A colorimetric fluorescent chemodosimeter based on diketopyrrolopyrrole and 1,3-indanedione for cysteine detection and cellular imaging in living cells

Sensors and Actuators B 205 (2014) 281–288

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A colorimetric fluorescent chemodosimeter based on diketopyrrolopyrrole and 1,3-indanedione for cysteine detection and cellular imaging in living cells Lingyun Wang ∗ , Jiqing Du, Derong Cao School of Chemistry and Chemical Engineering, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China

a r t i c l e

i n f o

Article history: Received 17 July 2014 Received in revised form 28 August 2014 Accepted 28 August 2014 Available online 8 September 2014 Keywords: Fluorescent probe Cysteine (Cys) Colorimetric Diketopyrrolopyrrole Protein Cellular imaging

a b s t r a c t A novel colorimetric fluorescent chemodosimeter (1) based on diketopyrrolopyrrole (DPP) and indanedione for the selective detection of cysteine (Cys) over glutathione (GSH) was synthesized, which was involved by the conjugate addition of Cys to ␣,␤-unsaturated ketones. The probe featured a fast response (a response time less than 2 min), excitation and emission in the visible region, dual-channel and high selectivity. Addition of Cys in PBS (pH = 7.4) to 1 in THF resulted in a rapid color change from purple to yellow together with appearance of a new absorption peak at 480 nm, while other amino acids did not induce any significant color change. Meanwhile, the Michael addition of Cys to 1 elicited 4.2-fold PL enhancement at 505 nm, which resulted in emission color change from deep red to yellow. Furthermore, 1 could be used as a fluorescent probe for detection Cys34 within BSA. In addition, the cellular imaging of human adult skin fibroblast cells indicated red fluorescence of 1 was present in the cytoplasm. The CCK-8 assay showed that the cytotoxicity of 1 was low. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Among the twenty amino acids commonly found in proteins, cysteine (Cys) is special in that it is present more often than other residues in functionally important locations. Elevated level of Cys in plasma is a vascular disease risk factor and is associated with neurotoxicity. On the contrary, deficiency of Cys may result in some serious diseases such as hematopoiesis decrease, leukocyte loss, and psoriasis. Additionally, altered level of Cys has been implicated in hyperhomocysteinemia, which has been linked to the increased risks of Alzheimer’s disease, neural tube defect, and osteoporosis. Thus, the detection of Cys continues to be of interest. Up to now, numbers of thiol-specific probes have been developed on the basis of various strategies [1], such as cleavage reactions [2], Michael addition reactions [3], metal complexes/ displaced coordination and others [4]. Among these strategies, Michael addition type probes attract many attentions. A number of excellent Michael acceptors have been exploited such as ␣,␤unsaturated aldehyde [5], ketone [6], diesters [7], malonitrile [8]

∗ Corresponding author. Tel.: +86 20 87110245; fax: +86 20 87110245. E-mail address: [email protected] (L. Wang). http://dx.doi.org/10.1016/j.snb.2014.08.090 0925-4005/© 2014 Elsevier B.V. All rights reserved.

and nitroolefin [9], etc. However, many of them are associated with some limitations, including a limited pH range, excitation and emission in the UV region (below 500 nm), especially a long response time. More importantly, only a few sensors able to detect Cys over GSH have been reported to date [10]. At the same time, the fluorophores used for thiol probes are limited to coumarin, fluorescein, rhodamine, or BODIPY [1]. Probes based on other fluorophores were rarely reported. On the other hand, diketopyrrolopyrrole (DPP) is a robust chromophore, but its application in thiol-specific fluorescent probes is very rare [8b]. So, much room is left to explore DPP-based probes to improve the sensing properties, such as achieving longer emission wavelength and rapid response time, etc. Furthermore, most of the thiol probes based on Michael addition are colorimetric or fluorescence probes [6,9,11], and a few thiol probes have been developed to combine the ratiometric and colorimetric fluorescence transductions. Inspired by elegant development of the thiol probes based on Michael addition, herein we designed probe 1 (Scheme 1) preferably with excitation and emission in the visible region with the intention as follows. (1) The indandione-derived vinyl moiety could be more reactive and enhance the sensitivity of the nucleophilic addition reaction between the vinyl group and Cys. (2) Electrondeficient diketopyrrolopyrrole (DPP) is selected as a chromophore due to its many advantageous features including its enhanced

