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Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 156 (2016) 1–8
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Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA Chao Gao a,b, Shu-yao Liu a,b, Xian Zhang a,b,⁎, Ying-kai Liu a,b, Cong-de Qiao a,b, Zhao-e Liu c a
School of Materials Science and Engineering, Qilu University of Technology, Jinan 250353, China Shandong Provincial Key Laboratory of Processing and Testing Technology of Glass and Functional Ceramics, Key Laboratory of Amorphous and Polycrystalline Materials, Shandong Provincial Key Laboratory of Fine Chemicals, Qilu University of Technology, Jinan 250353, China c Qilu Hospital of Shandong University, Jinan 250012, China b
a r t i c l e
i n f o
Article history: Received 28 February 2015 Received in revised form 16 November 2015 Accepted 18 November 2015 Available online 19 November 2015 Keywords: Synthesize Fluorescence imaging Heterocyclic DNA Two-photon
a b s t r a c t Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4- [4-(N-methyl)styrene] -benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye—DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye–DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Recently, the DNA detection using fluorescent probes was widely applied and has attracted a broad attention. Various fluorescent probes of DNA were explored, such as organic dyes [1], organic–inorganic hybrid materials [2] and inorganic nanoparticles [3]. Styryl heterocyclic and cyanine dyes were the hot topics in organic compounds. The cyanine dyes are a kind of conventional materials, but they gradually become obsolete because of some limitations such as easy photobleaching and short fluorescence lifetime [4]. Hence domestic and abroad scholars paid more attentions organic heterocyclic materials and have synthesized a series of compounds including thiophene, pyridine and so on [5–8]. Moreover, heterocyclic compounds are easily modified and interacted with DNA due to heteroatoms in molecular structure, such as oxygen and nitrogen etc. Meanwhile, they can be applied as potential two-photon(TP) materials with excellent photo-stabilization and adjusted colors [9,10]. At present, people pay more attentions to one-photon fluorescent probes for the applications in biological systems, but their disadvantages
⁎ Corresponding author at: School of Materials Science and Engineering, Qilu University of Technology, Jinan 250353, China. E-mail address: [email protected] (X. Zhang).
http://dx.doi.org/10.1016/j.saa.2015.11.014 1386-1425/© 2015 Elsevier B.V. All rights reserved.
are obvious, for example, the excitation wavelengths located in the range of 350–560 nm will cause more damages to the biological system because of their location beyond biological window (600–900 nm). Whereas TP laser scanning microscopy (TPM) in combination with suitable probes can avoid effectively the disadvantages and exhibit strong points such as deeper penetration [11],better localized excitation [12], much less photon damage and photobleaching [13],small absorption coefficient of light in tissue and lower tissue auto-fluorescence [14]. Although some TP fluorescent probes have been developed, most of them have poor TP absorption active cross-sections (Фδ), which limited the development of TPM. So it is very important to design and synthesis TP fluorescent probes with larger Фδ and good biocompatibility. In this paper, two styryl heterocyclic compounds were designed and synthesized with better TP properties and excellent biocompatibility by the reaction between methyl and formyl under piperidine as catalyst. Meanwhile, the heterocyclic pyridine iodate can increase significantly the water solubility and affinity to DNA [15]. Furthermore, the solvent effects of the dyes were investigated. The linear and nonlinear optical properties in free dye and dye—DNA complex were discussed, and quantum yields (Ф) and Фδ at some important points were calculated. The possible interaction mechanisms of dye-DNA were studied by a fluorescent method in the presence of NaCl and KI. Moreover, the toxicity of two probes was investigated by MTT assay, and the fluorescence imaging in living cells was finished by TP excitation.
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2. Experimental 2.1. Chemicals 1,10-phenanthroline (99%) was purchased from Acros Organics. 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Sigma. Other reagents were got from commercial suppliers. The solvents were purified according to Solvent Handbook III before use. Other chemicals were AR grade without further purification. The used Tris–HCl buffer solution (10 mmol/L, pH 7.2; KCl 100 mmol/L) was prepared according to the normal method. The calf thymus DNA (ctDNA) was provided by Shenggong biological engineering technology service (Shanghai) co., Ltd. This was a highly polymerized DNA which mainly contained double stranded form. The molecular weight is about 10 ~ 15 million Daltons. DNA from calt thymus included 42 mol% G–C and 58 mol% A–T, respectively. 2.2. Instruments Nuclear magnetic resonance spectra were obtained on a Bruker Avance 400 spectrometer with TMS internal standard as a reference for the chemical shifts. Infrared spectra were recorded on a Nicolet
NEXUS 670 FT-IR spectrometer using a liquid-nitrogen-cooled detector. The samples were mixed with KBr to form a disk. Elemental analyses were performed on a Perkin 2400 (II) autoanalyzer. The UV–vis-nearIR absorption spectra of probes were recorded on a Varian Cary 50 spectrophotometer. One-photon fluorescence spectra were obtained on an Edinburgh FLS920 spectrofluorimeter equipped with a 450 W Xe lamp. TP fluorescence spectra were noted on an OOIBASE32 spectrophotometer. The pump laser beam came from a mode-locked Ti:sapphire laser system with a pulse duration of 200 fs and a repetition rate of 76 MHz (Coherent Mira 900-D). All quartz cuvettes used in test were non-lighttight with 1 cm path length. TP fluorescent images were obtained on an Olympus FV300 confocal microscope with a 40× (0.75 numerical aperture) objective and a photomultiplier tube equipped with a femtosecond pulsed Ti:sapphire laser (Coherent Mira900-D). 2.3. Synthesis The synthetic route of the expected compounds was depicted in Scheme 1. 1,10-phenanthroline-5,6-dione (2) and 2-(4-formyl phenyl) imidazole [4,5-f][1,10] phenanthroline (Fmp) were obtained as described in literatures [16,17].
Scheme 1. The synthetic route of probe-1 and probe-2.
C. Gao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 156 (2016) 1–8 1 H NMR of 1,10-phenanthroline-5,6-dione (2) (CDC13, 400 MHz, TMS) δ (ppm): 9.05 (d, J = 4.9 Hz, 2 H), 8.44 (d, J = 7.2 Hz, 2 H), 7.53 (m, 2 H). 1 H NMR of Fmp (400 MHz, DMSO, TMS) δ (ppm):10.11 (s, 1 H), 9.05 (d, J = 2.8 Hz, 2 H), 8.95 (d, J = 9.28 Hz, 2 H), 8.51 (d, J = 8.12 Hz, 2 H), 8.15 (d, J = 8.2 Hz, 2 H), 7.86 (m, 2 H).
