A turn-on and colorimetric metal-free long lifetime fluorescent probe and its application for time-resolved luminescent detection and bioimaging of cysteine

Abstract

A turn-on and colorimetric metal-free long lifetime fluorescent probe for sensing Cys has been synthesized. Utilizing the long emission-lifetime of DCF-MPYM, the time-resolved luminescent assay of DCF-MPYM-thiol for sensing Cys was realized successfully. DCF-MPYM-thiol can realize confocal and time-resolved microscopy bioimaging for Cys in living cells.

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Biothiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play critical roles in essential biological molecules relevant to the growth of cells and tissues in living systems.¹ The levels of these biothiols are associated with many diseases, especially slow growth of children, liver damage, skin lesions, Alzheimer's and Parkinson's disease.² The detection of biothiols becomes very important and has been paid increasing attention in recent years.^2b,3 Fluorescent probes have the advantages of selectivity, sensitivity and accumulative effect, which play an important part in detection of analytes. Therefore, a lot of efforts has been directed to the development of fluorescent probes for biothiols.⁴

The lifetimes of traditional fluorescent probes are in the order of nanoseconds, thus, the fluorescence signals of them often suffer from the interference from background fluorescence because of their short lifetimes and scattered light in vivo and vitro applications.⁵ Compared with traditional fluorescent probes utilizing short-lifetime fluorophores, introduction of long-lived luminescence dyes is expected to exploit advantages in allowing detection with eliminating the short-lived background fluorescence and providing high signal-to-noise ratios.⁶ In our previous work, the photophysical characteristics of fluorescein derivative DCF-MPYM were studied.⁷ DCF-MPYM showed long-lived thermally activated delayed fluorescence (22.11 μs in deaerated ethanol) and then it can be used in time-resolved fluorescence imaging in living cells. Therefore, in order to eliminate the interference from the short-lived background fluorescence through time-resolved photoluminescent technique (TRPT), the particular property of long luminescence lifetime makes DCF-MPYM have the potential of synthesizing a new fluorescent probe for biothiols.

Colorimetric and ratiometric probes allow the measurement at two different wavelengths, which could provide a built-in correction for environmental effects and improve the sensitivity of detection.⁸ Thus, chemosensor exhibiting greatly ratiometric changes of absorption peak or emission intensity is an ideal selection for sensing. To image the distribution of biothiols in cellular processes, some relevant fluorescent probes have been reported.^8a Rusin and Strongin et al. firstly designed a fluorescent chemosensor for Cys/Hcy by colorimetric and fluorometric responses.⁹ But it only can achieve the intracellular colorimetric detection of Cys/Hcy. There are relatively few fluorescent probes which can realize the enhancement of emission intensity at two emission wavelengths and colorimetric detection of Cys/Hcy.

Herein, based on DCF-MPYM, it is very important to develop a new and excellent fluorescent probe with the ability to colorimetrically detect biothiols and eliminate the interference from the background fluorescence and scattered light. To make full use of the photophysical characteristics of DCF-MPYM, the ketene group that commonly utilized in the detection of Cys was introduced into DCF-MPYM. The probe DCF-MPYM-thiol for detecting Cys through Michael Addition was synthesized (Scheme 1). Thus, we expect that DCF-MPYM-thiol can overcome the drawbacks that common fluorescent Cys probes face. The structure of DCF-MPYM-thiol was characterized by ¹H, ¹³C NMR spectroscopy and high resolution mass spectra (ESI†).

Scheme 1 Synthetic route to the dye DCF-MPYM-thiol.

The sensing performances of probe DCF-MPYM-thiol for biothiols have been investigated by absorption and emission spectra. Firstly, considering environmental application, we tested the fluorescence spectra changes in pure PBS (phosphate buffer saline). The result showed that the fluorescence intensity had no obvious changes after addition of Cys for 1 h (Fig. S1†). But the emission intensity at 620 nm immediately had a 22-fold enhancement if 100 μL (25 mg mL⁻¹) BSA was added into the detection system (Fig. S2†). It showed that DCF-MPYM-thiol could react with Cys in PBS system. According to our previous report,⁷ we can propose that the product between probe DCF-MPYM-thiol and Cys may be the compound DCF-MPYM. But the poor solubility and fluorescence quenching of possible product made the fluorescence signal difficultly detected.