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CHO

CH2OH CH2OH

O

O

CH

O

CN 2 O

t-C4H9OK

O

O

t-C5H11ONa t-C5H11OH

NH2SO3H

O

O O

O

CN

C8H17Br

O

CH

O

CH

N H

CH

O O

O

3 C8H17

C8H17

N

N

CH

O

O

O

HCl/H2O

O

THF

OHC

C8H17

4

H N

CHO O

N

C8H17 C8H17

O

N

O

O

N

5 O

O O

O

N

C8H17

O

1 Scheme 1. Synthesis of probe 1.

vinyl moiety reactivity with Cys, strong fluorescence as well as its distinct rigid ring structure. (3) Two vinyl moieties are introduced to probe 1, which is favorable to accelerate reaction rate between probe 1 and Cys. Thus, a DPP–indanedione conjugate (1) as doubly activated Michael addition type probe for the colorimetric and fluorescent detection of Cys was developed, which was highly selective toward thiols over other amino acids with a fast response. Moreover, application of 1 in cellular imaging was demonstrated and discussed. 2. Experimental 2.1. Chemicals and instruments Nuclear magnetic resonance spectra were recorded on Bruker Avance III 400 MHz and chemical shifts are expressed in ppm using TMS as an internal standard. The UV–vis absorption spectra were recorded using a Helios Alpha UV–Vis scanning spectrophotometer. Fluorescence spectra were obtained with a Hitachi F-4500 FL spectrophotometer with quartz cuvette (path length = 1 cm). N-methyl-2-pyrrolidone (NMP) was dried with CaH2 and distilled under nitrogen atmosphere. Other solvents were obtained from commercially available resources without further purification. Probe 1 was synthesized according to our published literature [12]. The recognition between 1 in THF and different amino acids in PBS (pH 7.4) was investigated by UV–vis and fluorescence spectroscopy at room temperature. The stock solution of 1 and amino acids was at a concentration of 10.0 mM. After the 1 and amino acids with desired concentrations were mixed, they were measured by UV–vis and fluorescence spectroscopy.

humidified environment containing 5% CO2 . Before the experiment, the cells were precultured until confluence was reached. 2.2.2. Cell imaging The human adult skin fibroblast cells were seeded in the 12-well plate and cultured in H-DMEM with 10% FBS at 37 ◦ C in a humidified environment containing 5% CO2 . After 80% confluence, the medium was removed and the adherent cells were rinsed twice with 1 × PBS. The 1 in DMEM medium with FBS at 10 ␮M was then added to the culture plate. After incubation for 2 h, the cells were washed three times with 1 × PBS buffer and fixed with 4% paraform for 2 h at room temperature. The nuclei were stained by 4 ,6-diamidino2-phenylindole for 10 min. The cell monolayer was then washed twice with 1 × PBS buffer and imaged using inverted phase contrast fluorescence microscope (Nikon Eclipsc Ti-U) with imaging software (Nikon NIS Elements).

2.2. Cellular imaging

2.2.3. Cytotoxicity Cell viability was evaluated using the Cell Counting Kit-8 (CCK8) assay. CCK-8 was just as WST-8 to produce formazan in the presence of an electron mediator, and the amount of the formazan generated in cells was directly proportional to the number of living cells. The human adult skin fibroblast cells were seeded in 96-well plates at a density of 3000 cells per well. 1 at different concentrations (1 and 10 ␮M, respectively) was added into a 96-well plate. Meanwhile, cells culture with complete medium (H-DME with 10% FBS) were evaluated as a control. The human adult skin fibroblast cells were incubated in the medium under 5% CO2 in an incubator maintained at 37 ◦ C for 1, 2, 4 and 7 days, respectively. Then, 10 ␮L of the CCK-8 was added to each well of a 96-well plate incubated for additional 2 h. The absorbance was measured at 450 nm using a microplate reader (Varioskan Flash, Thermo Scientific). The assay was repeated three times.

