Dual Response Site Fluorescent Probe for Highly Sensitive Detection of Cys/Hcy and GSH In Vivo through Two Different Emission Channels

Much research has demonstrated that metabolic imbalances of biothiols are closely associated with the emergence of different types of disease. In view of the significant effect of biothiols, quantitative evaluation and discrimination of intracellular Cys/Hcy and GSH in complex biological environments is very important. In this study, probe CDS-NBD, synthesized by attaching 2,4-dinitrobenzenesulfonate (DNBS, site 1) and nitrobenzoxadiazole (NBD, site 2) as the highly sensitive and selective dual response site for thiols onto the coumarin derivative 7-hydroxycoumarin-4-acetic acid, exhibited large separation of the emission wavelengths, fast response, notable fluorescence enhancement, excellent sensitivity and selectivity to Cys/Hcy and GSH over other biological species. Additionally, CDS-NBD could make a distinction between two different fluorescent signals, GSH (an obvious blue fluorescence) and Cys/Hcy (a mixed blue-green fluorescence). Further study on imaging of Cys/Hcy and GSH in vivo by employing probe CDS-NBD could also be successfully achieved.


Introduction
Cysteine (Cys)/homocysteine (Hcy) and glutathione (GSH), as vital low-weight biomolecules containing a sulfhydryl group (-SH), play vital and irreplaceable roles in maintaining the appropriate redox status in diverse physiological processes [1][2][3]. Cys serves as a critical substrate for the synthesis of GSH and acetyl coenzyme A and participates in amino acid transport and detoxification of heavy metal poisoning in biological systems. Hcy is a methylene homolog of Cys, involved in constructing DNA and methylation catalyzed to produce Cys in organisms. As the most abundant of biothiols, the level of GSH is about 1-10 mmol/L in cells. It maintains the redox balance of cellular homeostasis against oxidative stress [4][5][6][7]. During recent years, much research has demonstrated that metabolic imbalances of biothiols are closely associated with the emergence of different types of disease [8][9][10]. The loss of Cys can cause growth retardation, metabolic disorders, type 2 diabetes, and neurodegenerative disorders. The elevated plasma of Hcy concentration in the serum is regarded as a root hazard element for cardiovascular disease and cognitive impairment in the elderly. Abnormal fluctuation of GSH is tightly relative to atherosclerosis, cancer, and Alzheimer's disease. In view of the significant effect of biothiols, quantitative evaluation and discrimination of intracellular Cys/Hcy and GSH in complex biological environments is very important.
Due to the above considerations, by using 2,4-dinitrobenzenesulfonate (DNBS, site 1) and nitrobenzoxadiazole (NBD, site 2) as the highly sensitive and selective dual re sponse site for thiols, a novel fluorescent probe CDS-NBD based on coumarin derivative 7-hydroxycoumarin-4-acetic Acid (7-HCA) for the detection of Cys/Hcy and GSH through two different emission channels is presented (Scheme 1). Probe CDS-NBD was a non fluorescent compound due to the PET process arising from the dual quenching site (2,4 dinitrobenzenesulfonate and NBD moiety). Cys/Hcy could react with site 1 and site 2 in probe CDS-NBD to generate the blue fluorescent substance Cou-OH and the green fluo rescent substance NBD-N-Cys or NBD-N-Hcy. However, GSH could not undergo the Smile rearrangement, so the blue fluorescent substance Cou-OH and the non-fluorescen substance NBD-S-GSH were obtained. Thus, probe CDS-NBD could make a distinction between GSH (an obvious blue fluorescence) and Cys/Hcy (a mixed blue-green fluores cence) by two different fluorescent signals (Scheme 2). In addition, dual-color fluorescence imaging experiments in living HeLa cells and zebrafish also demonstrated that CDS-NBD could serve as an effective tool for responding to Cys/Hcy and GSH.

Design of Probe CDS-NBD
Recently, single-molecule fluorescent probes with different reaction sites for simultaneously sensing a variety of related analytes in vitro and in vivo through two or more kinds of distinct fluorescence channels (Table S1) have attracted enormous attention [27][28][29]. In our design, 2,4-Dinitrobenzenesulfonate (site 1) [30] could serve as an excellent active site for identifying thiols (Cys, Hcy, and GSH) with high selectivity. Therefore, strong emission in the blue channel could be aroused after the reaction between site 1 in CDS-NBD and thiols. Moreover, the NBD group (site 2) [31] could easily react with Cys/Hcy to afford NBD-N-Cys/Hcy, enabling probe CDS-NBD to exhibit significant fluorescence changes at 557 nm. In contrast, no obvious green fluorescence signal was seen for GSH because the Smile rearrangement was forbidden. Furthermore, probe CDS-NBD adopted a dual-quenching strategy (site 1 and site 2), which could display the minimized background signal. For these reasons, we obtained a dual-site fluorescent probe CDS-NBD for simultaneously monitoring Cys/Hcy and GSH via two different emission channels. The HRMS, 13 C NMR, and 1 H NMR spectra of CDS-NBD are given in Figures S11-S16.