2.3.1. Procedure for the synthesis of 4-methyl-N-methyl iodized salt (1) 4-methyl pyridine (5.58 g, 0.16 mol) was added into a flask of 250 mL, and then iodomethane (12.5 g, 0.09 mol) was added dropwise. The mixture was heated to 50 °C and reacted for 2.5 h. The resultant solution was cooled and filtered. White crystals were obtained with a yield of 95%. 1H NMR (400 MHZ, DMSO, TMS) δ (ppm): 2.53 (s, 3 H), 4.23 (s, 3 H), 7.95 (d, J = 8.67 Hz, 2 H), 8.81 (d, J = 7.20 Hz, 2 H). 2.3.2. Procedure for the synthesis of 4-[4-(N-methyl)styrene]- imidazo [4,5f][1,10] phenanthroline-benzene iodated salt (probe-1) In a three-neck flask, Fmp (0.89 g, 2.74 mol) dissolved in chloroform (2 mL) was added to the solution of 4-methyl-N-methyl iodized salt 1 (0.675 g, 2.86 mol) in methanol (30 mL), and a few drops of piperidine as catalyzer were added. The mixture was heated to 60 °C and refluxed for 6 h, followed by cooling to room temperature and filtered. The solid residue was washed with ethanol and water for several times. The yellow crystals were obtained by crystallization in ethanol with a yield of 45%. 1H NMR (400 MHZ, DMSO, TMS) δ (ppm): 8.89 (m, 6 H), 8.50 (d, J = 4.62 Hz, 2 H), 8.39 (d, J = 3.12 Hz, 2 H), 8.10 (d, J = 4.0 Hz, 2 H), 7.85 (d, J = 1.80 Hz, 2 H), 7.82 (m, 3 H), 2.49 (s, 3 H). IR (KBr) ν/cm−1: 3371 (N–H),3041 (C–H),1452 (C–C, C–N),1182 (C–H),1072 (C–H),840 (C–H),738 (C–H). Calcd. for C27H20I N5: C, 59.90; H, 3.72; N, 12.94. Found C, 56.95; H, 3.69; N, 12.95. 2.3.3. Procedure for the synthesis of 4,4- [4-(N-methyl)styrene] -benzene iodated salt (probe-2) The probe-2 was synthesized according to the preparation method of probe-1with terephthalaldehyde and the compound 1. Brightly orange crystals were obtained with a yield of 56%. 1H NMR (400 MHz, DMSO, TMS) δ (ppm): 8.91 (m, 4 H), 8.28 (t, J = 10.0 Hz, 4 H), 8.07 (d, J = 7.2 Hz, 2 H), 7.87 (m, 4 H), 7.57 (d, J = 14.0 Hz, 2 H), 2.52 (s, 6 H). IR(KBr) ν/cm− 1: 3026 (C–H),1618 (C–C),1469 (C–N),1186 (C–H),840 (C–H). Calcd. for C22H22I2N2: C, 46.45; H, 3.88; N, 4.90. Found C, 46.50; H, 3.90; N, 4.92. 3. Results and discussion 3.1. Linear and nonlinear photophysical properties of two compounds in different solvents The data of the linear absorption, one- and two-photon fluorescent spectra and their related photophysical properties of probe-1 and probe-2 in various solvents were summarized in Table 1. The measured concentrations were 1 × 10− 5 mol/L for the linear spectra and 1 × 10− 4 mol/L for the nonlinear spectra, respectively. Ф and δФ of two compounds were determined with a reference method using coumarin 307 (Ф = 0.56, Фδ = 15.5 GM at 800 nm) as the standard under the same test condition [18]. In Table 1, the absorption peaks of two compounds showed an obvious blue-shift in the higher polar solvents except DMF. A slight red-shift for probe-1 was found in DMF (from 371 nm in acetone to 374 nm DMF). While an obvious red-shift for probe-2 was discovered (from 382 nm in acetone to 393 nm DMF). The cause may be attributed to the different structure. The formation of hydrogen bond for two imines greatly declined the conjugation of probe-1, which caused molecule bent and the blue-shift of absorption peaks. The fluorescent peaks of probe-1 clearly showed red-shifted with the increase of the solvent polarity. The Ф decreased slightly in higher polar solvents for every compound (from 18.16% in acetone to 6.11% in water for probe-1, from 14.26% in acetone to 7.07% in water
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Table 1 The data of the linear and nonlinear spectra and their related photophysical properties of probe-1 and probe-2 in different solvents (the concentrations in the linear and nonlinear measurements were 1 × 10−5 mol/L and 1 × 10−4 mol/L, respectively). Samples
Solvents
λamax(nm)
λoe max(nm)
Ф (%)
λte max (nm)
δ × Ф (GM)
Probe-1
Water DMF MeCN Ethanol Acetone Water DMF MeCN Ethanol Acetone
362 374 370 369 371 374 393 379 381 382
505 432 428 425 425 471 476 470 468 470
6.11 19.77 15.94 12.26 18.16 7.07 33.18 7.96 12.87 14.26
612 526/656 658 642 667 473 478 471 472 472
25.87 47.29 68.80 62.79 40.29 14.98 28.89 8.92 8.07 7.14
Probe-2
te λamax, λoe max and λmax are linear absorption and one- and two-photon fluorescence maximum peaks, respectively. The two-photon spectra were measured at 800 nm. Ф and δ are one-photon quantum yield and two-photon cross-section using coumarin307 as the standard. δ × Ф is the active two-photon absorption cross-section, 1 GM = 10−50 cm4 s
photo−1.
for probe-2), but the values were the maximum in DMF. These can be explained by the TICT (twisted intramolecular charge transfer) that the excited state of two compounds may possess a higher polarity than that of the ground state, because the solvatochromism is associated with the energy level redistribution. The increase of dipole-dipole interactions between the solute and solvent molecules led to the evident lowering of the energy levels [19]. But because of the higher viscosity of DMF, the process of TICT will be prevented and the fluorescence is protected. TP fluorescence emission wavelengths of probe-1 were obviously blue-shifted with the increase of the solvent polarity (from 667 nm in acetone to 612 nm in water), in addition, two fluorescence emission peaks in DMF were found. However, TP emission wavelengths of probe-2 have changed little. The δФ of probe-1 were enhanced with the higher solvent polarity, but the value in water was the smallest. The δФ changing trend of probe-2 in different solvents was coincident with that of probe-1 except in DMF. Meanwhile, the TP fluorescence emission wavelengths of probe-1 were obviously red-shifted in comparison to those by one-photon excitation. However, this changing trend was not found for probe-2. The reasons might be related with the solvent viscosity, hydrogen bond and re-absorption. Moreover, the different molecular structures of two compounds may play an important role [20–22].
