom by H2Sn. All the results of selectivity and competition tests fully confirm that CmNp-Sec is a highly selective fluorescence probe toward Sec and have the latent capability to track Sec in living cells. 3.3. Cytotoxicity, living cells imaging, and TP ratiometric imaging in tissues and zebrafish Encouraged by the above results, in order to further evaluate the imaging performance of TP rariometric fluorescent probe CmNp-Sec, this probe was used to detect Sec in biological samples, including cells,
Fig. 2. (a) Relative ratios of fluorescence intensity (I545/I463) of probe CmNp-Sec (1 μM) in 10 mM PBS buffers (1% DMSO, pH 7.4) to various analytes (50 μM). Legend: (1) Blank, (2) Sec, (3) K+; (4) Na+; (5) Zn2+; (6) Ca2+; (7) Mg2+; (8) F-; (9) Br-; (10) I-; (11) CO32-; (12) NO3-; (13) S2O32-; (14) ClO-; (15) SeO32-; (16) Ala; (17) Glu; (18) Arg; (19) Ser; (20) Lys; (21) Asp; (22) Gly; (23) Leu; (24) Ile; (25) Gln; (26) Tyr; (27) His; (28) Trp; (29) Thr; (30) Phe; (31) Asn; (32) Met; (33)Val; (34) GSH; (35) Cys; and (36) Glu. (b) Relative ratios of fluorescence intensity (I545/I463) of probe CmNp-Sec (1 μM) in 10 mM PBS buffers (1% DMSO, pH 7.4) to Sec (50 μM) in the presence of various bio-active substances (50 μM). Legend: (1) Sec, (2) Sec + K+; (3) Sec + Na+; (4) Sec + Zn2+; (5) Sec + Ca2+; (6) Sec + Mg2+; (7) Sec + F-; (8) Sec + Br-; (9) Sec + I-; (10) Sec + CO32-; (11) Sec + NO3-; (12) Sec + S2O32-; (13) Sec + ClO-; (14) Sec + SeO32-; (15) Sec + Ala; (16) Sec + Glu; (17) Sec + Arg; (18) Sec + Ser; (19) Sec + Lys; (20) Sec + Asp; (21) Sec + Gly; (22) Sec + Leu; (23) Sec + Ile; (24) Sec + Gln; (25) Sec + Tyr; (26) Sec + His; (27) Sec + Trp; (28) Sec + Thr; (29) Sec + Phe; (30) Sec + Asn; (31) Sec + Met; (32) Sec + Val; (33) Sec + GSH; (34) Sec + Cys; and (35) Sec + Glu. 5
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Fig. 3. Fluorescence images of HeLa cells stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging. (a) The cyan channel imaging, (b) the yellow channel imaging, (c) an overlay imaging of (a), (b) and the bright field imaging, (d) Pseudo color mode for ratiometric image; Fluorescence images of HeLa cells incubated with 25 μM Sec for 6 h, and then stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging. (e) The cyan channel imaging, (f) the yellow channel imaging, (g) an overlay imaging of (e), (f) and the bright field imaging, (h) Pseudo color mode for ratiometric image; Fluorescence images of HeLa cells incubated with 50 μM Sec for 6 h, and then stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging. (i) The cyan channel imaging, (j) the yellow channel imaging, (k) an overlay imaging of (i), (j) and the bright field imaging, (l) Pseudo color mode for ratiometric image. (m) Quantified relative fluorescence ratio of IYellow/ICyan of images analyzed using Nikon NIS Element software and presented as mean ± s.d, n = 3. The cyan channel and the yellow channel: (460-495) nm, and (530-560) nm, individually, λex = 458 nm, the scale bar is 20 μm.
4. Conclusion
probe CmNp-Sec had the potentiality to be an ideal tool for the quantification of cellular Sec. In order to evaluate the TP ratiometric imaging performance of CmNp-Sec in the rat liver tissues with deep-tissue penetration imaging and high resolution ratiometric imaging. The imaging thicknesses of the tissues were determined by confocal multiphoton microscopy (Olympus, FV1000) in the z-scan mode with 760 nm exaction (Fig. 4(a–c) and (g–i)). In the absence of Sec, CmNp-Sec was capable of tissue imaging at depths of 90 μm by TPM (Fig. 4(a–c)). Moreover, the tissues showed weak fluorescence intensity in the yellow (Fig. 4b) and bright fluorescence in the cyan channel (Fig. 4a). If were treated with different concentration of Sec (25 or 50 μM) for 6 h (Fig. 4g–i & m–o), tissue fluorescence images in two emission channels with different colors at 95 μm by TP imaging and the quantified relative fluorescence ratio of IYellow/ICyan of images were obtained (Fig. 4i, o & s). These data showed that CmNp-Sec had good tissue penetration and staining ability as well as quantified ratiometric two-color (cyan and yellow) imaging performance with less cross talk between channels. Finally, CmNp-Sec was further applied for imaging of Sec in living zebrafish. 5-day-old vertebrate zebrafishes were chosen as the experimental samples. Zebrafish was first incubated with CmNp-Sec (5 μM) for 30 min. As shown in Fig. 4(d–f), a bright fluorescence in the cyan channel could be observed without addition of Sec. A t the same time, as shown in Fig. 4(j–l & p–r), once the zebrafish was pretreated with 25 or 50 μM Sec for 6 h and then incubated with 5 μM CmNp-Sec for 30 min, the fluorescence intensity enhanced in the yellow channel (Fig. 4k & q) and the fluorescence intensity weakened in the cyan channel (Fig. 4j & p). After analyzing the fluorescence intensity of zebrafish imaging group (Fig. 4t), the quantified ability of CmNp-Sec in zebrafish model had also been confirmed. Combining these results together, these images proved that CmNp-Sec can be used to quantified detection of Sec in living cells, tissues and zebrafish.