In order to better observe the changes of spectra for DCF-MPYM-thiol, we adjusted the different ratios of PBS and CH₃CN, and then selected the most optimal detective condition (CH₃CN/PBS = 1/1, v/v) as a testing system at room temperature. The free probe DCF-MPYM-thiol showed no noticeable changes in detection system. Upon addition of Cys, Hcy and GSH to a solution of probe DCF-MPYM-thiol, the fluorescence intensity increased with prolonged reaction time, but the reaction of DCF-MPYM-thiol with Cys was nearly complete within 10 min (Fig. S3†). To Hcy and GSH, the reaction time of them should prolong more than 300 min to reach the maximal fluorescence intensity (Fig. S4–S6†). Therefore, Hcy/GSH cannot interfere with the detection of Cys within 10 min and all the following experiments were taken within 10 min to obtain highly sensitive and reproducible results (Fig. S7†). The reason of probe's high reactivity and selectivity toward Cys over Hcy and GSH may be attributed to the different nucleophilicities of the thiol groups, since the pK_a of Cys (8.30) is lower than those of Hcy (8.87) and GSH (9.20).¹⁰ Another possible factor could be steric effect, the thiol group of Cys is sterically less hindered than those of Hcy and GSH.¹¹

Thus, we do more researches to the typical biological thiol Cys. Upon addition of increasing concentrations of Cys, the absorption band of DCF-MPYM-thiol at 380 nm gradually deceased, whereas new absorption bands at 275 nm, 350 nm, 460 nm and 555 nm increased with isosbestic point at 400 nm. As shown in the inset of Fig. 1, an apparent solution color changes in the absorption spectrum from yellow to red thereby offering DCF-MPYM-thiol as a Cys-selective “naked-eye” chemosensor. Correspondingly in the emission spectrum, the fluorescence intensity at 625 nm increased with simultaneous increase at 525 in CH₃CN/PBS buffer = 1/1 solution with the increasing concentrations of Cys (Fig. 2). The emission color of probe DCF-MPYM-thiol changed from non-fluorescent to red emission (see the inset of Fig. 2). The ratiometric response of fluorescence intensities at 525 nm and 625 nm (F_625nm/F_525nm) enhanced from 0.4 to 6.5 with a final enhancement factor over 16.2 folds (Fig. S8†). The fluorescence titration studies with DCF-MPYM-thiol revealed a good linearity between the fluorescence intensity ratio and Cys concentration with a detection limit of approximately 4.31 × 10⁻⁶ M that allowed the quantification of Cys by a turn-on fluorescence method.

Fig. 1 Absorption spectral changes of DCF-MPYM-thiol (3.0 μM in CH₃CN/PBS buffer = 1/1) after incubation with different concentrations of Cys (0, 0.3, 0.6, 2.4, 4.2, 6.0, 7.8, 11.4, 16.8, 25.8, 43.8, 79.8, 133.8 and 187.8 μM). Each spectrum was recorded after 10 min. Inset: probe DCF-MPYM-thiol (3.0 μM) in the absence of Cys (left) and in the presence of 133.0 μM Cys (right).

Fig. 2 Fluorescence changes of DCF-MPYM-thiol (3.0 μM in CH₃CN/PBS buffer = 1/1) after incubation with different concentrations of Cys (0, 0.3, 0.6, 2.4, 4.2, 6.0, 7.8, 11.4, 16.8, 25.8, 43.8, 79.8, 133.8 and 187.8 μM). Each spectrum was recorded after 10 min, excitation at 485 nm. Inset shows the fluorescence changes of DCF-MPYM-thiol (3.0 μM) in the absence of Cys (left) and presence of 133.0 μM Cys (right), excited at a hand-held 365 nm UV lamp.

The possible mechanism for the detection of Cys may be similar with the reported literature by Chen's group^1c and our previous work.¹² However, to further prove the rationality of reaction mechanism, the plausible mechanism and mass spectra of final product were shown in Fig. S9 and S10,† respectively. Mass spectrometry analysis showed the DCF-MPYM and a cyclized product 5-oxo-1,4-thiazepane-3-carboxylic acid formed from the reaction between DCF-MPYM-thiol and Cys. The peak at 783.19 corresponding to the fluorescent compound DCF-MPYM and another peak at 174.01 corresponding to the cyclization product 5-oxo-1,4-thiazepane-3-carboxylic acid were clearly observed in Fig. S10.†

To investigate the probe's selectivity, the mixture of DCF-MPYM-thiol and Cys was treated with various biologically relevant analysts (such as represent proteins, metal ions and anions) and monitored by emission histograms. No noticeable changes of emission enhancement were observed upon addition of different proteins (Fig. S11†), metal ions (Fig. S12†) and anions (Fig. S13†).