3.2. The linear absorption and one-photon fluorescence of the probes interactions with ctDNA In order to check the possibility of the compound as DNA detection agents, the absorption and one-photon fluorescent spectra were investigated upon adding different volumes of ctDNA (3.5 × 10− 3 mol/L) to the probes in the Tris–HCl buffer solution (concentration: 1 × 10 − 5 mol/L). The mole ratios of ctDNA to probes were: 0,0.07,0.14,0.35,0.7,1.4,2.1,2.8,3.5,5.6,7.0,11,14,18,21,30,39,47,67,88, 111,150, respectively. The experimental results were obtained and depicted in Fig. 1 (for probe-1) and Fig. 2 (for probe-2). In Fig. 1, the maximum absorption wavelength(357 nm) of probe-1 did little change, while the absorbance was enhanced with ctDNA titrating. The fluorescent intensity began to increase significantly from 0 to 0.7, then declined and leveled off after the ratio of 1.4, and the changing trend of fluorescence emission wavelengths was coincident with that of the fluorescent intensity. In Fig. 2, the maximum absorption peaks of probe-2 were gradually red-shifted (from 377 nm to 391 nm) with ctDNA titrating. Meanwhile, the absorbance declined gradually and tended to stabilize after the ratio of 67. And the changing trend of fluorescence intensity of probe-2 was coincident with that of probe-1,
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Fig. 1. The UV absorption (a) and fluorescent spectra (b) of probe-1 with DNA titrating. The insets of I and II at (b) upper right corner are the fluorescent intensity and wavelengths evolution with ct-DNA concentrations, respectively.
but the maximum point of fluorescence intensity and the last stable point for probe-2 are at the ratios of 6 and 67, respectively. The mechanisms of dye-DNA may be explained with Twist intermolecular charge transfer (TICT). For the dyes, the intermolecular charge transfer (ICT) state could occur between the electron donor and the electron acceptor [23]. In water, the dipole molecule in the excited state interacted with the higher polar solvent more strongly than that in the ground state resulting in the larger dipole moment in the excited state, which could lead to charge separation resulting in formation of a TICT state. However, the formation of a TICT state was restricted when the dye was protected from DNA in water, and the fluorescence of the dye will be restored. However, the fluorescence will be quenched with the aggregation of dye molecules. In addition, the difference of the linear spectra for two dyes in details may be attributed to the different structures. Moreover, the binding constants between the probes and DNA were obtained by the one-photon fluorescent titration method according to Scatchard equation [24]: r=c f ¼ kn−kr
ð1Þ
where k and n refer to the binding constant and binding sites, respectively. Cf is the concentration of free dye. r is a ratio of Cb/[ct-DNA], and Cb is calculated by the following equation [25]: cb ¼ ct
h
i F−F 0 = F max − F 0
ð2Þ
where, ct is the total DNA concentration. F0 and F are the fluorescence intensity of the free dye and dye-DNA complex, respectively. Fmax is the saturated fluorescent intensity in DNA titration. According to the Eqs. (1) and (2), the fitted curves were shown in Fig. 3 (a) and (b). The obtained binding constants are 3.06 × 105 M−1 for probe-1 and 1.08 × 106 M−1 for probe-2, respectively. The value of probe-2 is slightly larger over that of probe-1 and comparable with the 106 M−1 value for DAPI [26], and the binding sites of two compounds are n = 0.86 (probe-1), 1.83 (probe-2). The cause may be attributed to more positive charges of probe-2 in comparison to probe-1, which increased the interaction of the dye-DNA. 3.3. The two-photon fluorescent spectra of the probes interaction with ctDNA The TP fluorescent spectra of two probes in Tris–HCl buffer solution were recorded with ctDNA titrating on Ti:sapphire laser at 800 nm. The concentrations of probes and ct-DNA were 1 × 10− 4 mol/L and 1 × 10−3 mol/L, respectively. The mole ratios of ctDNA to probes were: 0, 0.1, 0.2, 0.4, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 16.0, 25.0, 32, 43 and 67. The experimental results were obtained and depicted in Fig. 4 (a for probe-1 and b for probe-2). Fig. 4 showed that the shapes and the maximum emission wavelengths of TP fluorescent spectra for two compounds had hardly changed. The fluorescent intensity of probe-1 began to increase before the ratio of 0.2 (ctDNA/probe-1), and then declined and tended to stabilize after the ratio about 25. This trend was similar to that of one-photon
Fig. 2. The UV absorption (a) and fluorescent spectra (b) of probe-2 with DNA titrating. The insets of I and II at (a) upper right corner are the absorbance and the absorption maximum wavelengths evolution with ct-DNA concentrations, respectively. The inset of III at (b) upper right corner is the fluorescent intensity evolution with ct-DNA concentrations.
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Fig. 3. The fitted curves of probe-1(a) and probe-2(b) according to Scatchard equation on the basis of one-photon fluorescent DNA titration.
fluorescent spectra of probe-1. Meanwhile, the changing trend of fluorescent intensity for probe-2 was also almost coincident with that of probe-1, but its fluorescent intensity reached the maximum at the ratio of 12 and tended to be stable at the ratio of 16. The largest δФ of two probes with DNA titrating were 60.13 GM and 10.52 GM, respectively, which are far higher than those of some traditional DNA dyes (∼2 GM for DAPI). These indicated that they were possibly used to detect DNA by TP excitation, and the TP properties of probe-1 are superior to that of probe-2. 3.4. The possible interaction mechanisms between the probes and DNA In order to investigate the possible DNA–probe interaction mechanisms, the fluorescence of probe-ctDNA on the condition of NaCl was investigated. The concentrations of probes and ctDNA were 1 × 10−5 mol L−1 and 5.0 μg mL−1 in Tris–HCl buffer with pH 7.4. The concentrations of used NaCl were from 0.1 to 0.5 mol L−1. The changes of fluorescent intensity with adding NaCl were shown in Fig. 5 (a for probe-1 and b for probe-2). Generally, Na+ can partially close to phosphate of nucleotide skeleton. If the DNA–dye interaction is electrostatic, the change of fluorescent intensity will be disturbed with the increase of Na+ concentrations. Fig. 5(a) showed that fluorescent intensity of probe-1-DNA complex has slight changes with the addition of NaCl, which indicated that the electrostatic interaction did not play a major role for the probe-1-DNA action. However, the reductive fluorescence of probe-2 was obvious with the addition of ctDNA, which indicated that electrostatic interaction has an important contribution for the probe-2- DNA action [27].