In summary, we have developed a new TP-FRET-based TP ratiometric fluorescent probe CmNp-Sec for the selective detection of Sec, which is constructed by introducing DNB to a novel fluorophore (CmNp-OH). The CmNp-OH is fabricated with two-photon fluorophore cumarin derivative (as the donor) and a naphthalimide fluorophore (as an acceptor) through the connection of a non-conjugated linker. All the experiments conducted above indicate that CmNp-Sec owns rapid rariometric fluorescence response (10 min), quantitative determination in a wide range (0-50 μM) with a high sensitivity (LOD = 7.88 nM), selectivity toward Sec over other biothiols and representative amino acids. Additionally, Confocal microscopic fluorescence images of HeLa cells with CmNp-Sec revealed there were two distinct emission ranges at cyan channel and yellow channel in living cells. More impressively, the probe CmNp-Sec was successfully applied in tracking Sec in living tissues and zebrafish. We speculate that CmNp-Sec has the potentiality to find wide applications in biomedical diagnostics. Declaration of Competing Interest The authors declare no competing financial interest. Acknowledgments This work was supported by Natural Science Foundation of China (NSFC, Grants 21605046), Hunan Provincial Natural Science Foundation of China (No. 2017JJ3060), Science and Technology Innovation Platform and Talent Project of Hunan Province (2017TP1021). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jhazmat.2019.120918. 6
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Fig. 4. 2PFM images of a frozen liver tissue slice from a nude mouse model stained. Tissue imaging at depths of 90 μm by 2PFM in Z-scan mode upon excitation at 760 nm with femtosecond pulses. (a-c) liver tissue slice incubated with stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging: (a) The cyan channel imaging, (b) the yellow channel imaging and (c) pseudo color mode for ratiometric image; (g-i) liver tissue slice incubated with 25 μM Sec for 6 h, and then stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging: (g) The cyan channel imaging, (h) the yellow channel imaging and (i) pseudo color mode for ratiometric image; (m-o) liver tissue slice incubated with 50 μM Sec for 6 h, and then stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging: (m) The cyan channel imaging, (n) the yellow channel imaging and (o) pseudo color mode for ratiometric image. Fluorescence images of zebrafish incubated with stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging: (d) The cyan channel imaging, (e) the yellow channel imaging and (f) pseudo color mode for ratiometric image; (j-l) zebrafish incubated with 25 μM Sec for 6 h, and then stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging: (j) The cyan channel imaging, (k) the yellow channel imaging and (l) pseudo color mode for ratiometric image; (p-r) zebrafish incubated with 50 μM Sec for 6 h, and then stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging: (p) The cyan channel imaging, (q) the yellow channel imaging and (r) pseudo color mode for ratiometric image. (s-t) Quantified relative fluorescence ratio of IYellow/ICyan of images analyzed using Nikon NIS Element software and presented as mean ± s.d, n = 3(s for the images of tissues and t for the images of zebrafish). The cyan channel and the yellow channel: (460-495) nm, and (530-560) nm, individually, λex = 458 nm, the scale bar is 15 μm. 7′‐dimethylfluorescein as a fluorescent probe for selenols. Angew. Chem., Int. Ed. 45, 1810–1813. https://doi.org/10.1002/anie.200504299. Zhang, B., Ge, C., Yao, J., Liu, Y., Xie, H., Fang, J., 2015. Selective selenol fluorescent probes: design, synthesis, structural determinants, and biological applications. J. Am. Chem. Soc. 137, 757–769. https://doi.org/10.1021/ja5099676. Kong, F., Hua, B., Gao, Y., Xu, K., Pan, X., Huang, F., Zheng, Q., Chen, H., Tang, B., 2015. Fluorescence imaging of selenol in HepG2 cell apoptosis induced by Na2SeO3. Chem. Commun. 51, 3102–3105. https://doi.org/10.1039/C4CC06359G. Chen, H., Dong, B., Tang, Y., Lin, W., 2015. Construction of a near-infrared fluorescent turn-on probe for selenol and its bioimaging application in living animals. Chem.Eur. J. 21, 11696–11700. https://doi.org/10.1002/chem.201502226. Kong, F., Ge, L., Pan, X., Xu, K., Liu, X., Tang, B., 2016. A highly selective near-infrared fluorescent probe for imaging H2Se in living cells and in vivo. Chem. Sci. 7, 1051–1056. https://doi.org/10.1039/C5SC03471J. Li, M., Feng, W., Zhai, Q., Feng, G., 2017. Selenocysteine detection and bioimaging in living cells by a colorimetric and near-infrared fluorescent turn-on probe with a large stokes shift. Biosens. Bioelectron. 87, 894–900. https://doi.org/10.1016/j.bios.2016. 09.056. Feng, W., Li, M., Sun, Y., Feng, G., 2017. Near-infrared fluorescent turn-on probe with a remarkable large Stokes shift for imaging selenocysteine in living cells and animals. Anal. Chem. 89, 6106–6112. https://doi.org/10.1021/acs.analchem.7b00824. Areti, S., Verma, S.K., Bellareb, J., Raoa, C.P., 2016. Selenocysteine vs cysteine: tuning the derivatization on benzenesulfonyl moiety of a triazole linked dansyl connected glycoconjugate for selective recognition of selenocysteine and the applicability of the
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A TP-FRET-based fluorescent sensor for ratiometric visualization of selenocysteine derivatives in living cells, tissues and zebrafish
T
⁎
Xiongjie Zhaob, Gangqiang Yuana, Haiyuan Dinga, Liyi Zhoua, , Qinlu Lina a
Hunan Key Laboratory of Processed Food for Special Medical Purpose, National Engineering Laboratory for Deep Process of Rice and Byproducts, Hunan Key Laboratory of Grain-oil Deep Process and Quality Control, College of Food Science and Engineering, Central South University of Forestry and Technology, Changsha, Hunan, 41004, China b Department of Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Editor: D. Aga
Selenium is a biologically essential micronutrient element serving as an essential building block for selenoproteins (SePs), which is playing a key role in various cellular functions. Hence, it is of great significance to developing a reliable and rapid method for detection of Sec in biosystems. Compared with the previously reported probes that have been developed for selective detection of Sec, two-photon (TP) ratiometric Sec-specific probes would be advantageous for the NIR excitation and built-in correction of the dual emission bands. To quantitatively and selectively detect Sec over biothiols with rapid and sensitive response, we for the first time report a new fluorescence resonance energy transfer (FRET)-based TP ratiometric fluorescence probe CmNp-Sec, which was constructed by conjugating a TP fluorophore 6 (coumarin derivative with a D-π-A-structure) with a naphthalimide fluorophore 9 via a non-conjugated linker, and employed a 4-dinitrobenzene-ether (DNB) with a strong ICT effect as Sec responsive moiety. It exhibits quantitatively detect Sec in a wide range (0-50 μM) with a limit of detection of 7.88 nM within 10 min. More impressively, this probe can be conveniently used to detect Sec in living cells, tissues and zebrafish, demonstrating it has the latent capability in further biological applications.