In the reported literatures,¹³ time-resolved luminescent probes were always phosphorescent probes because of their long emission lifetime of phosphorescence signals. The long-lifetime signals can eliminate the interference from short-lived background fluorescence and scattered light.¹⁴ For example, Zhao and Huang et al. have utilized the long phosphorescence emission-lifetime of heavy-metal complexes to design an “OFF–ON” phosphorescent chemodosimeter,^13b which successfully realized the time-resolved luminescent assay in sensing Cys. It was the first report about the time-resolved luminescent detection of biothiols, which provided a helpful strategy for the further design of excellent probes with long emission lifetime suitable for time-resolved photoluminescent technique. However, there are still no reports about time-resolved luminescent probes for the detection of biothiols based on long lifetime metal-free organic fluorophores until now.

According to the above results, the reaction product of DCF-MPYM-thiol and Cys is namely the compound DCF-MPYM which exhibits excellent delayed fluorescence with a long emission lifetime of 22.11 μs in deaerated ethanol. Therefore, the long-lifetime characteristic of DCF-MPYM makes DCF-MPYM-thiol become a good time-resolved luminescent candidate for detecting biothiols in comparison with common fluorescent probes. To make full use of the advantages of DCF-MPYM, the time-resolved luminescent detection of biothiols was also introduced into our detection system. Thus, the time-resolved emission spectra (TRES) were recorded with increasing the concentrations of Cys (Fig. 3).

Fig. 3 Time-resolved emission spectra of the mixture of probe DCF-MPYM-thiol after addition of Cys. Time-gated photoluminescence spectrum was acquired after 0.100 ms delay of the mixture of probe DCF-MPYM-thiol (3.0 μM) before and after addition of different concentrations of Cys.

Compared with the work reported by Zhao and Huang et al.,^13b our method has the difference that another short-lived lifetime dye BODIPY should be utilized as a typical control compound. There is no need for introduction of another new dye in our detection system, the reason of which was explained by the steady-state emission spectra. As shown in Fig. 4, the emission of product DCF-MPYM has two peaks after the reaction of probe DCF-MPYM-thiol and Cys. After addition of 187.8 μM Cys, the normalized steady-state fluorescence spectrum (Fig. 4, black line) and time-resolved fluorescence spectrum (Fig. 4, red line) of the mixture were obtained at room temperature under air atmosphere, respectively. The weak short-wavelength emission (around 525 nm) in steady-state spectrum disappeared in the time-resolved fluorescence spectrum with a 100 μs delay. And the maximum long wavelength emission around 621 nm in the time-resolved fluorescence spectrum unexpectedly agreed closely with the maximum at 627 nm in the steady-state fluorescence spectrum. Therefore, it realizes time-resolved “turn-on” luminescence responsing to Cys, these results highlight the advantages of long-lifetime delayed fluorescence signal which can eliminate the interference from short-lived background fluorescence.

Fig. 4 Normalized steady-state and time-resolved fluorescence spectra of the mixture after the addition of 187.8 μM Cys, black line: steady-state fluorescence spectrum under air atmosphere (delay time, 0 s); red line: time-resolved fluorescence spectrum under air atmosphere. Delayed detection was carried out in phosphorescence mode (total decay time, 5 ms; delay time, 0.1 ms; gate time, 1.0 ms).

Considering the excellent sensing performances of probe DCF-MPYM-thiol to Cys in solution, to assess permeability and ability to monitor thiols in living cells, confocal fluorescence imagings for MCF-7 cells were tested. After incubation with probe DCF-MPYM-thiol (15 μM) at 37 °C for 120 min, MCF-7 cells displayed marked intracellular luminescence (Fig. 5). However, cells were incubated with the thiol-trapping regent N-ethylmaleimide (NEM). The confocal fluorescence images did not have significant fluorescence signal (Fig. S14†). Thus, the control experiment proved that probe DCF-MPYM-thiol could detect the Cys in the living cells. Meanwhile, the finally product of the reaction between probe DCF-MPYM-thiol and Cys is estimated to be compound DCF-MPYM. Our reported result indicated that compound DCF-MPYM had long lifetime, thus it can be utilized as a biolabel for visible-light excited time-gated luminescence bio-imaging applications (Fig. 6).