It is well known that iodide is a typical reagent to quench fluorescence. In order to further study the interaction mechanisms of probes–ctDNA, we investigated the fluorescence of the different systems including probe, probe–KI, probe–ctDNA and probe– ctDNA–KI, and the concentrations of probes, ct-DNA and KI were 1.0 × 10− 5 mol L− 1,5.0 μg mL− 1 and 0.02 mol L−1 in Tris–HCl buffer, respectively. The interactions between dyes and ctDNA are mainly noncovalent bond including outside binding, intercalation binding and groove binding [28–30]. Fig. 6 showed that the fluorescence of probe1 was obviously quenched by KI, but that of probe-1-ctDNA was partly reduced. It is likely that probe-1-ctDNA binding prevented the fluorescent quenching. In above discussion, outside binding by electrostatic interaction was unconspicuous. The reduction of absorbance, red-shift of absorption band and the formation of isobestic point are typical signs for intercalation binding [31], however those were not observed in Fig. 1. On the basis of the analyses for the absorption and fluorescent spectra, the main interaction mechanism of probe-1-DNA can be considered to groove binding. After adding KI, the fluorescence of probe-2 and probe-2–ctDNA was almost identical, which indicated that the interaction mechanism between probe-2 and DNA is mainly outside binding by electrostatic interaction, and it is coincident with the above conclusion. 3.5. MTT assay HeLa cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) containing high glucose supplemented with 10% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin. When in the proliferative
Fig. 4. The two-photon fluorescent spectra of probe-1(a) and probe-2(b) upon DNA titrating in Tris–HCl buffer at 800 nm with a concentration of 1 × 10−4 mol L−1. The insets of I at (a) and (b) upper right corner are the fluorescent intensity of two probes evolution with ct-DNA concentrations.
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Fig. 5. Effect of salt concentrations (a for probe-1 and b for probe-2). Conditions: Cprobe = 1.0 × 10−5 mol L−1; Cct-DNA = 5.0 μg mL−1; in Tris–HCl buffer solution with pH 7.4.
Fig. 6. The fluorescent spectra of KI quenching for probe-ctDNA systems, (a for probe-1 and b for probe-2). Conditions: Cprobe = 1.0 × 10−5 mol L−1; Cct-DNA = 5.0 μg mL−1; CKI = 0.02 mol L−1.
period, HeLa cells (∼5 × 105 cell/mL) were dispersed within replicate 96well microliter plates to a total volume of 100 μL/well and maintained at 37 °C in a 5% CO2/95% air incubator for 24 h. Then, the culture media was removed and the cells were incubated in culture medium containing the as prepared probe-1 and probe-2 with different concentrations (5, 10, 20, 40, 80 nM) for 48 h and washed with the culture medium. An amount of 100 μL of the new culture medium containing MTT (10 μL, 5 mg mL−1, PBS) was then added to each well, followed by incubating for 4 h to allow the formation of formazan dye. After removing the medium, 150 μL of DMSO was added to each well to dissolve the formazan crystals. Absorbance was measured at 490 nm in a Triturus microplate reader. The number of viable cells was determined by an MTT assay [32]. As shown in Fig. 7, the cellular viabilities were estimated to be greater than 90% after incubation for 48 h with the concentrations from 5 to 20 nM for probe-1 and from 5 to 40 nM for probe-2. In higher concentration of 80 nM, the viabilities were still over 70% (71% for probe-1 and 76% for probe-2). These results showed the as prepared probes are generally low toxicity for cellular imaging. Moreover, the toxicity of probe-1 is higher than that of probe-2, which may be attributed to the different interactions between dye and DNA. The groove binding of probe-1 and DNA increased the damage to bases on DNA in comparison to the electrostatic interaction of probe-2 [33].
with an Olympus FV300 confocal microscope. A Ti-sapphire laser (Coherent Mira900-D) was used to excite the probes at 800 nm. The fluorescent emission of two probes was collected using 450–650 nm for probe-1 and 450–550 nm for probe-2 band-pass filters. In Fig. 8 (a1, b1, c1),the position and shape stained by probe-1 were the same as that of nuclei in the DIC image, implying that the probe-1 can accurately label nuclei. But the position stained by probe-2 was not only in cell nuclei but also in the cytoplasm, which indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2. The
3.6. Fluorescence imaging The potential utilities of probes were proceeded to examine by the fluorescence imaging of DNA in living cells. HeLa cells were cultured in DMEM medium at 37 °C/5% CO2, and grown on glass cover slips before the preparation for imaging experiments. The cells were incubated with the probes solution (1 × 10−6 mol/L) for 30 min at room temperature and then washed with PBS for three times. The cells were imaged
Fig. 7. MTT assay of HeLa cells in the presence of different concentrations (5, 10, 20, 40, 80 nM) of probe-1 (black) and probe-2 (gray) for 48 h at 37 °C, respectively.
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Fig. 8. Two-photon fluorescence images of probe-1(a1) and probe-2 (a2) fluorescent images were incubated with 10 μm of probe-1 and probe-2 for 30 min and excited at 800 nm, respectively; (b1, b2) DIC photos; (c1, c2) merged images.