Keywords: Two-photon bioimaging selenocysteine ratiometric imaging zebrafish
Abbreviations: Sec, selenocysteine; SePs, selenoproteins; GSH, glutathione; Cys, cysteine; Glu, glutamate; TP, two-photon; NIR, near-infrared; DTT, dithiothreitol); EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; DMAP, 4-dimethylaminopyridine; DCM, dichloromethane solution; OP, one-photon; 2PFM, TP laser confocal imaging; DNB, 4-dinitrobenzene-ether; ICT, intramolecular charge transfer; FRET, fluorescence resonance energy transfer ⁎ Corresponding author. E-mail address: [email protected] (L. Zhou). https://doi.org/10.1016/j.jhazmat.2019.120918 Received 13 March 2019; Received in revised form 16 July 2019; Accepted 23 July 2019 Available online 29 July 2019 0304-3894/ © 2019 Published by Elsevier B.V.
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1. Introduction
2. Experimental
Selenium is a biologically essential micronutrient element playing a key role in various cellular functions in biosystems [1–3]. Despite numerous forms of selenium exist, such as hydrogen selenide, selenocysteine (Sec), selenite, selenophosphate, selenodiglutathione, and charged Sec-tRNA, Sec appears to carry out the majority of the function of the various Se-containing species in living systems, and is recognized as an essential building block for selenoproteins (SePs) [4]. SePs are related to many physiologically and pathologically processes and involved in numerous human disorders, such as inflammations, cancers, cardiovascular and neurodegenerative diseases [4–6]. Hence, in order to investigate the physiological function of Sec, it is of great significance to developing some reliable and rapid methods for the determination of Sec in vivo. It is well-known that fluorescent probes have their potential applications to serve as ideal tools in the field of bioimaging analysis and monitoring biological related analytes in biosystems, since they can offer less invasiveness, fast response, high selectivity and sensitivity, as well as high spatial and temporal resolution [7–8]. However, the similar chemical properties of Sec and biological thiols (including glutathione (GSH), cysteine (Cys), and homocysteine (Hcy)) make the design of Sec-specific fluorescence probes challenging. Additionally, the thiols are more abundant than Sec in living systems, which may bring potential interference in the detection of Sec. Therefore, to date, many efforts are part of the development of fluorescence probes for the determination of biothiols progressed fast in biosystems, while the reported for imaging physiological relevant Sec-specific fluorescent probes is still rare. The first Sec-specific fluorescent probe was developed based on the strategy of using the difference of pKa values between Sec (∼5.8) and biothiols (∼8.3) [9], while its application in biosystems was restricted due to the poor selectivity of this probe in physiological conditions (pH > 5.8). Impressively, in 2015, the first Sec-specific fluorescence probe that can be applied in physiological environment was reported by Zhang et al. [10]. Thereafter, some Secspecific fluorescent probes were developed [11–21]. However, the practical applications of these probes were limited since most of them were excited by UV or visible light, which may induce the photobleaching effect and shallow tissue penetration depth. In addition, the reported most of the Sec-specific fluorescent probes with a single fluorophore for single emission intensity change may be affected by instrumental efficiency, probe concentration and detecting conditions. Compared to these fluorescent probes in the visible region and single emission intensity change, it is well known that TP ratiometric fluorescent probe is a more favorable tool for image in biosystems, since it is excited under near-infrared (NIR) region resulting in deeper penetration depth (> 500 μm), minimized fluorescence background, less light scattering and tissue injury [22–26]. In addition, ratiometric fluorescence probes have the capability to eliminate interference from instrumental efficiency, detecting conditions, and the concentration of the probe by a built-in correction of the dual-emission band [22–25,27]. Therefore, it is highly desirable to develop a Sec-specific fluorescent probe with TP ratiometric fluorescence response, which would be advantageous in the qualitatively and quantitatively detection of the accurate concentration of Sec in biosystems. To the best of our knowledge, until now, no TP ratiometric fluorescent probe for the determination of Sec over other biothiols in biosystems has been reported. The design of the TP ratiometric probe which can qualitatively and quantitatively detect the accurate concentration of Sec is more challengeable. Herein, for the first time, a novel TP ratiometric fluorescence probe (CmNp-Sec) has been developed for the selective detection of Sec over biothiols with rapid TP ratiometric fluorescence response under physiological environment. In addition, fluorescence imaging experiments of Sec in living cells, tissues, and zebrafish demonstrate that this probe has the latent capability to monitor Sec in biosystems.
