A lysosome-targetable near infrared fluorescent probe for glutathione sensing and live-cell imaging

A lysosome-targetable near infrared fluorescent probe for glutathione sensing and live-cell imaging

Sensors & Actuators: B. Chemical 301 (2019) 127065 Contents lists available at ScienceDirect Sensors and Actuators B: Chemical journal homepage: www...

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Sensors & Actuators: B. Chemical 301 (2019) 127065

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

A lysosome-targetable near infrared fluorescent probe for glutathione sensing and live-cell imaging

T

Ziming Zheng, Yuchen Huyan, Hongjuan Li, Shiguo Sun, Yongqian Xu



Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling, Shaanxi, 712100, PR China

ARTICLE INFO

ABSTRACT

Keywords: Squaraine dye Enhanced self-quenching GSH Cell imaging Lysosomal organelle

A novel near-infrared self-quenching dye SQSS consisting of two SQ fluorophores and a cystamine linker was constructed for the selective detection of GSH. Owing to the enhanced aggregation-caused quenching (ACQ) and FRET-mediated self-quenching effects, the background fluorescence intensity of SQSS is very weak, providing relatively high signal-to-noise ratio. The disulfide linker of SQSS can be selectively cleaved by glutathione (GSH) rather than other biothiols to produce two SQ derivatives through cooperative intermolecular hydrogen bonds and specific electrostatic interactions. The exclusive interaction of GSH with SQSS interrupt the FRET selfquenching effects between two SQ fluorophores, thereby generating a significant turn on spectral response for detection of GSH in near infrared region. Cell imaging experiments showed that SQSS can monitor endogenous and exogenous GSH in tumor or normal cells. More importantly, colocalization experiments indicated that SQSS mainly located in the lysosome, an organelle tightly associated with GSH functions. With the excellent membrane permeability and lysosome-specificity, it is convinced that SQSS will be a powerful tool in the future research on the function of GSH in lysosomes.

1. Introduction Intracellular biothiols, glutathione (GSH), cysteine (Cys) and homocysteine (Hcy), play essential roles in many biological processes [1]. As the protectors of defense systems they can maintain the essential redox status of organism through the dynamic balance of reduced thiols and their oxidized forms. However, abnormal levels of biothiols are tightly associated with many diseases such as Alzheimer’s disease, liver injury [2], cardiovascular diseases, and neural tube defects [3,4]. The most abundant biothiol in biological systems is GSH, which is composed of a tripeptide of glutamic acid, cysteine, and glycine [5]. Glutathione plays a key role in preventing oxidative stress and maintaining redox balance through the reversible exchange reaction of disulfide bond. Glutathione plays a mediating role in many cellular functions, such as intracellular transduction, metabolism, gene regulation, immune response and detoxification. [6,7]. Accordingly, it is of great importance and interest to explore an efficient method to determine the concentration of GSH in biological samples for investigation and diagnosis of its related processes and diseases. In the past few decades, many analytical techniques such as HPLC, LC–MS, and capillary electrophoresis and so forth have been used for detection of GSH [8–17]. Although these techniques can be leveraged for GSH sensing, most of them suffer from inherent shortcomings, such ⁎

as expensive and complex equipments, complicated and time-consuming operating steps. In comparison, fluorescence probe techniques in conjunction of intracellular imaging hold great promise for the detection of GSH due to the advantages of low cost, high sensitivity, high selectivity, simplicity of operation and non-invasiveness [18–22]. Great efforts have been devoted in developing efficient fluorescence probes for sensing biothiols in living cells [23–33]. Among of them, the strong nucleophilicity of sulfhydryl is widely used to design biothiol fluorescence sensors with high selectivity and sensitivity. Disulfidethiol exchange reaction is such a kind of reaction that a disulfide bond can be easily attacked by thiols to generate two thiol derivatives, possessing a high specificity for biothiols sensing. Based on this disulfidethiol exchange reaction many biothiols fluorescent probes have been reported. A single fluorophore or multiple fluorophores were introduced into a fluorescent sensor, and the structure and emission properties of the fluorophore are changed after a disulfide-thiol exchange reaction triggered by mercapto group of the biothiol. However, their emission wavelengths of these probes are mostly concentrated in the range of 400–600 nm, which is not conducive to avoiding the background interference of biological auto-fluorescence during the process of imaging. In addition, due to the intrinsic limitation that most of biothiol can induce disulfide-thiol exchange reaction, it is difficult to achieve the specific recognition of GSH. More importantly, monitoring

Corresponding author. E-mail address: [email protected] (Y. Xu).

https://doi.org/10.1016/j.snb.2019.127065 Received 6 August 2019; Received in revised form 29 August 2019; Accepted 30 August 2019 Available online 31 August 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.