different fluorescence images of two probes may be attributed to diverse structures and interaction types [34,35]. 4. Conclusions Two styryl heterocyclic dyes of probe-1 and probe-2 were successfully synthesized. The linear and nonlinear photophysical properties in different solvents and in DNA titrating were investigated. The results implied that TP properties of probe-1 are superior to that of probe-2, and the change trends of fluorescent intensity were consistent for SPE and TPE with DNA titrating. The binding constant of probe-2-DNA (1.08 × 106 M−1) is slightly larger over that of probe-1 (3.06 × 105 M−1). These data showed that two compounds could be considered as efficient fluorescent probes for DNA detection by oneand two-photon excited method. The possible mechanisms of the probe–DNA interaction were studied by fluorescent spectra in the presence of NaCl and KI, which are mainly groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The results of MTT assay and the TP fluorescence imaging in living cells indicated that two probes are low toxicity, and the ability to locate in cell nuclei of probe-1 is superior to that of probe-2. Acknowledgments This work was supported by the National Natural Science Foundation of China (no. 51403111, 21276149), the Program of Young Teachers Growth Plan in Shandong Province, the Natural Science Foundation of Shandong Province (ZR2012EMM009, ZR2012EMZ003) and Program for Scientific Research Innovation Team in Colleges and Universities of Shandong Province. In addition, we are particularly grateful to Professor Yu from Shandong University for their help in fluorescent measuring.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA Chao Gao a,b, Shu-yao Liu a,b, Xian Zhang a,b,⁎, Ying-kai Liu a,b, Cong-de Qiao a,b, Zhao-e Liu c a
School of Materials Science and Engineering, Qilu University of Technology, Jinan 250353, China Shandong Provincial Key Laboratory of Processing and Testing Technology of Glass and Functional Ceramics, Key Laboratory of Amorphous and Polycrystalline Materials, Shandong Provincial Key Laboratory of Fine Chemicals, Qilu University of Technology, Jinan 250353, China c Qilu Hospital of Shandong University, Jinan 250012, China b
a r t i c l e
i n f o
Article history: Received 28 February 2015 Received in revised form 16 November 2015 Accepted 18 November 2015 Available online 19 November 2015 Keywords: Synthesize Fluorescence imaging Heterocyclic DNA Two-photon
a b s t r a c t Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4- [4-(N-methyl)styrene] -benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye—DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye–DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Recently, the DNA detection using fluorescent probes was widely applied and has attracted a broad attention. Various fluorescent probes of DNA were explored, such as organic dyes [1], organic–inorganic hybrid materials [2] and inorganic nanoparticles [3]. Styryl heterocyclic and cyanine dyes were the hot topics in organic compounds. The cyanine dyes are a kind of conventional materials, but they gradually become obsolete because of some limitations such as easy photobleaching and short fluorescence lifetime [4]. Hence domestic and abroad scholars paid more attentions organic heterocyclic materials and have synthesized a series of compounds including thiophene, pyridine and so on [5–8]. Moreover, heterocyclic compounds are easily modified and interacted with DNA due to heteroatoms in molecular structure, such as oxygen and nitrogen etc. Meanwhile, they can be applied as potential two-photon(TP) materials with excellent photo-stabilization and adjusted colors [9,10]. At present, people pay more attentions to one-photon fluorescent probes for the applications in biological systems, but their disadvantages
⁎ Corresponding author at: School of Materials Science and Engineering, Qilu University of Technology, Jinan 250353, China. E-mail address: [email protected] (X. Zhang).
http://dx.doi.org/10.1016/j.saa.2015.11.014 1386-1425/© 2015 Elsevier B.V. All rights reserved.
are obvious, for example, the excitation wavelengths located in the range of 350–560 nm will cause more damages to the biological system because of their location beyond biological window (600–900 nm). Whereas TP laser scanning microscopy (TPM) in combination with suitable probes can avoid effectively the disadvantages and exhibit strong points such as deeper penetration [11],better localized excitation [12], much less photon damage and photobleaching [13],small absorption coefficient of light in tissue and lower tissue auto-fluorescence [14]. Although some TP fluorescent probes have been developed, most of them have poor TP absorption active cross-sections (Фδ), which limited the development of TPM. So it is very important to design and synthesis TP fluorescent probes with larger Фδ and good biocompatibility. In this paper, two styryl heterocyclic compounds were designed and synthesized with better TP properties and excellent biocompatibility by the reaction between methyl and formyl under piperidine as catalyst. Meanwhile, the heterocyclic pyridine iodate can increase significantly the water solubility and affinity to DNA [15]. Furthermore, the solvent effects of the dyes were investigated. The linear and nonlinear optical properties in free dye and dye—DNA complex were discussed, and quantum yields (Ф) and Фδ at some important points were calculated. The possible interaction mechanisms of dye-DNA were studied by a fluorescent method in the presence of NaCl and KI. Moreover, the toxicity of two probes was investigated by MTT assay, and the fluorescence imaging in living cells was finished by TP excitation.
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2. Experimental 2.1. Chemicals 1,10-phenanthroline (99%) was purchased from Acros Organics. 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Sigma. Other reagents were got from commercial suppliers. The solvents were purified according to Solvent Handbook III before use. Other chemicals were AR grade without further purification. The used Tris–HCl buffer solution (10 mmol/L, pH 7.2; KCl 100 mmol/L) was prepared according to the normal method. The calf thymus DNA (ctDNA) was provided by Shenggong biological engineering technology service (Shanghai) co., Ltd. This was a highly polymerized DNA which mainly contained double stranded form. The molecular weight is about 10 ~ 15 million Daltons. DNA from calt thymus included 42 mol% G–C and 58 mol% A–T, respectively. 2.2. Instruments Nuclear magnetic resonance spectra were obtained on a Bruker Avance 400 spectrometer with TMS internal standard as a reference for the chemical shifts. Infrared spectra were recorded on a Nicolet
NEXUS 670 FT-IR spectrometer using a liquid-nitrogen-cooled detector. The samples were mixed with KBr to form a disk. Elemental analyses were performed on a Perkin 2400 (II) autoanalyzer. The UV–vis-nearIR absorption spectra of probes were recorded on a Varian Cary 50 spectrophotometer. One-photon fluorescence spectra were obtained on an Edinburgh FLS920 spectrofluorimeter equipped with a 450 W Xe lamp. TP fluorescence spectra were noted on an OOIBASE32 spectrophotometer. The pump laser beam came from a mode-locked Ti:sapphire laser system with a pulse duration of 200 fs and a repetition rate of 76 MHz (Coherent Mira 900-D). All quartz cuvettes used in test were non-lighttight with 1 cm path length. TP fluorescent images were obtained on an Olympus FV300 confocal microscope with a 40× (0.75 numerical aperture) objective and a photomultiplier tube equipped with a femtosecond pulsed Ti:sapphire laser (Coherent Mira900-D). 2.3. Synthesis The synthetic route of the expected compounds was depicted in Scheme 1. 1,10-phenanthroline-5,6-dione (2) and 2-(4-formyl phenyl) imidazole [4,5-f][1,10] phenanthroline (Fmp) were obtained as described in literatures [16,17].
Scheme 1. The synthetic route of probe-1 and probe-2.
C. Gao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 156 (2016) 1–8 1 H NMR of 1,10-phenanthroline-5,6-dione (2) (CDC13, 400 MHz, TMS) δ (ppm): 9.05 (d, J = 4.9 Hz, 2 H), 8.44 (d, J = 7.2 Hz, 2 H), 7.53 (m, 2 H). 1 H NMR of Fmp (400 MHz, DMSO, TMS) δ (ppm):10.11 (s, 1 H), 9.05 (d, J = 2.8 Hz, 2 H), 8.95 (d, J = 9.28 Hz, 2 H), 8.51 (d, J = 8.12 Hz, 2 H), 8.15 (d, J = 8.2 Hz, 2 H), 7.86 (m, 2 H).