Fig. 5 Fluorescence images of MCF-7 cells incubated without (b) or with (e) 15 μM DCF-MPYM-thiol and 40 μL BSA (10 mM) at 37 °C: (a) bright field image of cells, (b) fluorescent image excited at 488 nm, no fluorescence was observed; (c) overlay image of (a) and (b); After 120 min incubation with probe DCF-MPYM-thiol, (d) bright field image of cells, (e) fluorescent image excited at 488 nm, (f) overlay image of (d) and (e).

Fig. 6 (a) Bright-field, (b) time-gated luminescence (excited with 510–560 nm) images of MCF-7 immunostained with DCF-MPYM-thiol in PBS samples.

Conclusions

In summary, an excellent “TURN-ON” and colorimetric fluorescent chemosensor for sensing Cys based on metal-free organic fluorophore has been synthesized in this work. Importantly, utilizing the long emission-lifetime of the fluorescence signal of compound DCF-MPYM, the time-resolved luminescent assay of DCF-MPYM-thiol in sensing Cys was realized successfully, which can eliminate the interference from the short-lived background fluorescence. As far as we know, this is the first report about the time-resolved luminescent detection of biothiols based on metal-free organic fluorophore. It will be very helpful for the further design of probes with long emission lifetime suitable for time-resolved photoluminescent technique. Finally, probe DCF-MPYM-thiol can be successfully used for bioimaging Cys in living cells utilizing confocal and time-resolved microscopy imaging.

Acknowledgements

This work was supported financially by the science fund no. 2014A11GX030 from Dalian City in 2014, the NSF of China (21222605, 21006009, 21136002, and 21376039), the Fundamental Research Funds for the Central Universities of China, Program for New Century Excellent Talents in University and the Project-sponsored by SRF for ROCS, SEM.