2.1. Materials and instruments All chemical reagents were obtained from commercial suppliers and used without further purification. Water used in all experiments was doubly distilled and purified by a Millipore Milli-Q system (USA). Mass spectra were recorded by an LCQ Advantage ion trap mass spectrometer (Thermo Finnigan). NMR spectra were recorded on a Bruker DRX-400 spectrometer using TMS as an internal standard. UV-vis absorption spectra were recorded in 1.0 cm path length quartz cuvettes on a Hitachi U-4100 UV-vis spectrophotometer (Japan). The pH was measured with a PHS-3C pH meter. All fluorescence measurements with a G9800A fluorescence spectrometer with both excitation and emission slits fixed at 2.5 nm, respectively. Fluorescence imaging experiment was conducted on a confocal laser scanning microscope (Olympus, Japan) with 458 nm and 760 nm excitation. 2.2. Synthesis and characterization of fluorophore and probe 2.2.1. Synthesis of fluorophore CmNp-OH The obtained compound 6 and 9 were prepared according to the previous research [28,29,30,31,32] and used to synthesize TP fluorophore CmNp-OH, as showed in Scheme 1. Specifically, the dichloromethane solution (DCM, 10 mL) of 329 mg compound 6 (1 mmol), 271 mg compound 9 (1 mmol), 570 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 3 mmol), and 50 mg 4-dimethylaminopyridine (DMAP, 0.4 mmol) was stirred at ambient temperature for 4 h until the reaction was completed. The remaining solvent of DCM was then removed by a rotary evaporator. The residue was subsequently purified by column chromatography with DCM/MeOH = 50:1 (v/v) to obtain 273 mg yellow solid with a yield of 47%. 1 HNMR (400 MHz, d6-DMSO) δ(ppm): 9.02(s, 1 H), 8.58-8.55(d, J=12 Hz, 1 H), 8.43-8.42(d, J=4Hz, 1 H), 8.29-8.27(d, J=8Hz, 1 H), 8.05-8.04(d, J=4Hz, 1 H), 7.70-7.66(t, J=8Hz, 1 H), 7.52-7.51(d, J=4Hz, 1 H), 6.99-6.97(d, J=8Hz, 1 H), 6.76-6.74(d, J=8Hz, 1 H), 6.57(s, 1 H), 5.01-4.98(d, J=12 Hz, 2 H), 3.74-3.55(m, 12 H), 1.231.07(t, J=32 Hz, 6 H); 13 C NMR (100 MHz, d6-DMSO) δ(ppm):165.96, 164.53, 163.09, 158.99, 157.33, 152.06, 144.88, 131.68, 130.72, 130.02, 122.08, 116.30, 111.52, 110.08, 107.46, 96.66, 44.66, 12.77; LC-MS: m/z C32H30N4O7 calcd 582.21, found [C32H31N4O7]+ 583.3. 2.2.2. Synthesis of probe CmNp-Sec 100 mg CmNp-OH (0.17 mmol), 96 mg 2,4-dinitrofluorobenzene (0.51 mmol) and 70 mg potassium carbonate (0.51 mmol) were dissolved in 10 mL DCM and further reacted at 25 °C for 2 h. The reaction mixture was then rotary evaporated to remove the solvent. The crude product was subsequently purified with column chromatography by DCM/MeOH = 50:1 (v/v) to afford 69 mg yellowish solid with a yield of 54%. 1 HNMR (400 MHz, d6-DMSO) δ(ppm): 9.02(s, 1 H), 8.628.59(t, J=6.0 Hz, 2 H), 8.54-8.51(d, J=12.0 Hz, 2 H), 8.05-7.97(m, 2 H), 7.60-7.51(m, 3 H), 6.76-6.75(d, J=4.0 Hz, 1 H), 6.57(s, 1 H), 5.01-4.98(d, J=12.0 Hz, 2 H), 3.74-3.35(m, 12 H), 1.23-1.07(t, J=32.0 Hz, 6 H); 13 C NMR (100 MHz, d6-DMSO) δ(ppm):165.50, 163.46, 163.17, 155.84, 152.17, 140.66, 133.32, 124.34, 119.10, 115.63, 109.91, 108.01, 96.79, 44.67, 12.66; LC-MS: m/z C38H32N6O11 calcd 748.21, found [C38H33N6O11]+ 749.3. 2.3. Preparation of solutions and spectrophotometric measurements The preparation of a 1 × 10-3 M stock solution of probe is by dissolving right amount of CmNp-Sec in DMSO. The Sec was prepared by the reaction of equimolar selenocystine (Sec)2 with dithiothreitol (DTT) in 10 mM phosphate buffer solution (PBS) at 37 °C for 30 min and freshly used [9–10]. Other solutions of bio-relative analytes in selectivity and competition tests were prepared from dissolving 2
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Scheme 1. The synthetic route of TP-FRET-based ratiometric fluorophore CmNp-OH and fluorescent probe CmNp-Sec.
samples using a lens with a focus length of 3.0 cm. The emission was collected at an angle of 90° to the direction of the excitation beam to minimize the scattering. The emission signal was directed into a CCD (Princeton Instruments, Pixis 400B) coupled monochromator (IsoPlane160) with an optical fiber. A 750 nm short pass filter was placed before the spectrometer to minimize the scattering from the excitation light. The two-photon absorption (TPA) cross section (δ) of the sample (s) at each wavelength was calculated according to equation (2), and rhodamine B in CH3OH was used as the reference (r).
corresponding donors, including KCl, NaCl, CaCl2, ZnSO4, MgSO4, NaF, NaBr, KI, Na2CO3, NaNO3, Na2S2O3, NaClO, Na2SeO3, Ala, Glu, Arg, Ser, Lys, Asp, Gly, Leu, Ile, Gln, Tyr, His, Trp, Thr, Phe, Asn, Met, Val, Cys, Hcy and GSH in distilled water. The 10 mM PBS buffer was obtained by dissolving Na2HPO4 and NaH2PO4 in distilled water, and the pH was adjusted with NaOH and HCl solutions. Test samples of titration test were prepared by 2.0 mL volumetric flasks. Specifically, 0.20 mL of probe was firstly added into the volumetric flask and then diluted to approximately 1.80 mL by PBS solution. And then 0.20 mL of Sec solutions with different concentrations (0-2 mM) was added and finally fixed to 2.0 mL of liquid volume with PBS. The absorption and fluorescence spectra were recorded after reacting for 10 min at room temperature.