2.3.1. Procedure for the synthesis of 4-methyl-N-methyl iodized salt (1) 4-methyl pyridine (5.58 g, 0.16 mol) was added into a flask of 250 mL, and then iodomethane (12.5 g, 0.09 mol) was added dropwise. The mixture was heated to 50 °C and reacted for 2.5 h. The resultant solution was cooled and filtered. White crystals were obtained with a yield of 95%. 1H NMR (400 MHZ, DMSO, TMS) δ (ppm): 2.53 (s, 3 H), 4.23 (s, 3 H), 7.95 (d, J = 8.67 Hz, 2 H), 8.81 (d, J = 7.20 Hz, 2 H). 2.3.2. Procedure for the synthesis of 4-[4-(N-methyl)styrene]- imidazo [4,5f][1,10] phenanthroline-benzene iodated salt (probe-1) In a three-neck flask, Fmp (0.89 g, 2.74 mol) dissolved in chloroform (2 mL) was added to the solution of 4-methyl-N-methyl iodized salt 1 (0.675 g, 2.86 mol) in methanol (30 mL), and a few drops of piperidine as catalyzer were added. The mixture was heated to 60 °C and refluxed for 6 h, followed by cooling to room temperature and filtered. The solid residue was washed with ethanol and water for several times. The yellow crystals were obtained by crystallization in ethanol with a yield of 45%. 1H NMR (400 MHZ, DMSO, TMS) δ (ppm): 8.89 (m, 6 H), 8.50 (d, J = 4.62 Hz, 2 H), 8.39 (d, J = 3.12 Hz, 2 H), 8.10 (d, J = 4.0 Hz, 2 H), 7.85 (d, J = 1.80 Hz, 2 H), 7.82 (m, 3 H), 2.49 (s, 3 H). IR (KBr) ν/cm−1: 3371 (N–H),3041 (C–H),1452 (C–C, C–N),1182 (C–H),1072 (C–H),840 (C–H),738 (C–H). Calcd. for C27H20I N5: C, 59.90; H, 3.72; N, 12.94. Found C, 56.95; H, 3.69; N, 12.95. 2.3.3. Procedure for the synthesis of 4,4- [4-(N-methyl)styrene] -benzene iodated salt (probe-2) The probe-2 was synthesized according to the preparation method of probe-1with terephthalaldehyde and the compound 1. Brightly orange crystals were obtained with a yield of 56%. 1H NMR (400 MHz, DMSO, TMS) δ (ppm): 8.91 (m, 4 H), 8.28 (t, J = 10.0 Hz, 4 H), 8.07 (d, J = 7.2 Hz, 2 H), 7.87 (m, 4 H), 7.57 (d, J = 14.0 Hz, 2 H), 2.52 (s, 6 H). IR(KBr) ν/cm− 1: 3026 (C–H),1618 (C–C),1469 (C–N),1186 (C–H),840 (C–H). Calcd. for C22H22I2N2: C, 46.45; H, 3.88; N, 4.90. Found C, 46.50; H, 3.90; N, 4.92. 3. Results and discussion 3.1. Linear and nonlinear photophysical properties of two compounds in different solvents The data of the linear absorption, one- and two-photon fluorescent spectra and their related photophysical properties of probe-1 and probe-2 in various solvents were summarized in Table 1. The measured concentrations were 1 × 10− 5 mol/L for the linear spectra and 1 × 10− 4 mol/L for the nonlinear spectra, respectively. Ф and δФ of two compounds were determined with a reference method using coumarin 307 (Ф = 0.56, Фδ = 15.5 GM at 800 nm) as the standard under the same test condition [18]. In Table 1, the absorption peaks of two compounds showed an obvious blue-shift in the higher polar solvents except DMF. A slight red-shift for probe-1 was found in DMF (from 371 nm in acetone to 374 nm DMF). While an obvious red-shift for probe-2 was discovered (from 382 nm in acetone to 393 nm DMF). The cause may be attributed to the different structure. The formation of hydrogen bond for two imines greatly declined the conjugation of probe-1, which caused molecule bent and the blue-shift of absorption peaks. The fluorescent peaks of probe-1 clearly showed red-shifted with the increase of the solvent polarity. The Ф decreased slightly in higher polar solvents for every compound (from 18.16% in acetone to 6.11% in water for probe-1, from 14.26% in acetone to 7.07% in water
3
Table 1 The data of the linear and nonlinear spectra and their related photophysical properties of probe-1 and probe-2 in different solvents (the concentrations in the linear and nonlinear measurements were 1 × 10−5 mol/L and 1 × 10−4 mol/L, respectively). Samples
Solvents
λamax(nm)
λoe max(nm)
Ф (%)
λte max (nm)
δ × Ф (GM)
Probe-1
Water DMF MeCN Ethanol Acetone Water DMF MeCN Ethanol Acetone
362 374 370 369 371 374 393 379 381 382
505 432 428 425 425 471 476 470 468 470
6.11 19.77 15.94 12.26 18.16 7.07 33.18 7.96 12.87 14.26
612 526/656 658 642 667 473 478 471 472 472
25.87 47.29 68.80 62.79 40.29 14.98 28.89 8.92 8.07 7.14
Probe-2
te λamax, λoe max and λmax are linear absorption and one- and two-photon fluorescence maximum peaks, respectively. The two-photon spectra were measured at 800 nm. Ф and δ are one-photon quantum yield and two-photon cross-section using coumarin307 as the standard. δ × Ф is the active two-photon absorption cross-section, 1 GM = 10−50 cm4 s
photo−1.
for probe-2), but the values were the maximum in DMF. These can be explained by the TICT (twisted intramolecular charge transfer) that the excited state of two compounds may possess a higher polarity than that of the ground state, because the solvatochromism is associated with the energy level redistribution. The increase of dipole-dipole interactions between the solute and solvent molecules led to the evident lowering of the energy levels [19]. But because of the higher viscosity of DMF, the process of TICT will be prevented and the fluorescence is protected. TP fluorescence emission wavelengths of probe-1 were obviously blue-shifted with the increase of the solvent polarity (from 667 nm in acetone to 612 nm in water), in addition, two fluorescence emission peaks in DMF were found. However, TP emission wavelengths of probe-2 have changed little. The δФ of probe-1 were enhanced with the higher solvent polarity, but the value in water was the smallest. The δФ changing trend of probe-2 in different solvents was coincident with that of probe-1 except in DMF. Meanwhile, the TP fluorescence emission wavelengths of probe-1 were obviously red-shifted in comparison to those by one-photon excitation. However, this changing trend was not found for probe-2. The reasons might be related with the solvent viscosity, hydrogen bond and re-absorption. Moreover, the different molecular structures of two compounds may play an important role [20–22].