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lysosomal GSH is urgently needed because it is helpful for understanding the GSH functions in many biological processes such as GSHassociated proteolysis in lysosome and stabilization of lysosome membranes [34–36]. Therefore, the development of lysosome targetable GSH fluorescent probes with high selectivity and near-infrared fluorescence remains a challenge. Squaraines (SQ), typical near infrared dyes with emission at 650–900 nm, have attracted considerable attention in recent years because they have many advantages of lower photodamage, deeper tissue penetration and minimal fluorescence of background as used in biological systems [37–39]. Based on the outstanding characteristics of SQ dye, we designed a novel near-infrared fluorescent sensor SQSS based on squaraine dye for selective detection of GSH. In this sensor, two SQ fluorophores were closely linked through a disulfide bond. In the absence of GSH, The fluorescence intensity of SQSS is very weak due to the presence of high fluorescence resonance energy transfer (FRET) quenching effects between two SQ fluorophores. When GSH is added, a new disulfide generates through the exchange between thiol of GSH and disulfide bond in SQSS, resulting in the separation of the original two SQ fluorophores in SQSS. The initial FRET quenching effects is interrupted and the fluorescence intensity of SQ fluorophore gradually recovers (Scheme 1). Specifically, the structural feature of SQSS has the following advantages: 1) the aggregation degree of dyes was greatly increased due to the linking of two SQ fluorophores in close distance, effectively reducing the background fluorescence through aggregationcaused quenching (ACQ) and FRET-induced self-quenching; 2) The linking mode itself through disulfide bond is conducive to aggregation of hydrophobic SQ [40,41]; 3) The positively charged SQ provides electrostatic interactions with negatively charged GSH, effectively increasing the selectivity for GSH. With the excellent cell membrane permeability and NIR emission property, SQSS has been successfully applied to exogenous and endogenous GSH imaging in live cells. More importantly, SQSS can specifically target lysosome, demonstrating the potential capability for studying the function of GSH in lysosomes.

Reagent Co., Ltd. (Shanghai, China), Wolsen Biological Reagent Company (Xiaan, China) and used without further purification unless otherwise stated. The compound SQSS and its referenced complex SQCC in which disulfide bond was replaced by carbon-carbon bond were synthesized according to the reported literature [40,41]. Using TMS as internal standard, the 1H and 13C NMR spectra were recorded on Bruker Avance III 500 spectrometer in Germany. Mass spectrometry was carried out using LCQ fleet mass spectrometer (USA). Absorption and emission spectra were collected by Shimadzu 1750 Ultraviolet Visible spectrophotometer and Japan RF-5301 fluorescence spectrophotometer, respectively. Cell imaging was performed using FV1000MPE laser scanning confocal microscopy in Olympus (Japan). 2.2. General procedure for assay Stock solutions including various amino acids such as Ala, Val, Gly, Arg, Gln, Glu, His, Lys, Pro, Met, Phe, Trp, Ser, Thr, Hcy, Cys and GSH were prepared using deionized water and the concentrations were 1.0 × 10−2 M. A stock solution of SQSS or SQCC with the concentration of 1.0 × 10−2 M was prepared in DMSO and then diluted using 10 mM PBS buffer solution with pH 7.4. All measured solutions for UV–vis absorption and fluorescence spectra were PBS solution (10 mM, pH 7.4) with 20% MeCN (v/v) at room temperature. 2.3. Calculation of limit of detection Limit of detection LOD was calculated according to standard equation: LOD = K×S1/S, where K is coefficient 3, S is the slope obtained from calibration curve, while S1 is the statistical result of standard derivation of the blank solution. 2.4. Experiments in live cells 2.4.1. Cell culture and imaging HepG2 (human hepatocellular carcinoma cells) were inoculated in a 35 mm glass dish (nest) and incubated for 24 h in a 37 0C incubator with 10% fetal bovine serum, 50 units/ml penicillin and 50 units/ml streptomycin culture medium (Dulbecco’s modified Eagle’s solution). Cells were treated with 500 μM N-ethyl maleimide (NEM) for 0.5 h in a PBS buffer (10 mM, pH 7.4) at 37°C. The remaining NEM was washed