Design of Probe CDS-NBD
Recently, single-molecule fluorescent probes with different reaction sites for simultaneously sensing a variety of related analytes in vitro and in vivo through two or more kinds of distinct fluorescence channels (Table S1) have attracted enormous attention [27][28][29]. In our design, 2,4-Dinitrobenzenesulfonate (site 1) [30] could serve as an excellent active site for identifying thiols (Cys, Hcy, and GSH) with high selectivity. Therefore, strong emission in the blue channel could be aroused after the reaction between site 1 in CDS-NBD and thiols. Moreover, the NBD group (site 2) [31] could easily react with Cys/Hcy to afford NBD-N-Cys/Hcy, enabling probe CDS-NBD to exhibit significant fluorescence changes at 557 nm. In contrast, no obvious green fluorescence signal was seen for GSH because the Smile rearrangement was forbidden. Furthermore, probe CDS-NBD adopted a dual-quenching strategy (site 1 and site 2), which could display the minimized background signal. For these reasons, we obtained a dual-site fluorescent probe CDS-NBD for simultaneously monitoring Cys/Hcy and GSH via two different emission channels. The HRMS, 13 C NMR, and 1 H NMR spectra of CDS-NBD are given in Figures S11-S16.

Spectral Performances
At first, we studied the fluorescence spectra of probe CDS-NBD (10.0 µM) by adding Cys/Hcy and GSH in PBS (20 mM, pH 7.4, containing 20% DMSO). CDS-NBD (10.0 µM) had essentially no signal both in the blue and green channel ( Figure 1). However, when Cys/Hcy/GSH (0.0-60.0 µM) were added, a significant fluorescence signal at 470 nm ( Figure 1a,d,g) under the 355 nm excited condition was observed, corresponding to that of Cou-OH. Importantly, fluorescence intensity was evidently increased (46-fold for Cys, 35-fold for Hcy, and 40-fold for GSH) after responding to the above three thiols (60.0 µM). As shown in Figure 1c Figure 1h). These satisfactory sensing data of CDS-NBD for three thiols proved that CDS-NBD could report Cys/Hcy and GSH with desirable sensitivity through the blue and green emission channels.
Biosensors 2022, 12, x FOR PEER REVIEW 4 of 12 557 nm was found under the 450 nm excited condition, corresponding to that of NBD-N-Cys/Hcy. Additionally, the fluorescence intensity at 557 nm showed persistent and proportional enhancement along with the concentration of Cys/Hcy (0.0-60.0 μM). In stark contrast, CDS-NBD (10.0 μM) in response to GSH (60.0 μM) displayed little impact on the emission intensity at 557 nm ( Figure 1h). These satisfactory sensing data of CDS-NBD for three thiols proved that CDS-NBD could report Cys/Hcy and GSH with desirable sensitivity through the blue and green emission channels.

Selectivity Studies
To evaluate the specific sensing property of probe CDS-NBD towards biothiols, the selectivity of probe CDS-NBD (10.00 µM) was assessed by incubation with biologically related species. For emission at 470 nm ( Figure 2a), Cys, Hcy, and GSH exhibited similar distinct changes of fluorescence enhancement, while obvious emission enhancement at 557 nm (Figure 2b) was detected by incubation of Cys/Hcy (60.00 µM) in comparison to that of GSH (60.00 µM). The results indicated that miscellaneous amino acids (including 100.00 µM for Asp, Ala, Arg, His, Glu, Gln, Gly, Leu, Ile, Met, Lys, Phe, Pro, Tyr, Thr) and biologically relevant ions (including 100.00 µM for K + , Zn 2+ , Mg 2+ , Na + , Ca 2+ , NO 3 − , of fluorescence intensity change, F I was the fluorescence intensity of probe CDS-NBD (10.00 µM) reacted with Cys (60.00 µM) + interfering substances (100.00 µM), F r is the fluorescence intensity of probe CDS-NBD (10.00 µM) reacted with Cys (60.00 µM)). In other words, the probe CDS-NBD can serve as a monitoring tool for distinguishing and detecting corresponding biothiols (Cys/Hcy and GSH) over other biological species by different fluorescence signals.