δ = δr(SsΦrφrCr)/(SrΦsφsCs) (2) where S is the integrated fluorescence intensity, Φ is the fluorescence quantum yield, C is the concentration of sample (s) and reference (r), and φ is the collection efficiency of the experimental setup. The uncertainty in the measurement of cross sections is ∼15%. The detailed calculation is given in Fig. S5 in Supplementary Materials.
2.4. Fluorescent quantum yields and TP absorption active cross section of CmNp-Sec The quantum yield of CmNp-Sec was calculated by comparison with rhodamine 6 G (R = 0.95 in ethanol) as a reference using the following equation (1):
2.5. Selectivity and competition test Test samples of selectivity and competition assays were prepared in a series of 2 mL volumetric flasks. Specifically, 0.20 mL of probe (100 μM) was firstly added into the volumetric flask and then diluted to approximately 1.80 mL by PBS solution. Subsequently, 0.10 mL of 1.0 mM other bio-relative substance (including KCl, NaCl, CaCl2, ZnSO4, MgSO4, NaF, NaBr, KI, Na2CO3, NaNO3, Na2S2O3, NaClO, Na2SeO3, Ala, Glu, Arg, Ser, Lys, Asp, Gly, Leu, Ile, Gln, Tyr, His, Trp, Thr, Phe, Asn, Met, Val, Cys, Hcy and GSH) and 0.10 mL of Sec solutions (1.0 mM) was successively added into the flask. The solution mixture was finally fixed to 2.0 mL of solution volume with PBS buffer. All the fluorescence spectra were collected after reacting for 10 min at room
ΦF = IAR(n/nR)2ΦFR/IRA (1) Where F is the quantum yield, I is the integrated area under the fluorescence spectra, A is the absorbance, n is the refractive index of the solvent, and R refers to the reference rhodamine 6 G. The two-photon excited fluorescence was measured by using a Ti: sapphire femtosecond oscillator (Spectra Physics Mai Tai) as the excitation source. The output laser pulses have a tunable central wavelength from 690 nm to 1020 nm with pulse duration of less than 100 fs and a repetition rate of 80.5 MHz. The laser beam was focused onto the 3
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band of CmNp-OH centered at 419 nm, while the absorbance spectra of CmNp-Sec peaked at 406 nm under the experiment condition. After treated with 10 equivalent Sec, the probe CmNp-Sec exhibitd an absorption peak at 419 nm, which meant that the probe had reacted with Sec to release fluorophore. This phenomenon also could be confirmed by the HPLC analysis of CmNp-OH, CmNp-Sec and CmNp-Sec after treated with Sec. As shown in Fig.S1, the retention time of CmNp-OH (black line) and CmNp-Sec (pink line) were 3.4 min and 7.8 min respectively. Meanwhile, the HPLC spectrum of the sample of CmNp-Sec after treated with equivalent Sec (blue line) exhibited two absorbance peaks at 3.4 min and 7.8 min, which means that the CmNp-OH fluorophore has been generated upon added with Sec. In addition, we next tested the fluorescence response time of CmNpSec toward Sec. As shown in Fig. S2, upon added with 50 μM Sec into the solution of 1 μM probe, the emission at 545 nm was increased dramatically at first and gradually reached a stable state over 10 min. Moreover, the fluorescence spectra of CmNp-Sec with the addition of different concentration of Sec were next examined in pH 7.4 DMSO/ PBS (1:99, v/v). As shown in Fig. 1A(b) and B(a), the probe showed a strong blue fluorescence and exhibited an emission band peaked at 463 nm, which was ascribed to the fluorescence signal of cumarin moiety since the FRET process was blocked due to the ICT effect of DNB. However, upon added with Sec, the fluorescence signal of the probe changed to yellow and exerted a new center at 545 nm due to the release of naphthalimide fluorophore with the recovery FRET effect, and the signal at 545 nm gradually increased with the concentration of Sec in the range of 0-200 μM. Impressively, a good linear relationship (R2 = 0.9932) between fluorescence intensity ratios I545/I463 with the concentration of Sec in the range of 0-50 μM was achieved with a regression equation of I545/I463 = 0.064 [Sec] μM + 0.215. The limit of determination (LOD) was calculated as low as 7.88 nM. The titration experiment results indicated that the probe CmNp-Sec had the potentiality to quantitatively detect minute amount of Sec in aqueous solution. Furthermore, in order to verify the FRET process in the probe, we next examined the life time of the probe before and after reacted with Sec. As shown in Fig. S3, the life time of donor in probe was obvious longer than that of the donor in the solution of probe after addition of Sec. The results indicated that there was an efficient energy transfer of FRET process in the probe. In addition, we further measured the two-photon cross section (δ) of the probe solution to confirm the TP properties of this new probe by utilizing a Ti: sapphire femtosecond oscillator (Spectra Physics Mai Tai) as the excitation source. As shown in Fig. S4, the TP absorption cross action spectra of the probe exhibited δmax values of 182.2 GM at 760 nm under the experiment condition indicating the probe owned excellent TP properties and was potentially useful to imaging in deep tissue. The high selectivity to the target analyte over other potential competing species is an important index to evaluate the performance of a new probe, especially for a bioimaging analytical probe with potential application in complex biological systems. Therefore, we next examined the selectivity of the probe under simulative physiological condition. As depicted in Fig. 2a, representing cations (K+, Na+, Ca2+, Zn2+ and Mg2+), anions (F-, Br-, I-, CO32-, NO3-, S2O32-, ClO- and SeO32-), amino acids (Ala, Glu, Arg, Ser, Lys, Asp, Gly, Leu, Ile, Gln, Tyr, His, Trp, Thr, Phe, Asn, Met and Val) and biothiols (GSH, Cys and Glu) rendered no interference on the fluorescent response of CmNp-Sec in aqueous solutions. Meanwhile, the competition test was further conducted under simulative physiological environment. As showed in Fig. 2b, when the target analyte Sec coexisted with other potentially competing species, the ratio of fluorescence intensity I545/I463 exhibited no remarkable changes. Moreover, the selectivity and competition tests of probe CmNp-Sec toward nitroreductase (NTR), H2S and H2Sn were next conducted. As shown in Fig. S5, the addition of NTR and H2Sn could not cause remarkable interference in the selectivity experiments. Meanwhile, the addition of H2S for 10 min triggered only a small fluorescence enhancement that had nearly no interference to Sec detection.