2. Experiment section 2.1. Materials and instruments All chemicals are purchased from Shanghai Mayer Chemical

Scheme 1. (a) Chemical structure of the bis-squaraine dye. (b) Schematic illustration of the mechanism of GSH sensing. 2

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3. Results and discussion 3.1. Molecule design The nucleophilicity of the biothiols enable to cleave a disulfide bond to generate two thiols, which can be harnessed to turn on biothiols fluorescent sensor through the destruction of FRET quenching effect. In this work, a thiol specific cleavable disulfide linker was utilized to conjugate two SQ molecules in the bis-squaraine dye SQSS. The distance between two SQ molecules is in the range of Förster radius, which makes one SQ act as a fluorescence quencher for the other. For comparison, a non-activatable bis-squaraine dye SQCC which using an alkyl chain to replace the disulfide linker as a control was also prepared. 3.2. Self-quenching studies of the two dyes We first studied the fluorescence self-quenching properties of the bis-squaraine dyes under the detection condition. We defined the selfquenching efficiency of the dye molecules as [(I0-I)/I0], in which I0 and I stand for the fluorescence intensity of the individual SQ molecules and the dye molecules under the test system, respectively. By studying the fluorescence spectra of SQ, SQSS and SQCC (Fig. S1), we obtained the fluorescence self-quenching efficiency for SQSS and SQCC was 93.1% and 90.3%, respectively. SQSS or SQCC has two “SQ units” in molecule structure. In order to exclude the ACQ effect produced by concentration, we studied the fluorescence spectrum of SQ with 2-fold concentration of SQSS or SQCC (Fig. S2). In this case, the quenching efficiency for SQSS and SQCC was calculated to be 59.6% and 52.2%, respectively. Compared with SQ, the quenching efficiency of SQSS or SQCC undoubtedly comes from FRET self-quenching. The experimental data showed that the fluorescence of the two bis-squaraine dyes was highly quenched. Correspondingly, the background fluorescence of the probe itself is relatively low, which is conducive to improving the signal-to-noise ratio of detection. For strong FRET self-quenching effect, the distance of two fluorophores should be in the best range from 10 to 100 Å. To estimate the distance of two SQ fluorophores in the bis-squaraine dyes precisely, the optimized geometry molecular models of SQSS and SQCC were achieved by using the Chem3D software package (ChemOffice, USA), in which energy minimization was performed via molecular mechanics (MM2 force field). The distance of two SQ fluorophores in SQSS and SQCC were 14.3 Å and 15.0 Å, respectively, which is in favor of the intramolecular FRET self-quenching (Fig. S3).

Fig. 1. (a) Time-dependent fluorescence spectra change of SQSS (10 μM) upon treatment with GSH (10 μM) in MeCN/PBS (v/v = 20/80, 10 mM, pH 7.4). (b) The plot of fluorescence intensity change of SQSS at 665 nm versus incubation time in the presence of GSH (10 μM), λex =610 nm.

out with PBS buffer and cultured in PBS buffer at 37°C for 2 h with 10 μM SQSS. After washing with fresh DMEM, the cells treated by sensor was further cultured in fresh DMEM containing 100 μM glutathione for 2 h, and the cells were imaged by confocal microscopy. The excitation wavelength is 630 nm and the emission path is 635–700 nm. All images were obtained under identical experimental parameters to minimize the impact of possible changes in fluorescence intensity.