2.2.1. Cell culture Human adult skin fibroblast cells were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (H-DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin streptomycin at 37 ◦ C in a

2.2.4. Statistical analysis Statistical analysis was performed using a standard Student’s t-test with a minimum confidence level of 0.05 for significant statistical difference. All experiments were performed in triplicate.

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283

1.6

3. Results and discussions

0

3.1. Synthesis and structural characterization

3.2. UV–vis response of Cys to 1 To exploit its sensing behavior, we firstly examined the time dependent changes in the absorption spectra of 1 (20 ␮M) upon reaction with Cys (10 equiv). Free 1 showed two major absorption peaks at 359 and 553 nm in THF. The addition of Cys in PBS solution (pH 7.4) elicited not only a significant absorption increase around 480 nm but also dramatic absorption decreases around 359 and 553 nm (Fig. S1). Moreover, the response of 1 to Cys was found to be very fast. After 120 s, the UV–vis spectra of 1 reached a plateau, indicating the completion of the reaction. These results indicated high reactivity was the unique feature of 1. As we know, the conjugate 1,4-addition of thiols to ␣,␤-unsaturated ketones to form thioethers is normally performed under drastic conditions, such as with bases [15] or Lewis acids [16] as catalysts at elevated temperature. Herein, 1,4-addition of probe 1 and Cys could be conducted in THF/water at room temperature without the use of any catalyst. More importantly, high reactivity of probe 1 was impressive as many reported Cys probes require high equivalents of Cys and long reaction time to reach a maximal spectral signal. For example, the complete reaction times of 6 h for malononitrile containing DPP probe and Cys [8b], 30 min for coumarin–quinoline conjugate with Cys [3d] were reported, respectively. A monochlorinated BODIPY ratiometric fluorescent sensor completed its reaction with Cys within 15 min at 37 ◦ C by thiol − halogen nucleophilic substitution [2e]. Guo’s group [9b] reported nitroolefin-based coumarin probe got maximal spectral signals in the presence of 100 equiv of Cys. To get insight into the reaction of Cys with 1, the absorption spectra of 1 in THF upon titration with Cys in PBS solution (pH 7.4) were recorded. As shown in Fig. 1, addition of an increasing amount of Cys (0–120 equiv) to a solution of probe 1 elicited a gradual decrease of the absorption peaks at 359 and 553 nm and a progressive increase of a new absorption band centered at 480 nm. A 73 nm hypsochromic shift suggested the addition reaction of Cys to the C C bond of the probe 1, thus reducing of the ␲-conjugation framework. Meanwhile, two isosbestic points at 413 and 501 nm were observed, which implied new species with less conjugation were formed.

Abs.

1.2

As we know, during the sensing processes, both thiols and probe are maintained at very low concentrations, and effective collisions of them decrease relatively, which directly leads to a low reaction rate and a long response time [13]. Therefore, it is significant to consider accelerating the sensing processes by some feasible means during probe design. Compared with the general Michael acceptors, doubly activated vinyl moieties, in which two electron-withdrawing groups are attached to the C C group, are more reactive [14], and allow the Michael addition reaction to occur under mild conditions. Clearly, to improve the sensitivity, it is necessary to enhance the reactivity of the probe to Cys. We reasoned that this could be accomplished by increasing the electrophilicity of the ␤-carbon. With this in mind, we decided to introduce electron withdrawing and sterically hindered 1,3-indanedione as well as electron-deficient DPP moiety to afford probe 1. Thus, it is anticipated that probe 1 contained a sterically hindered and electrophilic center that would undergo a selective reaction with Cys over GSH and would possess highly reactivity to Cys. Probe 1 (Scheme 1) was prepared from p-cyanobenzaldehydeas as a starting material in four steps and subsequently by an aldoltype condensation reaction with 1,3-indanedione to yield a purple solid, according to our published literature [12].

0.8

413nm 0.4

120eqv 501nm

0.0 400

500

600

700

Wavelength (nm) Fig. 1. UV–vis spectral changes of 1 in THF (20 ␮M) with the increasing concentrations of Cys in PBS (pH = 7.4).