temperature. 2.6. Cell cytotoxic assays and imaging Cytotoxicity of probe CmNp-Sec was evaluated by standard MTT assay. One-photon (OP) fluorescence images of HeLa cells were obtained by an Olympus FV1000-MPE multiphoton laser scanning confocal microscope (Japan). The OP excitation wavelength was fixed at 458 nm. The fluorescent emissions wavelength was recorded at (460495) nm, and (530-560) nm, individually. 2.7. Fluorescence imaging in tissues and living zebrafish Rat liver frozen slices were used for the tissue imaging, which was prepared by vibrating-blade microtome to get the tissue with a flat side. The slices were firstly incubated with 5 μM CmNp-Sec in an incubator at 37 °C for 30 min. Prior to TP laser confocal fluorescence imaging (2PFM), the slices were washed with PBS three times to remove the excessive amount of probe and incubated with Sec (25 or 50 μM) for 6 h. 2PFM images (with a magnification at 10×) were collected upon excitation at 760 nm with a pulse laser. The 5-d old wild type zebrafish was kindly gifted from the school of life sciences, Hunan Normal University. For the control group, zebrafish was only incubated with CmNp-Sec (5 μM) for 30 min, then imaged after washing by PBS buffer three times, and for the experimental group, zebrafish was pretreated with Sec (25 or 50 μM) for 6 h, subsequently incubated with CmNp-Sec for 30 min after washing by PBS buffer three times, then imaged with confocal fluorescence imaging upon excitation at 458 nm. 3. Results and discussion 3.1. Design and synthesis In the present work, the design approach of TP ratiometric fluorescence probe CmNp-Sec is by introducing 4-dinitrobenzene-ether (DNB) to a novel fluorophore (CmNp-OH). DNB has been selected as the Sec response site due to its chemoselectivity to Sec and the intramolecular charge transfer (ICT) effect to quench the fluorescence of naphthalimide. As for the design of the fluorophore, CmNp-OH is constructed base on the strategy of fluorescence resonance energy transfer (FRET) by conjugating a coumarin moiety with a naphthalimide fluorophore via a non-conjugated linker. The response mechanism is shown in Scheme 1. Specifically, at first, the fluorescence of naphthalimide moiety has been quenched by DNB, and the fluorescence of CmNp-Sec exhibits only blue emission which is ascribed to the fluorescent signal of cumarin moiety. Upon addition of Sec, the fluorescence signal of naphthalimide has been recovered, and a bright yellow emission can be obtained. Additionally, the 7-(diethylamino) cumarin3-carboxylic acid fluorophore exerts excellent TP properties and is selected as a two-photon fluorophore scaffold, because a 7-(diethylamino) coumarin-based fluorophore has been reported to possess TP properties with the maximum absorption cross section (δmax) value of 164.7 GM at 760 nm [33]. Thus, by using the Sec-dependent nucleophilic ability to release CmNp-O-, a concentrate-dependent TP ratiometric fluorescence response can be observed when the probe is treated with Sec. The CmNp-OH and CmNp-Sec are synthesized according to the route depicted in Scheme 1, and fully characterized by 1H NMR, 13C NMR and mass spectrometry (details see experimental section and Supplementary Materials). 3.2. Spectroscopic properties and selectivity of CmNp-Sec With prepared fluorophore and probe in hand, we firstly investigated the spectral properties of CmNp-OH and CmNp-Sec in pH 7.4 DMSO/PBS (1:99, v/v). As showed in Fig. 1A(a), the absorption 4
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Fig. 1. A: (a) Normalized absorption spectra of CmNp-OH, CmNp-Sec and CmNp-Sec after treatment with 10 equivalent Sec, (b) Fluorescence emission spectra (excitation at 385 nm) of the probe (1 μM) with varied concentrations of Sec (0, 2, 4, 8, 10, 15, 20, 25, 30, 40, 50, 75, 100, and 200 μM, respectively), and (c) The linear relationship between fluorescence intensity ratios I545/I463 of 1 μM CmNp-Sec and Sec concentrations (0-50 μM) in 10 mM PBS buffers (1% DMSO); B: Sensing mechanism of probe CmNp-Sec for Sec and the fluorescence photographs of 1 μM CmNp-Sec before (a) and after (b) treatment 10 equivalent Sec in 10 mM PBS buffers (1% DMSO), λex = 365 nm.