3.2. The linear absorption and one-photon fluorescence of the probes interactions with ctDNA In order to check the possibility of the compound as DNA detection agents, the absorption and one-photon fluorescent spectra were investigated upon adding different volumes of ctDNA (3.5 × 10− 3 mol/L) to the probes in the Tris–HCl buffer solution (concentration: 1 × 10 − 5 mol/L). The mole ratios of ctDNA to probes were: 0,0.07,0.14,0.35,0.7,1.4,2.1,2.8,3.5,5.6,7.0,11,14,18,21,30,39,47,67,88, 111,150, respectively. The experimental results were obtained and depicted in Fig. 1 (for probe-1) and Fig. 2 (for probe-2). In Fig. 1, the maximum absorption wavelength(357 nm) of probe-1 did little change, while the absorbance was enhanced with ctDNA titrating. The fluorescent intensity began to increase significantly from 0 to 0.7, then declined and leveled off after the ratio of 1.4, and the changing trend of fluorescence emission wavelengths was coincident with that of the fluorescent intensity. In Fig. 2, the maximum absorption peaks of probe-2 were gradually red-shifted (from 377 nm to 391 nm) with ctDNA titrating. Meanwhile, the absorbance declined gradually and tended to stabilize after the ratio of 67. And the changing trend of fluorescence intensity of probe-2 was coincident with that of probe-1,
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Fig. 1. The UV absorption (a) and fluorescent spectra (b) of probe-1 with DNA titrating. The insets of I and II at (b) upper right corner are the fluorescent intensity and wavelengths evolution with ct-DNA concentrations, respectively.
but the maximum point of fluorescence intensity and the last stable point for probe-2 are at the ratios of 6 and 67, respectively. The mechanisms of dye-DNA may be explained with Twist intermolecular charge transfer (TICT). For the dyes, the intermolecular charge transfer (ICT) state could occur between the electron donor and the electron acceptor [23]. In water, the dipole molecule in the excited state interacted with the higher polar solvent more strongly than that in the ground state resulting in the larger dipole moment in the excited state, which could lead to charge separation resulting in formation of a TICT state. However, the formation of a TICT state was restricted when the dye was protected from DNA in water, and the fluorescence of the dye will be restored. However, the fluorescence will be quenched with the aggregation of dye molecules. In addition, the difference of the linear spectra for two dyes in details may be attributed to the different structures. Moreover, the binding constants between the probes and DNA were obtained by the one-photon fluorescent titration method according to Scatchard equation [24]: r=c f ¼ kn−kr
ð1Þ
where k and n refer to the binding constant and binding sites, respectively. Cf is the concentration of free dye. r is a ratio of Cb/[ct-DNA], and Cb is calculated by the following equation [25]: cb ¼ ct
h
i F−F 0 = F max − F 0
ð2Þ
where, ct is the total DNA concentration. F0 and F are the fluorescence intensity of the free dye and dye-DNA complex, respectively. Fmax is the saturated fluorescent intensity in DNA titration. According to the Eqs. (1) and (2), the fitted curves were shown in Fig. 3 (a) and (b). The obtained binding constants are 3.06 × 105 M−1 for probe-1 and 1.08 × 106 M−1 for probe-2, respectively. The value of probe-2 is slightly larger over that of probe-1 and comparable with the 106 M−1 value for DAPI [26], and the binding sites of two compounds are n = 0.86 (probe-1), 1.83 (probe-2). The cause may be attributed to more positive charges of probe-2 in comparison to probe-1, which increased the interaction of the dye-DNA. 3.3. The two-photon fluorescent spectra of the probes interaction with ctDNA The TP fluorescent spectra of two probes in Tris–HCl buffer solution were recorded with ctDNA titrating on Ti:sapphire laser at 800 nm. The concentrations of probes and ct-DNA were 1 × 10− 4 mol/L and 1 × 10−3 mol/L, respectively. The mole ratios of ctDNA to probes were: 0, 0.1, 0.2, 0.4, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 16.0, 25.0, 32, 43 and 67. The experimental results were obtained and depicted in Fig. 4 (a for probe-1 and b for probe-2). Fig. 4 showed that the shapes and the maximum emission wavelengths of TP fluorescent spectra for two compounds had hardly changed. The fluorescent intensity of probe-1 began to increase before the ratio of 0.2 (ctDNA/probe-1), and then declined and tended to stabilize after the ratio about 25. This trend was similar to that of one-photon
Fig. 2. The UV absorption (a) and fluorescent spectra (b) of probe-2 with DNA titrating. The insets of I and II at (a) upper right corner are the absorbance and the absorption maximum wavelengths evolution with ct-DNA concentrations, respectively. The inset of III at (b) upper right corner is the fluorescent intensity evolution with ct-DNA concentrations.
C. Gao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 156 (2016) 1–8
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Fig. 3. The fitted curves of probe-1(a) and probe-2(b) according to Scatchard equation on the basis of one-photon fluorescent DNA titration.
fluorescent spectra of probe-1. Meanwhile, the changing trend of fluorescent intensity for probe-2 was also almost coincident with that of probe-1, but its fluorescent intensity reached the maximum at the ratio of 12 and tended to be stable at the ratio of 16. The largest δФ of two probes with DNA titrating were 60.13 GM and 10.52 GM, respectively, which are far higher than those of some traditional DNA dyes (∼2 GM for DAPI). These indicated that they were possibly used to detect DNA by TP excitation, and the TP properties of probe-1 are superior to that of probe-2. 3.4. The possible interaction mechanisms between the probes and DNA In order to investigate the possible DNA–probe interaction mechanisms, the fluorescence of probe-ctDNA on the condition of NaCl was investigated. The concentrations of probes and ctDNA were 1 × 10−5 mol L−1 and 5.0 μg mL−1 in Tris–HCl buffer with pH 7.4. The concentrations of used NaCl were from 0.1 to 0.5 mol L−1. The changes of fluorescent intensity with adding NaCl were shown in Fig. 5 (a for probe-1 and b for probe-2). Generally, Na+ can partially close to phosphate of nucleotide skeleton. If the DNA–dye interaction is electrostatic, the change of fluorescent intensity will be disturbed with the increase of Na+ concentrations. Fig. 5(a) showed that fluorescent intensity of probe-1-DNA complex has slight changes with the addition of NaCl, which indicated that the electrostatic interaction did not play a major role for the probe-1-DNA action. However, the reductive fluorescence of probe-2 was obvious with the addition of ctDNA, which indicated that electrostatic interaction has an important contribution for the probe-2- DNA action [27].