For the purpose of evaluating selectivity of 1 to Cys, the absorption spectral changes of 1 upon addition of various amino acids in PBS solution (pH 7.4) were examined. It could be found probe 1 was selective for Cys over other amino acids (Pro, Arg, Tyr, Thr, Lys, Phe, His, Val, NAc, Trp, Glu, Ala, Leu, Ser, Ile, Met, Asp). Importantly, the potentially competing thiol, glutathione (GSH), gave rise to relatively insignificant changes in the absorption spectrum and was more time-consuming (within 5 min). The absorption peaks of 1 at 359 and 553 nm were slightly decreased, and a new band centered at 505 nm appeared in presence of GSH (Fig. 2a). Probe 1 showed selective detection and more rapid addition reactivity of Cys over GSH, which could be assigned to two possible reasons as follows. Firstly, the GSH is a tripeptide in which the thiol is located in the middle of the molecule. The inherent reactivity of the SH group is thus expected to be reduced as the result of steric shielding. This should be particularly true for nucleophilic reactions involving 1 with hindered 1,3-indanedione moiety. Secondly, in Cys the SH proton is relatively acidic (lower pKa value for the sulfhydryl group compared to other thiols) in aqueous media [17]. As a consequence, the thiolate/thiol ratio is higher for Cys at neutral pH than GSH. This difference is expected to translate into greater reactivity in nucleophilic reactions. More importantly, the color change from purple to yellow can be clearly observed by the naked eye in presence of Cys, while other amino acids did not induce any significant color change. Thus, naked-eye selective detection of Cys became possible (Fig. 2b). 3.3. Fluorescence response of Cys to 1 The Cys sensing property was further examined through fluorescent titration studies. As shown in Fig. 3, 1 showed two weak emission peaks at 505 and 667 nm upon excitation at 500 nm. Upon addition of Cys to 1, the PL intensity at 505 nm was enhanced gradually, whereas the emission at 667 nm was almost unchanged. Finally, emission intensity at 505 nm was enhanced by 4.20-fold and the I505 /I667 ratio changed from 2.43 to 10.22 after addition of 120 ␮M Cys. Because Michael addition of Cys with 1 really interrupted the ␲-conjugation of the molecule, the fluorescence enhancement at 505 nm of 1 upon addition of Cys was normal. Correspondingly, the emission color of the solution changed from deep red to light-yellow. Thus, 1 was a ratiometric fluorescence probe. To the best of our knowledge, thiol probes that showed the simultaneous ratiometric and colorimetric spectral changes were rarely reported [11a,8b], and this was the second ratiometric fluorescent thiol probe that was based on DPP other than coumarin. Recently, a chlorinated coumarin–hemicyanine dye was used as a Cys

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Fig. 4. MALDI-TOF-MS of probe 1 in presence of Cys in PBS (pH 7.4).

Fig. 2. (a) UV–vis absorption and (b) color changes of 1 in THF (20 ␮M) upon addition of 10 equiv of each amino acid in PBS (pH 7.4).

Arg, Tyr, Thr, Lys, Phe, His, Val, NAC, Trp, Glu, Ala, Leu, Ser, Ile, Met, Asp and GSH were carried out. Most of these amino acids did not cause significant changes in the fluorescence intensity at 505 nm of 1, as shown in Fig. S2. Another important feature of 1 is its high selectivity toward the Cys in presence of other competitive anions. Changes of fluorescence spectra of 1 (1 ␮M) caused by Cys (100 equiv) and other amino acids (100 equiv) were recorded in Fig. S2. As can be seen, these competitive species, did not lead to any significant interference. In the presence of these amino acids, the Cys still produced similar optical spectral changes. These results showed that the selectivity of probe 1 toward Cys was not affected by the presence of other amino acids. The detection limit of 1 for Cys was calculated based on the fluorescence titration data according to a reported method [18]. Under optimal conditions, calibration graphs for the determination of Cys were constructed. The enhanced fluorescence intensity at 505 nm showed a good linear relationship with the concentration of Cys in the range of 0 ␮M–8 ␮M (R2 = 0.996), as shown in Fig. S3. The detection limit for Cys was determined as 1.21 ␮M based on S/N = 3. 3.4. Sensing mechanism of probe 1 toward Cys As shown in Scheme 2, the thiol of Cys can react with 1 via a Michael-type addition reaction to produce the corresponding adduct (1-Cys). Although the above spectral studies have indicated that, as designed, the sensing response of the probe to Cys was most likely due to the formation of the 1-Cys adduct, we provided further line of evidence to corroborate this. The matrix assisted laser desorption ionization/time of flight mass spectrum (MALDITOF-MS, Fig. 4) of 1 treated with Cys in PBS solution (pH 7.4) revealed signals consistent with the formation of the expected 1-Cys adduct (m/z 1065.952 [1 + 2Cys]). The strong peak at m/z 860.979 [1 + 2H2 O] was also observed. 3.5. Fluorescent labeling of protein containing free Cys residue