3.3. Response of SQSS to GSH To investigate the reaction kinetics of SQSS to GSH, we incubated 10 μM SQSS with GSH (10 μM), and the fluorescence spectra were measured at different time intervals. As shown in Fig. 1, the fluorescence intensity of SQSS at 665 nm increased significantly with time of duration, and reached the plateau within 2 h. But for SQCC, there is no obvious spectra change with duration time in the presence of GSH (Fig. S4). Next, we investigated the relationship between GSH concentration and the emission of the probes. SQSS was incubated with GSH at different concentrations for 2 h, and corresponding absorption and fluorescence spectra were obtained (Fig. 2). With increasing GSH concentration, the fluorescence intensity increased. As 10 μM GSH was added, about 2.7-fold increment of fluorescence intensity can be found (Fig. 2a). As to the absorption spectra, the absorption band at 596 nm that is assigned to the aggregates of SQSS showed a sharp decrease along with the increase of GSH concentration, indicating that the aggregates of SQSS reduced (Fig. 2b). The cleavage of disulfide linker

2.4.2. Cytotoxicity Metabolic activity of HepG2 cells was evaluated by 3-(4,5-dimethylthiazole-2-yl) -2,5-diphenyltetrazolium ammonium bromide (MTT) assay. HepG2 cells were incubated into 96-well plate (nest), and the incubation intensity was 8 × 104 cell mL−1. After 24 h of culture, the medium was replaced by a probe suspension with a concentration of 0–20 μm (0.1% (v/v) dimethyl sulfoxide as co-solvent). After incubation at 37°C for 24 h, fresh DMEM was used to replace the medium. After incubation at 37°C, the medium was washed twice with PBS buffer solution, and the freshly prepared MTT (0.5 mg mL−1) solution (100 μL) was added to the medium. MTT medium solution was cultured in a 37°C incubator for 3 h and then carefully removed. Then 100 μL DMSO is added to each hole, and the plate is shaken slightly to dissolve all the precipitates. The absorbance of MTT at 570 nm is monitored by Genios Tecan, a microplate reader. Cell viability is reflected by the ratio of absorbance of cells cultured with probe suspension to cells cultured only in medium.

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fluorescence intensity of SQSS at 665 nm, a good linear line can be obtained, indicating the possibility of probe SQSS for GSH quantification. The LOD was calculated to be about 0.15 μM (Fig. 2c). The pH value of the solution is very important for the exchange reaction of dithiol. In order to study the effect of pH, the fluorescence intensity of SQSS (10 μM) at 665 nm in the absence and presence of 100 μM GSH at different pH conditions was observed. As shown in Fig. S6, the probe SQSS itself showed negligible fluorescence intensity change in the pH range from 3.9 to 10.8. While, there was significant increase in pH range from 3.9 to 7 in the presence of GSH. At pH value 3.9, no obvious fluorescence enhancement could be observed, which may be attributed to the low nucleophilic activity of protonated glutathione that decomposes disulfide bonds in acidic conditions. But in pH range of 7 to 9, the pH value showed no effect on the fluorescence intensity. These results suggest that SQSS can detect GSH under the physiological pH conditions. 3.4. Study on the possible mechanism of turn on fluorescence response for GSH In order to get insight into the response mechanism, we checked the reaction product of the probe SQSS with GSH under the test system through mass spectrometry analysis. As seen in Fig. S7, the fragment peak at 762.42 is attributed to the product SQSSG of which GSH was conjugated to SQ through disulfide bond after the disulfide bond of SQSS was cut apart by GSH. The results demonstrated that SQSS underwent disulfide-thiol exchange reaction with GSH, and the disulfide bonds in the probe molecules were destroyed. 3.5. Selectivity and competition of SQSS toward GSH over other amino acids Selectivity is an important parameter for probes in practical applications. The spectral responses of SQSS toward GSH over other amino acids were further investigated. As shown in Fig. 3, only GSH exhibited significant enhancement at 665 nm. The other amino acids studied did not cause detectable spectral changes, except that Cys caused considerable slight interference (almost 0.5 times enhancement). Competitive experiments were conducted in the presence of 1 equivalent of GSH and 2 equivalents of other amino acids. SQSS can still trigger turnon fluorescence response to GSH without any other interference (Fig. S8). It should be noted that most fluorescent probes based on disulfidethiol exchange reaction often lack selectivity for GSH over Cys and Hcy, [25,26,30–32] but SQSS possesses a high affinity toward GSH over the other two biothiols. We presumed that possible reason of the high selectivity is attributed to the following two points. Firstly, the obvious