It is well known that iodide is a typical reagent to quench fluorescence. In order to further study the interaction mechanisms of probes–ctDNA, we investigated the fluorescence of the different systems including probe, probe–KI, probe–ctDNA and probe– ctDNA–KI, and the concentrations of probes, ct-DNA and KI were 1.0 × 10− 5 mol L− 1,5.0 μg mL− 1 and 0.02 mol L−1 in Tris–HCl buffer, respectively. The interactions between dyes and ctDNA are mainly noncovalent bond including outside binding, intercalation binding and groove binding [28–30]. Fig. 6 showed that the fluorescence of probe1 was obviously quenched by KI, but that of probe-1-ctDNA was partly reduced. It is likely that probe-1-ctDNA binding prevented the fluorescent quenching. In above discussion, outside binding by electrostatic interaction was unconspicuous. The reduction of absorbance, red-shift of absorption band and the formation of isobestic point are typical signs for intercalation binding [31], however those were not observed in Fig. 1. On the basis of the analyses for the absorption and fluorescent spectra, the main interaction mechanism of probe-1-DNA can be considered to groove binding. After adding KI, the fluorescence of probe-2 and probe-2–ctDNA was almost identical, which indicated that the interaction mechanism between probe-2 and DNA is mainly outside binding by electrostatic interaction, and it is coincident with the above conclusion. 3.5. MTT assay HeLa cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) containing high glucose supplemented with 10% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin. When in the proliferative
Fig. 4. The two-photon fluorescent spectra of probe-1(a) and probe-2(b) upon DNA titrating in Tris–HCl buffer at 800 nm with a concentration of 1 × 10−4 mol L−1. The insets of I at (a) and (b) upper right corner are the fluorescent intensity of two probes evolution with ct-DNA concentrations.
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Fig. 5. Effect of salt concentrations (a for probe-1 and b for probe-2). Conditions: Cprobe = 1.0 × 10−5 mol L−1; Cct-DNA = 5.0 μg mL−1; in Tris–HCl buffer solution with pH 7.4.
Fig. 6. The fluorescent spectra of KI quenching for probe-ctDNA systems, (a for probe-1 and b for probe-2). Conditions: Cprobe = 1.0 × 10−5 mol L−1; Cct-DNA = 5.0 μg mL−1; CKI = 0.02 mol L−1.
period, HeLa cells (∼5 × 105 cell/mL) were dispersed within replicate 96well microliter plates to a total volume of 100 μL/well and maintained at 37 °C in a 5% CO2/95% air incubator for 24 h. Then, the culture media was removed and the cells were incubated in culture medium containing the as prepared probe-1 and probe-2 with different concentrations (5, 10, 20, 40, 80 nM) for 48 h and washed with the culture medium. An amount of 100 μL of the new culture medium containing MTT (10 μL, 5 mg mL−1, PBS) was then added to each well, followed by incubating for 4 h to allow the formation of formazan dye. After removing the medium, 150 μL of DMSO was added to each well to dissolve the formazan crystals. Absorbance was measured at 490 nm in a Triturus microplate reader. The number of viable cells was determined by an MTT assay [32]. As shown in Fig. 7, the cellular viabilities were estimated to be greater than 90% after incubation for 48 h with the concentrations from 5 to 20 nM for probe-1 and from 5 to 40 nM for probe-2. In higher concentration of 80 nM, the viabilities were still over 70% (71% for probe-1 and 76% for probe-2). These results showed the as prepared probes are generally low toxicity for cellular imaging. Moreover, the toxicity of probe-1 is higher than that of probe-2, which may be attributed to the different interactions between dye and DNA. The groove binding of probe-1 and DNA increased the damage to bases on DNA in comparison to the electrostatic interaction of probe-2 [33].
with an Olympus FV300 confocal microscope. A Ti-sapphire laser (Coherent Mira900-D) was used to excite the probes at 800 nm. The fluorescent emission of two probes was collected using 450–650 nm for probe-1 and 450–550 nm for probe-2 band-pass filters. In Fig. 8 (a1, b1, c1),the position and shape stained by probe-1 were the same as that of nuclei in the DIC image, implying that the probe-1 can accurately label nuclei. But the position stained by probe-2 was not only in cell nuclei but also in the cytoplasm, which indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2. The
3.6. Fluorescence imaging The potential utilities of probes were proceeded to examine by the fluorescence imaging of DNA in living cells. HeLa cells were cultured in DMEM medium at 37 °C/5% CO2, and grown on glass cover slips before the preparation for imaging experiments. The cells were incubated with the probes solution (1 × 10−6 mol/L) for 30 min at room temperature and then washed with PBS for three times. The cells were imaged
Fig. 7. MTT assay of HeLa cells in the presence of different concentrations (5, 10, 20, 40, 80 nM) of probe-1 (black) and probe-2 (gray) for 48 h at 37 °C, respectively.
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Fig. 8. Two-photon fluorescence images of probe-1(a1) and probe-2 (a2) fluorescent images were incubated with 10 μm of probe-1 and probe-2 for 30 min and excited at 800 nm, respectively; (b1, b2) DIC photos; (c1, c2) merged images.
different fluorescence images of two probes may be attributed to diverse structures and interaction types [34,35]. 4. Conclusions Two styryl heterocyclic dyes of probe-1 and probe-2 were successfully synthesized. The linear and nonlinear photophysical properties in different solvents and in DNA titrating were investigated. The results implied that TP properties of probe-1 are superior to that of probe-2, and the change trends of fluorescent intensity were consistent for SPE and TPE with DNA titrating. The binding constant of probe-2-DNA (1.08 × 106 M−1) is slightly larger over that of probe-1 (3.06 × 105 M−1). These data showed that two compounds could be considered as efficient fluorescent probes for DNA detection by oneand two-photon excited method. The possible mechanisms of the probe–DNA interaction were studied by fluorescent spectra in the presence of NaCl and KI, which are mainly groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The results of MTT assay and the TP fluorescence imaging in living cells indicated that two probes are low toxicity, and the ability to locate in cell nuclei of probe-1 is superior to that of probe-2. Acknowledgments This work was supported by the National Natural Science Foundation of China (no. 51403111, 21276149), the Program of Young Teachers Growth Plan in Shandong Province, the Natural Science Foundation of Shandong Province (ZR2012EMM009, ZR2012EMZ003) and Program for Scientific Research Innovation Team in Colleges and Universities of Shandong Province. In addition, we are particularly grateful to Professor Yu from Shandong University for their help in fluorescent measuring.
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