Fig. 3. Fluorescence spectral changes of 1 (1 ␮M) in THF with the increasing concentrations of Cys in PBS (pH 7.4). Excitation wavelength of 1 was 500 nm.

sensor and showed a fluorescence OFF−ON effect, but not ratiometric fluorescence changes [9b]. To evaluate the selectivity of 1 for detection of Cys, fluorescence changes caused by the addition of other amino acids including Pro,

Since 1 can act as a dual-channel, high selective, immediacy and sensitive optical probe for Cys, it was further employed to detect free Cys residues within proteins. Herein, bovine serum albumin (BSA) was used as a template protein because it had only one free cysteine residue at position 34 (Cys34 ). The sensing processes were monitored with UV–vis and fluorescence spectra. As shown in Fig. 5, the absorption peaks of 1 at 359 and 553 nm disappeared following the formation of a new band centered at 480 nm in presence of BSA.

L. Wang et al. / Sensors and Actuators B 205 (2014) 281–288

NH3

O

C8H17 N

O

OOC

O Cys

O

N

O

O

O

S O

C8H17

O

1

O

S N

C8H17

O

C8H17

O

N

285

COO H3N

1-Cys

Scheme 2. The proposed reaction between probe 1 with Cys. 0.5

BSA 1+BSA 1

Absorbtance

0.4

0.3

480 nm

0.2

0.1

0.0 400

500

600

700

Wavelength (nm) Fig. 5. UV–vis spectra of probe 1 (10 ␮M) toward BSA (2 equiv) in PBS (pH 7.4).

7000

500

2.2 eq. FL intensity

FL intensity

6000 5000 4000 3000 600 Wavelength (nm)

2000 1000

700

0

0 500

600

700

Wavelength (nm) Fig. 6. Fluorescence titrations of probe 1 (10 ␮M) in THF toward BSA (0–2.2 equiv) in PBS (pH 7.4) upon excitation at 480 nm.

Moreover, with addition of BSA, a gradual fluorescent enhancement also emerged (Fig. 6). The emission intensity at 485 and 579 nm was enhanced by 21.35 and 4.40-fold after adding 50 ␮M Cys, respectively. These results were in an agreement with the observations in the case of Cys. Meanwhile, remarkable colorimetric and fluorescent color changes were observed. As shown in Fig. 7, BSA was colorless and initially gave a blue emission. After the addition of probe 1, a bright light-yellow emission emerged along with a visible color change to yellow. This indicated that the probe 1 conferred selective covalent binding to the Cys34 residue within BSA. However, compared to the fast response of probe 1 toward free Cys, this sensing process was more time-consuming (about 5 min), which might be mainly attributed to the conserved microenvironment around Cys34 . It is ˚ well known that the Cys34 residue was buried in a crevice (6 A) within domain I, in which it stayed in a unique microenvironment

Fig. 7. (a) Colorimetric and (b) fluorescent responses of probe 1 (10 ␮M) in THF after the addition of native BSA (0.1 mM) in PBS (pH 7.4).