Response Time and pH Effect
In order to evaluate the real-time performance of a reactive fluorescent probe, the response dynamics of probe CDS-NBD with biothiols was conducted by testing the changes of fluorescence intensity with time under two different emissions at 470 nm and 557 nm. In fact, free probe CDS-NBD (10.00 µM) was non-emissive no matter how long it took owing to the dual quenching site in its structure. After the addition of three biothiols (60.00 µM) to the solution of probe CDS-NBD (10.00 µM) at 470 nm (Figure 2e), sharp increments of similar fluorescence intensities were observed. The reaction rate of Cys was slightly quicker than Hcy and GSH during the initial 100 s, and the response rate of probe CDS-NBD toward three biothiols was all speedy, which can rapidly maximize the fluorescence intensity in 350 s. It is notable that the fluorescence behavior of the solution at 557 nm emission wavelength (Figure 2f) was silent following GSH addition under the same conditions. In contrast, probe CDS-NBD showed fast fluorescence response to other biothiols (Cys and Hcy), which obtained saturation within 350 s. Therefore, 350 s was selected as the optimization time for the detection of biothiols in the following experiments.
As a significant influence factor in application environments, the effect of pH on the system was also investigated by examining the interactions of probe CDS-NBD toward Cys, Hcy, and GSH at different pH values from 2.0 to 12.0. As shown in Figures S8 and S9, in the absence of three biothiols, probe CDS-NBD (10.00 µM) was pH insensitive along with a wide pH range (from 2.0 to 11.0) at 470 nm and 557 nm. Upon addition of Cys and Hcy (60.00 µM), distinct enhancement of fluorescence signals at the emission of 470 nm and 557 nm were achieved within the pH 6.0-9.0 range, respectively. By contrast, the probe CDS-NBD (10.00 µM) only became sensitive at 470 nm toward GSH (60.00 µM) with pH values changed from 6.0 to 9.0, while the addition of GSH elicited inappreciable fluorescence changes at 557 nm between pH 2.0 and 12.0. Fortunately, the physiological environment (pH = 7.4) placed exactly in the excellent scope of probe detection toward biothiols, suggesting that probe CDS-NBD could be capable of sensing and differentiating biothiols in more complex environments.

Reaction Mechanism
To attest to the sensing mechanism of CDS-NBD toward thiols in this paper, we first used HRMS analyses to certify the reaction products between CDS-NBD and GSH/Cys. In addition, this mechanism was also investigated by spectral analyses. As is shown in Figures S1-S3, the control compound 7-HCA and Cou-OH had a similar fluorescence spectrum. Meantime, the emission spectra of the control compounds NBD-N-Bu were quite similar to the emission spectra of NBD-N-Cys. These findings robustly proved the sensing mechanism of CDS-NBD toward thiols in Scheme 2.

Cellular Imaging
Using classical MTT assays, the cell cytotoxicity of probe CDS-NBD was first evaluated. As displayed in Figure S10, HeLa cells pre-incubated with 30.0 µM CDS-NBD for 24 h remained at over 93% of cell viability, indicating CDS-NBD had good biocompatibility and low cytotoxicity. Subsequently, to image biothiols in vitro, we attempted to choose HeLa cells as the test objects ( Figure 3). As expected, CDS-NBD (10.0 µM)-loaded HeLa cells exhibited bright dual-fluorescence signals in blue (Figure 3(A2)) and green channels (Figure 3(A1)). In contrast, much weaker emission was observed from the above-mentioned channels (Figure 3(B1,B2)) after CDS-NBD (10.0 µM) was added to the NEM-treated HeLa cells (1.0 mM). These data supported that CDS-NBD could effectively image endogenous thiols in living HeLa cells. What is more, we also captured remarkable fluorescence signals in green (Figure 3(C1,D1)) and blue channels (Figure 3(C2,D2)) when HeLa cells pre-dealed with 1.0 mM N-ethylmaleimide (NEM, a typical biothiols depletion reagent, covalent sulfide bonds with sulfhydryls, enabling them to be permanently blocked to prevent disulfide bond formation) was successively stained with CDS-NBD (10.0 µM) and Cys/Hcy (100.0 µM).  (Figure 3(E1,E2)). Overall, CDS-NBD had good biocompatibility and could serve as an enabling tool for sensing biothiols in living cells by monitoring the changes of dual-fluorescence signals in blue and green channels.
HeLa cells (1.0 mM). These data supported that CDS-NBD could effectively image endogenous thiols in living HeLa cells. What is more, we also captured remarkable fluorescence signals in green ( Figure 3C1,D1) and blue channels ( Figure 3C2,D2) when HeLa cells pre-dealed with 1.0 mM N-ethylmaleimide (NEM, a typical biothiols depletion reagent, covalent sulfide bonds with sulfhydryls, enabling them to be permanently blocked to prevent disulfide bond formation) was successively stained with CDS-NBD (10.0 μM) and Cys/Hcy (100.0 μM). Further, HeLa cells pre-treated with N-ethylmaleimide (1.0 mM), CDS-NBD (10.0 μM), and GSH (100.0 μM) demonstrated only strong blue fluorescence emission ( Figure 3E1,E2). Overall, CDS-NBD had good biocompatibility and could serve as an enabling tool for sensing biothiols in living cells by monitoring the changes of dualfluorescence signals in blue and green channels.