tissues and zebrafish. HeLa cells were chosen as the model cell line. At first, the cytotoxicity experiment showed that the CmNp-Sec was nearly nontoxic to living cells under our experimental conditions (Fig. S6). Next, after incubation with CmNp-Sec (5 μM) at 37 °C for 30 min, followed by excitation at 458 nm for imaging, the cells showed weak fluorescence intensity in the yellow channel (Fig. 3b) and bright fluorescence signal in the cyan channel (Fig. 3a). At the same time, tested group with addition of 25 or 50 μM Sec (6 h) and 5 μM CmNpSec for another 30 min, showed brighter fluorescence intensity in yellow channel (Fig. 3f & j, respectively) accompanying with weaker cyan emission intensity (Fig. 3e & i, respectively). The higher resolution of pseudo color mode for ratiometric images (Fig. 3h & k) and quantified relative fluorescence ratio of IYellow/ICyan of images analyzed using Nikon NIS Element software (Fig. 3l) were obtained indicating the
However, in the competition tests, NTR and H2Sn could render an attenuated enhancement of emission intensity after addition of Sec, which might be caused by the consumption of probe due to the reduction of nitro group by NTR or nucleophilic substitution of F atom by H2Sn. All the results of selectivity and competition tests fully confirm that CmNp-Sec is a highly selective fluorescence probe toward Sec and have the latent capability to track Sec in living cells. 3.3. Cytotoxicity, living cells imaging, and TP ratiometric imaging in tissues and zebrafish Encouraged by the above results, in order to further evaluate the imaging performance of TP rariometric fluorescent probe CmNp-Sec, this probe was used to detect Sec in biological samples, including cells,
Fig. 2. (a) Relative ratios of fluorescence intensity (I545/I463) of probe CmNp-Sec (1 μM) in 10 mM PBS buffers (1% DMSO, pH 7.4) to various analytes (50 μM). Legend: (1) Blank, (2) Sec, (3) K+; (4) Na+; (5) Zn2+; (6) Ca2+; (7) Mg2+; (8) F-; (9) Br-; (10) I-; (11) CO32-; (12) NO3-; (13) S2O32-; (14) ClO-; (15) SeO32-; (16) Ala; (17) Glu; (18) Arg; (19) Ser; (20) Lys; (21) Asp; (22) Gly; (23) Leu; (24) Ile; (25) Gln; (26) Tyr; (27) His; (28) Trp; (29) Thr; (30) Phe; (31) Asn; (32) Met; (33)Val; (34) GSH; (35) Cys; and (36) Glu. (b) Relative ratios of fluorescence intensity (I545/I463) of probe CmNp-Sec (1 μM) in 10 mM PBS buffers (1% DMSO, pH 7.4) to Sec (50 μM) in the presence of various bio-active substances (50 μM). Legend: (1) Sec, (2) Sec + K+; (3) Sec + Na+; (4) Sec + Zn2+; (5) Sec + Ca2+; (6) Sec + Mg2+; (7) Sec + F-; (8) Sec + Br-; (9) Sec + I-; (10) Sec + CO32-; (11) Sec + NO3-; (12) Sec + S2O32-; (13) Sec + ClO-; (14) Sec + SeO32-; (15) Sec + Ala; (16) Sec + Glu; (17) Sec + Arg; (18) Sec + Ser; (19) Sec + Lys; (20) Sec + Asp; (21) Sec + Gly; (22) Sec + Leu; (23) Sec + Ile; (24) Sec + Gln; (25) Sec + Tyr; (26) Sec + His; (27) Sec + Trp; (28) Sec + Thr; (29) Sec + Phe; (30) Sec + Asn; (31) Sec + Met; (32) Sec + Val; (33) Sec + GSH; (34) Sec + Cys; and (35) Sec + Glu. 5
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Fig. 3. Fluorescence images of HeLa cells stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging. (a) The cyan channel imaging, (b) the yellow channel imaging, (c) an overlay imaging of (a), (b) and the bright field imaging, (d) Pseudo color mode for ratiometric image; Fluorescence images of HeLa cells incubated with 25 μM Sec for 6 h, and then stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging. (e) The cyan channel imaging, (f) the yellow channel imaging, (g) an overlay imaging of (e), (f) and the bright field imaging, (h) Pseudo color mode for ratiometric image; Fluorescence images of HeLa cells incubated with 50 μM Sec for 6 h, and then stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging. (i) The cyan channel imaging, (j) the yellow channel imaging, (k) an overlay imaging of (i), (j) and the bright field imaging, (l) Pseudo color mode for ratiometric image. (m) Quantified relative fluorescence ratio of IYellow/ICyan of images analyzed using Nikon NIS Element software and presented as mean ± s.d, n = 3. The cyan channel and the yellow channel: (460-495) nm, and (530-560) nm, individually, λex = 458 nm, the scale bar is 20 μm.
4. Conclusion
probe CmNp-Sec had the potentiality to be an ideal tool for the quantification of cellular Sec. In order to evaluate the TP ratiometric imaging performance of CmNp-Sec in the rat liver tissues with deep-tissue penetration imaging and high resolution ratiometric imaging. The imaging thicknesses of the tissues were determined by confocal multiphoton microscopy (Olympus, FV1000) in the z-scan mode with 760 nm exaction (Fig. 4(a–c) and (g–i)). In the absence of Sec, CmNp-Sec was capable of tissue imaging at depths of 90 μm by TPM (Fig. 4(a–c)). Moreover, the tissues showed weak fluorescence intensity in the yellow (Fig. 4b) and bright fluorescence in the cyan channel (Fig. 4a). If were treated with different concentration of Sec (25 or 50 μM) for 6 h (Fig. 4g–i & m–o), tissue fluorescence images in two emission channels with different colors at 95 μm by TP imaging and the quantified relative fluorescence ratio of IYellow/ICyan of images were obtained (Fig. 4i, o & s). These data showed that CmNp-Sec had good tissue penetration and staining ability as well as quantified ratiometric two-color (cyan and yellow) imaging performance with less cross talk between channels. Finally, CmNp-Sec was further applied for imaging of Sec in living zebrafish. 5-day-old vertebrate zebrafishes were chosen as the experimental samples. Zebrafish was first incubated with CmNp-Sec (5 μM) for 30 min. As shown in Fig. 4(d–f), a bright fluorescence in the cyan channel could be observed without addition of Sec. A t the same time, as shown in Fig. 4(j–l & p–r), once the zebrafish was pretreated with 25 or 50 μM Sec for 6 h and then incubated with 5 μM CmNp-Sec for 30 min, the fluorescence intensity enhanced in the yellow channel (Fig. 4k & q) and the fluorescence intensity weakened in the cyan channel (Fig. 4j & p). After analyzing the fluorescence intensity of zebrafish imaging group (Fig. 4t), the quantified ability of CmNp-Sec in zebrafish model had also been confirmed. Combining these results together, these images proved that CmNp-Sec can be used to quantified detection of Sec in living cells, tissues and zebrafish.