Fig. 2. (a) Emission spectral changes of SQSS (10 μM) with increasing concentrations of GSH (0–10 μM). (b) UV–vis absorption change of SQSS upon addition of GSH (0–10 μM). Experiments were performed in MeCN/PBS (v/ v = 20/80, 10 mM, pH 7.4). (c) Linear plot showing the fluorescence intensity of SQSS at 665 nm as a function of incremental concentrations of GSH, λex=610 nm.

triggered by GSH releases more SQ molecules. Without GSH, the fluorescence of SQSS is very weak due to the strong FRET selfquenching process from one SQ molecule to the other. After addition of GSH, the sulfydryl of GSH interacts with disulfide bond to cut off the linker, which destructs the FRET self-quenching effect of two connected SQ molecules. For SQCC, there is no activated group for GSH, thus a negligible increase in fluorescence intensity was observed (Fig. S5). In addition, according to the change of GSH concentration and

Fig. 3. The relative fluorescence intensity changes of SQSS (10 μM) in phosphate buffer (10 mM, pH 7.4) with 20% MeCN toward various amino acids and GSH (0.5 mM), λex=610 nm. 4

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Fig. 4. CLSM images of SQSS (10 μM) labeled HepG2 cells either (a) untreated or pre-treated with (b) NEM (500 μM for 30 min), or (c) NEM (500 μM for 30 min) and sequential addition of GSH (500 μM for 120 min). Excitation wavelength is at 630 nm for the red channel (640–700 nm). Scale bar corresponds to 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

structural differences between GSH and other biological biothiols should be the main reason of different reactivity with SQSS. GSH has two amide units and a longer flexible skeleton than Cys and Hcy, which provides the possibility of intermolecular interactions with SQSS, such as hydrogen bonds. For example, the amide unit of GSH can form hydrogen bonds with imine group of SQSS through CeH⋯N and CeH⋯O mode. Secondly, the electrostatic interaction between the two carbonate anions of GSH and the thiazole cations of SQSS may be stronger than that with Hcy and Cys.

3.6. Cellular imaging Before investigating the biological application of SQSS, the cytotoxicity of SQSS to living cells was evaluated. HepG2 cells were cultured with different concentrations of SQSS ranged from 0 to 20 μM for 24 h. As the concentration of SQSS is about 10 μM, the cells survival rate determined by standard MTT assay was calculated to be nearly 98%, indicating that SQSS demonstrates minimal cytotoxicity (Fig. S9). As the intracellular GSH concentration is in the millimolar range Fig. 5. Determination of intracellular localization of SQSS by confocal microscopy. HepG2 cells were incubated with SQSS (10 μM) for 2 h and then co-incubated with (a) Hoechst 33342 (λex=405 nm, λem = 415–430 nm), (b) Rh123 (λex=488 nm, λem = 500 –520 nm) and (c) LysoTracker® Green DND-26 (λex=488 nm, λem = 500 –520 nm) for 30 min at 37 °C, respectively. Scale bar =20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

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(1–10 mM) in human body [42], SQSS is expected to be used for imaging of endogenous and exogenous GSH. After incubation with SQSS at 37 °C for 2 h, a strong red fluorescence emission is observed for HepG2 cells (Fig. 4a), reflecting that the cleavage of disulfide linker of SQSS by intracellular GSH took place. To provide further evidence that the fluorescence turn-on response comes from thiol-triggered cleavage of disulfide bond, the HepG2 cells were pretreated using N-ethylmaleimide (NEM) before incubation with SQSS. NEM is regarded as a strong GSH blocking agent which can react with GSH to form a derivative GSNEM [43]. As expected, weak fluorescence was observed after 2 h (Fig. 4b). After treatment of 500 μM GSH for 2 h, the strong red fluorescence of probe gradually recovered (Fig. 4c). Likewise, the similar phenomenon was also observed in human hepatic HL-7702 cells (Fig. S10). These results implied that SQSS could be efficiently uptaken and capable of detecting exogenous and endogenous GSH in live cells. To study the intracellular location of SQSS, colocalization experiments of SQSS were further performed (Fig. 5). The commercial lysosomal tracker LysoTracker® Green DND-26, mitochondrial tracker Rhodamine 123 and nuclei staining dye Hoechst 33342 were used. The Pearson correction coefficient between SQSS and these trackers are 0.93, 0.38 and 0.21, respectively. These results indicated that SQSS was mainly located in the lysosomal organelle. The reason for lysosomespecificity of SQSS perhaps is that the two weakly alkaline imine groups tend to aggregate in acidic lysosomes. In lysosomes, thiols are closely associated with proteolysis which reduces disulphides [34,35]. Morecover, GSH was proved to be relevant with the stabilization of lysosome membranes [36]. Therefore, in view of the characteristics of selective accumulation in lysosome, SQSS could be considered as a convincing tool in the research on the related GSH functions in lysosomes.