Zebrafish Imaging
Motivated by the above results, using probe CDS-NBD, zebrafish imaging was next carried out on four-day-old zebrafish. As is shown in Figure 4A1

Instruments and Reagents
Unless otherwise noted, all reagents and solvents were purchased from Chinese com mercial suppliers and used for experiments without further purification. Nuclear mag netic resonance (NMR) spectra of probe CDS-NBD were collected using a Bruker Avanc 600 MHz spectrometer. UV-vis absorption and fluorescence spectra through a Shimadz UV-2450 spectrophotometer and HITACHI F-4600 fluorescence spectrophotometer. Fluo rescence imaging was recorded using a Zeiss LSM710 Wetzlar (German) laser scannin confocal microscope. High-resolution mass spectra (HR-MS) data of synthesized ne compounds were measured with AB Sciex TripleTOF 4600.

Instruments and Reagents
Unless otherwise noted, all reagents and solvents were purchased from Chinese commercial suppliers and used for experiments without further purification. Nuclear magnetic resonance (NMR) spectra of probe CDS-NBD were collected using a Bruker Avance 600 MHz spectrometer. UV-vis absorption and fluorescence spectra through a Shimadzu UV-2450 spectrophotometer and HITACHI F-4600 fluorescence spectrophotometer. Fluorescence imaging was recorded using a Zeiss LSM710 Wetzlar (German) laser scanning confocal microscope. High-resolution mass spectra (HR-MS) data of synthesized new compounds were measured with AB Sciex TripleTOF 4600.

Cell Experiments
The HeLa cells were cultured and grown in DMEM containing 10% FBS with a humidified atmosphere of 5% CO 2 overnight for cell attachment. The standard MTT method was applied to evaluate the cytotoxicity of probe CDS-NBD with disparate concentrations (

Zebrafish Experiments
The 4-day-old zebrafish were purchased from Eze-Rinka Company (Nanjing, China) and cultured in E3 medium on a light and dark cycle (13/11 h). Five experimental groups were established with different treatments, the same as cell imaging. In the first experimental group, zebrafish were only stained with probe CDS-NBD ( 13   After stirring at room temperature for 4 h, the resulting solution was extracted three times with CH 2 Cl 2 and washed to neutrality with saturated salt water. The combined organic layers were subjected to concentrate under reduced pressure and purified on silica gel chromatography with CH 2 Cl 2 /EtOAc (20:1) as eluent to obtain a faint yellow powder (51.9 mg, 72.1%). 1

Conclusions
In summary, we constructed a novel fluorescent probe CDS-NBD based on 7-HCA for the detection of Cys/Hcy and GSH through two different emission channels by using DNBS and NBD as the highly sensitive and selective dual response sites for thiols. CDS-NBD had a large separation of the emission wavelengths, fast response, notable fluorescence enhancement, and excellent sensitivity and selectivity. Importantly, probe CDS-NBD could make a distinction between GSH (blue fluorescence) and Cys/Hcy (mixed blue-green fluorescence) by two different fluorescent signals. Furthermore, dual-color fluorescence imaging experiments in living HeLa cells and zebrafish also demonstrated that CDS-NBD could serve as a useful tool for the detection of Cys/Hcy and GSH.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/bios12111056/s1, Table S1: The reported fluorescent probes based on dual-site for thiols, Figure S1: UV-vis absorption spectra of probe CDS-NBD (black) and reacted with Cys (red), Hcy (green) and GSH (blue) in PBS buffer, Figure S2: UV-vis absorption (black) and fluorescence (blue) spectra of 7-HCA in PBS buffer, Figure S3: UV-vis absorption (black) and fluorescence (green) spectra of NBD-N-Bu in PBS buffer, Figure S4  Data Availability Statement: All data generated or analyzed during this study are included in this published article (and its Supplementary Information files) or are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.