In summary, we have developed a new TP-FRET-based TP ratiometric fluorescent probe CmNp-Sec for the selective detection of Sec, which is constructed by introducing DNB to a novel fluorophore (CmNp-OH). The CmNp-OH is fabricated with two-photon fluorophore cumarin derivative (as the donor) and a naphthalimide fluorophore (as an acceptor) through the connection of a non-conjugated linker. All the experiments conducted above indicate that CmNp-Sec owns rapid rariometric fluorescence response (10 min), quantitative determination in a wide range (0-50 μM) with a high sensitivity (LOD = 7.88 nM), selectivity toward Sec over other biothiols and representative amino acids. Additionally, Confocal microscopic fluorescence images of HeLa cells with CmNp-Sec revealed there were two distinct emission ranges at cyan channel and yellow channel in living cells. More impressively, the probe CmNp-Sec was successfully applied in tracking Sec in living tissues and zebrafish. We speculate that CmNp-Sec has the potentiality to find wide applications in biomedical diagnostics. Declaration of Competing Interest The authors declare no competing financial interest. Acknowledgments This work was supported by Natural Science Foundation of China (NSFC, Grants 21605046), Hunan Provincial Natural Science Foundation of China (No. 2017JJ3060), Science and Technology Innovation Platform and Talent Project of Hunan Province (2017TP1021). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jhazmat.2019.120918. 6
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Fig. 4. 2PFM images of a frozen liver tissue slice from a nude mouse model stained. Tissue imaging at depths of 90 μm by 2PFM in Z-scan mode upon excitation at 760 nm with femtosecond pulses. (a-c) liver tissue slice incubated with stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging: (a) The cyan channel imaging, (b) the yellow channel imaging and (c) pseudo color mode for ratiometric image; (g-i) liver tissue slice incubated with 25 μM Sec for 6 h, and then stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging: (g) The cyan channel imaging, (h) the yellow channel imaging and (i) pseudo color mode for ratiometric image; (m-o) liver tissue slice incubated with 50 μM Sec for 6 h, and then stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging: (m) The cyan channel imaging, (n) the yellow channel imaging and (o) pseudo color mode for ratiometric image. Fluorescence images of zebrafish incubated with stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging: (d) The cyan channel imaging, (e) the yellow channel imaging and (f) pseudo color mode for ratiometric image; (j-l) zebrafish incubated with 25 μM Sec for 6 h, and then stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging: (j) The cyan channel imaging, (k) the yellow channel imaging and (l) pseudo color mode for ratiometric image; (p-r) zebrafish incubated with 50 μM Sec for 6 h, and then stained with CmNp-Sec (5 μM) for 30 min for ratiometric imaging: (p) The cyan channel imaging, (q) the yellow channel imaging and (r) pseudo color mode for ratiometric image. (s-t) Quantified relative fluorescence ratio of IYellow/ICyan of images analyzed using Nikon NIS Element software and presented as mean ± s.d, n = 3(s for the images of tissues and t for the images of zebrafish). The cyan channel and the yellow channel: (460-495) nm, and (530-560) nm, individually, λex = 458 nm, the scale bar is 15 μm. 7′‐dimethylfluorescein as a fluorescent probe for selenols. Angew. Chem., Int. Ed. 45, 1810–1813. https://doi.org/10.1002/anie.200504299. Zhang, B., Ge, C., Yao, J., Liu, Y., Xie, H., Fang, J., 2015. Selective selenol fluorescent probes: design, synthesis, structural determinants, and biological applications. J. Am. Chem. Soc. 137, 757–769. https://doi.org/10.1021/ja5099676. Kong, F., Hua, B., Gao, Y., Xu, K., Pan, X., Huang, F., Zheng, Q., Chen, H., Tang, B., 2015. Fluorescence imaging of selenol in HepG2 cell apoptosis induced by Na2SeO3. Chem. Commun. 51, 3102–3105. https://doi.org/10.1039/C4CC06359G. Chen, H., Dong, B., Tang, Y., Lin, W., 2015. Construction of a near-infrared fluorescent turn-on probe for selenol and its bioimaging application in living animals. Chem.Eur. J. 21, 11696–11700. https://doi.org/10.1002/chem.201502226. Kong, F., Ge, L., Pan, X., Xu, K., Liu, X., Tang, B., 2016. A highly selective near-infrared fluorescent probe for imaging H2Se in living cells and in vivo. Chem. Sci. 7, 1051–1056. https://doi.org/10.1039/C5SC03471J. Li, M., Feng, W., Zhai, Q., Feng, G., 2017. Selenocysteine detection and bioimaging in living cells by a colorimetric and near-infrared fluorescent turn-on probe with a large stokes shift. Biosens. Bioelectron. 87, 894–900. https://doi.org/10.1016/j.bios.2016. 09.056. Feng, W., Li, M., Sun, Y., Feng, G., 2017. Near-infrared fluorescent turn-on probe with a remarkable large Stokes shift for imaging selenocysteine in living cells and animals. Anal. Chem. 89, 6106–6112. https://doi.org/10.1021/acs.analchem.7b00824. Areti, S., Verma, S.K., Bellareb, J., Raoa, C.P., 2016. Selenocysteine vs cysteine: tuning the derivatization on benzenesulfonyl moiety of a triazole linked dansyl connected glycoconjugate for selective recognition of selenocysteine and the applicability of the
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