close to three ionizable residues: Asp38 , His39 , and Tyr84 [19]. Such a conserved motif prevented Cys34 from reacting with probe 1 efficiently. 3.6. Cellular imaging The application of 1 in cellular imaging was investigated by fluorescence images. In these experiments, human adult skin fibroblast cells were individually incubated in culture medium with 1 (10 ␮mol/L) for 0.5 h, and then imaged, as shown in Fig. 8. The nuclei were stained with 4 ,6-diamidino-2-phenylindole, which was a fluorescent dye that gave out blue emission, as shown in Fig. 8(B). The endocytic 1 of human adult skin fibroblast cells was then evaluated by using inverted phase contrast fluorescence microscope, the results of which were shown in Fig. 8(C). Red fluorescence was obviously observed in the cytoplasm of the human adult skin fibroblast cells. The appearance of red fluorescence in the cytoplasm region of the cells surrounding the nucleus indicated that 1 was

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Fig. 8. Human adult skin fibroblast cells (20× magnification): (A) brightfield image; (B) fluorescence images of cells nuclei stained by 4 ,6-diamidino-2-phenylindole; (C) fluorescence images of cells stained by 1 (10 ␮M) excited at 340–380 nm; (D) overlap image of (B) and (C).

1 to 7 days, suggesting that the cells proliferated in all groups. Meanwhile, there were no statistically differences between control group and two test groups (1 and 10 ␮mol/L) at the same time point. The low cytotoxicity of 1 ensured its application in real practice as a long time fluorescent agent for cellular imaging when necessary.

control (Negative CTR) 1uM 10 uM DMSO (Prositive CTR)

1.8 1.5

OD 450 nm

1.2 0.9

4. Conclusions

0.6 0.3 0.0 Day 1

Day 2

Day 4

Day 7

Fig. 9. Metabolic viability of human adult skin fibroblast cells after incubation with 1 at different concentrations for 1, 2, 4 and 7 days, respectively.

successfully endocytosed by cell lines and accumulated in the entire cell cytoplasm. In Fig. 8, the fluorescence image (D) was the overlaying images of (B) and (C).

In summary, we developed a new Cys-specific fluorescent “turn on” probe 1 that underwent a Michael-type reaction with Cys. Probe 1 showed a rapid response and selectivity for detection of Cys over GSH. Addition of Cys in PBS (pH = 7.4) to 1 in THF resulted in a rapid color change from purple to yellow and emission color from red to yellow, while other amino acids did not induce any significant color change. This indicated naked-eye selective detection of Cys using 1 became possible. Furthermore, the practical utility of the probe in fluorescent detection in the microenvironment of Cys34 within BSA was demonstrated. Cellular imaging results indicated 1 could be utilized as a fluorescent probe for cellular imaging of human adult skin fibroblast cells, where red fluorescence was observed in the cytoplasm. Moreover, CCK-8 assay showed 1 had a low cytotoxicity.

3.7. Cell viability Acknowledgements The cell viability was assayed to estimate the toxicity of 1 quantitatively by CCK-8 assay, in which the formation of formazan dye depended on the mitochondria activity. The data of OD 450 was directly proportional to the number of living cells. Fig. 9 showed the viability of human adult skin fibroblast cells after treatment with 1 (1 and 10 ␮mol/L) for a period of 1, 2, 4 and 7 days. Results indicated that 1 had a good biocompatibility on human adult skin fibroblast cells. The number of cells increased significantly from

The supports by National Natural Science Foundation of China (No. 21274045), the Pearl River in Guangzhou city of Nova of Science and Technology Special Funded Projects (No. 2012J2200009), the Fundamental Research Funds for the Central Universities (2013ZZ067), the Natural Science Foundation of Guangdong Province (10351064101000000) and National Basic Research Program of China (2012CB720801) are gratefully acknowledged.

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design and synthesis of functional water-soluble conjugated polymers and exploration of their applications in chemosensors, biosensors and optoelectronic. She has over 40 peer-reviewed publications.

Biographies

Jiqing Du received the BSc in Chemistry in Shenzhen University, China, in 2012. Currently, he is a graduate student in South China University of Technology, China.

Lingyun Wang received her PhD degree in Chemistry from Sun Yat-sen University, China, in 2006. After post-doctoral training at the South China University of Technology (SCUT), she joined SCUT where she is currently an assistant professor in the School of Chemistry and Chemical Engineering. Her current research focuses on the

Derong Cao is a professor in the School of Chemistry and Chemical Engineering, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, China. His research interest focuses on organic synthesis and materials science.