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4. Conclusion In conclusion, we have successfully developed a bis-squaraine dye SQSS for detecting GSH with high sensitivity and selectivity in vitro and in vivo. Thanks to the favorable attributes of high biocompatibility, excellent membrane permeability, and near infrared emission, SQSS has been utilized for imaging of the endogenous and exogenous GSH in live cells. More attractively, SQSS possesses a high lysosome-specificity, which may contribute to a better understanding of GSH in lysosomes. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No.21676218, 21978241, 21878249 and 21476185) and Natural Science Foundation of Shaanxi Province Science and Technology (2019JM-173). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.127065. References [1] R.O. Ball, G. Courtney-martin, P.B. Pencharz, The in vivo sparing of methionine by cysteine in sulfur amino acid requirements in animal models and adult humans, J. Nutr. 136 (2006) 1682S–1693S. [2] S. Shahrokhian, Lead phthalocyanine as a selective carrier for preparation of a cysteine-selective electrode, Anal. Chem. 73 (2001) 5972–5978. [3] H. Refsum, P.M. Ueland, O. Nygård, S.E. Vollset, Homocystein and cardiovascular disease, Annu. Rev. Med. 49 (1998) 31–62. [4] H. Refsum, A.D. Smith, P.M. Ueland, E. Nexo, R. Clarke, J. Mcpartlin, C. Johnston, F. Engbaek, J. Schneede, C. Mcpartlin, J.M. Scott, Facts and recommendations about total homocysteine determinations: an expert opinion, Clin. Chem. 50 (2004) 3–32. [5] A. Meister, Glutathione metabolism and its selective modification, J. Biol. Chem. 263 (1988) 17205–17208. [6] H. Tapiero, D.M. Townsend, K.D. Tew, The antioxidant role of selenium and selenocompounds, Biomed. Pharmacother. 57 (2003) 134–144.

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Yuchen Huyan obtained her B.S. degree from Yan'an University (China). Subsequently, he joined the group of Prof. Yongqian Xu at Northwest A&F University as a master’s student. Her research interests focus on developing fluorescent chemosensors. Hongjuan Li obtained her Ph.D. from Shaanxi Normal University (China) in 2010. She is currently an associate professor at the College of Chemistry & Pharmacy, Northwest A&F University, China. Her current research interests mainly focus on the development of functional nanocomposites and fluorescent chemosensor based on layered double hydroxide materials. Shiguo Sun obtained his Ph.D. degree in 2003 from Dalian University of Technology. He is currently a professor and doctoral supervisor in the college of Chemistry & Pharmacy, Northwest A&F University, China. His research interests include functional molecules with special light or electrochemistry properties, self-assembly chemistry of carbon nanotube/graphene material for DNA and protein sensing, drug delivery and fluorescence tracing, and electrochemiluminescence and fluorescent sensors. Yongqian Xu received his Ph.D. in Applied Chemistry from the Dalian University of Technology (China) in 2007. In 2008, he worked as a postdoctoral fellow at The University of Akron (USA). He is currently a professor at the College of Chemistry & Pharmacy, Northwest A&F University, China. His current research interests mainly focus on supramolecular self-assembly and fluorescent chemosensors.

Ziming Zheng obtained his B.S. degree from Nanjing Forestry University (China). Subsequently, he joined group of Prof. Yongqian Xu at Northwest A&F University as a master’s student. His research interests focus on developing fluorescent chemosensors based on near infrared squaraine dyes.

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