Notes and references

1. (a) X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010, 39, 2120; (b) Y. Zhou and J. Yoon, Chem. Soc. Rev., 2012, 41, 52; (c) H. Wang, G. Zhou, H. Gai and X. Chen, Chem. Commun., 2012, 48, 8341.
2. (a) O. García-Beltrán, N. Mena, E. G. Pérez, B. K. Cassels, M. T. Nuñez, F. Werlinger, D. Zavala, M. E. Aliaga and P. Pavez, Tetrahedron Lett., 2011, 52, 6606; (b) T.-K. Kim, D.-N. Lee and H.-J. Kim, Tetrahedron Lett., 2008, 49, 4879.
3. (a) S. Y. Lim, K. H. Hong, D. I. Kim, H. Kwon and H. J. Kim, J. Am. Chem. Soc., 2014, 136, 7018; (b) W. Lin, L. Yuan, Z. Cao, Y. Feng and L. Long, Chem.–Eur. J., 2009, 15, 5096; (c) J. Liu, Y.-Q. Sun, Y. Huo, H. Zhang, L. Wang, P. Zhang, D. Song, Y. Shi and W. Guo, J. Am. Chem. Soc., 2013, 136, 574.
4. (a) X. Tang, W. Liu, J. Wu, W. Zhao, H. Zhang and P. Wang, Tetrahedron Lett., 2011, 52, 5136; (b) Q.-Q. Wu, Z.-F. Xiao, X.-J. Du and Q.-H. Song, Chem.–Asian J., 2013, 8, 2564; (c) Y. Yuan, R. T. Kwok, G. Feng, J. Liang, J. Geng, B. Z. Tang and B. Liu, Chem. Commun., 2014, 50, 295.
5. (a) X. Peng, Z. Yang, J. Wang, J. Fan, Y. He, F. Song, B. Wang, S. Sun, J. Qu and J. Qi, J. Am. Chem. Soc., 2011, 133, 6626; (b) S. Zhang, T. Wu, J. Fan, Z. Li, N. Jiang, J. Wang, B. Dou, S. Sun, F. Song and X. Peng, Org. Biomol. Chem., 2012, 11, 555.
6. (a) N. Weibel, L. J. Charbonnière, M. Guardigli, A. Roda and R. Ziessel, J. Am. Chem. Soc., 2004, 126, 4888; (b) B. Song, G. Wang, M. Tan and J. Yuan, J. Am. Chem. Soc., 2006, 128, 13442; (c) B. Song, G. Wang and J. Yuan, Chem. Commun., 2005, 3553; (d) Z. Dai, L. Tian, Y. Xiao, Z. Ye, R. Zhang and J. Yuan, J. Mater. Chem. B, 2013, 1, 924.
7. (a) X. Xiong, F. Song, J. Wang, Y. Zhang, Y. Xue, L. Sun, N. Jiang, P. Gao, L. Tian and X. Peng, J. Am. Chem. Soc., 2014, 136, 9590; (b) X. Xiong, F. Song, S. Sun, J. Fan and X. Peng, Asian J. Org. Chem., 2013, 2, 145.
8. (a) J. Shi, Y. Wang, X. Tang, W. Liu, H. Jiang, W. Dou and W. Liu, Dyes Pigm., 2014, 100, 255; (b) G. Chen, F. Song, J. Wang, Z. Yang, S. Sun, J. Fan, X. Qiang, X. Wang, B. Dou and X. Peng, Chem. Commun., 2012, 48, 2949.
9. O. Rusin, N. N. S. Luce, R. A. Agbaria, J. O. Escobedo, S. Jiang, I. M. Warner, F. B. Dawan, K. Lian and R. M. Strongin, J. Am. Chem. Soc., 2004, 126, 438.
10. (a) H. S. Jung, K. C. Ko, G.-H. Kim, A.-R. Lee, Y.-C. Na, C. Kang, J. Y. Lee and J. S. Kim, Org. Lett., 2011, 13, 1498; (b) L. Yuan, W. Lin and Y. Yang, Chem. Commun., 2011, 47, 6275; (c) W. Lin, L. Yuan, Z. Cao, Y. Feng and L. Long, Chem.–Eur. J., 2009, 15, 5096; (d) Y.-Q. Sun, M. Chen, J. Liu, X. Lv, J.-f. Li and W. Guo, Chem.–Eur. J., 2011, 47, 11029.
11. M. Zhang, Y. Wu, S. Zhang, H. Zhu, Q. Wu, L. Jiao and E. Hao, Chem.–Eur. J., 2012, 48, 8925.
12. X. Xiong, F. Song, G. Chen, W. Sun, J. Wang, P. Gao, Y. Zhang, B. Qiao, W. Li and S. Sun, Chem.–Eur. J., 2013, 19, 6538.
13. (a) M. Liu, Z. Ye, C. Xin and J. Yuan, Anal. Chim. Acta, 2013, 761, 149; (b) Y. Tang, H. R. Yang, H. B. Sun, S. J. Liu, J. X. Wang, Q. Zhao, X. M. Liu, W. J. Xu, S. B. Li and W. Huang, Chem.–Eur. J., 2013, 19, 1311; (c) G. Zhang, J. Chen, S. J. Payne, S. E. Kooi, J. Demas and C. L. Fraser, J. Am. Chem. Soc., 2007, 129, 8942; (d) Q. Zhao, X. Zhou, T. Cao, K. Y. Zhang, L. Yang, S. Liu, H. Liang, H. Yang, F. Li and W. Huang, Chem. Sci., 2015, 6, 1825; (e) K. Y. Zhang, J. Zhang, Y. Liu, S. Liu, P. Zhang, Q. Zhao, Y. Tang and W. Huang, Chem. Sci., 2015, 6, 301.
14. (a) S. Liu, H. Liang, K. Y. Zhang, Q. Zhao, X. Zhou, W. Xu and W. Huang, Chem. Commun., 2015, 51, 7943; (b) Q. Zhao, Y. Liu, Y. Cao, W. Lv, Q. Yu, S. Liu, X. Liu, M. Shi and W. Huang, Adv. Opt. Mater., 2015, 3, 233; (c) W. Xu, X. Zhao, W. Lv, H. Yang, S. Liu, H. Liang, Z. Tu, H. Xu, W. Qiao, Q. Zhao and W. Huang, Adv. Healthcare Mater., 2014, 3, 658; (d) H. Shi, H. Sun, H. Yang, S. Liu, G. Jenkins, W. Feng, F. Li, Q. Zhao, B. Liu and W. Huang, Adv. Funct. Mater., 2013, 23, 3268; (e) Y. Ma, S. Liu, H. Yang, Y. Wu, H. Sun, J. Wang, Q. Zhao, F. Li and W. Huang, J. Mater. Chem. B, 2013, 1, 319.

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Footnote

† Electronic supplementary information (ESI) available: Time course of the fluorescence response of different kinds of thiols, plausible mechanism, synthesis of DCF-MPYM-thiol. See DOI: 10.1039/c5ra08539j

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